ABL Fusion Protein Research Articles

Getting to Know the Molecular Landscape of R/R Chronic Myeloid Leukemia: NGS-Based Profiling of Myeloid Mutations Beyond BCR/ABL

Introduction: Even in the tyrosine kinase inhibitors (TKIs) era, there's still a subgroup of patients with chronic myeloid leukemia (CML) with poor response to treatment and therefore worse prognosis. The most frequent cause of resistance to treatment is the mutation in ABL fusion protein reported in 15-25% of the failures to TKIs. Nevertheless, there are other several factors not clearly described yet in published data, including myeloid mutations assessed by Next-Generation Sequence (NGS) beyond BCR-ABL kinase domain mutations. Methods: We performed a multicenter, open, prospective trial to identify the frequency and clinical implications of myeloid mutations. We included all patients with relapsed/refractory CML to any TKI defined as failure to achieve cytogenetic response at 3 months or decrease <10% of BCR/ABL at 6 months or progression of CML after any response achieved by TKI. Patients with intolerance or non-adherence to treatment were excluded. For adherence validation, the MMAS-8 test was applied. We calculated a sample of 29 patients. Recruitment began in February 2021 and the sample was completed in August 2022. DNA was extracted from a bone marrow blood in EDTA with the automatized equipment, Maxwell 16 DNA purification Kits. The evaluation of DNA was performed by massive parallel sequencing of 40 genes frequently mutated in myeloid malignancies identifying ABL mutations and simultaneous mutations in other genes. Results: Twenty-nine patients were evaluated in 16 centers from February 2021 to August 2022; seventeen were men. Six patients were diagnosed from 2002 to 2009, seventeen from 2010 to2019 and 6 after 2020. At time of diagnosis, the mean age was 45 years ± 14.8, two were in accelerated phase (AP-CML) and the rest were diagnosed in chronic phase (CP-CML). Sokal score was achievable in 24 cases being low, intermediate and high in 6, 15 and 3 cases respectively. In karyotype, two patients showed minor chromosomal aberrations and another one had complex karyotype. The study was performed at 68.1 months from diagnosis; 9 cases had relapse and the rest didn't achieve molecular responses. All the treatment failures were identified by PCR-BCR/ABL by the exception of two cases that presented blast phase (BP-CML). At recruitment, 23 cases were in CP-CML (2 in its second), 4 cases in AP-CML and 2 in BP-CML. The patients had a mean of 4 previous lines of treatment. The NGS panel showed the next results: Sixteen mutations were found in eleven patients (37.9%). As expected the most frequently mutated gene was ABL in four cases. The mutations involved were G252H, T315I, E255V, Y253H. Nevertheless, there were more patients with mutations in genes other than ABL with seven cases. Three mutations were repeated in two occasions: DNMT3A, ASXL-1 y TP53. The other mutations detected were RUNX1, ATM, PFH6, SF3B1, y WT1. Patients with mutations detected had higher Sokal score compared to non-mutated ones, 0.96 vs 1.05 points, without statistical significance. The only variable that showed statistical value for the presence or absence of mutations was the platelet count at diagnosis (281,200 vs 478,523) Conclusion: This study shows us that there are more mutations associated with therapeutic failure in patients with CML in addition to those in the ABL domain. Knowing these mutations can support the diagnosis, prognosis, establish target therapies and even guide the decision of a bone marrow transplant. Studies with larger populations are required to corroborate the data presented here to generate correlations the clinical and prognostic relevance of myeloid mutations, both at the time of treatment failure and perhaps at diagnosis.

Chromosomal Instability in Chronic Myeloid Leukemia: Mechanistic Insights and Effects.

Simple SummaryChronic myeloid leukemia is a disease diagnosed by the presence of the Philadelphia chromosome, which leads to the BCR::ABL fusion oncoprotein and overactive tyrosine kinase activity. Multiple other genetic aberrations and chromosomal changes make the disease very heterogeneous, and these changes increase as the disease becomes more aggressive. Understanding the cause and effects of chromosomal instability in CML might help to improve treatment options and monitoring of patients with advanced phases of CML.The most recent two decades have seen tremendous progress in the understanding and treatment of chronic myeloid leukemia, a disease defined by the characteristic Philadelphia chromosome and the ensuing BCR::ABL fusion protein. However, the biology of the disease extends beyond the Philadelphia chromosome into a nebulous arena of chromosomal and genetic instability, which makes it a genetically heterogeneous disease. The BCR::ABL oncoprotein creates a fertile backdrop for oxidative damage to the DNA, along with impairment of genetic surveillance and the favoring of imprecise error-prone DNA repair pathways. These factors lead to growing chromosomal instability, manifested as additional chromosomal abnormalities along with other genetic aberrations. This worsens with disease progression to accelerated and blast phase, and modulates responses to tyrosine kinase inhibitors. Treatment options that target the genetic aberrations that mitigate chromosome instability might be a potential area for research in patients with advanced phase CML.

COL-3-Induced Molecular and Ultrastructural Alterations in K562 Cells.

Tetracycline-3 (4-dedimethylamino sancycline, COL-3) is a non-antibiotic tetracycline derivative. COL-3 exerts potent anti-metalloproteinase activity and its antitumor effects have been reported both in vitro and in vivo. In this study, we investigated the mechanisms of COL-3-induced cytotoxicity in a chronic myeloid leukemia cell line, K562, characterized by the BCR–ABL fusion protein. COL-3 induced K562 cell death in a concentration-dependent manner with an IC50 of 10.8 µg/mL and exhibited features of both apoptosis and necrosis. However, flow cytometry analysis revealed that necrotic cells dominated over the early and late apoptotic cells upon treatment with COL-3. Transmission electron microscopy analysis in combination with Western blotting (WB) analysis revealed early mitochondrial swelling accompanied by the early release of cytochrome c and truncated apoptosis inducing factor (tAIF). In addition, ultrastructural changes were detected in the endoplasmic reticulum (ER). COL-3 affected the levels of glucose-regulated protein-94 (GRP94) and resulted in m-calpain activation. DNA double strand breaks as a signature for DNA damage was also confirmed using an antibody against γH2AX. WB analyses did not demonstrate caspase activation, while Bcl-xL protein remained unaffected. In conclusion, COL-3-induced cell death involves DNA damage as well as mitochondrial and ER perturbation with features of paraptosis and programmed necrosis.

Current Management of Chronic Myeloid Leukemia Myeloid Blast Phase.

Despite the major advancements in the management of chronic phase (CP) chronic myeloid leukemia (CML), blast crisis (BC) remains a major therapeutic challenge. BC can be myeloid, lymphoid, or mixed lineage with myeloid BC being the most common type. BC in CML is mediated by aberrant tyrosine kinase activity of the BCR::ABL fusion protein. The introduction of BCR::ABL tyrosine kinase inhibitor (TKI) has been a gamechanger in the treatment of CML and there has been a significant reduction in the incidence of BC. The main treatment goal in BC is to achieve a second CP and consolidate that with an allogeneic stem cell transplantation (SCT) in eligible patients. The outcomes in BC remain dismal even in the current era. In this review, we provide an overview of the biology and current therapeutic approach in myeloid BC.

Characteristics of BCR-ABL gene variants in patients of chronic myeloid leukemia.

BackgroundDepending on breakpoints of rearrangement different types of BCR–ABL fusion protein can be generated in patients of chronic myeloid leukemia (CML). The aim of this study is to observe frequencies of major transcripts in CML patients by reverse transcriptase polymerase chain reaction (RT-PCR) and their hematological features at the time of presentation.Materials and methodsThis cross sectional study was performed at Molecular Lab of Riphah International University, Islamabad from January to June 2019. Consecutive peripheral blood samples of 70 newly diagnosed CML patients in chronic phase were analyzed by RT-PCR to detect different BCR–ABL transcripts. Routine blood cell counts were assessed by an automated hematology analyzer.ResultsAll samples expressed typical BCR–ABL rearrangement. Expression of either e14a2 or e13a2 transcript was detected in 38 (54%) and 30 (43%) patients, respectively. Coexpression of e13a2 + e14a2 was found in 2 (3%) patients. The mean total leukocyte count was higher in group expressing e13a2 (P = 0.01). Higher mean platelet count was noted in patients with e14a2 transcript, but this difference was statistically insignificant (P = 0.1). The association of male gender was observed with the group exhibiting e14a2 (P = 0.01). There was no statistically significant association between transcript type and different ranges of age, hemoglobin levels, and platelet and total leukocyte counts (P > 0.05).Conclusione14a2 transcript was most common transcript in CML patients. Patients exhibiting e13a2 subgroup presented with significantly higher mean white blood cell count at the time of presentation. Significantly higher proportion of male patients was found to express e14a2 transcript over e13a2.

CAY10683 and imatinib have synergistic effects in overcoming imatinib resistance via HDAC2 inhibition in chronic myeloid leukemia.

Imatinib (IM) is utilized for targeting the BCR–ABL fusion protein and as such, chronic myeloid leukemia (CML) is considered to be a curable disorder for which patients can achieve a long survival. However, 15–20% CML cases end up with IM resistance that will develop into the accelerated stage and eventually the blast crisis, thereby restricting the treatment choices and giving rise to a dismal survival rate. Histone deacetylases (HDACs) have been identified to modulate the oncogene as well as tumor suppressor gene activities, and they play crucial parts in tumorigenesis. It is found recently that IM combined with HDAC inhibitors (HDACi) can serve as a promising means of overcoming IM resistance in CML cases. Santacruzamate A (CAY10683) has been developed as one of the selective and powerful HDACi to resist HDAC2. Therefore, in this study, we aimed to examine whether CAY10683 combined with IM could serve as the candidate antitumor treatment for CML cases with IM resistance. The influences of CAY10683 combined with IM on the cell cycle arrest, apoptosis, and viability of CML cells with IM resistance were investigated, and it was discovered that the combined treatment exerted synergistic effects on managing the IM resistance. Moreover, further studies indicated that CAY10683 combined with IM mainly exerted synergistic effects through inhibiting HDAC2 in K562-R and LAMA84-R cells with IM resistance. Besides, the PI3K/Akt signal transduction pathway was found to mediate the HDAC2 regulation of CML cells with IM resistance. Eventually, it was also discovered, based on the xenograft mouse model, that the combined treatment dramatically suppressed CML proliferation in vivo. To sum up, findings in the current study indicate that CAY10683 combined with IM can be potentially used as the candidate treatment for CML with IM resistance.

Computational dissection of allosteric inhibition of the SH2 domain of Bcr-Abl kinase by the monobody inhibitor AS25.

The deregulated breakpoint cluster region (Bcr)-Abelson tyrosine kinase (Abl) fusion protein represents an attractive pharmacological target for the treatment of chronic myeloid leukemia (CML). The high affinity of monobody AS25 was designed to target the Src homology 2 (SH2) domain of Bcr-Abl, leading to allosteric inhibition of Bcr-Abl through formation of protein-protein interactions. An I164E mutation in the SH2 domain disrupts AS25 binding to the SH2 domain of Bcr-Abl. The detailed mechanisms, however, remain to be unresolved. Here, molecular dynamics (MD) simulations and binding free energy calculations were performed to explore the conformational and energetic differences between the wild-type (WT) complexes of Bcr-Abl SH2 domain and AS25 (SH2WT-AS25) as well as the mutated complexes (SH2I164E-AS25). The results revealed that I164E mutation not only caused an increase in the conformational flexibility of SH2-AS25 complexes, but also weakened the binding affinity of AS25 to SH2. The comparative binding modes of SH2-AS25 complexes between WT and the I164E mutant were comprehensively analyzed to unravel the disruption of hydrophobic and hydrogen bonding interactions in the interface of the SH2-AS25 complex triggered by the I164E mutation. The results obtained may help to design the next generation of higher affinity Bcr-Abl SH2-specific peptide inhibitors.

Identification of TKI-Sensitive Point Mutations That Activate c-ABL Kinase Activity and Transformation Potential and Confer in Vitro Resistance to the Allosteric ABL Inhibitor GNF-5

Introduction:Pathologically activated tyrosine kinases represent attractive therapeutic targets, with deregulated ABL kinase representing one of the best-validated examples.While physiologic activation of c-ABL is tightly controlled by intramolecular interactions, ABL fusion proteins are constitutively activated by oligomerization driven by the fusion partner, as well as by loss of kinase autoinhibition due to removal of an autoinhibitory myristate site at the N-terminus of c-ABL (Nagar et al, Cell, 2003). Recent routine genomic tumor sequencing of various human malignancies has identified point mutations of unknown significance in numerous kinases, including c-ABL, and it is expected that the increasingly common practice of tumor genome sequencing will provide new opportunities for tailored targeted therapy. To maximize therapeutic benefit and avoid unnecessary cost and toxicity, it will be critical to distinguish "driver" from "passenger" mutations. We therefore sought to: (i) test the transforming potential of select clinically detected c-ABL mutants, (ii) prospectively identify novel additional activating c-ABL point mutations, and (iii) determine if active mutant c-ABL isoforms retain sensitivity to ABL TKIs.Results:Two of four clinically identified c-ABL mutations tested caused a dramatic increase in ABL kinase activity and transformed Ba/F3 cells to growth factor independence. A mutagenesis screen of c-ABL identified six additional transforming point mutations in the SH3 and kinase domains, including one that has been reported clinically in breast invasive carcinoma (cBioPortal.org). Relative to BCR-ABL, all c-ABL mutants showed heightened sensitivity to imatinib. Allosteric site inhibitors targeting the myristate-binding pocket in the BCR-ABL kinase domain (KD) have been shown to inhibit BCR-ABL kinase activity, purportedly by mimicking binding of the c-ABL myristate group and establishing c-ABL-like autoinhibitory constraints (Hantschel, Haematologica, 2012). While only two c-ABL mutations from the screen were near the allosteric site, all mutants exhibited decreased sensitivity to the allosteric inhibitor GNF-5. Recent studies have shown that derivative compounds of GNF-5 targeting this site in BCR-ABL are subvertable by on-target resistance mutations. We tested whether three BCR-ABL mutations described to confer in vitro, and in one case clinical, resistance to ABL001 (currently undergoing clinical trial evaluation) could impact c-ABL kinase activity presumably by perturbing this autoinhibitory pocket. In the context of c-ABL, all three ABL001-resistant mutations activated ABL kinase, transformed Ba/F3 cells, and conferred substantial resistance to allosteric inhibition relative to native BCR-ABL. These findings raise the possibility that c-ABL mutations may play a role in resistance to emerging allosteric ABL inhibitors. Importantly, previous work suggested that the genomic breakpoint on chromosome 9 in one third of CML cases is located 5' to ABL exon 1b, which would be predicted to result in simultaneous KD mutation expression in BCR-ABL and c-ABL (Uphoff et al, Leuk Res, 1999). To definitively demonstrate that co-expression of KD mutations in c-ABL and BCR-ABL can occur in primary patient samples, we analyzed RNA from five CML patients harboring a dominant BCR-ABL T315I mutation, and identified a heterozygous c-ABL 1b T315I mutation in one case.Conclusions:We have identified eight novel activating point mutations in c-ABL (three found in clinical isolates) that are potential therapeutic targets of ABL TKIs. Activating c-ABL mutants, including T315I, confer substantial resistance to the allosteric ABL inhibitor GNF-5 regardless of proximity to the inhibitor-binding site, implying resistance mechanisms beyond mere steric hindrance. Thus, mutations may induce distal conformational changes by disrupting a kinase conformation required for allosteric compound inhibition. In a subset of CML cases, c-ABL point mutants are co-expressed from the same allele as BCR-ABL, suggesting that the location of the chromosome 9 breakpoint may negatively impact clinical responsiveness to allosteric ABL inhibitors. Sequence analysis of c-ABL in patients with resistance to allosteric ABL inhibitors is required to validate c-ABL point mutants as a clinically important mechanism of resistance to this emerging class of targeted therapeutics. DisclosuresOff Label Use: the use of ABL TKIs for malignancies associated with point mutations in c-ABL. Shah:Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding; Plexxikon Inc.: Research Funding.

Non-steady-state hematopoiesis regulated by the C/EBPβ transcription factor.

Steady-state hematopoiesis responds to extracellular stimuli to meet changing demands and also to pathologically altered intracellular signaling. Granulocyte production increases following infection or in response to cytokine stimulation, and activation of the CCAAT/enhancer-binding protein β (C/EBPβ) transcription factor is required for such stress-induced granulopoiesis, whereas C/EBPα plays a critical role in maintaining steady-state granulopoiesis. Different roles of these C/EBP transcription factors in different modes of hematopoiesis are evolutionally conserved from zebrafish to humans. In addition to reactions against infections, C/EBPβ is responsible for cancer-driven myelopoiesis, which promotes cancer progression, at least in part, by abrogating the immune response in the cancer microenvironment. The BCR–ABL fusion protein activates emergency-specific pathway of granulopoiesis by upregulating C/EBPβ. This in turn causes chronic phase chronic myeloid leukemia, which is characterized by myeloid expansion. The C/EBPβ transcription factor also plays a role in other hematological malignancies of both myeloid and lymphoid lineage origin. Thus, elucidation of the upstream and downstream networks surrounding C/EBPβ will lead to the development of novel therapeutic strategies for diseases mediated by non-steady-state hematopoiesis.

C-Terminal Domain of ABL Family Kinases, ABL and ARG, Defines Their Distinct Leukemogenic Activities in Vivo

ARG (ABL2) is a member of ABL family kinases and highly homologous to ABL (ABL1) except the C-terminal domain adjacent to the kinase domain. TEL/ARG that consists of ARG fused to TEL (ETV6) has been found in AML M3, M4 or T-ALL patients, with additional chromosomal abnormalities of t(15;17)(q12;q21), inv(16)(p13;q12) or t(1;10;12)(q25;q23;p13) translocation, respectively. The structure of TEL/ARG is similar to that of TEL/ABL, which has been found in patients with T-ALL, B-ALL, AML and CML. TEL mediates homo-oligomerization of these fusion proteins, TEL/ABL and TEL/ARG, resulting in constitutive activation of the tyrosine kinases. Although ABL fusion proteins such as BCR/ABL and TEL/ABL have been intensively investigated, the involvement of TEL/ARG in leukemogenesis is not fully elucidated yet.We have recently reported that in vitro transforming activity of TEL/ARG was significantly lower than that of TEL/ABL although their kinase activities were almost identical. Interestingly, the in vitro transforming activities of C-terminus-swapped mutants, TEL/ABL with C-terminal domain of ARG [TEL-ABL (ARG-C)] or TEL/ARG with C-terminal domain of ABL [TEL/ARG (ABL-C)], were comparable to those of TEL/ARG or TEL/ABL, respectively, while kinase activities in the swapped mutants were not altered. These results suggest that C-termini of ABL family kinases contain some functional domain that defines their distinct transforming activities. The purpose of this study is to compare the in vivo leukemogenic activities of TEL/ABL and TEL/ARG, and evaluate the impact of the C-terminal domains.First, we investigated whether TEL/ABL or TEL/ARG caused leukemia in mice. Each fusion gene together with GFP gene was retrovirally transduced into the bone marrow cells harvested from C57BL/6 mice treated with 5-fluorouracil, and the transduced cells were transplanted into lethally irradiated mice. Similar to BCR/ABL, transplantation of TEL/ABL-transduced cells induced rapid myeloproliferative status accompanied by hepatomegaly and/or splenomegaly, and all the recipient mice died within 33 days after transplantation, indicating the development of myeloid leukemia. In contrast, the recipient mice transplanted with TEL/ARG-transduced cells did not develop myeloid leukemia but infiltrative mastocytosis, and died around 200 days after transplantation (Figure 1). Hemophagocytic mast cells accumulating in the bone marrow, and mast cells circulating in the peripheral blood were also observed in these mice.Next we investigated the roles of C-terminal domains of ABL and ARG in their in vivo leukemogenic activities. C-terminus-swapped mutants, TEL/ABL (ARG-C) and TEL/ARG (ABL-C) were retrovirally transduced into bone marrow cells and the transduced cells were transplanted as described above. Intriguingly, TEL/ABL (ARG-C) mutant failed to cause myeloproliferative status or leukemia at day 153 (Figure 2A). On the other hand, TEL/ARG (ABL-C) induced lethal myeloid leukemia in 4 out of 13 mice (30.8%) within 111 days after transplantation (Figure 2B). Collectively, the in vivo phenotypes induced by TEL/ABL (ARG-C) or TEL/ARG (ABL-C) resembled those induced by TEL/ARG or TEL/ABL, respectively. Mastocytosis, a characteristic of TEL-ARG-induced phenotype, has not been observed so far in any of the recipients of TEL/ABL (ARG-C) or TEL/ARG (ABL-C).In conclusion, these results indicate that C-terminal domain of ABL family kinases defines their distinct leukemogenic activities in vivo through modulating both proliferation and differentiation. Notably, C-terminus of ARG strongly suppressed the in vivo leukemogenic activity of TEL/ABL without impairing the tyrosine kinase activity. Further clarification of the molecular mechanisms underlying the suppressive activity of C-terminus of ARG will lead to development of a novel therapeutic strategy, especially for patients with CML harboring mutations, which are resistant to tyrosine kinase inhibitors. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Exposure of neuroblastoma cell lines to imatinib results in the upregulation of the CDK inhibitor p27KIP1 as a consequence of c-Abl inhibition

Imatinib mesylate is a tyrosine kinase inhibitor with selectivity for abelson tyrosine-protein kinase 1 (c-Abl), breakpoint cluster region (Bcr)-Abl fusion protein (Bcr-Abl), mast/stem cell growth factor receptor Kit (c-Kit), and platelet-derived growth factor receptor (PDGFR). Previous studies demonstrated that imatinib in the low micromolar range exerted antiproliferative effects on neuroblastoma cell lines. However, although neuroblastoma cells express c-Kit and PDGFR, the imatinib concentrations required to achieve significant growth inhibitory effects (≥10μM) are substantially higher than those required for inhibition of ligand-induced phosphorylation of wild type c-Kit and PDGFR (≤1μM), suggesting that additional mechanisms are responsible for the antitumor activity of imatinib on these cells. In this study, we show that treatment of neuroblastoma cell lines with 1–15μM imatinib resulted in a dose dependent inhibition of 5-bromo-2′-deoxyuridine (BrdU) incorporation into newly synthesized DNA. The antiproliferative effect of imatinib was dependent on the upregulation of the cyclin-dependent kinase (CDK) inhibitor p27KIP1 in the nuclear compartment as a result of increased p27KIP1 protein stability. We demonstrate that the mechanism of p27KIP1 stabilization relied on inhibition of p27KIP1 phosphorylation on tyrosine residues by c-Abl. We provide evidence that in neuroblastoma cell lines a significant fraction of cellular c-Abl is phosphorylated on Tyr-245, consistent with an open and active conformation. Notably, exposure to imatinib did not affect Tyr-245 phosphorylation. Given the low affinity of active c-Abl for imatinib, these data provide a molecular explanation for the relatively high imatinib concentrations required to inhibit neuroblastoma cell proliferation.

Nilotinib for the Frontline Treatment of Chronic Myeloid Leukemia Carrying the p230 Transcript: Dream or Reality?

OPINION article Front. Oncol., 03 February 2014Sec. Hematologic Malignancies https://doi.org/10.3389/fonc.2014.00017

Evolution of High-Affinity Peptide Probes to Detect the SH3 Domain of Cancer Biomarker BCR–ABL

The BCR–ABL fusion protein is closely associated with the pathological progression of chronic myelogenous leukaemia and some other myeloproliferative diseases, which has long been recognized as one of the most important cancer biomarkers in the tumor diagnosis community. The SH3 domain of BCR–ABL is a small, conserved protein module that specifically recognizes and binds proline-rich peptide fragments. In the current study, we used a synthetic strategy to discover new peptide probes with high affinity binding to the BCR–ABL SH3 domain. In the procedure, a sequence-based machine learning predictor was developed based on a set of affinity-known SH3 binders, and the predictor was then used to guide the evolutional optimization of numerous virtual peptides to enrich high binding potency for the SH3 domain. Subsequently, a evolved peptide population was generated, from which ten peptides with the highest affinity scores were selected and their interaction free energies with SH3 domain were characterized systematically using a combination of molecular dynamics simulation and binding free energy analysis. Consequently, four peptides were suggested as promising candidates and their affinities toward SH3 domain were assayed; two peptides, APTYTPPPPP and APTYAPPPPP, were identified to have potent binding capability with dissociation constants K d of 3 and 8 μM, respectively. Further, the structural basis and energetic property of SH3 domain in complex with APTYTPPPPP were examined in detail, revealing a non-specific interaction in SH3–peptide recognition that should render a broad ligand spectrum for the domain.

Overcoming resistance in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is characterized by the BCR–ABL fusion protein produced as a result of the reciprocal translocation between a portion of the ABL tyrosine kinase gene on chromosome 9 and a region within the BCR gene on chromosome 22. The BCR–ABL oncoprotein displays constitutively elevated tyrosine kinase activity that drives the pathogenesis of CML by activating multiple signaling pathways including the RAS/MAPK, PI3K/AKT and JAK2/STAT5 pathways. The development of tyrosine kinase inhibitors (TKIs) aimed at BCR–ABL revolutionized the treatment of CML. Imatinib was the first of the TKIs to become available after it was approved by the US FDA in 2002 based upon data from the IRIS. Indeed, imatinib was associated with unprecedented rates of hematologic, cytogenetic and molecular responses in comparison with IFN-a with complete hematologic response (CHR) rates of 97%, complete cytogenetic response (CCyR) rates of 82% and major molecular response (MMR) rates on the international scale (IS) of 86% [1]. Treatment with imatinib also resulted in significantly improved rates of freedom from progression in comparison with IFN-a with an 8-year freedom from progression to accelerated or blast phase of 92% and afforded patients who were able to remain on treatment an 8-year overall survival (OS) of 85% [1]. However, imatinib use has been complicated by the development of resistance. Resistance led to 24% of patients in IRIS failing to achieve a CCyR at 18 months, which represented treatment failure [2]. Resistance also led 17% of patients on imatinib in IRIS to develop relapsed disease and 7% to develop progressive disease [2]. These numbers are, in fact, likely to be underestimates given the number of patients in the IRIS study who came off the trial and were not ultimately included in analysis. Treatment failure and disease progression due to resistance is also seen in patients treated with the subsequently approved second-generation TKIs, dasatinib, nilotinib and bosutinib. The etiology of resistance to TKIs is multifactorial and involves BCR–ABL independent and dependent mechanisms. Insufficient plasma levels of imatinib due to noncompliance, drug–drug interactions or binding to acute-phase inflammatory proteins can all result in resistance. It is known that trough plasma levels of imatinib are important for good clinical outcomes, and it has been demonstrated that patients with high imatinib exposure have better rates of CCyR, MMR and event-free survival [3]. Imatinib and all three of the second-generation TKIs are substrates for, and are extensively metabolized by, the cytochrome P450 enzymes including CYP3A4. As there are multiple drugs, food products and supplements that either induce or inhibit CYP3A4 activity, the potential for drug interactions is high. Alpha 1 acid glycoprotein has been identified as an acutephase inflammatory product that binds directly to imatinib and is frequently “As the number of drugs available for the treatment of chronic myeloid leukemia has expanded, so have the strategies for overcoming or avoiding resistance.”

Impact of chromosomal translocation and genomic instability on personalized medicine.

structural exchanges between heterologous chromosomes. We recently reviewed the critical impact of chromosomal translocations on cancer diagnosis, prognosis and treatment [4]. Specific chromosomal translocations are often closely associated with subtypes of leukemia and lymphoma, which has greatly facilitated the diagnosis and stratification of patients for certain types of treatments [4]. A classic example is the t(9;22) translocation, which generates the BCR–ABL fusion product in chronic myelo genous leukemia (CML) patients [5]. The cancer cell-specific BCR–ABL fusion protein is a target of the therapeutic agent STI-571 (Gleevec), which induces remission in the vast majority of CML patients [6]. The efficacy of Gleevec in CML is an early and key success that cancer research has delivered in personalized medicine. The Gleevec story also exemplifies the essential contribution of basic research, which enabled discovery of the molecular mechanisms of leukemo genesis and development of targeted therapies for the disease. Another success of individualized therapy in leukemia is the use of all-trans retinoic acid [7] and arsenic trioxide [8–10] in acute promyelocytic leukemia patients harboring the t(15;17) translocation [11]. The t(15;17) translocation fuses a portion of the PML gene to the RARA gene to encode the PML–RARA fusion protein [12]. Combined therapy of all-trans retinoic acid and arsenic trioxide induces a high rate of remission and survival in the vast majority of acute promyelocytic leukemia patients [13]. Many clinics now routinely screen for these translocations, performing karyotyping and cytogenetic analyses of leukemia or lymphoma cells before a specific treatment is identified. These successful medical practices owe credit to the extensive basic and The concept of personalized medicine The term ‘personalized medicine’, in the context used today, first appeared in a Wall Street Journal article in 1999 (already implicating its potential economic impact), which later appeared in the Oncologist [1]. In 2008, the President’s Council of Advisors on Science and Technology published a report, ‘Priorities for Personalized Medicine’, which provided a comprehensive definition of personalized medicine and highlighted its potential to influence healthcare and economics [101]. In recent years, personalized medicine has gained much wider recognition in the scientific community [2]. An ongoing paradigm shift from standard medicine (one-size-fits-all) to personalized medicine can be attributed to two important factors. First is the increasing incidence of chronic diseases such as cancer or metabolic diseases that tend to display more variation in clinical presentation, rather than acute infectious diseases such as those caused by bacteria. Second are rapid advances in new ‘omics’ technologies, including genomics, proteomics and metabolomics, which enable better characterization of individual variation and profiling of clinical differences at the molecular level. Cancer research has been at the forefront of personalized medicine, largely because of the low treatment response rate in a majority of cancers, the intrinsic heterogeneity of cancers, successful examples of targeted cancer therapy, and recent advances in genomic approaches for cancer pathology [3].

Cumulative clinical experience from a decade of use: imatinib as first-line treatment of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a malignant disease that originates in the bone marrow and is designated by the presence of the Philadelphia (Ph+) chromosome, a translocation between chromosomes 9 and 22. Targeted therapy against CML commenced with the development of small-molecule tyrosine kinase inhibitors (TKIs) exerting their effect against the oncogenic breakpoint cluster region (BCR)-ABL fusion protein. Imatinib emerged as the first successful example of a TKI used for the treatment of chronic-phase CML patients and resulted in significant improvements in response rate and overall survival compared with previous treatments. However, a significant portion of patients failed to respond to the therapy and developed resistance against imatinib. Second-generation TKIs nilotinib and dasatinib were to have higher efficiency in clinical trials in imatinib- resistant or intolerant CML patients compared with imatinib. Identification of novel strategies such as dose escalation, drug combination therapy, and use of novel BCR-ABL inhibitors may eventually overcome resistance against BCR-ABL TKIs. This article reviews the history of CML, including the treatment strategies used prediscovery of TKIs and the preclinical and clinical data obtained after the use of imatinib, and the second-generation TKIs developed for the treatment of CML.

1093P - BCR ABL Fusion Protein Detection by a Modern Approach: Fluroscent Immuno Bead Assay

ABSTRACT Introduction Chronic Myeloid Leukemia (CML) is probably the most extensively studied human malignancy. CML patients (90-95%) harbor the Philadelphia (Ph) chromosome, a shortened chromosome 22, resulting from a reciprocal translocation t (9; 22) (q34; q11) between the long arms of chromosome 9 and 22, fusion ABL proto-oncogene on chromosome 9 with the BCR gene on chromosome 22. BCR-ABL fusion gene plays a key role in pathogenesis of the disease. Approximately 15-30% of adult and 2-5% of childhood ALL and Materials and methods Study Period: June 2009- February 2012 Test Specimen Peripheral blood or Bone marrow from 242 CML patient (from hospital registry of a tertiary cancer institute of Eastern India) Normal (as negative control): Peripheral blood from 20 individuals BCR-ABL positive cell line: K562 FISH was performed using (for 100 samples) directly labeled dual color prob for detection of BCR-ABL as standard laboratory protocol. Isolation of WBC were done by lysis buffer. Then lysate treatment were done to release the intracellular BCR-ABL protein. After fluorescent immune bead assay were performed by captured bead and detector reagent. After forming a sandwich complex (if sample contain targeted protein), they were analysed by flowcytometry method. Result In 242 cases 166 (68.6%) were positive for BCR ABL fusion protein by Flowcytometric Bead Assay. Approximately 96% cases were observed as positive by both techniques FISH and Flowcytometric Bead Assay. Conclusion We conclude that the Flowcytometry Immuno Bead Assay is a faster, easier and reliable technique for detection of BCR ABL protein in leukemic cells and shows promise for serial evaluation of patients undergoing treatment. The main advantage of immune bead assay is not dependence on the breakpoint position in the BCR gene. Moreover this technique is based on protein translation label. Disclosure All authors have declared no conflicts of interest.

Direct Binding Assay for the Detection of Type IV Allosteric Inhibitors of Abl

Abelson (Abl) tyrosine kinase is an important cellular enzyme that is rendered constitutively active in the breakpoint cluster region (BCR)-Abl fusion protein, contributing to several forms of leukemia. Although inhibiting BCR-Abl activity with imatinib shows great clinical success, many patients acquire secondary mutations that result in resistance to imatinib. Second-generation inhibitors such as dasatinib and nilotinib can overcome the majority of these mutations but fail to treat patients with an especially prevalent T315I mutation at the gatekeeper position of the kinase domain. However, a combination of nilotinib with an allosteric type IV inhibitor was recently shown to overcome this clinically relevant point mutation. In this study, we present the development of a direct binding assay that enables the straightforward detection of allosteric inhibitors which bind within the myristate pocket of Abl. The assay is amenable to high-throughput screening and exclusively detects the binding of ligands to this unique allosteric site.

Structure, regulation, signaling, and targeting of abl kinases in cancer.

Abl kinases are prototypic cytoplasmic tyrosine kinases and are involved in a variety of chromosomal aberrations in different cancers. This causes the expression of Abl fusion proteins, such as Bcr-Abl, that are constitutively activated and drivers of tumorigenesis. Over the past decades, biochemical and functional studies on the molecular mechanisms of Abl regulation have gone hand in hand with progression of our structural understanding of autoinhibited and active Abl conformations. In parallel, Abl oncoproteins have become prime molecular targets for cancer therapy, using adenosine triphosphate (ATP)-competitive kinase inhibitors, such as imatinib. Abl-targeting drugs serve as a paradigm for our understanding of kinase inhibitor action, specificity, and resistance development. In this review article, I will review the molecular mechanisms that are responsible for the regulation of Abl kinase activity and how oncogenic Abl fusions signal. Furthermore, past and ongoing efforts to target Abl oncoproteins using ATP-competitive and allosteric inhibitors, as well as future possibilities using combination therapy, will be discussed.

Inhibition of small GTPase RalA regulates growth and arsenic-induced apoptosis in chronic myeloid leukemia (CML) cells

Chronic myelogenous leukemia (CML) results from the transformation of a primitive hematopoietic cell by the bcr–abl gene. RalA, one of the Ras superfamily of small GTPases, is a downstream molecule of bcr–abl fusion protein in ras signaling pathway, but its role in CML is poorly understood. Here, we first detected RalA level in CML cells, which is highly expressed and distributed mainly in the cytoplasm and/or partially in endomembrane. Next, siRNA was used to deplete RalA expression for elucidating its function. The results showed that siRNA RalA effectively inhibited cell viability, induced apoptosis and enhanced sensitivity of arsenic trioxide (ATO), and there are some synergistic effects of anti-CML between RalA siRNA and ATO. Finally, we found that ATO also could downregulate protein level of bcr–abl in K562 and KCL-22. Our research provides evidence that RalA might also serve as linchpin modulators in leukemia, and combinatorial therapies of dual inhibition of bcr–abl and ras signaling pathways have a great potential in treatment of CML.