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How can Chronic myeloid Leukaemic drugs reduce the production of the Philadelphia genotype?

How can Chronic myeloid Leukaemic drugs reduce the production of the Philadelphia genotype?


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How can Chronic Myeloid Leukaemic drugs (Tyrosine kinase inhibitors, e.g. imatinib, etc.) that act by inhibiting bind of ATP to the active site of the BCR-ABL1 protein actually reduce the prevalence of the Philadelphia chromosome? (For example, optimal response to TKIs in 3 months would be Ph+ <35% and/or BCR-ABL1 <10%.) What I am actually asking is how a drug that targets the result of the oncogene (the BCR-ABL1 protein) can have an effect on the source (ie. genotype of the cells), as CML is a clonal haematopoietic stem cell disorder?

An example Reference paper: Apperley JF. Chronic myeloid leukaemia. Lancet. 2015 Apr 11;385(9976):1447-59. doi: 10.1016/S0140-6736(13)62120-0. Epub 2014 Dec 5. PMID: 25484026.


The key is that TKIs, such as Imatinib, are a therapy rather than a cure

Per Wikipedia, Imatinib acts as a competitive inhibitor of the otherwise constitutively-active BCR-ABL fusion protein, rather than the fusion gene. By binding the kinase domain, BCR-ABL is unable to phosphorylate downstream effectors that result in unchecked proliferation. As long as a patient continues to take Imatinib, BCR-ABL activity should be blocked.

In the past few years (i.e, long after imatinib's discovery) there has been a lot of research into exactly how hematopoetic stem cells, which reside in the marrow and create leukemic blasts, work. Most HSCs remain in a quiescent state, but it's not particularly well-known how frequently they turn over. Recent CML studies have shown that after ~5 years, a large portion of CML patients can safely taper the the drug. Presumably this is related to HSC turnover, but frankly as a leukemia molecular biologist rather than a physician, I'm not the best font of clinical knowledge.


Chronic myeloid leukemia: mechanisms of blastic transformation

1 Department of Molecular Virology, Immunology and Medical Genetics and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA. 2 Division of Hematology-Oncology, Department of Internal Medicine, University of California at San Diego, La Jolla, California, USA. 3 Department of Haematology, Imperial College at Hammersmith Hospital, London, United Kingdom. 4 Department of Microbiology and Immunology, Temple University, Philadelphia, Pennsylvania, USA.

Address correspondence to: Danilo Perrotti, Department of Molecular Virology, Immunology and Medical Genetics and Comprehensive Cancer Center, The Ohio State University, 892 Biomedical Research Tower, 460 West 12th Avenue, Columbus, Ohio 41230, USA. Phone: 614.292.3255 Fax: 614.688.4181 E-mail: [email protected] Or to: Tomasz Skorski, Department of Microbiology and Immunology, School of Medicine, Temple University, 3400 N. Broad Street, MRB 548, Philadelphia, Pennsylvania 19140, USA. Phone: 215.707.9157 Fax: 215.707.9160 E-mail: [email protected]

Find articles by Perrotti, D. in: JCI | PubMed | Google Scholar

1 Department of Molecular Virology, Immunology and Medical Genetics and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA. 2 Division of Hematology-Oncology, Department of Internal Medicine, University of California at San Diego, La Jolla, California, USA. 3 Department of Haematology, Imperial College at Hammersmith Hospital, London, United Kingdom. 4 Department of Microbiology and Immunology, Temple University, Philadelphia, Pennsylvania, USA.

Address correspondence to: Danilo Perrotti, Department of Molecular Virology, Immunology and Medical Genetics and Comprehensive Cancer Center, The Ohio State University, 892 Biomedical Research Tower, 460 West 12th Avenue, Columbus, Ohio 41230, USA. Phone: 614.292.3255 Fax: 614.688.4181 E-mail: [email protected] Or to: Tomasz Skorski, Department of Microbiology and Immunology, School of Medicine, Temple University, 3400 N. Broad Street, MRB 548, Philadelphia, Pennsylvania 19140, USA. Phone: 215.707.9157 Fax: 215.707.9160 E-mail: [email protected]

Find articles by Jamieson, C. in: JCI | PubMed | Google Scholar

1 Department of Molecular Virology, Immunology and Medical Genetics and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA. 2 Division of Hematology-Oncology, Department of Internal Medicine, University of California at San Diego, La Jolla, California, USA. 3 Department of Haematology, Imperial College at Hammersmith Hospital, London, United Kingdom. 4 Department of Microbiology and Immunology, Temple University, Philadelphia, Pennsylvania, USA.

Address correspondence to: Danilo Perrotti, Department of Molecular Virology, Immunology and Medical Genetics and Comprehensive Cancer Center, The Ohio State University, 892 Biomedical Research Tower, 460 West 12th Avenue, Columbus, Ohio 41230, USA. Phone: 614.292.3255 Fax: 614.688.4181 E-mail: [email protected] Or to: Tomasz Skorski, Department of Microbiology and Immunology, School of Medicine, Temple University, 3400 N. Broad Street, MRB 548, Philadelphia, Pennsylvania 19140, USA. Phone: 215.707.9157 Fax: 215.707.9160 E-mail: [email protected]

Find articles by Goldman, J. in: JCI | PubMed | Google Scholar

1 Department of Molecular Virology, Immunology and Medical Genetics and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA. 2 Division of Hematology-Oncology, Department of Internal Medicine, University of California at San Diego, La Jolla, California, USA. 3 Department of Haematology, Imperial College at Hammersmith Hospital, London, United Kingdom. 4 Department of Microbiology and Immunology, Temple University, Philadelphia, Pennsylvania, USA.

Address correspondence to: Danilo Perrotti, Department of Molecular Virology, Immunology and Medical Genetics and Comprehensive Cancer Center, The Ohio State University, 892 Biomedical Research Tower, 460 West 12th Avenue, Columbus, Ohio 41230, USA. Phone: 614.292.3255 Fax: 614.688.4181 E-mail: [email protected] Or to: Tomasz Skorski, Department of Microbiology and Immunology, School of Medicine, Temple University, 3400 N. Broad Street, MRB 548, Philadelphia, Pennsylvania 19140, USA. Phone: 215.707.9157 Fax: 215.707.9160 E-mail: [email protected]

Find articles by Skorski, T. in: JCI | PubMed | Google Scholar

The BCR-ABL1 oncoprotein transforms pluripotent HSCs and initiates chronic myeloid leukemia (CML). Patients with early phase (also known as chronic phase [CP]) disease usually respond to treatment with ABL tyrosine kinase inhibitors (TKIs), although some patients who respond initially later become resistant. In most patients, TKIs reduce the leukemia cell load substantially, but the cells from which the leukemia cells are derived during CP (so-called leukemia stem cells [LSCs]) are intrinsically insensitive to TKIs and survive long term. LSCs or their progeny can acquire additional genetic and/or epigenetic changes that cause the leukemia to transform from CP to a more advanced phase, which has been subclassified as either accelerated phase or blastic phase disease. The latter responds poorly to treatment and is usually fatal. Here, we discuss what is known about the molecular mechanisms leading to blastic transformation of CML and propose some novel therapeutic approaches.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by excessive accumulation of apparently normal myeloid cells. It occurs with an annual incidence of 1.0–1.5 per 100,000 persons. CML occurs very rarely in children. In the Western world, the median age of onset is 50–60 years, which reflects the average age of the population. Although symptoms at presentation may include lethargy, weight loss, unusual bleeding, sweats, anemia, and splenomegaly, in more developed countries, 50% of patients are asymptomatic and are diagnosed as a consequence of blood tests performed for unrelated reasons. More than 90% of CML patients are diagnosed when their disease is in a relatively early phase known as the chronic phase (CP).

CML-CP is characterized by the presence of the Philadelphia (Ph) chromosome and the oncogene that it encodes in the vast majority of myeloid cells and some lymphocytes. The Ph chromosome results from a (922)(q34q11) reciprocal translocation that juxtaposes the c-abl oncogene 1 (ABL1) gene on chromosome 9 with the breakpoint cluster region (BCR) gene on chromosome 22, generating the BCR-ABL1 fusion oncogene with greatly enhanced ABL1 kinase activity. It is generally accepted that acquisition of the BCR-ABL1 oncogene is the initiating event in the genesis of CML-CP, despite various lines of evidence suggesting that, at least in some cases, hematopoiesis may already be clonal before the acquisition of the Ph chromosome ( 1 ). It is believed that acquisition of the BCR-ABL1 gene occurs initially in a single HSC that gains a proliferative advantage and/or aberrant differentiation capacity over its normal counterparts, giving rise to the expanded myeloid compartment ( 2 ).

Most CML-CP patients are currently treated with one of three drugs designed to block the enzymatic action of the BCR-ABL1 tyrosine kinase. The first of these to be developed was imatinib. Recent karyotype analyses show that 60%–70% of patients achieve complete disappearance of Ph-positive marrow cells and maintain exclusively Ph-negative bone marrow cells (a state designated as a complete cytogenetic response [CCyR]) 5 years after initiating imatinib treatment. The incidence of progression to a more advanced phase of leukemia in patients responding to imatinib is extremely low beyond the first two years ( 3 ). However, a small number of patients fail to respond to imatinib (primary resistance), while others respond initially and then lose their response (secondary resistance) ( 4 ). The reasons for imatinib resistance in CML-CP patients are poorly understood. Primary resistance may be related, at least in part, to the intrinsic heterogeneity of the disease (e.g., different BCR-ABL1 levels) in different patients and to the survival of variable numbers of quiescent cells from which the more mature leukemia cells are derived during CP ( 5 ). Secondary resistance may have a wide range of causes, of which the best characterized is the acquisition of mutations in the BCR-ABL1 kinase domain (such as the T315I mutation) ( 6 ).

In the last few years, two new tyrosine kinase inhibitors (TKIs), dasatinib and nilotinib, have become available, both of which are more potent in vitro inhibitors of the BCR-ABL1 kinase than imatinib. Both of these “second-generation” TKIs are effective at inducing or restoring CCyR in 40%–50% of patients who appear to have failed primary treatment with imatinib. However, approximately 20% of patients presenting with CML-CP fail to respond to both imatinib and a subsequent second-generation TKI their prognosis is poor because of a higher risk of disease progression.

Before the advent of BCR-ABL1 TKIs, all patients with CML-CP progressed spontaneously to advanced phase CML after a median interval of approximately 5 years. The advanced phase is divided into an initial accelerated phase (AP), during which patients may still respond to treatment for some months or sometimes years, and a subsequent more aggressive blastic phase (BP). Patients with CML-BP have a median survival of approximately 6 months. Some patients progress directly to BP without an intermediate AP. The precise definitions of these three phases have been much debated in recent years ( 3 , 7 ).

The biological basis of BP is poorly understood. Although the majority of patients have a myeloblastic phenotype, approximately 25% of CML-BP patients show a pre–B lymphoblastic cell phenotype ( 8 ). Occasional cases of T lymphoblastic transformation have been identified ( 9 ). These findings lend support to the notion that the BCR-ABL1 oncogene arises in a primitive cell, namely a leukemia stem cell (LSC), not yet committed to either myeloid or lymphoid differentiation. Conversely the blastic clone may originate either at the level of the multipotent LSC or at the level of a more committed leukemia progenitor cell (LPC). Here, we discuss genetic and epigenetic mechanisms leading to the transition of CML-CP into CML-BP and propose some novel therapeutic modalities that might prevent malignant progression.

In the past, CML-BP was often treated with drugs used for acute leukemias, but patients usually relapsed within a few months. The introduction of TKIs has improved prognosis to some degree. The majority of CML-BP patients not previously treated with TKIs do initially respond to treatment with these agents, either alone or in combination with conventional chemotherapeutic drugs, but most still relapse within a few months of achieving a seemingly complete hematologic or even cytogenetic response. Therefore, any CML-BP patient who does respond to modern therapy should proceed, if possible, to allogeneic stem cell transplant prior to relapse. In the 1990s, the results of allografting for CML-BP patients were not impressive — only 5%–10% of patients experienced long-term, leukemia-free survival ( 10 ) — but the use of a TKI after transplant may improve these results.

Extrapolating from the good clinical outcomes of treating CML-CP with TKIs and the dismal responses achieved in treating CML-BP, one might reasonably conclude that the best approach to CML-BP would be prevention. Indeed, it appears today that continued use of TKIs to treat CML-CP may prevent BP in a large proportion of patients, but 15%–20% of patients, most of whom will have been classified as nonresponders, may progress to BP ( 11 ). Indeed, the GIMEMA Working Party (Italian Group for Adult Hematologic Diseases) reported that the detection of TKI-resistant BCR-ABL1 mutations in CML-CP is associated with a greater likelihood of disease progression ( 12 ). These patients may possess genetic/epigenetic abnormalities distinct from the patients with nonmutated BCR-ABL1, the appearance of which could be influenced by the duration of the BCR-ABL1–induced signals. Furthermore, the ability of TKIs to render residual CML cells “inactive” rather than to eradicate them entirely suggests that BP might still occur occasionally even in “responding” patients.

However, as a minority of patients will still progress to CML-BP, the routine use of TKIs may need to be supplemented with other agents, any of which might prevent BP. Possible examples are antioxidants ( 13 ), which protect against cancer-causing DNA mutations farnesyl transferase inhibitors ( 14 ), which inhibit RAS signaling hydroxychloroquine ( 15 ), which inhibits autophagy in some situations sonic hedgehog pathway antagonists ( 16 , 17 ), which impair self-renewal pathways only when used in combination with TKIs and activators of protein phosphatase 2A (PP2A) ( 18 ), which targets BCR-ABL1 and other downstream oncogenic signaling cascades.

At present, the molecular mechanisms underlying disease progression are still uncertain, but most likely involve activation of oncogenic factors and/or inactivation of tumor suppressors ( 19 ). A plausible assumption is that BP is a multistep, time-dependent process initiated by both BCR-ABL1–dependent and –independent DNA damage associated with inefficient and unfaithful DNA repair in CML-CP that, if facilitated by an increased level of BCR-ABL1 activity, leads to selection of one or more CML-BP clones.

The genetic lesions observed in CML-BP patients in the past and now since the introduction of TKIs mostly include the presence of additional chromosomes, gene deletions, gene insertions, and/or point mutations (including BCR-ABL1 mutations) ( 20 – 22 ), but patterns differ in myeloblastic and lymphoblastic transformations ( 23 ). At the molecular level, the most common mutations detectable (other than those in the BCR-ABL1 kinase domain) occur at the loci of the tumor suppressor genes P53 (20%–30% of cases) and the runt-related transcription factor gene (RUNX1) (38% of cases) in myeloid BP and at the loci of cyclin-dependent kinase inhibitor 2A/2B (CDKN2A/B) (50% of cases) and Ikaros transcription factor (IKZF1) (55% of cases) in lymphoid BP ( 22 , 24 – 28 ). As specific CML-BP–associated genetic alterations are relatively common, no one lesion occurs in the majority of CML-BP patients, and it is unlikely that any one specific secondary genetic aberration can be defined as the “culprit” causing disease progression. More likely, CML-BP results from the accumulation of a critical number or combination of different mutations.

Epigenetic changes are dependent mostly on the pleiotropic effect of constitutive BCR-ABL1 activity ( 19 , 29 ), the levels of which start to increase in CML-AP ( 30 ). In support of this suggestion, expression studies revealed that BCR-ABL1 dramatically perturbs the CML transcriptome ( 31 ), resulting in altered expression of genes, some of which (e.g., PRAME, MZF1, EVI-1, WT1, and JUN-B) might play a role in BP ( 19 , 32 – 34 ). Nonetheless, the posttranscriptional, translational, and posttranslational effects of high BCR-ABL1 levels result in the constitutive activation of factors with reported mitogenic, antiapoptotic, and antidifferentiation activity (e.g., MAPK ERK1/2 , MYC, JAK2, YES-1, LYN, hnRNP-E2, MDM2, STAT5, BMI-1, and BCL-2) and inhibition of major key regulators of cellular processes, such as those regulated by the tumor suppressors p53, CCAAT/enhancer binding protein-α (C/EBPα), and PP2A ( 19 , 29 , 35 ). Interestingly, a signature based on six genes (NOB1, DDX47, IGSF2, LTB4R, SCARB1, and SLC25A3) was recently found to accurately discriminate early from late CP, CP from AP, and CP from BP ( 36 ) however, the biological role of these genes in disease progression is still unknown.

Thus, it is highly plausible that unrestrained and increasing BCR-ABL1 activity promotes and/or contributes to clonal evolution, thereby leading to CML-BP ( 37 ). This might occur at the level of LSCs, which display innate or acquired TKI resistance, and/or at the level of an LPC population that might have developed resistance and expanded during TKI therapy ( 38 , 39 ).

Because there is a direct correlation between levels of BCR-ABL1, the frequency of clinically relevant BCR-ABL1 mutations ( 40 , 41 ), and the differentiation arrest of myeloid progenitors ( 42 ), it is likely that disease progression is triggered by the “right” combinations of genetic and epigenetic abnormalities (Figure 1). Thus, we can speculate that prevention or effective treatment of CML-BP will only be achieved if novel therapeutic strategies can be developed that are capable of interfering with the biological processes currently considered critical for the leukemic behavior of CML-BP progenitors.

BCR-ABL1–dependent pathways to blastic transformation. Schematic representation of the potential BCR-ABL1–dependent molecular mechanisms leading to CML disease progression.The relatively high BCR-ABL1 expression/activity in CML-CP CD34 + CD38 – stem cells and/or CD34 + early progenitors compared with more committed progenitors, which is further markedly increased in CML-BP CD34 + progenitors results in the following: enhancement of proliferation/survival pathways increased genomic instability and activation of pathways leading to a block in myeloid differentiation, acquisition of the ability to self renew, and inhibition of tumor suppressors with broad cell regulatory functions. BAD, BCL2 antagonist of cell death DNA-PKcs, DNA-dependent protein kinase, catalytic subunit FOXO, forkhead box O IK6, Ikaros 6 miR-328, microRNA-328 MLH1, mutL homolog 1 PMS2, postmeiotic segregation increased 2 RAD51, RecA homolog in Escherichia coli RAD52, RAD52 homolog (Saccharomyces cerevisiae) Shh, Sonic Hedgehog wnt/β-catenin, wingless-int1/beta-catenin.

According to the basic concept, LSCs should represent the most primitive cell able to initiate leukemia in animal xenograft limiting dilution experiments, to display self-renewal capacity, and to proliferate and differentiate ( 43 , 44 ). In CML-CP, LSCs are located in the self-renewing Lin – CD34 + CD38 – population, but not in the non–self-renewing Lin – CD34 + CD38 + population ( 45 ), indicating that, in contrast to other oncogenes (e.g., MOZ-TIF2 and MLL-ENL) BCR-ABL1 cannot confer self-renewal properties ( 46 , 47 ). While most human LSC research has focused on the Lin – CD34 + compartment, a recent study suggests that a Lin – CD34 – fraction of CML-CP cells also engrafts immunodeficient mouse strains, underscoring the complexity of the LSC compartment ( 48 ).

However, in CML-BP, Lin – CD34 + CD38 + granulocyte-macrophage progenitors (GMPs) that overexpress BCR-ABL1 behave like LSCs ( 49 ), suggesting that the acquisition of self renewal in GMPs may depend on epigenetic and/or genetic alterations caused by elevated expression of BCR-ABL1. Thus, LSCs in CML-BP patients may reside in at least 3 different subsets: Lin – CD34 + CD38 – and Lin – CD34 + cells remaining from CML-CP and the disease-driving Lin – CD34 + CD38 + GMPs.

Progression to BP is marked by overexpression of BCR-ABL1 in committed progenitors, leading to a multiplicity of genetic and epigenetic events. These cell type– and context-specific molecular events serve to enhance survival and self renewal, leading to impaired differentiation and generation of CML-BP LSCs. To date, the cell type– and context-specific effects of BCR-ABL1 overexpression have not been clearly elucidated in human stem cells, nor has the effect of the microenvironment on LSC maintenance. It seems, however, that increased BCR-ABL1 expression does play a critical role in promoting the genetic instability that drives progression to BP and the molecular evolution of LSCs in CML.

BCR-ABL1 overexpression and aberrant splicing. BCR-ABL1 induces alterations in pre-mRNA splicing in hematopoietic progenitor cells that result in aberrant adhesion, differentiation, survival, and self renewal as well as therapeutic resistance. Ectopic BCR-ABL1 expression in human bone marrow and cord blood CD34 + cells results in induction of factors involved in mRNA processing, export, and translation ( 50 , 51 ). Interestingly, the metabolism of several mRNAs has been found to be altered in CML-BP ( 51 ). Among these, BCR-ABL1 induces alternative splicing of proline-rich tyrosine kinase 2 (PYK2) mRNA, thereby increasing expression of the β1-integrin–responsive PYK2 kinase, which in turn may contribute to aberrant adhesion of CML-BP progenitors ( 50 ).

Likewise, BCR-ABL1–induced aberrant splicing might play an important role in those cases of CML-BP without deletion of the IKZF1 gene ( 25 ). Indeed, a recent study suggests that BCR-ABL1 may inhibit differentiation and contribute to lymphoid CML-BP by promoting the production of a dominant negative splice isoform (IK6) of IKZF1, a transcription factor gene involved in pre–B cell differentiation ( 52 ). When this aberrant, non–DNA-binding splice isoform, IK6, was silenced in Ph-positive pre–B cells using siRNA or its production reduced by imatinib treatment, differentiation along the B cell lineage was partially restored ( 52 ). Notably, alternative splicing was also observed for BCR-ABL1. Aberrant BCR-ABL1 mRNA splicing results in the generation of transcripts harboring a 35-kb insertion between ABL1 domain exons 8 and 9, resulting in a frameshift with a truncation that, like IK6 expression, is associated with imatinib resistance ( 53 , 54 ).

Finally, BCR-ABL1 overexpression is associated with mis-splicing of glycogen synthase kinase 3β (GSK3β) ( 55 ), a key component of the β-catenin destruction complex, leading to enhanced self renewal of GMPs that behave like LSCs ( 49 ). Lentiviral overexpression of wild-type GSK3β in CML-BP progenitors inhibits their capacity to engraft leukemia in immunocompromised mice ( 55 ).

Stem cell self renewal. Self renewal refers to division without differentiation and is a property normally ascribed to long-term HSCs. In mouse models, loss of junB/AP-1 enhances HSC proliferation and myeloid progenitor expansion, setting the stage for BP ( 56 ). In CML-BP, committed progenitors subvert this stem cell property of self renewal, lack the capacity to regulate it, and are able to propagate leukemia more readily. Various self-renewal pathways — including Wnt/β-catenin, sonic hedgehog, and Notch signaling — have been implicated in the generation and maintenance of CML-BP LSCs. Indeed, BCR-ABL1–independent ( 57 ) and –dependent ( 49 , 58 ) mechanisms both seem to contribute to the acquisition of self renewal by CD34 + CD38 + CD45RA + CD123 + Lin – CML-BP GMPs. In fact, CML-BP is associated with accumulation of β-catenin, a key stem cell self-renewal mediator, in the nucleus of GMPs, thereby endowing them with self-renewal potential ( 49 , 59 ). BCR-ABL1 stabilizes β-catenin through phosphorylation of tyrosines 86 and 654, which inhibits binding to axin/GSK3β, thereby enabling binding to T cell factor 4 (TCF4) and activation of transcription.

By inhibiting BCR-ABL1, imatinib prevents tyrosine phosphorylation of β-catenin and thus prevents nuclear translocation and transcriptional activation ( 58 ). Loss of β-catenin in a CML mouse model impairs self renewal of both normal HSCs and CML-BP LSCs, although the effects of decreased nuclear β-catenin on human normal HSC and CML-BP LSC maintenance remains to be established in xenograft models ( 60 ).

Decreased expression of functional GSK3β enhances CML progenitor self renewal by activating β-catenin and by elevating levels of sonic hedgehog pathway–mediators such as GLI family zinc finger 1 (GLI1) and GLI2 ( 32 , 55 ). Recently, two independent studies demonstrated that overexpression of smoothened homolog (Drosophila) (Smo), an essential activator of sonic hedgehog signaling, enhanced LSC maintenance in mouse models of CML ( 16 , 17 ). Conversely, Smo inhibition reduced LSC, but not normal HSC engraftment ( 16 , 17 ), suggesting that the sonic hedgehog pathway is preferentially utilized by LSCs for self renewal.

Another recent study confirmed that sonic hedgehog signaling is dispensable for normal adult mouse HSC function, suggesting the possibility of targeting leukemic GMP without damaging residual HSCs ( 61 ). These findings provide the impetus for preclinical testing of a combination of Smo and BCR-ABL1 inhibitors to determine whether LSCs can be eradicated both in vitro and in xenogeneic transplantation models.

LSC survival. Resistance to apoptosis, an intrinsic property of normal HSCs, is also a hallmark of LSCs. In vivo inactivation of Dok-1 or Dok-2 decreases apoptosis, resulting in a myeloproliferative disorder ( 62 ). Moreover, the promyelocytic leukemia (PML) gene, a tumor suppressor that was first shown to be deregulated in acute promyelocytic leukemia, was recently found to play a pivotal role in LSC maintenance in a CML mouse model ( 63 ). Other investigators demonstrated that enhanced progenitor cell survival driven by B cell leukemia/lymphoma 2 (BCL2) and BCR-ABL1 overexpression promoted CML-BP development in a transgenic mouse model ( 64 ), underscoring the importance of resistance to apoptosis in BP evolution.

Although extensive SNP marker analyses demonstrated that a SNP (rs1801018) in the BCL2 gene was associated with susceptibility to CML ( 65 ), the role of BCL2 in CML-BP progenitor survival remains to be elucidated. In CML-BP cell lines, expression levels of BCL2-interacting mediator of cell death (BIM), a proapoptotic BCL2 family member, are low and can be induced by BCR-ABL1 inhibition ( 66 ). In recent studies, induction of apoptosis correlated with the magnitude and duration of BCR-ABL1 kinase inhibition ( 67 ). Transient, potent BCR-ABL1 inhibition was associated with BIM activation and induction of apoptosis, underscoring the importance of BCR-ABL1 gene dosage in regulating apoptotic responses ( 67 ). In addition, JAK2-mediated activation of LYN kinase through the suppressor of variegation, enhancer of zeste, and Trithorax (SET)/PP2A/SHP1 pathway ( 68 ) may be important in promoting CML-BP LSC survival during imatinib therapy and disease progression. Pharmacologic inhibition of JAK2 induced apoptosis in imatinib-resistant CML-BP cells to a greater degree than in normal progenitors ( 68 ). Recently, targeted inhibition of arachidonate 5-lipoxygenase (ALOX5) with a 5-lipoxygenase inhibitor was shown to impair LSC survival in a CML mouse model, a finding that warrants further investigation into the role of ALOX5 in CML-BP pathogenesis ( 69 ).

Interestingly, a recent study has shown that imatinib induces autophagy in CML-BP primitive progenitors through a mechanism that is independent of imatinib-induced, caspase-dependent apoptosis but is associated with ER stress and is suppressed by intracellular Ca 2+ depletion ( 15 ). Suppression of autophagy genes enhanced imatinib-induced death of Ph-positive cells ( 15 ). Critically, the combination of TKIs with autophagy inhibitors resulted in killing of CML LSCs ( 15 ). Thus, autophagy inhibitors may enhance the therapeutic effects of TKIs in the treatment of CML-BP.

Since the late 1980s, when BCR-ABL1 was identified as a constitutively active tyrosine kinase, an impressive series of in vitro and in vivo studies have indicated a direct causal role of BCR-ABL1 activity in the acquisition of the molecular changes that characterize the phenotype of CML-BP progenitors ( 19 ).

In vivo resistance and in vitro sensitivity of CML-BP progenitors to TKI treatment: a biological paradox. Compelling research shows that CML-CP LSCs are resistant to imatinib as a result of various collaborating factors. These factors include quiescence, high BCR-ABL1 levels, lack of “oncogene addiction,” increased activity of the drug efflux pumps ATP-binding cassette sub-family B member 1 (ABCB1) and transporter G2 (ABCG2), and decreasing organic cation transporter 1 (OCT1) expression ( 5 , 70 , 71 ).

In CML-BP, increased BCR-ABL1 expression ( 49 , 72 ) accounts for activation of pathways transducing mitogenic, antiapoptotic signals and for differentiation arrest of the Ph-positive progenitors ( 42 , 49 , 73 , 74 ). However, BCR-ABL1–independent mechanisms (e.g., LYN kinase–dependent mechanisms) also contribute to disease progression and imatinib resistance in some CML-BP cases with no BCR-ABL1 amplification/overexpression ( 75 – 78 ). In this regard, the paradoxical in vitro and in vivo response of CML-BP progenitors to TKIs needs to be taken into consideration. While most CML-BP patients do not show long-term responses to TKIs and relapse within 12–24 months, CML-BP progenitors from these patients are still sensitive to the proapoptotic effects of imatinib when administered ex vivo. Thus, it is possible that the bone marrow environment elicits BCR-ABL1–independent signals conferring TKI resistance and sustaining in vivo survival of CML-BP blasts. In this scenario, BCR-ABL1–dependent and –independent signals likely synergize in inducing and maintaining the CML-BP phenotype. Furthermore, from this consideration, the concept emerges clearly that TKI treatment, especially at high dosage, might exert a selective pressure allowing clonal expansion of genetically unstable CML-BP progenitor cell clones that are more prone to acquire secondary chromosomal abnormalities and/or clinically relevant mutations in the BCR-ABL1 oncogene itself and, likely, in other kinases targeted by TKIs. As CML-BP is also characterized by the loss of function of tumor suppressors, a rational and alternative therapeutic approach might envision the use of drugs capable of reactivating a tumor suppressor or suppressors.

Pharmacologic reactivation of the PP2A tumor suppressor gene. The notion that the serine-threonine phosphatase PP2A is inhibited in several types of cancer, through mechanisms that either involve the loss of expression/activity of one or more subunits or the enhanced expression of the endogenous PP2A inhibitors SET ( 79 , 80 ) and cancerous inhibitor of PP2A (CIP2A) ( 81 ), led to the recognition of PP2A as a true tumor suppressor. In fact, loss of PP2A activity plays a central role in the pathophysiology of BCR-ABL1–driven leukemias. PP2A activity is slightly reduced in CML-CP CD34 + progenitors but becomes markedly inhibited in CML-BP through the BCR-ABL1 dose- and kinase-dependent induction of SET ( 74 , 82 ) (Figure 2). Remarkably, several targets that are shared by BCR-ABL1 and PP2A are either essential for BCR-ABL1 leukemogenesis or are altered in CML-BP ( 19 ).

BCR-ABL1 and PP2A interplay. (A) In CML-BP and Ph-positive ALL CD34 + progenitors, p210 and p190 BCR-ABL1 oncoproteins inhibit PP2A activity by inducing hnRNP-A1, which, in turn, enhances expression of SET. In BCR-ABL–positive myeloid progenitor cells, suppression of PP2A phosphatase activity is required for sustained activation of mitogenic and survival signals. (B) Restored PP2A activity, achieved by treatment with PP2A activators (e.g., Forskolin or FTY720), impairs in vitro and in vivo wild-type and T315I BCR-ABL1 leukemogenesis by antagonizing the effects of BCR-ABL1 on its downstream signal transducers (not shown) and promoting SHP-1–mediated BCR-ABL1 inactivation and proteasome-dependent degradation.

Restoration of PP2A activity, either by chemical PP2A activators (e.g., forskolin and FTY720) (Figure 2) or by interfering with SET/PP2A interplay, promotes Src homology region 2–domain phosphatase 1 (SHP-1) tyrosine phosphatase–dependent BCR-ABL1 dephosphorylation (inactivation) which, in turn, triggers its degradation ( 74 , 82 , 83 ). Notably, SHP-1 expression is diminished in most leukemias and lymphomas ( 84 , 85 ). Restoring normal PP2A activity induces marked apoptosis of CD34 + CML (CP and BP) progenitors and suppresses in vivo leukemogenesis regardless of sensitivity to imatinib/dasatinib ( 74 , 82 ) (Figure 2).

Loss of PP2A activity is also a feature of imatinib/dasatinib-insensitive CD34 + CD38 – BCR-ABL1 + HSCs from CML (CP and BP) patients ( 57 ). Clonogenic, colony-forming cell (CFC)/replating, long-term culture–initiating cell (LTC-IC), and CFSE-mediated cell division–tracking assays revealed that FTY720 suppresses survival and self renewal and triggers apoptosis of BCR-ABL1 + stem cells in a BCR-ABL1 kinase–independent and β-catenin–mediated manner ( 57 ). Notably, normal quiescent stem cells are not sensitive to FTY720 ( 57 ).

Because of the central role of PP2A in the regulation of survival, proliferation, self renewal, and differentiation of CML stem/progenitor cells, it is highly plausible that its loss of function contributes to BP. In this scenario, PP2A may have the role of a “gatekeeper,” as its activation may control and restrain BCR-ABL1 expression/activity, whereas its inhibition allows increased oncogene activity and induces a cascade of events that promotes disease development and progression. Thus, incorporating PP2A-activating drugs into current therapeutic protocols for CML-BP and imatinib/dasatinib-resistant (including T315I) patients has not only the potential to treat CML-BP but also to eradicate CML at the stem cell level.

Blastic transformation is phenotypically associated with the clonal expansion of the GMP pool ( 49 ), members of which have lost the ability to differentiate in response to cytokine stimuli. If we exclude the 20%–30% of CML-BP cases with P53 mutations ( 28 ), the 11% of CML-BP cases with GATA-binding protein 2 (GATA2) mutations ( 86 ), and the 1%–2% of CML-BP cases with the t(321)(q26q22) and t(711)(p15p15) translocations associated with expression of AME (AML-1 [acute myeloid leukemia 1], MDS/EVI1 [Myelodysplastic syndrome–associated gene 1]) ( 87 ) and NUP98-HOXA9 ( 88 ) chimeric proteins, we can safely state that impaired myeloid maturation of Ph-positive GMPs is the consequence of increased BCR-ABL1 dosage. Indeed, low BCR-ABL1 levels allow G-CSF–induced granulocytic maturation, while high oncogene expression impedes differentiation of Lin – progenitors ( 89 ).

BCR-ABL1 levels and C/EBPα inhibition. Different genetic and epigenetic mechanisms may act alone or in cooperation to enhance BCR-ABL1 expression and activity. Among them, BCR-ABL1 gene amplification ( 90 , 91 ), increased BCR promoter activity ( 92 ), decreased miR-203 expression ( 93 ), impaired PP2A activity ( 74 ), and genetic/epigenetic inhibition of SHP-1 phosphatase ( 74 , 94 ) may all account for increased BCR-ABL1 expression/activity observed during disease progression ( 72 ). Interestingly, restoration of PP2A activity in myeloid precursors expressing high BCR-ABL1 levels restores G-CSF–driven differentiation ( 74 ), suggesting that PP2A loss of function might play a central role in impairing maturation of Ph-positive GMPs.

The inhibitory effect of high BCR-ABL1 levels on differentiation depends on marked downregulation of C/EBPα ( 42 ), a transcription factor essential for granulocytic differentiation. The importance of the loss of C/EBPα activity as a central mechanism leading to differentiation arrest of myeloid CML blasts is supported by evidence that ectopic C/EBPα expression induces maturation of differentiation-arrested BCR-ABL1 + myeloid precursors and CD34 + CML-BP progenitors ( 42 , 95 , 96 ) and that a CML-BP–like process emerges in mice transplanted with BCR-ABL1–transduced Cebpa-null fetal liver cells ( 97 ). In CD34 + CML-BP GMPs, loss of C/EBPα does not depend on CEBPA gene mutations ( 98 ), but results from the BCR-ABL1 dose–dependent induction of the RNA-binding protein heterogenous nuclear ribonucleoprotein E2 (hnRNP-E2) that, upon interaction with the CEBPA upstream open reading frame (uORF)/spacer element, inhibits CEBPA translation ( 42 ). hnRNP-E2 expression is high in CD34 + CML-BP progenitors, where it suppresses C/EBPα and inhibits differentiation ( 42 ). Highlighting the importance of loss of C/EBPα expression in CML-BP, coexpression of BCR-ABL1 and AME also suppresses CEBPA translation and induces accumulation of blasts through activation of the CEBPA uORF-binding protein calreticulin ( 99 , 100 ). Notably, C/EBPβ is also repressed in CML-BP ( 101 ), suggesting that loss of C/EBP activity contributes to differentiation arrest and aggressive behavior of CML-BP cells. In this regard, suppression of C/EBP proteins in CML-BP may also depend on BCR-ABL1–induced preferentially expressed antigen in melanoma (PRAME) expression, which impairs myeloid differentiation when ectopically expressed in CD34 + progenitors ( 34 ).

The BCR-ABL1/hnRNP-E2/MAPK inhibitory pathway. The ability of hnRNP-E2 to suppress C/EBPα requires the constitutive activation of the MAPKs ERK1 and ERK2 ( 89 , 102 ), which directly increases hnRNP-E2 stability ( 89 ). This is consistent with the observation that enhanced expression of various RNA-binding proteins is among the many imatinib-sensitive changes found in myeloid CML-BP ( 51 ). The involvement of ERK1/2 in the regulation of hnRNP-E2 is not surprising, as constitutive MAPK activation is readily detectable in CD34 + CML-BP ( 102 ), while CML-CP progenitors show transient MAPK activation in response to mitogenic/survival signals induced by extracellular growth factors ( 103 ). Accordingly, levels of activated ERK1/2 in the absence of exogenous cytokines were similar in normal and CD34 + CML-CP progenitors and were not affected by imatinib ( 103 ). Graded BCR-ABL1 expression correlates with a progressive increase in ERK1/2 activity ( 102 ), and ERK1/2 suppression rescues C/EBPα expression and allows G-CSF–driven maturation of differentiation-arrested progenitors expressing high BCR-ABL1 levels ( 89 ). Thus, constitutive ERK1/2 activation in CML-BP is not only essential for transduction of mitogenic/survival signals but also promotes the activation of antidifferentiation signals leading to translational ( 42 ) and, perhaps, posttranslational ( 104 ) inactivation of C/EBPα. Notably, a decrease in monophosphorylated ERK2 in imatinib-responsive but not -resistant patients suggests that ERK signaling may be important for transformation of TKI-resistant CML ( 105 ).

miR-328: a molecular relay in CML disease progression. A few miRNAs are aberrantly regulated in CML ( 93 , 106 , 107 ), but their involvement in disease progression is unclear. Interestingly, the correct functioning of the BCR-ABL1/MAPK/hnRNP-E2 inhibitory axis requires the inhibition of miR-328, which, otherwise, would bind hnRNP-E2 and prevent its interaction with CEBPA mRNA, thus restoring CEBPA mRNA translation. Loss of miR-328 occurs in CD34 + CML-BP but not CML-CP myeloid progenitors, and forced miR-328 expression at levels resembling those observed in CML-CP rescues C/EBPα expression and reverses the CML-BP–like leukemia to a disease that resembles a myeloproliferative disorder in mice transplanted with BCR-ABL1–expressing myeloid precursors ( 108 ).

Genomic instability usually results from an aberrant cellular response to enhanced DNA damage. In CML cells, these mechanisms can be modulated by BCR-ABL1 kinase (Figure 3) or may be kinase-independent.

BCR-ABL1 regulates DNA damage and DNA repair, the 2 major components of genomic instability. BCR-ABL1–positive leukemia cells accumulate more DNA lesions, such as 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), and DNA DSBs induced by ROS, AID, and genotoxic agents (e.g., γ-radiation, cisplatin, mitomycin C, hydroxyurea, and UV light) in comparison with normal cells. In addition, BCR-ABL1 inhibits MMR and stimulates mutagenic NER to generate point mutations including those causing TKI resistance. Moreover, BCR-ABL1 activates unfaithful DSB repair mechanisms, HRR, NHEJ, and SSA, which contribute to chromosomal aberrations. The effect of BCR-ABL1 on base excision repair (BER) and O( 6 )-methylguanine–DNA methyltransferase (MGMT) is not known. Altogether, elevated levels of DNA damage combined with inefficient/unfaithful DNA repair cause genomic instability in CML-CP and facilitate CML-BP.

Enhanced DNA damage. Much endogenous DNA damage arises from ROS such as superoxide radical anion (•O2 – ), which may lead to the production of hydrogen peroxide (H2O2) and hydroxyl radical (•OH). BCR-ABL1–transformed cell lines and CD34 + CML cells contain, on average, 2–6 times more ROS than their normal counterparts (CML-BP > CML-CP > normal) ( 37 , 109 , 110 ) the mitochondrial respiratory chain, enhanced glucose uptake, and NADPH oxidase may play a role in this phenomenon ( 111 ). ROS can cause damage to all nucleobases and deoxyribose residues in DNA and free nucleotides, generating oxidized bases and DNA double-strand breaks (DSBs) ( 112 ). The number of oxidative “hits” to DNA per normal human cell per day is about 10 4 , and normal cells contain approximately 50 DSBs per cell per cell cycle. CD34 + CML cells display 3–8 times more oxidized nucleobases and 4–8 times more DSBs ( 37 , 109 , 110 ).

DNA damage could also be directly induced by ionizing radiation and genotoxic drugs, which are used as part of a conditioning regimen in hematopoietic transplantation for CML patients. BCR-ABL1–positive cells, in comparison with normal cells, accumulate more irradiation- and drug-induced DNA lesions, thus generating more chromosomal aberrations ( 113 , 114 ).

Unfaithful and inefficient DNA repair. Unfaithful and/or inefficient repair of ROS-induced oxidized DNA bases and DSBs may lead to a variety of point mutations and chromosomal aberrations ( 115 ). CD34 + CML cells display a malfunctioning mismatch repair (MMR) pathway, which can facilitate accumulation of point mutations ( 116 ) (Figure 3). BCR-ABL1 also promotes mutagenic nucleotide excision repair (NER) ( 117 ) and stimulates DSB repair, but the fidelity of the repair mechanisms (homologous recombination repair [HRR], nonhomologous end-joining [NHEJ], and single-strand annealing [SSA]) is compromised ( 37 , 110 , 118 , 119 ). In BCR-ABL1–positive cells, point mutations were introduced during usually faithful HRR, extensive nucleobase loss was associated with NHEJ, and enhanced SSA generated large deletions. Overexpression and tyrosine phosphorylation of RAD51, a key element in HRR responsible for strand invasion and pairing, may result in aberrant HRR. Deregulation of DNA ligase IIIα, Werner helicase/exonuclease, and Artemis may contribute to excessive loss of DNA bases during NHEJ in BCR-ABL1–positive cells.

Consequences of genomic instability in CML. Genomic instability is probably responsible for two major problems in CML: TKI resistance and disease progression ( 13 ). Both phenomena could be induced by accumulation of point mutations and additional chromosomal aberrations in CML-CP cells irreversibly changing their phenotype toward that in CML-BP.

BCR-ABL1 point mutations have been detected in 50%–90% of patients displaying resistance to imatinib, including approximately 23% of imatinib-naive patients ( 120 ). Moreover, second-generation TKI treatment in imatinib-resistant cases led to selection of additional resistance mutations ( 121 ).

TKI-resistant BCR-ABL1 mutants exhibit altered kinase activity, substrate utilization, and transformation potency and are associated with clonal cytogenetic evolution, which may have an impact on disease progression ( 120 , 122 ). Accordingly, BCR-ABL1 kinase mutations are associated with greater likelihood of disease progression, which suggests enhanced genomic instability in these cells ( 12 ).

Accumulation of various chromosomal aberrations and mutations is believed to be responsible for the transition of a relatively benign CP to aggressive BP ( 13 ). The frequency of additional chromosomal abnormalities is approximately 7% in CML-CP and increases to 40%–70% in the advanced phases of disease, as evaluated by standard cytogenetic analysis ( 23 ).

Numeric chromosomal changes are detected at a 50-fold higher frequency and structural changes at a 12-fold higher frequency in CML-BP, in comparison with CML-CP. More sensitive comparative genomic hybridization (CGH) and SNP analyses detect multiple genetic aberrations already in CP, but CML-BP patients have much more complex karyotypes ( 123 , 124 ). This observation suggests that genomic instability is an early event in CML. Patients from the pre-imatinib and imatinib era display similar types of genetic aberrations ( 125 ).

These aberrations involve acquisition of major alterations, such as the following: (a) the acquisition of additional chromosomes (e.g., +Ph, +8, +19) (b) the acquisition of isochromosome i(17q) (c) the acquisition of t(117), which is associated with loss of p53 (d) the acquisition of t(121), which affects RUNX1 (which is also known as AML1) (e) the acquisition of t(321), which generates the AML-1/EVI-1 fusion protein (a negative transcriptional regulator and cell signaling modulator) (f) the acquisition of t(711), which produces the NUP98-HOXA9 fusion protein that causes aberrant self renewal and (g) the acquisition of translocations and inversions associated with AML/myelodysplasia (e.g., inv[3] and t[1517]). In addition, minor genetic aberrations such as loss of heterozygosity (LOH) at 14q32, homozygous mutations/deletions of pRB, inactivating point mutations in P53 and in interferon consensus sequence binding protein (ICSBP, which encodes an interferon regulatory transcription factor with leukemia-suppressor activity), gain-of-function mutations in GATA-2 (which regulates myelomonocytic differentiation) and RAS (small GTP-binding signal transduction protein), and mutations in a zinc finger transcription factor PR domain containing 16 (PRDM16, mutated in myelodysplastic syndrome and AML) have been also detected. Numerous SNPs have been reported in additional genes regulating cell differentiation, such as ICSBP, GATA-3, and AML1 in myeloid CML-BP ( 86 ) however, these results await confirmation.

In addition, mutations in CDKN2A/B and IKZF1 facilitate CML-CP progression to CML-lymphoid BP ( 24 , 25 ). Moreover, BCR-ABL1–mediated stimulation of activation-induced cytidine deaminase (AID) leads to a hypermutator phenotype, CML-lymphoid BP, and imatinib resistance ( 126 ).

Experimental findings support the conclusion that genetic aberrations contribute to malignant progression of CML. For example, loss of p53 led to a CML-BP–like disorder in mice ( 127 ). CDKN2A gene loss enhanced oncogenicity in mouse models of BCR-ABL1–induced ALL ( 128 ). Coexpression of BCR-ABL1 and NUP98-HOXA9 caused CML-BP–like disease in mice ( 129 ). GATA-2 gain-of-function mutations, partial deletions of PMRD16 and RUNX1, and expression of RUNX1-PMRD16 detected in CML-myeloid BP may disturb myelomonocytic differentiation, strongly suggesting their involvement in acute myeloid transformation ( 86 , 130 ).

Moreover, genetic aberrations associated with CML-BP progression likely play a role in TKI resistance ( 131 ), causing a high risk of treatment failure ( 132 ). For example, additional chromosomal aberrations, loss of P53, and CDKN2A and RUNX1 abnormalities may be responsible for disease persistence under imatinib treatment ( 128 , 133 – 135 ).

BCR-ABL1 kinase–dependent and –independent genomic instability in CML-CP LSCs and/or LPCs. The (922) translocation that results in the formation of the Ph chromosome may be a random event or may result from preexisting conditions associated with genomic instability in HSCs. Therefore, additional genetic aberrations accumulated during the course of CML may be promoted by BCR-ABL1 kinase and also by a preexisting abnormality responsible for the formation of t(922)(q34q11). The former statement is supported by reports that BCR-ABL1 kinase–positive cells acquire more oxidative DNA lesions than normal counterparts in response to endogenous ROS and genotoxic treatment ( 109 , 110 ) and that BCR-ABL1 can inhibit some DNA repair mechanisms (MMR) and stimulate other mechanisms (NER, HRR, NHEJ, and SSA) at the cost of their fidelity ( 136 ) (Figure 3). However, the latter speculation about preexisting abnormality cannot be ruled out because chromosome abnormalities were detected in t(922)(q34q11)-negative metaphases appearing during imatinib therapy in patients with newly diagnosed CML-CP ( 121 ).

Genomic instability in CML-CP most likely occurs in the LSC-enriched CD34 + CD38 – population and/or the LPC-rich CD34 + population because TKI-resistant BCR-ABL1 mutants and chromosomal aberrations were detected in both subpopulations ( 38 , 41 , 114 ). As CML-CP can progress to either myeloid or lymphoid BP (sometimes a mixed myeloid/lymphoid phenotype) and chromosomal abnormalities are documented in both phenotypes ( 137 ), this suggests that genomic instability may occur at the LSC and/or LPC level. Mutations acquired by the LSCs are likely to be passed on to successive generations of LPCs. On the other hand, genetic aberrations acquired by CML-CP LPCs may “upgrade” them to the status of CML-BP LSCs ( 49 ).

Altogether, we postulate that elevated levels of DNA damage combined with unfaithful/inefficient DNA repair may generate mutations and chromosomal aberrations in CML-CP LSCs and/or LPCs, causing resistance to TKIs and progression toward CML-BP. These mechanisms at least partially depend on BCR-ABL1 kinase. Since LSCs, in contrast to LPCs, are not sensitive to TKIs, LSCs may be “ticking time bombs,” eventually exploding to produce a TKI-resistant LPC clone that may evolve into a CML-BP clone.

Genomic instability in CML cells in the era of TKIs. BCR-ABL1 kinase induces genomic instability ( 13 ) therefore, imatinib and other TKIs should prevent accumulation of additional genetic changes in CML cells. In fact, imatinib reduced ROS, oxidative DNA damage, point mutations, and other genetic aberrations in BCR-ABL1–positive cells ( 109 , 110 , 138 ). Nevertheless, imatinib-treated CML patients continue to accumulate point mutations (including those causing resistance to other TKIs) and chromosomal aberrations ( 21 , 121 , 130 , 139 ).

There are several possible explanations for persistent genomic instability during TKI treatment. First, although TKIs inhibit BCR-ABL1 kinase activity in CML-CP LPCs, their effectiveness in CML-CP LSCs is questionable. The effect of TKIs on BCR-ABL1 kinase–induced signaling may be obscured by growth factors, usually resulting in incomplete inhibition or even stimulation of signaling pathways, such as those involving STAT5, AKT, and MAPKs ( 140 , 141 ). Therefore, TKIs cannot completely eliminate the effects of BCR-ABL1 kinase and may not effectively inhibit genomic instability. Second, imatinib may exert mutagenic activity to induce centrosome and chromosome aberrations ( 142 ). The appearance of cytogenetic aberrations in t(922)(q34q11)-negative cells following imatinib therapy supports this hypothesis ( 143 ). Third, if CML-CP cells display an active preexisting genomic instability responsible for generation of t(922), this process should be BCR-ABL1 kinase independent and will continue generating errors despite treatment ( 121 ). This speculation implicates BCR-ABL1 kinase–dependent and –independent genomic instability in CML cells.

Prevention of genomic instability in CML-CP to improve therapeutic effects of TKIs and antagonize CML-BP. The majority of CML-CP patients at diagnosis do not have mutations or a “critical” combination of aberrations causing either TKI resistance or disease progression. However, a cohort of TKI-treated patients still develops mutations and chromosomal aberrations even though imatinib efficiently antagonizes genomic instability in experimental CML models. Given the fact that BCR-ABL1–negative patients, as assessed by reverse transcriptase PCR, may contain up to 10 6 CML cells in their body, that CML-CP patients can have approximately 5 × 10 7 CD34 + cells displaying innate imatinib resistance ( 144 ), and that even imatinib-sensitive CD34 + LPCs can still undergo up to 1–3 cell cycles in the presence of the drug and growth factors before eventually being eliminated ( 71 ), prevention of genomic instability may be critical for a better therapeutic effect or even eradication of CML.

ROS cause oxidative DNA damage resulting in both clinically relevant BCR-ABL1 mutations and chromosomal aberrations often detected in CML-BP (i.e., aneuploidy, translocations, and truncations) ( 109 , 113 ). Antioxidants diminished ROS-mediated oxidative DNA damage and reduced the appearance of TKI-resistant mutations and chromosomal aberrations ( 37 , 109 , 110 ). Because the combination of imatinib and an antioxidant exerted a synergistic/additive antimutagenic effect ( 109 ), it is possible that the combination of TKI and antioxidants may prevent CML-BP by reducing the appearance of TKI-resistant clones and accumulation of a “critical” combination of genetic aberrations.

To date, there is strong evidence supporting the idea that the level of BCR-ABL1 kinase activity plays a pivotal role in almost all CML patients undergoing progression and that BCR-ABL1–induced genetic/chromosomal abnormalities can predispose to transformation and/or markedly influence the aggressiveness of the blast crisis progenitor cell clone. However, there are several crucial and burning questions that remain to be answered. What controls BCR-ABL1 expression and activity during progression? Does malignant progression originate from CML-CP LSCs and/or LPCs? Is the acquisition of self renewal, impaired differentiation, and increased genomic instability of CML-BP stem and/or progenitor cells solely a BCR-ABL1–dependent effect? A possible scenario might envision a BCR-ABL1 autoregulatory loop that amplifies signals that positively influence BCR-ABL1 gene transcription and enhance its protein stability. Likewise, it is highly plausible that, in CML-CP, BCR-ABL1–induced genomic aberrations and/or BCR-ABL1–independent preexisting genetic lesions function as “amplifiers” of a genetically unstable phenotype and thereby predispose CML to blastic transformation by affecting stemness, survival, proliferation, differentiation, and/or genome stability of the Ph-positive bone marrow stem and progenitor cells.

This work was supported in part by grants from the National Cancer Institute (CA095512 to D. Perrotti CA123014 and CA133646 to T. Skorski) and the US Army and CML Research Program (W81XWH-07-1-0270 to D. Perrotti). D. Perrotti is a Scholar of The Leukemia and Lymphoma Society. C. Jamieson is funded by California Institute for Regenerative Medicine (CIRM) grants. We thank Paolo Neviani and Stephen Lee (The Ohio State University) and Elisabeth Bolton (Temple University) for editorial and/or graphical assistance.

Conflict of interest: Catriona Jamieson has research grants from Pfizer, Coronado Biosciences, and Celgene and has also consulted for Bristol-Myers Squibb and Wintherix.

Citation for this article: J Clin Invest. 2010120(7):2254–2264. doi:10.1172/JCI41246.


The National Cancer Intelligence Network (NCIN) have this week released a report which has shown that deaths from Chronic Myeloid Leukaemia have halved since TKIs were introduced in 2001.
The report states that “What’s even more promising is that, in the last four years, second and third generations of these drugs have been developed. We believe more and more CML patients have been receiving TKI’s and we’d predict that the improvements in survival should be even greater in the future” – However, we know of the challenges patients will face on long term access to dasatinib, especially with no firm long term commitment to the future of the CDF. If we take into account the success of these new drugs over the last 4 years I predict that survival figure will sky-rocket. More reason to ensure access to all of the drugs for everyone.
Kris

IMPROVED TREATMENTS FOR CHRONIC MYELOID LEUKAEMIA HAVE DRAMATICALLY INCREASED SURVIVAL

Survival for people diagnosed with Chronic Myeloid Leukaemia (CML) has risen by nearly half, with around 58 per cent of people surviving their disease for at least five years compared with only around 40 per cent in the late 1990s, according to a new report from the National Cancer Intelligence Network (NCIN), published today (Wednesday)*.

The improvements are largely down to a family of drugs called Tyrosine Kinase Inhibitors (TKIs) which have now become the standard treatment for the disease. The first of these was imatinib (Glivec), which was licensed in 2001.

The Northern and Yorkshire Cancer Registry and Information Service (NYCRIS), on behalf of the NCIN Haematology Site Specific Clinical Reference Group (SSCRG), looked at the rates of people in England getting, dying from and surviving a range of different blood cancers between 1995 and 2008. And it is the first national study in England to look at survival for different types of leukaemia.

For patients diagnosed with CML, researchers found that the chance of surviving the disease for at least five years after diagnosis rose from 41 per cent to 57 per cent in men and from 38 per cent to 59 per cent in women between the late 1990s and the early 2000s**.

CML is a relatively rare form of leukaemia*** that mostly affects older people, with around 700 cases diagnosed in the UK every year****.

Dr Robin Ireland, chair of the SSCRG at the NCIN, said: “It’s really exciting to see the enormous difference new drugs can make in treating cancer. And, as this new data shows, TKI’s can be considered a revolutionary treatment for Chronic Myeloid Leukaemia.”

“Basic research has given us a greater biological understanding of cancer tumours, which has led to the development of successful targeted cancer drugs that are now the first line treatment for CML. TKIs target cancer cells by blocking the molecules they make, which stops them from multiplying. These drugs have completely changed the outlook for patients with this disease and it’s the first example of our improved understanding of cell molecular biology leading to the design of a specific inhibitor of the disease.

”Dr Steven Oliver, Haematological Cancer Epidemiology Lead at NYCRIS and lead author of the report, said: “This report shows that, although the number of people developing Chronic Myeloid Leukaemia hasn’t changed much since 2001, survival from the disease has greatly improved.”

“What’s even more promising is that, in the last four years, second and third generations of these drugs have been developed. We believe more and more CML patients have been receiving TKI’s and we’d predict that the improvements in survival should be even greater in the future.”

“Chris Carrigan, head of the National Cancer Intelligence Network (NCIN), said: “Being able to link data on the diagnosis, treatment and outcomes for cancer patients allows us to identify where improved cancer care is having an effect on peoples lives. The improvements in survival demonstrated here highlight the difference that effective treatments can make.”

*Haematological Malignancies in England: cancers diagnosed 2001 – 2008.Note that analyses of deaths from blood cancers for 2001-2008 were also reported, along with 5-year survival figures for patients diagnosed with different blood cancers in 1995-1999 compared with those diagnosed in 2000-2003.

**Absolute change in CML 5-year survival rates, in England, 1995-1999 compared with 2000-2003. Male rates 40.7% and 56.9% female rates 38.4% and 58.7%, in 1995-1999 and 2000-2003 respectively.

***Leukaemias are a group of malignant diseases that affect the production of white blood cells – the body’s guards against foreign cells and infections. The different types of leukaemia affect different organs and vary in how quickly they spread. Different types of leukaemia affect different age groups and there are some wide differences in survival found across the age groups in certain forms of the disease.

**** There were 710 cases of CML registered in 2010 in the UK (Cancer Research UK Statistical Information Team)The NICE guidance recommends standard-dose imatinib as the first-line treatment for CML.


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Highlights

The vascular considerations for the clinical care of patients with chronic myelogenous leukemia have been addressed by mechanisms of toxicities of each tyrosine kinase inhibitor.

By defining the various cardiovascular and metabolic effects of tyrosine kinase inhibitor, the care and outcome of patients with chronic myelogenous leukemia will be dramatically improved.

Basic insights into toxicities of novel cancer therapies may serve as a new platform for investigation in vascular biology and a new translational research opportunity in vascular medicine.

TYROSINE Kinase Inhibitors Used in Chronic Myelogenous Leukemia: Overview

Overactivation of kinases can lead to a number of pathological processes including cancer. In chronic myelogenous leukemia (CML), one such kinase (ABL1 [Abelson 1 kinase]) becomes constitutively active as a result of a chromosomal translocation, juxtaposing the BCR (breakpoint cluster region) gene with the ABL kinase gene and genesis of the deregulated BCR-ABL1 kinase. Inhibitors of ABL1 kinase have resulted in remarkable efficacy in treating CML. In 2019, cardiovascular care has emerged as a crucial element of CML patient care for several reasons. First, patients with CML are now expected to have near-normal life expectancy and may succumb to other noncancer morbidities and mortality. Second, numerous kinase inhibitors (KI) used for the treatment of CML are associated with vascular disease. Increasingly, cardiologists and vascular medicine physicians who specialize in oncology care (cardio-oncologists) play a critical role in CML care. In this review, we will discuss vascular aspects of care for patients with CML. We will also discuss the emerging data on the mechanisms of vascular sequelae from CML tyrosine KI (TKI) therapy and the implications for vascular medicine and vascular biology.

Please see www.ahajournals.org/atvb/atvb-focus for all articles published in this series.

Kinase and TKI

Kinases are enzymes that transfer phosphate groups from ATP to specific protein or lipid substrates, leading to fundamental regulation of cell signaling. Over 500 kinases have been discovered, which are divided structurally into 2 groups: serine/threonine kinases and tyrosine/tyrosine-like kinases. 1 KI inhibit either the ATP-binding pocket (where the ATP binds) or the allosteric pocket (where the phosphorylated protein binds) and thus prevent kinase activity itself (Figure 1). While most KI are reversible, some KI (eg, ibrutinib) bind to the proximal binding pocket with a reactive nucleophilic cysteine covalently and thus irreversibly. However, most KI are steady-state competitive enzyme inhibitors with respect to ATP and either directly or indirectly interact with the ATP pocket. 2

Figure 1. Mechanism of action of BCR (breakpoint cluster region)-ABL (Abelson 1 kinase) tyrosine kinase and tyrosine kinase inhibitor (TKI) in chronic myelogenous leukemia (CML). BCR-ABL tyrosine kinase in CML (left). TKI in treatment of CML (right). P indicates phosphate.

Because of the high similarity of the ATP-binding sites across the entire kinome, small molecular KI can bind >1 kinase receptor, thus decreasing selectivity. For this reason, many KI occupy both the spaces where the ATP adenine group binds but also adjacent regions (including occasionally the adjacent allosteric pocket). 3 Nevertheless, even with this approach, the selectivity of approved KI is not absolute. Imatinib—the first approved KI for CML—inhibits not only the ABL1 tyrosine kinase relevant to CML but also the tyrosine kinase receptor c-KIT (CD117), aberrantly activated in gastrointestinal stromal tumors, resulting in another drug indication for imatinib. 4,5

Kinases also play a critical role in cardiac, vascular, and metabolic homeostasis thus, altered activity of kinases in the cardiovascular systems can lead to pathology and various effects on the vasculature. For example, the kinase target of a specific KI may have an oncogenic role if altered in a cancer cell but also a critical role, unaltered, in a myocyte or an endothelial cell. Inhibition of that kinase, as an unintended consequence, can lead to vascular disease. Such on-target toxicity is in contrast to off-target toxicity where the promiscuous nature of the KI can lead to inhibition of structurally similar or related kinase receptor(s), which can cause disease. In addition, the effects of KI on the cardiovascular system may not be entirely pathological and may actually be beneficial. We will review these concepts in the case of CML and TKI used to treat CML below.

TKI—Inhibiting ABL1 Kinase for the Treatment of CML

Five small-molecule TKI have been approved to date for the treatment of CML. 6 Imatinib was initially developed as a PDGFR (platelet-derived growth factor receptor) inhibitor but was also found to inhibit other tyrosine kinases, such as ABL1 and c-KIT (the stem cell factor receptor). 6 The IRIS trial (International Randomized Interferon Versus STI571) demonstrated the unprecedented efficacy of imatinib in newly diagnosed CML. Remarkably, the longer term overall survival of patients treated with imatinib was close to 90%, establishing imatinib as a benchmark treatment for CML. In 2001, imatinib became the first small-molecule TKI approved by the Food and Drug Administration (FDA) and has revolutionized the treatment of CML and other leukemias where the ABL1 tyrosine kinase is constitutively active. 2

Since this initial historic approval, a number of later generation TKI were tested and approved for the treatment of CML. These included dasatinib, nilotinib, bosutinib, and ponatinib. The impetus for introducing new TKI in this space was driven by several factors. First, although imatinib dramatically altered the natural history of CML, >20% of patients with CML were either unable to tolerate or developed resistance to imatinib. Second, the newer TKI were shown to be more potent inhibitors of ABL1 tyrosine kinase and could offer both salvage and augmented primary response. In head-to-head clinical trials with imatinib, dasatinib and nilotinib resulted in faster and greater proportion of molecular response, as well as protection from early progression. Although this still has not resulted in better overall survival from trial data, both drugs were approved for front-line therapy. In 2017, bosutinib obtained similar FDA approval for front-line treatment of CML.

Despite the success of TKI therapy in CML, specific kinase domain mutations emerged in the leukemic clones driving resistance to TKI therapy. The most impactful and notorious is the T315I mutation where threonine substitution for isoleucine at a key contact point severely affected TKI inhibition and was the major determinant of TKI resistance within the leukemic clone. 7 Ponatinib was developed as a multitargeted TKI with potent activity against the T315I mutation, as well as the spectrum of other ABL1 kinase mutations. In a phase 2 trial (called the PACE study), ponatinib showed considerable response in patients who had failed other TKI, leading to drug approval via the FDA accelerated approval program in 2012. 8 However, 1 year later, ponatinib prescribing authorization was transiently suspended in the United States by the FDA because of emerging recognition of cardiovascular risks with the drug and need for updated label warnings. Not surprisingly the vascular/cardiovascular toxicities noted with ponatinib heralded closer assessment of cardiac, vascular, and metabolic issues with all other TKI used for CML treatment (Table).

Cardiovascular Risks Associated With TKI Used in CML—Clinical Studies

Despite considerable efficacy in patients who had failed other TKI, the early phase 2 trial with ponatinib showed considerable vascular toxicity. At a median follow-up of 12 months in the PACE trial, 6% of patients had coronary events, 3% had cerebrovascular events, and 4% had peripheral vascular events. At 28 months, cumulative events were 10%, 7%, and 7%, respectively. Indeed, retrospective analysis of the PACE database suggests a higher risk in patients with cardiovascular risk factors or cardiovascular disease, as well as a signal for cardiovascular events occurring in a ponatinib dose-dependent manner. 9 In addition, at least a quarter of patients developed hypertension after initiating treatment. A cardiovascular signal for toxicity was also seen in a trial involving ponatinib as first-line therapy, which led to trial suspension. The aggregate data strongly suggested that ponatinib is associated with higher risk of cardiovascular adverse events.

The cardiovascular risk profile of ponatinib brought into focus vascular and metabolic effects of other TKI used in CML. Certain cardiac signals become apparent early in drug approval for each therapy. For example, a small percentage of patients treated with both dasatinib and nilotinib were noted to have QT prolongation, although no clear risk of ventricular arrhythmias was identified. For this reason, QT assessment via an electrocardiogram was recommended for nilotinib. In the case of dasatinib, dyspnea observed in a fraction of patients led the US FDA to issue a warning and recommend that patients be evaluated for signs and symptoms of cardiopulmonary disease before and during dasatinib treatment. The cause of dyspnea appears to be multifactorial. In initial studies with dasatinib, a significant fraction of patients had pleural effusions. In addition to pleural effusion, dasatinib is associated with pericardial effusion. The pathogenesis is unclear but is felt to be a capillary leak–like syndrome. 10 More concerning, reports of pulmonary hypertension were subsequently noted with dasatinib. In 2012, the French Pulmonary Hypertension Registry reported 9 severe cases of dasatinib-associated pulmonary hypertension. On diagnosis, patients had moderate-to-severe precapillary pulmonary hypertension, with severe symptoms and hemodynamic compromise with 2 patients dying in follow-up. 11 The incidence of dasatinib-associated pulmonary hypertension is felt to be at least 3% based on data from randomized trials. However, none of these trials systematically screened for pulmonary hypertension.

Vascular events, including cardiac, cerebral, and peripheral events, have emerged as the biggest cardiovascular safety concern in patients with CML. Before ponatinib-associated vascular complications became obvious late in 2013, initial case reports or case series of vascular events had been reported with nilotinib. A multicenter analysis of 179 patients revealed 11 patients (6.2%) who developed severe peripheral artery disease involving lower limbs. Eight patients required invasive therapy, and 4 patients required amputation. More comprehensive appreciation of vascular events in CML patients was derived from clinical trials where newer TKI were compared with imatinib for efficacy. Although these trials were not designed to assess cardiovascular safety, an increased risk of vascular events was observed with newer TKI compared with imatinib. A 3-year follow-up of the ENESTnd trial (where nilotinib was compared with imatinib in front-line therapy) suggested a higher incidence of vascular events in patients treated with nilotinib compared with imatinib. 12 These data were more striking at 5-year follow-up 28 of 279 (10%) patients treated with nilotinib at 300 mg twice per day, 44 of 277 patients (15.9%) treated with nilotinib at 400 mg twice per day, and 7 (2.5%) of 280 patients treated with imatinib 400 once per day had cardiovascular events. 13 In these analyses, cardiovascular events were defined as ischemic heart disease, ischemic cerebrovascular disease, and peripheral artery disease. These data suggest that nilotinib-associated toxicity occurs in all arterial beds fewer venous events were noted. Five-year follow-up report of the DASISION (Dasatinib Versus Imatinib Study in Treatment-Naive Chronic Myeloid Leukemia Patients Trial where dasatinib was compared with imatinib in the front-line setting) noted a 5% risk of arterial ischemic events in patients on dasatinib compared with 2% risk for imatinib. 14

In contrast, long-term studies of patients treated with imatinib have generally shown net neutral or possibly favorable effects on vascular disease risk. Arguably, the best data come from head-to-head prospective studies where the efficacy of imatinib is compared with other TKI where cardiovascular event rates are lower in the imatinib arms of the studies. What remains unknown is the baseline cardiovascular risk profile of patients with CML irrespective of therapy or as they commence therapy. A current ongoing prospective study (NCT03045120) is assessing the baseline cardiovascular risk factors and follow-up cardiovascular risks of a real-world population of CML patients on chronic TKI therapy.

Cardiovascular Toxicities of TKI: Mechanistic Studies

The cardiovascular events observed in CML patients treated with TKI have led to mechanistic studies interrogating the vascular effects of each TKI, with some insight gained to date. Additional unanswered questions include the impact the CML diagnosis itself poses on risk of vascular events, as well as the potential reversibility of any pathology stemming from either CML or TKI therapy as CML treatment paradigms shift toward a focus on finite therapy and treatment-free remission (cessation of therapy in deep remission).

Nilotinib was found to upregulate proatherogenic adhesion proteins on human endothelial cells, including ICAM1 (intercellular adhesion molecule 1), VCAM1 (vascular cell adhesion protein 1), and E-selectin, which collectively can recruit inflammatory cells and platelets and promote vascular events. Increased expression of adhesion molecules is mediated through reduction in the level of miR-3121-3p, which additionally leads to upregulation of IL (interleukin)-1β. It is proposed that the miR-3121-3p/IL-1β axis could be a potential target to prevent vascular events in patients with CML determined to have a high risk. 15 Whole blood samples from nilotinib-treated CML patients demonstrated increased platelet adhesion. These patients also showed increased expression of markers of endothelial and platelet activation, including plasma soluble P- and E-selectin, sICAM-1, sVCAM-1, TNF (tumor necrosis factor)-α, IL-6 levels, and endogenous thrombin potential levels in vivo, despite being on daily low-dose aspirin. 16 Treatment of atherogenic (ApoE −/− [apolipoprotein E]) mice with nilotinib increases atherosclerotic buildup and blocked reperfusion and angiogenesis in a hindlimb ischemia model of arterial occlusion. 16 In mouse model studies, nilotinib remarkably enhanced thrombus growth and stability in damaged mesenteric arterioles and the carotid artery. 17

Further interrogation of effects of nilotinib on other atherosclerosis risk factors has shown mixed results. A prospective clinical study showed that nilotinib doubles the levels of low-density lipoprotein cholesterol (bad cholesterol) but also raises high-density lipoprotein cholesterol levels. 18 In atherosclerosis (APOE*3Leiden.CETP mouse)-prone mice, nilotinib had no significant effect on cholesterol levels. 19 On the contrary, early clinical trials with nilotinib showed hyperglycemia and development of prediabetes or worsening of glycemic control in a subset of patients—an obvious risk factor for atherosclerosis. 20,21 From patient studies, there is no consensus with respect to the mechanisms of vascular toxicity associated with nilotinib. However, deep phenotyping in specific patients with vascular studies can be informative. 22,23 Vessel wall magnetic resonance imaging in a case of nilotinib-associated cerebral stroke revealed diffuse concentric thickening of the intracranial artery walls, which is not consistent with ordinary atherosclerosis (which often manifests with eccentric thickening). 23 In addition, a study involving 159 patients on imatinib or nilotinib showed a higher incidence of abnormal ankle-brachial index (ABI) in patients treated with nilotinib. Abnormal ABI in patients treated with first- and second-line nilotinib was 26% and 35.7%, respectively, compared with 6.3% for first-line imatinib. 24 These studies are informative because an abnormal ABI is a sensitive and specific test for not only peripheral artery disease but also systemic polyvascular atherosclerosis. 25

The mechanisms of vascular toxicities associated with ponatinib may be different than nilotinib. Because of the promiscuity of ponatinib, multiple kinases (and thus pathways) in the vasculature can be affected. For example, unlike nilotinib, ponatinib is a potent inhibitor of VEGF (vascular endothelial growth factor) receptors (eg, kdr), leading to increased blood pressure, like other TKI with potent anti-VEGF properties. 5,26 Ponatinib has significant detrimental effects on cultured endothelial cells in vitro, both inhibiting proliferation and inducing apoptosis. Ponatinib also has direct prothrombotic effects increasing platelet activation and adhesion. A recent series of elegant experiments suggest that ponatinib vascular toxicity involves platelet adhesion followed by secondary microvascular angiopathy. 27 Ultrasound molecular imaging demonstrates 5- to 6-fold increased signal for glycoprotein-Ibα–mediated and VWF (von Willebrand factor)-mediated endothelial cell and platelet adhesion in ponatinib-treated mice compared with vehicle-treated controls. These effects were accentuated in atherosclerosis-prone (ApoE −/− ) mice and were present in both large arteries, as well as the microcirculation, the latter producing ventricular dysfunction. 27 Given this elegant study, It is most likely that any degree of ventricular dysfunction seen with ponatinib is due to an indirect effect, rather than a direct effect on cardiomyocytes, as has been demonstrated in other models. 28

Separate studies have been performed to interrogate the mechanisms of pulmonary hypertension associated with dasatinib. Treatment of rats with dasatinib led to a dose-dependent endothelial cell apoptosis and dysfunction via increased mitochondrial reactive oxygen species production. Over time, dasatinib attenuated pulmonary vasoconstriction following hypoxia exposure and increased susceptibility to experimental pulmonary hypertension. 29 In a separate study, a Rho-KI blunted the change in pulmonary pressures observed after dasatinib treatment, providing a potential for developing treatments. 30 In both studies, the pulmonary effects were specific to dasatinib since they were not observed with other TKI used in CML treatment and consistent with clinical data.

Implications for Patient Care: Preventive and Treatment Strategies

In 2019, cardiovascular health has emerged as an important consideration in all patients with CML irrespective of CML therapy. All patients with CML should undergo general cardiovascular risk assessment and examination before starting TKI therapy. Given the disparate effects of TKI on vascular disease, cardiovascular and metabolic factors (such as hypertension, hyperlipidemia, and diabetes mellitus) should be assessed at various times following start of treatment (Figure 2). ABI is a sensitive and specific test for systemic atherosclerosis and offers an informative noninvasive diagnostic test for patients starting specific TKI. A crucial point to clarify is whether baseline cardiovascular risk profile should definitively influence or dictate the choice of TKI selected for treatment. Patients considered high risk based on comorbidities or TKI selection may benefit from more frequent monitoring and from involvement of a cardiologist, vascular medicine specialist, or a cardio-oncologist to optimize primary and secondary prevention. This is particularly relevant for patients on ponatinib treatment. A simple ABCDE algorithm is an established means to reduce cardiovascular events in the general population and maybe particularly relevant to the CML population (Figure 3). 31

Figure 2. Clinical recommendations for assessment of cardiovascular toxicity in individuals with chronic myelogenous leukemia (CML) receiving tyrosine kinase inhibitors. ✓ Recommended ✗ as clinically indicated assess the QT prolongation. The FDA requires QT monitoring with nilotinib. Specifically, “Nilotinib prolongs the QT interval and requires ECGs to monitor the QTc at baseline, 7 days after initiation, and periodically thereafter, and following any dose adjustments.” If there are any signs in favor of pulmonary arterial hypertension like dyspnea, consider echocardiogram as first line of screening. HgbA1C indicates glycated hemoglobin and TKI, tyrosine kinase inhibitor.

Figure 3. ABCDE approach, which has been proposed to reduce cardiovascular (CV) disease in patients with chronic myelogenous leukemia receiving tyrosine kinase inhibitor treatment. ABI indicates ankle-brachial index ACE, angiotensin-converting enzyme ARB, angiotensin receptor blocker BB, β-blocker BP, blood pressure dp-CCB, dihydropyridine calcium channel blocker HTN, hypertension and ndp-CCB, nondihydropyridine calcium channel blocker.

Future Directions

In the future, better defining the various cardiovascular and metabolic effects of TKI will further advance the care and outcome of CML patients already dramatically improved by availability of TKI. Importantly, prospective, systematic cardiovascular toxicity assessment will be important in any future clinical trial in CML. Additionally, incorporation of adjudication of adverse events by independent experts (cardio-oncologists) could provide more reliable and clear safety data. Further study of real-world populations will allow better understanding of underlying cardiovascular risk factors and cardiovascular disease, as well as the effects of CML itself and CML therapy. Finally, better delineation of mechanisms of underlying toxicities will be critical in terms of personalized preventive and treatment strategies for patients, knowing that the clear mechanisms by which TKI induce cardiovascular toxicity are still unknown and need to be subject to further investigation. These future directions are important because even today newer TKI are being introduced for CML therapy. For example, ABL001 (asciminib) is an allosteric inhibitor of BCR-ABL is currently being tested as a more effective therapy in CML. Indeed, the cardiovascular health of patients with CML may be a more imperative topic in the future. 32 Thus, it is strongly recommended to consider cardiovascular screening and follow-up in the future clinical trials on new TKIs including ABI measurement, serial electrocardiograms, and baseline and periodic echocardiography.

Table. Food and Drug Administration–Approved TKIs for Treatment of Chronic Myelogenous Leukemia by Date, Their Molecular Target, and Their Effect on the Vasculature


Discussion

In this study, we assessed trends in the disease burden of CML based on GBD study, providing valuable epidemiologic information for health promotion and disease prevention. Though the ASIR, ASDR, and age-standardized DALYs rate generally declined, the worldwide disease burden of CML appeared to be stable due to population growth in developing countries and population aging in developed countries [17]. From 1990 to 2017, incidence cases decreased by 34.9% in high SDI quintiles but increased by over 60% in low SDI, low-middle SDI, and middle SDI quintiles. Similarly, Andean Latin America, Central Sub-Saharan Africa, South Asia, and Western Sub-Saharan Africa with lower SDI noticed the fastest growth in the incidence cases, death cases, and DALYs. Besides, India had the most incidence cases, death cases, and DALYs of CML in the globe, leading to the highest disease burden, which primarily due to a vast population base. Developed countries generally obtained remarkable achievements in reducing the disease burden due to CML [9, 18]. TKI-based therapy was the foremost reason for the profound improvement of survival in CML patients [7]. Currently, developing and low-income countries gradually introduced novel drugs like imatinib [19]. However, CML patients in these countries still had difficulties accessing TKI-based therapy or may receive delayed therapy, thanks to the limited availability of novel drugs [20]. Additionally, the high cost of medication and monitoring was another challenge for patients in low-income countries [21, 22]. Generic drug application and international patient assistance programs can reduce the economic burden of CML patients in countries with limited resources.

It is worth mentioning that treatment-free remission (TFR) is a new goal for many CML patients, as TKI-based therapy requires lifelong treatment and significantly aggravates economic burden [23, 24]. A prospective trial in France first revealed some CML patients with a stable deep molecular response could cease their TKI therapy safely without relapsing, known as TFR [25]. Since then, multiple clinical trials were conducted to explore the criteria of cessation attempts and triggers for resuming TKI therapy [26, 27]. In most studies, the molecular recurrence rate of TKI cessation was about 50%. Meanwhile, at least three years of TKI therapy and one-year deep molecular response were the criteria of cessation attempts, and loss of major molecular response met the triggers for resuming TKI therapy. The financial burden of health agencies became more massive based on lifelong TKI treatment with increasing cases of CML patients around the world. Besides, lifelong therapy is also associated with adverse events related to therapy, resulting in lower life quality and a growing disease burden of CML patients [28]. Women with childbearing potential must use effective contraception during TKI therapy and avoid breastfeeding due to evidence of teratogenicity. TFR successfully achieved in some CML patients can help to reduce disease burden. However, it should be emphasized that frequent monitoring and planned follow-up were essential requirements for discontinuing TKI therapy. Countries with inadequate resources might face challenges in ensuring necessary monitoring and effective drugs thus, improving survival rate rather than TFR was the primary goal of CML patients in these countries [29]. Developed countries could conduct more research to establish the TFR criterion, which can be a critical initiative in reducing CML's disease burden.

The epidemiologic trend of CML differed in age and sex, which was significant for policymakers [30]. Aging, related to a decrease of hematopoietic stem cell function, was an essential factor associated with leukemogenesis [31, 32]. Moreover, CML survival was age-related, and age was an essential factor for treatment options [33, 34]. Thus, higher SDI quintiles with population aging showed a more substantial proportion of older patients over 70 years old. Males had a higher risk to CML than females, and the male to female ratio fluctuated between 1.2 and 1.7 [35, 36]. In general, females had a better survival rate of CML than males, which was in line with other malignant diseases [35, 37]. Better general longevity and hormonal status of females and environmental and genetic factors might impact the age distribution of CML patients [38, 39]. However, in the low SDI quintiles, females had a higher age-standardized rate of deaths and DALYs than males in 2017. Previous studies also found that women in low-income countries tended to have higher mortality due to inadequate opportunities for screening and early treatment [40, 41]. Besides, the trend changes in smoking, obesity and physical inactivity in these countries may also contribute to the increasing disease burden of females. Prioritizing the reduction of known disease risk factors in low-income countries may be an effective intervention to reduce the health burden. Given the scarcity of resources, the support and commitment of the international community are also imperative.

Smoking was always the leading potential factor contributing to CML deaths and DALYs, based on data in the GBD study, though its contribution ratio decreased. Attributable deaths and DALYs due to smoking descended most quickly in high SDI quintiles and declined slightly in the other four SDI quintiles. These findings emphasized the urgent need to strengthen smoking control to reduce the CML burden. A previous study revealed that smoking might be an adverse prognostic factor, shortening survival time and contributing to CML's progression [42]. The smoking prevalence declined in most countries during the research, especially in high-income countries [43]. The ratio of former smokers to current smokers in middle age was a reliable indicator of smoking cessation the ratio was about one in developed countries, implying partial successful quitting [44]. Worryingly, low-income and middle-income countries, with approximately 80% of all smokers living in, had far fewer former smokers than current smokers [44]. Actually, the smoking population in many developing countries has boomed as their affordability for cigarettes since 1990. Reviews of comprehensive cessation programs highlight higher prices are particularly useful [45]. Besides, non-price interventions include warning labels, media campaigns, and assistance with smoking cessation could also increase quit rates [46]. High body mass index was the second attributable risk factor after smoking its contribution ratio declined slightly in high SDI and high-middle SDI quintiles but increased in the other three SDI quintiles. Prospective studies also indicated a positive correlation between obesity and leukemia mortality [47, 48]. In the past decade, the obesity epidemic has leveled off in developed countries by contrast, an increasing trend was still evident in developing countries [49, 50]. The obesity burden seemed to shift to the poor [51]. Since the twenty-first century, urbanization was a critical driver in obesity increasing in developing countries production of cheap vegetable oils, allowing increasing consumption of energy- and fat-rich diet at low income, and sedentary lifestyles also forced the obesity epidemic [50, 51]. Providing consultation and screening in the health care system, and reducing the marketing of sugary beverages and nonessential high‐calorie foods could help reduce attributable CML deaths.

A recent study also assessed the global burden of acute myeloid leukemia (AML) based on the GBD study. The significant difference in the global burden between CML and AML was a downward trend in CML and an upward trend in AML [52]. In 2017, high SDI quintiles tended to have a lower incidence and death rate of CML than low SDI quintiles, while AML was the opposite. CML's disease risk was similar to AML in terms of age and sex distribution, which was increased by age and a higher chance in males. As for attributable risks of CML and AML, the trend was consistent, and smoking was the leading attributable risk factor, followed by high body mass index. TKI-based therapy has drastically changed the landscape of CML survival, while novel effective therapies are incredibly urgent for AML patients [52]. Policymakers could pay more attention to reducing the CML burden in lower SDI countries. Besides, establishing the TFR criterion could be a critical initiative in reducing CML's disease burden.

Some unavoidable limitations did exist in this study. The data from the GBD study could fill the gap since actual data of CML burden was unavailable. The accuracy of our study depended on the quality of data in the GBD study. Additionally, differences in data acquiring and data source quality in the GBD study could be inevitable. Minor fluctuation in age-standardized rates may be associated with adjustments of disease screening strategies instead of real changes. GBD study lacked data about race, which can help better explain the distribution of CML.


Author Summary

Targeted therapy using imatinib, nilotinib or dasatinib has become standard treatment for chronicle myeloid leukemia. A minority of patients, however, fail to respond to treatment or relapse due to drug resistance. One primary driving factor of drug resistance are point mutations within the driving oncogene. Laboratory studies have shown that different leukemic mutants respond differently to different drugs, so a promising way to improve treatment efficacy is to combine multiple targeted therapies. We build a mathematical model to predict the dynamics of different leukemic mutants with imatinib, nilotinib and dasatinib, and employ optimization techniques to find the best treatment schedule of combining the three drugs sequentially. Our study shows that the optimally designed combination therapy is more effective at controlling the leukemic cell burden than any monotherapy under a wide range of scenarios. The structure of the optimal schedule depends heavily on the mutant types present, growth kinetics of leukemic cells and drug toxicity parameters. Our methodology is an important step towards the design of personalized optimal therapeutic schedules for chronicle myeloid leukemia.

Citation: He Q, Zhu J, Dingli D, Foo J, Leder KZ (2016) Optimized Treatment Schedules for Chronic Myeloid Leukemia. PLoS Comput Biol 12(10): e1005129. https://doi.org/10.1371/journal.pcbi.1005129

Editor: Natalia L. Komarova, University of California Irvine, UNITED STATES

Received: April 13, 2016 Accepted: September 2, 2016 Published: October 20, 2016

Copyright: © 2016 He et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: JF is supported by NSF grants DMS-1224362 and DMS-134972. KZL is supported by NSF grants CMMI-1362236 and CMMI-1552764. JZ was supported by NSF grant CMMI-1362236. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.


Bone marrow microenvironment (BMM)

Most leukaemia evolves from HSPCs in the BM. HSPCs are defined by their microenvironment, namely they can only maintain their function (self-renewal, differentiation and maturation) under very specific microenvironment conditions. Most cellular components of the BMM undergo age-related phenotypic evolution due to programmed ageing which is the consequence of hormonal changes [ 49 and chronic inflammation (inflammaging) [ 50 . The exact changes occurring in the human BMM are reviewed elsewhere [ 51 . These changes create new niches and therefore new HSPCs that maintain their function under the new environment. Leukaemia can be broadly divided into age related and age unrelated. CML, CLL, MDS, MPNs and cytogenetic normal AMLs are age-related leukaemia. Whilst AMLs due to core binding factor leukaemia MLL translocations and some CML and ALL subtypes are either age unrelated or enriched in younger individuals [ 52 . In age-related leukaemia, PreL-HSPCs and LSCs evolve from aged HSPCs and therefore will share many phenotypes regardless of their genetic background. Another possible crosstalk between the microenvironment and leukaemia evolution can be mediated by germline genetic aberrations in the BMM. Rodent models have repeatedly identified genetic alterations in BM niches cells which can promote leukaemogenesis [ 53-56 . In some of these studies, an increase in inflammatory cytokine production by BM stromal cells is involved in leukaemia evolution either by altering HSPCs kinetics [ 57 or promoting direct mutagenesis in HSPCs [ 58 . In these models, the most commonly involved cells are stroma cells and the molecular mechanisms include Notch and Wnt signalling alterations, ribosome biogenesis, and RNA processing, which lead BM stroma remodelling deregulation of inflammatory signals. Such mechanisms might be relevant for human leukaemia evolution if germline variants of BMM will pose a selective pressure on mutated preL-HSPCs. It is less likely that somatic variations in the BMM will provide such a selective pressure as a more generalized effect, since all or most BMM cells should be involved to produce such a selective pressure.

Another important component of the BMM is mature blood cells. It is becoming clearer that the accumulation of mutated mature blood cells originating from preL-HSPCs can have pathophysiologic consequences on atherosclerosis through monocytes [ 59, 60 but also on leukaemogenesis through the effect of myeloid cells on intestinal permeability [ 61 . The relevance of other mutated blood cells on leukaemia evolution has not been well studied yet.

The BMM is constantly changing in the leukaemogenesis process. Changes occur either due to germline determinants, age-related changes and later once preL-HSPCs and LSCs evolve they can also modulate the BMM. The effect of CML-LSCs on normal HSCs is mediated through IL-6 [ 62 . In AML, LSCs inhibit haematopoiesis by secreting SCF [ 63 . Remodelling of the BMM by leukaemic clones is reported in B-cell acute lymphoblastic leukaemia B-ALL [ 64 , CML [ 65 AML [ 66 and MPN [ 67 . Mechanisms of the crosstalk between LSCs and BMM include secretion of exosomes containing amphiregulin, which activates the epidermal growth factor (EGFR) pathway in mesenchymal stromal cells by CML-LSCs. This interaction increases secretion of IL-8 facilitating adhesion of CML-LSCs to mesenchymal stromal cells and favouring their survival [ 68 . Another possible crosstalk suggests an interplay between AML-LSCs and mesenchymal adipose tissue to create a unique microenvironment that supports the metabolic demands and survival of a distinct LSC subpopulation expressing CD36 (which is a fatty acid transporter) [ 69 .

Other changes to the BMM during leukaemia evolution can be introduced by radio/chemotherapy and BM transplant. All such changes will consequently introduce new selective pressures which will increase genetic diversity as most ancestral clones will still survive as not all niches are changed, and therefore, ancestral cells can still better survive under old niches. It is important to stress that changes in the BMM will give selective advantage to existing cells with specific phenotypes. Therefore, for the eradication of preL-HSPCs/LSCs uncovering fitness phenotypes is needed.


New approach to fighting chronic myeloid leukemia

Chronic myeloid leukemia develops when a gene mutates and causes an enzyme to become hyperactive, causing blood-forming stem cells in the bone marrow to grow rapidly into abnormal cells. The enzyme, Abl-kinase, is a member of the "kinase" family of enzymes, which serve as an "on" or "off" switch for many functions in our cells. In chronic myeloid leukemia, the hyperactive Abl-kinase is targeted with drugs that bind to a specific part of the enzyme and block it, aiming to ultimately kill the fast-growing cancer cell. However, treatments are often limited by the fact that the cancer cells can adapt to resist drugs. EPFL scientists have identified an alternative part of Abl-kinase on which drugs can bind and act with a reduced risk of drug resistance. Their work is published in Nature Communications.

Abl-kinase and leukemia

Abl-kinase can turn "on" molecules that are involved in many cell functions including cell growth. In chronic myeloid leukemia, the chromosome that contains the gene for Abl-kinase swaps a section with another chromosome, causing what is known as the "Philadelphia chromosome." When this mutation takes place in the blood stem cells in the bone marrow, Abl-kinase fuses with another protein, turning into a deregulated, hyperactive enzyme. This causes large numbers of blood-forming stem cells to grow into an abnormal type of white blood cell, which gives rise to chronic myeloid leukemia.

To treat this type of leukemia we use drugs that specifically bind and block a part of Abl-kinase called the "active site." As the name suggests, this is the part of the enzyme that binds molecules to turn them on. Therefore, blocking the active site with a drug stops the hyperactivity of Abl-kinase caused by the Philadelphia mutation and slow down or even abolishes the production of abnormal cancerous blood cells. The problem is that targeting the active site of Abl-kinase often causes the cancer cells to adapt and develop drug resistance, making them harder to kill.

An indirect path against resistance

A team of researchers led by Oliver Hantschel at EPFL (ISREC) has now discovered a new way to indirectly inhibit the activity of Abl-kinase. The scientists systematically made small, strategic mutations to Abl-kinase that caused its 3D structure to change. Then they tested each mutant version of the enzyme to see if its function would change.

Hantschel's team built on previous studies showing that Abl-kinase is indirectly controlled by another part of itself called the "SH2 region," which is located close to the active site. Normally, the SH2 region regulates the active site by opening and closing it. But under the Philadelphia mutation, that regulation is lost. What the scientists discovered was that when the Philadelphia mutation takes effect, the SH2 region actually "clamps" open the active site of Abl-kinase and forces it to go into overdrive.

The discovery provides a first-ever picture of the molecular events surrounding the hyperactivity of Abl-kinase. By blocking the SH2 region, it is possible to modulate the activity of the enzyme, and perhaps stop the growth of leukemic tumors. And since because the SH2 region is common to other kinases, it is likely that effect could extend to other types of cancers as well, particularly those characterized by abnormal kinase activity. Finally, Oliver Hantschel expects that this approach could overcome the problem of tumor drug resistance, as it might offer an alternative way to inhibit the enzyme and mutations of rapidly growing tumor cells may be less likely to occur.


Conclusions

The activation of the NKG2D immunoreceptor is expressed in cytotoxic lymphocytes, thus the reactivity of NK cells is stimulated following recognition of NKG2DL. The BCR-ABL gene directly controls the expression of NKG2DL and the antitumor reactivity of NK cells depends directly on the quantity of these receptors on the cell surface. In CML, production of sMICA inhibits the anti-leukemic action of NK cells and favors tumor survival. During treatment with tyrosine kinase inhibitors the expression of NKG2DL is modulated which may favor the action of NK cells.


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