Advances in the structural biology, design and clinical development of Bcr-Abl kinase inhibitors for the treatment of chronic myeloid leukaemia

The constitutively activated Abl tyrosine kinase domain of the chimeric Bcr-Abl oncoprotein is responsible for the transformation of haematopoietic stem cells and the symptoms of chronic myeloid leukaemia (CML). Imatinib targets the tyrosine kinase activity of Bcr-Abl and is a first-line therapy for this malignancy. Although highly effective in chronic phase CML, patients who have progressed to the advanced phase of the disease frequently fail to respond to imatinib or develop resistance to therapy and relapse. This is often due to the emergence of clones expressing mutant forms of Bcr-Abl, which exhibit a decreased sensitivity towards inhibition by imatinib. Considerable progress has recently been made in understanding the structural biology of Abl and the molecular basis for resistance, facilitating the discovery and development of second generation drugs designed to combat mutant forms of Bcr-Abl. The first of these compounds to enter clinical development were BMS-354825 (BristolMyersSquibb) and AMN107 (Novartis Pharma) and, from Phase I results, both of these promise a breakthrough in the treatment of imatinib-resistant CML. Recent advances with these and other promising classes of new CML drugs are reviewed.     

Colocalization of USP1 and РН domain of Bcr-Abl oncoprotein in terms of chronic myeloid leukemia cell rearrangements

The development of chronic myeloid leukemia (CML) is the result of a reciprocal translocation between chromosomes 9 and 22 due to the emergence of Philadelphia chromosome. The product of this mutation is a hybrid oncoprotein Bcr-Abl. According to the results of mass spectrometric analysis, USP1 protein was identified as a potential candidate for interaction with the PH domain Bcr-Abl oncoprotein. Due to the deubiquitination properties, USP1 protein can prevent proteasomal degradation of Bcr-Abl oncoprotein in a cell and, consequently, contribute to its accumulation, and the progression of the disease. In this work, creating the genetic constructs, we detected the USP1 protein localization in the cell. Also, a nuclear colocalization of USP1 protein with PH domain of Bcr-Abl oncoprotein in HEK293T cells was shown. The results are important for understanding the implications of the Philadelphia chromosome emergence, and the development of new methods for CML treatment, since the recent techniques are not always effective due to the emergence of numerous mutations that cause drug resistance and relapse of the disease.     

Functional phosphoproteomic analysis reveals cold-shock domain protein A to be a Bcr-Abl effector-regulating proliferation and transformation in chronic myeloid leukemia

One proposed strategy to suppress the proliferation of imatinib-resistant cells in chronic myeloid leukemia (CML) is to inhibit key proteins downstream of Bcr-Abl. The PI3K/Akt pathway is activated by Bcr-Abl and is specifically required for the growth of CML cells. To identify targets of this pathway, we undertook a proteomic screen and identified several proteins that differentially bind 14-3-3, dependent on Bcr-Abl kinase activity. An siRNA screen of candidates selected by bioinformatics analysis reveals cold-shock domain protein A (CSDA), shown previously to regulate cell cycle progression in epithelial cells, to be a positive regulator of proliferation in a CML cell line. We show that Akt can phosphorylate the serine 134 residue of CSDA but, downstream of Bcr-Abl activity, this modification is mediated through the activation of MEK/p90 ribosomal S6 kinase (RSK) signaling. Inhibition of RSK, similarly to treatment with imatinib, blocked proliferation specifically in Bcr-Abl-positive leukemia cell lines, as well as cells from CML patients. Furthermore, these primary CML cells showed an increase in CSDA phosphorylation. Expression of a CSDA phospho-deficient mutant resulted in the decrease of Bcr-Abl-dependent transformation in Rat1 cells. Our results support a model whereby phosphorylation of CSDA downstream of Bcr-Abl enhances proliferation in CML cells to drive leukemogenesis.     

Changes in the proteome associated with the action of Bcr-Abl tyrosine kinase are not related to transcriptional regulation

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease, the hallmark of which is the Bcr-Abl protein tyrosine kinase (PTK). Without intervention the disease progresses from a benign chronic phase to a rapidly fatal blast crisis. To identify the molecular mechanisms underlying disease progression we used two-dimensional gel electrophoresis on a model we have previously described using the expression of a conditional mutant of Bcr-Abl PTK in a multipotent stem cell line, FDCP-Mix. Long term exposure of FDCP-Mix cells to Bcr-Abl mimics disease progression in CML. Four major differences were observed as a consequence of long term exposure to the Bcr-Abl PTK compared with cells exposed short term. The proteins were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry-generated peptide mass fingerprint data and liquid chromatography-tandem mass spectrometry-generated sequence information. Leukotriene A4 hydrolase, an enzyme known to be deregulated in CML, was found to be up-regulated. Annexin VI, vacuolar ATP synthase catalytic subunit A, and mortalin were found to be down-regulated. Poly(A) PCR cDNA analysis showed there was no correlation between the protein expression changes and mRNA levels. Western blot analysis also indicated no change in the levels of mortalin or leukotriene A4 hydrolase, indicating that post-translational events may modify protein content of the specific spots. Leukotriene B4 levels (product of leukotriene A4 hydrolase) were, however, reduced in cells exposed long term to Bcr-Abl activity. This study demonstrates the potential of proteomic analysis to define novel effects of oncogenes.     

Atypical activation of signaling downstream of inactivated Bcr-Abl mediates chemoresistance in chronic myeloid leukemia

Chronic myeloid leukemia (CML) epitomises successful targeted therapy, where inhibition of tyrosine kinase activity of oncoprotein Bcr-Abl1 by imatinib, induces remission in 86% patients in initial chronic phase (CP). However, in acute phase of blast crisis, 80% patients show resistance, 40% among them despite inhibition of Bcr-Abl1 activity. This implies activation of either Bcr-Abl1- independent signalling pathways or restoration of signalling downstream of inactive Bcr-Abl1. In the present study, mass spectrometry and subsequent in silico pathway analysis of differentiators in resistant CML-CP cells identified key differentiators, 14–3-3ε and p38 MAPK, which belong to Bcr-Abl1 pathway. Their levels and activity respectively, indicated active Bcr-Abl1 pathway in CML-BC resistant cells, though Bcr-Abl1 is inhibited by imatinib. Further, contribution of these components to resistance was demonstrated by inhibition of Bcr-Abl1 down-stream signalling by knocking-out of 14–3-3ε and inhibition of p38 MAPK activity. The observations merit clinical validation to explore their translational potential.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00647-x.     

Targeting the silent minority: emerging immunotherapeutic strategies for eradication of malignant stem cells in chronic myeloid leukaemia

Standard allogeneic stem cell transplantation (alloSCT) has provided a cure for chronic myeloid leukaemia (CML) over the last 25 years, but is only an option for a minority of patients. It was hoped that the introduction of imatinib mesylate (IM), a specific tyrosine kinase inhibitor that targets the Bcr-Abl oncogene product, would provide long-term remission or even cure for those patients without a donor, but studies have shown that IM does not eliminate leukaemic stem cells in CML patients. To overcome this problem of molecular persistence, research is underway to combine reduced intensity stem cell transplant or non-donor-dependent immunotherapies with IM with the aim of increasing cure rate, reducing toxicity and improving quality of life. The alternative approach is to combine IM or second-generation agents with other novel drugs that interrupt key signalling pathways activated by Bcr-Abl. This article will focus on the latest immunotherapy and molecularly targeted therapeutic options in CML and how they may be combined to improve the outcome for CML patients in the future.     

Expression of interferon consensus sequence binding protein (ICSBP) is downregulated in Bcr-Abl-induced murine chronic myelogenous leukemia-like disease, and forced coexpression of ICSBP inhibits Bcr-Abl-induced myeloproliferative disorder

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder resulting from the neoplastic transformation of a hematopoietic stem cell. The majority of cases of CML are associated with the (9;22) chromosome translocation that generates the bcr-abl chimeric gene. Alpha interferon (IFN-alpha) treatment induces hematological remission and prolongs life in 75% of CML patients in the chronic phase. It has been shown that mice deficient in interferon consensus sequence binding protein (ICSBP), a member of the interferon regulatory factor family, manifest a CML-like syndrome. We have shown that expression of Bcr-Abl in bone marrow (BM) cells from 5-fluorouracil (5-FU)-treated mice by retroviral transduction efficiently induces a myeloproliferative disease in mice resembling human CML. To directly test whether icsbp can function as a tumor suppressor gene, we examined the effect of ICSBP on Bcr-Abl-induced CML-like disease using this murine model for CML. We found that expression of the ICSBP protein was significantly decreased in Bcr-Abl-induced CML-like disease. Forced coexpression of ICSBP inhibited the Bcr-Abl-induced colony formation of BM cells from 5-FU-treated mice in vitro and Bcr-Abl-induced CML-like disease in vivo. Interestingly, coexpression of ICSBP and Bcr-Abl induced a transient B-lymphoproliferative disorder in the murine model of Bcr-Abl-induced CML-like disease. Overexpression of ICSBP consistently promotes rather than inhibits Bcr-Abl-induced B lymphoproliferation in a murine model where BM cells from non-5-FU-treated donors were used, indicating that ICSBP has a specific antitumor activity toward myeloid neoplasms. We also found that overexpression of ICSBP negatively regulated normal hematopoiesis. These data provide direct evidence that ICSBP can act as a tumor suppressor that regulates normal and neoplastic proliferation of hematopoietic cells.     

The NH(2)-terminal coiled-coil domain and tyrosine 177 play important roles in induction of a myeloproliferative disease in mice by Bcr-Abl

Bcr-Abl, a fusion protein generated by t(9;22)(q34;q11) translocation, plays a critical role in the pathogenesis of chronic myelogenous leukemia (CML). It has been shown that Bcr-Abl contains multiple functional domains and motifs and can disrupt regulation of many signaling pathways and cellular functions. However, the role of specific domains and motifs of Bcr-Abl or of specific signaling pathways in the complex in vivo pathogenesis of CML is not completely known. We have previously shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces a myeloproliferative disorder (MPD) in mice resembling human CML. We have also shown that the Abl kinase activity within Bcr-Abl is essential for Bcr-Abl leukemogenesis, yet activation of the Abl kinase without Bcr sequences is not sufficient to induce MPD in mice. In this study we investigated the role of Bcr sequences within Bcr-Abl in inducing MPD using this murine model for CML. We found that the NH(2)-terminal coiled-coil (CC) domain was both essential and sufficient, even though not efficient, to activate Abl to induce an MPD in mice. Interestingly, deletion of the Src homology 3 domain complemented the deficiencies of the CC-deleted Bcr-Abl in inducing MPD in mice. We further demonstrated that the Grb2 binding site at Y177 played an important role in efficient induction of MPD. These studies directly demonstrated the important roles of Bcr sequences in induction of MPD by Bcr-Abl.     

Leukemia stem cells: the root of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by a chromosome translocation that generates the Bcr-Abl oncogene encoding a constitutive kinase activity. Despite remarkable success in controlling CML at chronic phase by Bcr-Abl tyrosine kinase inhibitors (TKIs), a significant proportion of CML patients treated with TKIs develop drug resistance due to the inability of TKIs to kill leukemia stem cells (LSCs) that are responsible for initiation, drug resistance, and relapse of CML. Therefore, there is an urgent need for more potent and safer therapies against leukemia stem cells for curing CML. A number of LSCassociated targets and corresponding signaling pathways, including CaMKII-γ, a critical molecular switch for co-activating multiple LSC-associated signaling pathways, have been identified over the past decades and various small inhibitors targeting LSC are also under development. Increasing evidence shows that leukemia stem cells are the root of CML and targeting LSC may offer a curable treatment option for CML patients. This review summarizes the molecular biology of LSC and itsassociated targets, and the potential clinical application in chronic myeloid leukemia.     

Bcr-Abl with an SH3 deletion retains the ability To induce a myeloproliferative disease in mice, yet c-Abl activated by an SH3 deletion induces only lymphoid malignancy

The bcr-abl oncogene plays a critical role in the pathogenesis of chronic myelogenous leukemia (CML). The fusion of Bcr sequences to Abl constitutively activates the Abl protein tyrosine kinase. We have recently shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces in mice a myeloproliferative disease resembling human CML and that Abl kinase activity is essential for Bcr-Abl to induce a CML-like myeloproliferative disease. However, it is not known if activation of the Abl kinase alone is sufficient to induce a myeloproliferative disease. In this study, we examined the role of the Abl SH3 domain of Bcr-Abl in induction of myeloproliferative disease and tested whether c-Abl activated by SH3 deletion can induce a CML-like disease. We found that Bcr-Abl with an Abl SH3 deletion still induced a CML-like disease in mice. In contrast, c-Abl activated by SH3 deletion induced only lymphoid malignancies in mice and did not stimulate the growth of myeloid colonies from 5-fluorouracil-treated bone marrow cells in vitro. These results indicate that Bcr sequences in Bcr-Abl play additional roles in inducing myeloproliferative disease beyond simply activating the Abl kinase domain and that functions of the Abl SH3 domain are either not required or redundant in Bcr-Abl-induced myeloproliferative disease. The results also suggest that the type of hematological neoplasm induced by an abl oncogene is influenced not only by what type of hematopoietic cells the oncogene is targeted into but also by the intrinsic oncogenic properties of the particular abl oncogene. In addition, we found that DeltaSH3 c-Abl induced less activation of Akt and STAT5 than did Bcr-Abl, suggesting that activation of these pathways plays a critical role in inducing a CML-like disease.     

Characterization of p87C3G, a novel, truncated C3G isoform that is overexpressed in chronic myeloid leukemia and interacts with Bcr-Abl

A novel C3G isoform, designated p87C3G, lacking the most amino terminal region of the cognate protein has been found to be overexpressed in two CML cell lines, K562 and Boff 210, both expressing Bcr-Abl p210. p87C3G expression is also highly augmented in patients diagnosed with chronic myeloid leukemia (CML) Ph+, in comparison with healthy individuals, and returns to basal levels after treatment with STI571. p87C3G co-immunoprecipitates with both CrkL and Bcr-Abl in CML cell lines and co-immunoprecipitation between p87C3G and Bcr-Abl was also detected in primary cells from CML patients. These interactions have been confirmed by in vitro pull down experiments. The interaction between p87C3G and Bcr-Abl involves the SH3-binding domain of p87C3G and the SH3 domain of Abl and depends mostly on the first polyproline region of p87C3G. Furthermore, we also demonstrated that p87C3G is phosphorylated in vitro by a Bcr-Abl-dependent mechanism. These results indicate that p87C3G overexpression is linked to CML phenotype and that p87C3G may exert productive functional interactions with Bcr-Abl signaling components suggesting the implication of this C3G isoform in the pathogenesis of chronic myeloid leukemia.     

Growth arrest of BCR-ABL positive cells with a sequence-specific polyamide-chlorambucil conjugate

Chronic myeloid leukemia (CML) is characterized by the presence of a constitutively active Abl kinase, which is the product of a chimeric BCR-ABL gene, caused by the genetic translocation known as the Philadelphia chromosome. Imatinib, a selective inhibitor of the Bcr-Abl tyrosine kinase, has significantly improved the clinical outcome of patients with CML. However, subsets of patients lose their response to treatment through the emergence of imatinib-resistant cells, and imatinib treatment is less durable for patients with late stage CML. Although alternative Bcr-Abl tyrosine kinase inhibitors have been developed to overcome drug resistance, a cocktail therapy of different kinase inhibitors and additional chemotherapeutics may be needed for complete remission of CML in some cases. Chlorambucil has been used for treatment of B cell chronic lymphocytic leukemia, non-Hodgkin's and Hodgkin's disease. Here we report that a DNA sequence-specific pyrrole-imidazole polyamide-chlorambucil conjugate, 1R-Chl, causes growth arrest of cells harboring both unmutated BCR-ABL and three imatinib resistant strains. 1R-Chl also displays selective toxicities against activated lymphocytes and a high dose tolerance in a murine model.     

A direct binding site for Grb2 contributes to transformation and leukemogenesis by the Tel-Abl (ETV6-Abl) tyrosine kinase

A direct binding site for the Grb2 adapter protein is required for the induction of fatal chronic myeloid leukemia (CML)-like disease in mice by Bcr-Abl. Here, we demonstrate direct binding of Grb2 to the Tel-Abl (ETV6-Abl) fusion protein, the product of complex (9;12) chromosomal translocations in human leukemia, via tyrosine 314 encoded by TEL exon 5. A Tel-Abl point mutant (Y314F) and a splice variant without TEL exon 5 sequences (Deltae5) lacked Grb2 interaction and exhibited decreased binding and phosphorylation of the scaffolding protein Gab2 and impaired activation of phosphatidylinositol 3-kinase, Akt, and extracellular signal-regulated kinase/mitogen-activated protein kinase in hematopoietic cells. Tel-Abl Y314F and Deltae5 were unable to transform fibroblasts to anchorage-independent growth and were defective for B-lymphoid transformation in vitro and lymphoid leukemogenesis in vivo. Previously, we demonstrated that full-length Tel-Abl induced two distinct myeloproliferative diseases in mice: CML-like leukemia similar to that induced by Bcr-Abl and a novel syndrome of small-bowel myeloid infiltration endotoxemia and hepatic and renal failure. Lack of the Grb2 binding site had no effect on development of small bowel syndrome but significantly attenuated the induction of CML-like disease by Tel-Abl. These results suggest that direct binding of Grb2 is a common mechanism contributing to leukemogenesis by oncogenic Abl fusion proteins.     

Cell-penetrating SH3 domain blocker peptides inhibit proliferation of primary blast cells from CML patients.

Bcr-Abl contributes prominently to the development of most chronic myeloid leukemias (CMLs). Prior work has identified the adapter protein CRKL as a major substrate of the Bcr-Abl tyrosine kinase. CRKL can also bind via its first SH3 domain [SH3(1)] to specific sequences in Bcr-Abl. Cell-penetrating peptides were developed that bind with high affinity and selectivity to the SH3(1) domain of CRKL. They disrupt Bcr-Abl-CRKL complexes and strongly reduce the proliferation of primary CML blast cells and cell lines established from Bcr-Abl-positive patients. Activation-specific antibodies against phosphorylated MAP kinase (MAPK) showed that MAPK activity is down-regulated in blast cells treated with the CRKLSH3(1) blocker peptides. We conclude that the Bcr-Abl-CRKL complexes are largely dependent on the CRKLSH3(1) domain, that the central mitogenic cascade is down-regulated as a consequence of the disruption of CRKLSH3(1) interactions, and that CRKL therefore contributes to the proliferation of CML blast cells.     

A novel tubulin polymerization inhibitor,MPT0B206, downregulates Bcr-Abl expression and induces apoptosis in imatinib-sensitive and imatinib-resistant CML cells

Imatinib, a Bcr-Abl-specific inhibitor, is effective for treating chronic myeloid leukemia (CML), but drug resistance has emerged for this disease. In this study, we synthesized a novel tubulin polymerization inhibitor, MPT0B206 (N-[1-(4-methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-formamide), and demonstrated its apoptotic effect and mechanism in imatinib-sensitive K562 and imatinib-resistant K562R CML cells. Western blotting and immunofluorescence microscopy showed that MPT0B206 induced microtubule depolymerization in K562 and K562R cells. MPT0B206 inhibited the growth of these cells in a concentration- and time-dependent manner. It did not affect the viability of normal human umbilical vein endothelial cells. MPT0B206 induced G2/M cell cycle arrest and the appearance of the mitotic marker MPM-2 in K562 and K562R cells, which is associated with the upregulation of cyclin B1 and the dephosphorylation of Cdc2. Treatment of K562 and K562R cells with MPT0B206 induced apoptosis and reduced the protein levels of procaspase-9 and procaspase-3 and increased caspase-3 activity and PARP cleavage. MPT0B206 also reduced the levels of the antiapoptotic proteins Mcl-1 and Bcl-2 and increased the level of the apoptotic protein Bax. Additional experiments showed that MPT0B206 markedly downregulated Bcr-Abl mRNA expression and total and phosphorylated Bcr-Abl protein levels and inhibited the phosphorylation of its downstream proteins STAT5, MAPK, and AKT, and the protein level of c-Myc in K562 and K562R cells. Furthermore, MPT0B206 triggered viability reduction and apoptosis in CML cells carrying T315I-mutated Bcr-Abl. Together, these results suggest that MPT0B206 is a promising alternative for treating imatinib-resistant CML.     

Cotreatment with apicidin overcomes TRAIL resistance via inhibition of Bcr-Abl signaling pathway in K562 leukemia cells

TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic cytokine that is capable of inducing apoptosis in a wide variety of cancer cells but not in normal cells. Although many cancer cells are sensitive to TRAIL-induced apoptosis, chronic myeloid leukemia (CML) develops resistance to TRAIL. In this study, we investigated whether apicidin, a novel histone deacetylase inhibitor, could overcome the TRAIL resistance in CML-derived K562 cells. Compared to treatment with apicidin or TRAIL alone, cotreatment with apicidin and TRAIL-induced apoptosis synergistically in K562 cells. This combination led to activation of caspase-8 and Bcl-2 interacting domain (Bid), resulting in the cytosolic accumulation of cytochrome c from mitochondria as well as an activation of caspase-3. Treatment with apicidin resulted in down-regulation of Bcr-Abl and inhibition of its downstream target, PI3K/AKT-NF-κB pathway. In addition, apicidin decreased the level of NF-κB-dependent Bcl-x_L, leading to caspase activation and Bid cleavage. These results suggest that apicidin may sensitize K562 cells to TRAIL-induced apoptosis through caspase-dependent mitochondrial pathway by regulating expression of Bcr-Abl and its related anti-apoptotic proteins. Therefore, the present study suggests that combination of apicidin and TRAIL may be an effective strategy for treating TRAIL-resistant Bcr-Abl expressing CML cells.