The antiproliferative activity of kinase inhibitors in chronic myeloid leukemia cells is mediated by FOXO transcription factors

Chronic myeloid leukemia (CML) is initiated and maintained by the tyrosine kinase BCR-ABL which activates a number of signal transduction pathways, including PI3K/AKT signaling and consequently inactivates FOXO transcription factors. ABL-specific tyrosine kinase inhibitors (TKIs) induce minimal apoptosis in CML progenitor cells, yet exert potent antiproliferative effects, through as yet poorly understood mechanisms. Here, we demonstrate that in CD34+ CML cells, FOXO1 and 3a are inactivated and relocalized to the cytoplasm by BCR-ABL activity. TKIs caused a decrease in phosphorylation of FOXOs, leading to their relocalization from cytoplasm (inactive) to nucleus (active), where they modulated the expression of key FOXO target genes, such as Cyclin D1, ATM, CDKN1C, and BCL6 and induced G1 arrest. Activation of FOXO1 and 3a and a decreased expression of their target gene Cyclin D1 were also observed after 6 days of in vivo treatment with dasatinib in a CML transgenic mouse model. The over-expression of FOXO3a in CML cells combined with TKIs to reduce proliferation, with similar results seen for inhibitors of PI3K/AKT/mTOR signaling. While stable expression of an active FOXO3a mutant induced a similar level of quiescence to TKIs alone, shRNA-mediated knockdown of FOXO3a drove CML cells into cell cycle and potentiated TKI-induced apoptosis. These data demonstrate that TKI-induced G1 arrest in CML cells is mediated through inhibition of the PI3K/AKT pathway and reactivation of FOXOs. This enhanced understanding of TKI activity and induced progenitor cell quiescence suggests that new therapeutic strategies for CML should focus on manipulation of this signaling network

AMN107 (nilotinib): a novel and selective inhibitor of BCR-ABL

Chronic myelogenous leukaemia (CML) and Philadelphia chromosome positive (Ph+) acute lymphoblastic leukaemia (ALL) are caused by the BCR-ABL oncogene. Imatinib inhibits the tyrosine kinase activity of the BCR-ABL protein and is an effective, frontline therapy for chronic-phase CML. However, accelerated or blast-crisis phase CML patients and Ph+ ALL patients often relapse due to drug resistance resulting from the emergence of imatinib-resistant point mutations within the BCR-ABL tyrosine kinase domain. This has stimulated the development of new kinase inhibitors that are able to over-ride resistance to imatinib. The novel, selective BCR-ABL inhibitor, AMN107, was designed to fit into the ATP-binding site of the BCR-ABL protein with higher affinity than imatinib. In addition to being more potent than imatinib (IC50<30 nM) against wild-type BCR-ABL, AMN107 is also significantly active against 32/33 imatinib-resistant BCR-ABL mutants. In preclinical studies, AMN107 demonstrated activity in vitro and in vivo against wild-type and imatinib-resistant BCR-ABL-expressing cells. In phase I/II clinical trials, AMN107 has produced haematological and cytogenetic responses in CML patients, who either did not initially respond to imatinib or developed imatinib resistance. Dasatinib (BMS-354825), which inhibits Abl and Src family kinases, is another promising new clinical candidate for CML that has shown good efficacy in CML patients. In this review, the early characterisation and development of AMN107 is discussed, as is the current status of AMN107 in clinical trials for imatinib-resistant CML and Ph+ ALL. Future trends investigating prediction of mechanisms of resistance to AMN107, and how and where AMN107 is expected to fit into the overall picture for treatment of early-phase CML and imatinib-refractory and late-stage disease are discussed

Myeloid Leukemia

This book comprises a series of chapters from experts in the field of diagnosis and treatment of myeloid leukemias from all over the world, including America, Europe, Africa and Asia. It contains both reviews on clinical aspects of acute (AML) and chronic myeloid leukemias (CML) and original publications covering specific clinical aspects of these important diseases. Covering the specifics of myeloid leukemia epidemiology, diagnosis, risk stratification and management by authors from different parts of the world, this book will be of interest to experienced hematologists as well as physicians in training and students from all around the globe

Doctor of Philosophy

dissertationChronic myeloid leukemia (CML) is identified by the unique reciprocal chromosomal translocation involving BCR and ABL1, the fusion of which generates a constitutively active tyrosine kinase. Of critical importance for kinase function is oligomerization of multiple BCR-ABL1 proteins, facilitated by the N-terminal coiled-coil (CC) domain in BCR. While antineoplastic therapies have historically been dominated by small molecule drugs with a broad impact on cancer, recently there has been a shift toward small molecule targeted therapeutics, which was led by the development of imatinib. Imatinib, a tyrosine kinase inhibitor (TKI), was rationally developed for the treatment of CML. Although imatinib has been extremely successful in disease modification and increasing overall survival, it, like many of the subsequently developed TKIs, is subject to failure when mutations in the BCR-ABL1 kinase domain (the target of TKIs) occur, or the cell loses its dependence on the BCR-ABL1 protein. We have broken from the small molecule development track and instead focused on peptide-based inhibition of the upstream oligomerization event in CML pathogenesis. We previously described the anticancer activity of a dimerization inhibitor derived from the CC domain, called CCmut2/3 (representing two different versions of coiled-coil inhibitors). Driven by the positive results in previous studies, we proposed the following overarching hypothesis: Differential manipulation of domains within one BCRABL1 protein; or parallel manipulation of multiple pathways within one iv BCR-ABL1-containing cell will lead to a potent therapy which may overcome TKIresistant disease. Here we examine this hypothesis to determine the efficacy of the CCmut2/3 for broad-spectrum CML disease. In one study we observed that use of the CCmut2 in concert with one of several selective leukemia-specific secondary pathway inhibitors enhances the apoptotic potential and limits the proliferative capacity of K562 BCR-ABL1-containing cells. Another study describes the broad anticancer inhibitory potential of CCmut3 in cells with varying mutational status in the BCR-ABL1 kinase domain. Finally we investigate the potential of CCmut3 in the context of human disease with a series of ex vivo inquires using patient samples. This dissertation focuses on demonstrating efficacy of CCmut2/3 as a front-line CML therapy against several cell lines including those with wild-type and mutant BCRABL

ReSETting PP2A tumour suppressor activity in blast crisis and imatinib-resistant chronic myelogenous leukaemia

The deregulated kinase activity of p210-BCR/ABL oncoproteins, hallmark of chronic myelogenous leukaemia (CML), induces and sustains the leukaemic phenotype, and contributes to disease progression. Imatinib mesylate, a BCR/ABL kinase inhibitor, is effective in most of chronic phase CML patients. However, a significant percentage of CML patients develop resistance to imatinib and/or still progresses to blast crisis, a disease stage that is often refractory to imatinib therapy. Furthermore, there is compelling evidence indicating that the CML leukaemia stem cell is also resistant to imatinib. Thus, there is still a need for new drugs that, if combined with imatinib, will decrease the rate of relapse, fully overcome imatinib resistance and prevent blastic transformation of CML. We recently reported that the activity of the tumour suppressor protein phosphatase 2A (PP2A) is markedly inhibited in blast crisis CML patient cells and that molecular or pharmacologic re-activation of PP2A phosphatase led to growth suppression, enhanced apoptosis, impaired clonogenic potential and decreased in vivo leukaemogenesis of imatinib-sensitive and -resistant (T315I included) CML-BC patient cells and/or BCR/ABL+ myeloid progenitor cell lines. Thus, the combination of PP2A phosphatase-activating and BCR/ABL kinase-inhibiting drugs may represent a powerful therapeutic strategy for blast crisis CML patients

Investigation into the relevance of BCR-ABL for the survival of cancer stem cells in chronic myeloid leukaemia

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder of the haemopoietic stem cell, defined by the Philadelphia chromosome (Ph) - the outcome of a balanced, reciprocal translocation between the long arms of chromosomes 9 and 22. The novel fusion oncogene generated on chromosome 22 as a result of this translocation is called BCR-ABL. In the majority of patients, this oncogene transcribes a 210-kDa constitutively active protein tyrosine kinase, often referred to as p210BCR-ABL, which is necessary for the transformation of the disease. The introduction of the orally available, tyrosine kinase inhibitor (TKI) - imatinib mesylate (IM) - marked a major advance in CML treatment in terms of efficacy and tolerability and has now become the first line of therapy. IM acts by competing with ATP to block ABL-kinase activity, resulting in the selective apoptosis induction of BCR-ABL+ cells. However, despite the success of IM as standard therapy for CML, only a small proportion of patients obtain a complete molecular response, where they become negative for BCR-ABL transcripts by RTPCR. It is hypothesised that this minimal residual disease is the result of a primitive quiescent subpopulation of leukaemic cells, which may be the cause for relapse at a later date. Another major clinical concern is the observation of molecular resistance in IM-treated patients. Proposed mechanisms of resistance include BCR-ABL amplification, decreased intracellular IM concentrations caused by drug efflux proteins such as multi drug resistance-1 and the development of point mutations within the ABL-kinase domain. In an attempt to overcome IMresistance, a second generation of BCR-ABL inhibitors has been developed. Dasatinib (BMS-354825, Sprycel) is a TKI developed for the treatment of IM resistant or -intolerant patients with Ph+ leukaemias, which has a 325-fold greater potency than IM against cells expressing wild-type BCR-ABL, and is effective against all IM-resistant BCR-ABL mutants tested in vitro, except T315I. Previously, we showed that dasatinib induced durable inhibition of BCR-ABL and impressive clearance of Ph+ cells, however, the primitive quiescent cell population did not appear to undergo apoptosis even after several days TKI exposure. Therefore, it was still not clear whether early CML progenitor cells depend on BCR-ABL for their growth and survival. In this study we have attempted to determine whether CML stem cells are dependent on BCR-ABL TK activity for their survival and/or proliferation using dasatinib treatment and aimed to characterise the cells which survived drug exposure. We found that 10% of the CML cells were able to survive the dasatinib treatment. We also showed that maximal BCR-ABL TK inhibition was achieved in the surviving CML cells, both in the bulk population of cells and the more problematic primitive stem cell population. Those cells which survived the dasatinib treatment were found to be primitive, residing mainly in the undivided cell fraction and the very early cell divisions. Since these BCR-ABL TK-inhibited, resistant cells were also able to grow when re-cultured in cytokines and form longterm culture-initiating cell (LTC-IC) colonies; these data suggested that ~10% of primitive CD34+ CML cells are not addicted to BCR-ABL TK activity for their survival. This also suggested that these primitive, resistant CML cells appeared to survive and proliferate by BCR-ABL-independent mechanisms. Therefore, the next experiments were then designed to investigate the cellular process of autophagy as a potential means of primitive CML cell survival. Analysis of the properties of CD34+ CML cells which remained viable following dasatinib treatment, revealed the existence of cytoplasmic autophagic structures determined by electron microscopy and significantly increased autophagosome-asociated LC3-II, particularly in the cells cultured without growth factors (GF)s. This suggested that autophagy is induced following GF deprivation of CML cells and is significantly increased within these cells, upon BCR-ABL inhibition following dasatinib treatment. Furthermore, we also found that the inhibition of autophagy greatly potentiated the effect of TKI treatment on the reduction of primitive CML progenitor cells, in terms of the effective eradication of functionally defined colony forming cells and LTC-ICs. Overall, this thesis has shown for the first time that the most TKI-resistant primitive CML cells are likely to be independent of BCR-ABL TK activity for their proliferation and/or survival. Furthermore, we have shown that these resistant CML stem cells rely on the BCR-ABL independent autophagy process for survival in response to stressful conditions, such as, GF deprivation and TKI treatment

A personalised medicine approach for ponatinib-resistant chronic myeloid leukaemia.

BACKGROUND: Chronic myeloid leukaemia (CML) is characterised by the presence of a fusion driver oncogene, BCR-ABL1, which is a constitutive tyrosine kinase. Tyrosine kinase inhibitors (TKIs) are the central treatment strategy for CML patients and have significantly improved survival rates, but the T315I mutation in the kinase domain of BCR-ABL1 confers resistance to all clinically approved TKIs, except ponatinib. However, compound mutations can mediate resistance even to ponatinib and remain a clinical challenge in CML therapy. Here, we investigated a ponatinib-resistant CML patient through whole-genome sequencing (WGS) to identify the cause of resistance and to find alternative therapeutic targets. PATIENTS AND METHODS: We carried out WGS on a ponatinib-resistant CML patient and demonstrated an effective combination therapy against the primary CML cells derived from this patient in vitro. RESULTS: Our findings demonstrate the emergence of compound mutations in the BCR-ABL1 kinase domain following ponatinib treatment, and chromosomal structural variation data predicted amplification of BCL2. The primary CD34(+) CML cells from this patient showed increased sensitivity to the combination of ponatinib and ABT-263, a BCL2 inhibitor with a negligible effect against the normal CD34(+) cells. CONCLUSION: Our results show the potential of personalised medicine approaches in TKI-resistant CML patients and provide a strategy that could improve clinical outcomes for these patients