Epigenetic dysregulation in chronic myeloid leukaemia: A myriad of mechanisms and therapeutic options

The onset of global epigenetic changes in chromatin that drive tumor proliferation and heterogeneity is a hallmark of many forms cancer. Identifying the epigenetic mechanisms that govern these changes and developing therapeutic approaches to modulate them, is a well-established avenue pursued in translational cancer medicine. Chronic myeloid leukemia (CML) arises clonally when a hematopoietic stem cell (HSC) acquires the capacity to produce the constitutively active tyrosine kinase BCR-ABL1 fusion protein which drives tumor development. Treatment with tyrosine kinase inhibitors (TKI) that target BCR-ABL1 has been transformative in CML management but it does not lead to cure in the vast majority of patients. Thus novel therapeutic approaches are required and these must target changes to biological pathways that are aberrant in CML − including those that occur when epigenetic mechanisms are altered. These changes may be due to alterations in DNA or histones, their biochemical modifications and requisite ‘writer’ proteins, or to dysregulation of various types of non-coding RNAs that collectively function as modulators of transcriptional control and DNA integrity. Here, we review the evidence for subverted epigenetic mechanisms in CML and how these impact on a diverse set of biological pathways, on disease progression, prognosis and drug resistance. We will also discuss recent progress towards developing epigenetic therapies that show promise to improve CML patient care and may lead to improved cure rates

The chronic myeloid leukemia stem cell: stemming the tide of persistence

Chronic myeloid leukaemia (CML) is caused by the acquisition of the tyrosine kinase BCR-ABL1 in a haemopoietic stem cell (HSC), transforming it into a leukaemic stem cell (LSC) that self-renews, proliferates and differentiates to give rise to a myeloproliferative disease. While tyrosine kinase inhibitors (TKI) that target the kinase activity of BCR-ABL1 have transformed CML from a once fatal disease to a manageable one for the vast majority of patients, only ~10% of those who present in chronic phase (CP) can discontinue TKI treatment and maintain a therapy-free remission. Strong evidence now shows that CML LSC are resistant to the effects of TKIs and they persist in all patients on long-term therapy, where they may promote acquired TKI resistance, drive relapse or disease progression and inevitably represent a bottleneck to cure. Since their discovery in patients almost two decades ago, CML LSC have become a well-recognised exemplar of the cancer stem cell and have been characterised extensively with the aim of developing new curative therapeutic approaches based on LSC eradication. This review summarises our current understanding of many of the pathways and mechanisms that promote the survival of the CP CML LSC and how they can be a source of new gene coding mutations that impact in the clinic. We also review recent pre-clinical approaches that show promise to eradicate the LSC, and future challenges on the path to cure

Eradication of chronic myeloid leukemia stem cells: a novel mathematical model predicts no therapeutic benefit of adding G-CSF to imatinib

Imatinib mesylate induces complete cytogenetic responses in patients with chronic myeloid leukemia (CML), yet many patients have detectable BCR-ABL transcripts in peripheral blood even after prolonged therapy. Bone marrow studies have shown that this residual disease resides within the stem cell compartment. Quiescence of leukemic stem cells has been suggested as a mechanism conferring insensitivity to imatinib, and exposure to the Granulocyte-Colony Stimulating Factor (G-CSF), together with imatinib, has led to a significant reduction in leukemic stem cells in vitro. In this paper, we design a novel mathematical model of stem cell quiescence to investigate the treatment response to imatinib and G-CSF. We find that the addition of G-CSF to an imatinib treatment protocol leads to observable effects only if the majority of leukemic stem cells are quiescent; otherwise it does not modulate the leukemic cell burden. The latter scenario is in agreement with clinical findings in a pilot study administering imatinib continuously or intermittently, with or without G-CSF (GIMI trial). Furthermore, our model predicts that the addition of G-CSF leads to a higher risk of resistance since it increases the production of cycling leukemic stem cells. Although the pilot study did not include enough patients to draw any conclusion with statistical significance, there were more cases of progression in the experimental arms as compared to continuous imatinib. Our results suggest that the additional use of G-CSF may be detrimental to patients in the clinic

Oscillations in a maturation model of blood cell production.

We present a mathematical model of blood cell production which describes both the development of cells through the cell cycle, and the maturation of these cells as they differentiate to form the various mature blood cell types. The model differs from earlier similar ones by considering primitive stem cells as a separate population from the differentiating cells, and this formulation removes an apparent inconsistency in these earlier models. Three different controls are included in the model: proliferative control of stem cells, proliferative control of differentiating cells, and peripheral control of stem cell committal rate. It is shown that an increase in sensitivity of these controls can cause oscillations to occur through their interaction with time delays associated with proliferation and differentiation, respectively. We show that the characters of these oscillations are quite distinct and suggest that the model may explain an apparent superposition of fast and slow oscillations which can occur in cyclical neutropenia. © 2006 Society for Industrial and Applied Mathematics

Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL-T315I

Background: Chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphatic leukemia (Ph + ALL) are caused by the t(9;22), which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase "escapes" the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs) Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the 'gatekeeper' mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia. Methods: The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC) from Ph + ALL-patients. Results: Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner. Conclusions: Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors

Investigating the SRC kinase HCK functions in Chronic Myelogenous Leukemia using chemical genetics methods.

The hallmark of chronic myelogenous leukemia (CML) is a chromosomal translocation between the c-abl gene (chromosome 9) and the bcr gene (chromosome 22). This event gives rise to BcrAbl, a chimeric protein with constitutive tyrosine kinase activity that drives the pathogenesis of the disease. Imatinib, a Bcr-Abl kinase inhibitor is the frontline therapy in CML. Although imatinib is very effective in the chronic phase of CML, patients in advanced stages develop resistance. An increased understanding of the signaling pathways implicated in CML pathogenesis and imatinib resistance is critical to the development of improved therapies. Previous work in our laboratory found that A-419259, a broad-spectrum Src family kinase (SFK) inhibitor induces growth arrest and apoptosis in CML cells, suggesting that SFKs are required for Bcr-Abl transformation of myeloid progenitors. Additionally, Hck couples BcrAbl to Stat5 activation in myeloid cells, which may contribute to survival. Furthermore, studies on samples from some imatinib-resistant patients found increased expression and activity of Hck and Lyn. In this dissertation, using two chemical genetic methods, I addressed the contribution of Hck to Bcr-Abl signaling and imatinib resistance. To explore the individual contribution of Hck to Bcr-Abl signaling, I developed an A419259-resistant mutant of Hck (Hck-T338M). Expression of Hck-T338M fully protected K562 CML cells from A-419259-induced apoptosis, an effect that correlated with sustained Stat5 activation. In addition, the Hck-T338M partially protected CML cells against the growth inhibition induced by A-419259. These studies suggest that Hck plays a non-redundant role as a key downstream survival partner for Bcr-Abl.I also tested whether Hck overexpression was sufficient to induce imatinib resistance in CML cells. For this study, I developed a mutant of Hck (Hck-T338A) that is uniquely sensitive to NaPP1, an analog of the generic SFK inhibitor pyrrazolo-pyrimidine 1. Overexpression of Hck or Hck-T338A in K562 cells induced resistance to imatinib-dependent apoptosis and growth arrest. Furthermore, NaPP1 reversed imatinib resistance in K562-Hck-T338A cells, suggesting that Hck-induced imatinib resistance requires Hck kinase activity. Taken together, my work validates Hck as a target for the development of apoptosis-inducing drugs and that are likely to be effective in imatinib-resistant patients

Chronic myeloid leukemia-clinical, experimental and health economic studies with special reference to imatinib treatment

CML is a malignant disease that originates in the bone marrow stem cell, carrying the Philadelphia chromosome with the BCR-ABL fusion gene. This gene translates into an active tyrosine kinase, Bcr-Abl, affecting hematopoiesis, particularly resulting in increased numbers of white blood cells in the peripheral blood. Left untreated, CML progresses from a silent chronic phase (CP) to a life-threatening blastic phase (BP). After the millennium shift imatinib was introduced for the treatment of CML. Specifically targeting the Bcr-Abl oncoprotein, it was the first tyrosine kinase inhibitor (TKI) employed in cancer. It induced spectacular responses among CML-CP patients, strikingly reducing the risk of disease progression, combined with excellent tolerability. In this thesis we have studied various aspects of imatinib treatment in CML. In a cohort of 45 newly diagnosed CML-CP patients initiated on imatinib, we consecutively assessed treatment responses by FISH, PCR and chromosome banding analysis (CBA). In a landmark analysis, an early favourable response, defined as less than 10% BCR-ABL-positive cells by FISH after 3 months of treatment, was identified as a predictive marker of an improved long-term clinical outcome. Among evaluable patients 51% achieved this response. A large majority, 95% of such responders, reached complete cytogenetic response within 12 months and 100% an event-free survival at 48 months. We assessed the effect of imatinib treatment on neutrophil leukotriene (LT) signaling to evaluate its possible role as a clinical biomarker predictive of treatment response. Increased LT signaling has previously been suggested as a driver of leukocytosis in CML. The activity and expression of LTC4S, catalyzing formation of LTC4 from LTA4, were determined in neutrophils from 11 CML-CP patients during their initial phase of imatinib treatment, and the results related to the parallel development of BCR-ABL-expression. CD16+ neutrophils were isolated, their LTC4S activity measured and LTC4S expression determined at the protein and mRNA levels. In parallel, BCR-ABL expression was assessed by bone marrow CBA and by FISH on peripheral blood cells, including a combined May Grünewald Giemsa staining and FISH technique (MGG-FISH) to score neutrophilic cells. An aberrant expression of LTC4S in CML neutrophils was typically found, but it was rapidly normalized after initiation of imatinib treatment, later paralleled by a decreasing expression of BCR-ABL. The findings indicate that increased expression and activity of LTC4S in CML is a down-stream step of BCR-ABL activity, i.e. the Bcr-Abl protein directly or indirectly causes an upregulation of LTC4S. It is possible that an early evaluation of LTC4S expression during imatinib treatment could serve as a more rapid way of assessing treatment response than the current methods identifying BCR-ABL expression through CBA, FISH or qRT- PCR. We also defined real life outcome of patients with CML in Sweden during four decades and related the relative survival (RS) patterns to imatinib treatment and other management strategies. We assessed trends in survival and short-and long-term excess mortality among all patients (n=3,173) regardless of clinical trial enrollment. Patients were categorized into five age groups (79 years) and five calendar periods (1973- 1979, 1980-1986, 1987-1993, 1994-2000 and 2001-2008). We found that throughout all calendar periods, age was a strong predictor of survival, with superior survival for the youngest patients. In analyses including age and period of diagnosis, RS improved with calendar period in all age groups, but most markedly in patients younger than 79 years of age, particulary those 70-79 years of age. Survival among all age groups was greatest in the last calendar period, mainly as a result of an increasing use of imatinib. However, elderly patients still do poorly. The Swedish CML registry data show that patients diagnosed 2002-2008, at the age of 70-79 years received TKI in 66% and patients >80 years in only 18% of the cases. Finally, we compared the costs during the last decades with earlier decades treatment regimens and related the costs to the expected improved survival. Using Swedish real world national data from CML patients diagnosed in the country from 1973 to 2008 (n=1,778), we evaluated the incremental cost-effectiveness ratio (ICER) between three periods associated with broad implementation of imatinib (III), interferon-α and allogeneic stem cell transplantation (II), and symptomatic treatment (I), respectively. We observed substantial health gains over time, paralleled by increased treatment costs. The mean survival was 2.9, 9.2 and 18.5 years during periods I-III, respectively. The resulting ICER was £45 700 per QALY gained comparing periods III and II using a societal perspective. In a separate analysis by groups of age at diagnosis showed lower ICERs for individuals <50 years at diagnosis: £38 500 for the societal perspective. Since the prevalence of CML patients is increasing and assuming that 75% of each incident cohort was to receive imatinib at current prices, the imatinib budget would need to double by 2050. A future potential discontinuation of imatinib for selected excellent responders would reduce the ICER per QALY gained. Reduced drug cost of imatinib linked to the patent expiry of the drug will probably have a greater impact on ICER per QALY. An estimated price reduction of 80% (global competition) or 30% (expected change for biological drugs) would be associated with an ICER of £20 000 and £36 000, respectively, per QALY gained

A combined biomarker and clinical panel for chronic graft versus host disease diagnosis.

Whilst many chronic graft versus host disease (cGVHD) biomarkers have been previously reported, few have been verified in an independent cGVHD cohort. We aimed to verify the diagnostic accuracy of previously reported markers of cGVHD in a multi-centre Chronic GVHD Consortium. A total of 42 RNA and 18 protein candidate biomarkers were assessed amongst 59 cGVHD cases and 33 matched non-GVHD controls. Total RNA was isolated from PBMC, and RNA markers were quantified using PCR. Serum protein markers were quantified using ELISA. A combined 3 RNA biomarker (IRS2, PLEKHF1 and IL1R2) and 2 clinical variables (recipient CMV serostatus and conditioning regimen intensity) panel accurately (AUC 0.81) segregated cGVHD cases from controls. Other studied RNA and protein markers were not confirmed as accurate cGVHD diagnostic biomarkers. The studied markers failed to segregate higher risk cGVHD (per overall NIH 0-3 score, and overlap versus classic cGVHD status). These data support the need for multiple independent verification studies for the ultimate clinical application of cGVHD diagnostic biomarkers