BCR-ABL Kinase Domain Mutations Research Articles

Ponatinib in Chronic-Phase Chronic Myeloid Leukemia Patients: Final Report from a Phase 1 Trial

Ponatinib in Chronic-Phase Chronic Myeloid Leukemia Patients: Final Report from a Phase 1 Trial

Downregulation of Mir-146a-5p, Mir-99b-5p, Mir-143-3p, Mir-10a-5p and Mir-151a-3p Associated with PI3K/AKT, p53, NF-Kb, and Fanconi Anemia/BRCA Signaling Pathways Are Observed in Imatinib-Resistant Chronic Myeloid Leukemia Patients without Detectable BCR-ABL kinase Domain Mutations

IntroductionChronic myeloid leukemia (CML) patients who do not achieve landmark responses are considered imatinib (IM)-resistant. IM-resistance can be due to BCR-ABL kinase domain (KD) mutations, although many IM-resistant patients do not harbor KD mutations. The pathogenesis of this phenomenon is unclear. Aberrations in BCR-ABL independent downstream signaling pathways, namely PI3K/AKT, p53, NF-kB and Fanconi anaemia (FA)/BRCA, have been implicated. MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression and are notoriously promiscuous, with one miRNA regulating many mRNAs. In recognition of this, we sought to identify dysregulated miRNAs associated with the above pathways in patients with IM-resistant CML.MethodsEight patients with chronic phase CML who demonstrated primary resistance to IM and tested negative for KD mutations via Sanger sequencing were enrolled. Two healthy volunteers constituted the control group. Peripheral blood samples were taken and miRNA extracted from the white cells. MiRNA profiling was performed using miRNA sequencing (miRNA-seq) of the human microRNAome. MiRNA expression analyses (miEA) were performed on 4 sets of miRNAs associated with 4 different signaling pathways derived from publicly available databases: 237 miRNAs for PI3K/AKT, 223 miRNAs for p53, 221 miRNAs for NF-kB, and 126 miRNAs for FA/BRCA. MiRNA expression levels of the patients were compared with normal controls to obtain the differential expression of the miRNAs. MiRNAs that occur in all 4 pathways were further investigated in silico for their putative gene targets (Tarbase v7.0). Gene targets which were commonly linked with 3 different miRNAs were identified.ResultsMiRNA-seq yielded a total of 790 miRNAs. MiEA showed downregulation of 5 miRNAs in all 4 signaling pathways above: miR-146a-5p, miR-99b-5p, miR-143-3p and miR-10a-5p, miR-151a-3p. MiR-146a-5p and miR-99b-5p were the first and second most downregulated miRNAs in all 4 pathways. Putative gene targets that were commonly modulated by 3 miRNAs were: CDK6 (miR-143-3p, miR-10a-5p, miR-151a-3p), MDM2 (miR-146a-5p, miR-143-3p, miR-10a-5p), PTGS2 (miR-146a-5p, miR-143-3p, miR-10a-5p) and REV3L (miR-146a-5p, miR-143-3p, miR-151a-3p), as shown in Table 1.ConclusionsThe PI3K/AKT pathway is activated in response to IM-exposure to IM-naïve cells, implying a role in IM resistance. As cell cycle modulators, both p53 and NF-kB pathways are regulated within the PI3K/AKT pathway via AKT. FANCD2 is a central FA/BRCA pathway regulator and its transcription is maintained by AKT/mTOR via enhancement of NF-kB activity. The complexity of the interactions above has impeded efforts in identifying a unifying target in counteracting IM resistance. Thus, it is of significance that our results showed 5 differentially expressed miRNAs that consistently featured in the above signaling pathways, 4 of which remarkably influence PI3K/AKT and p53.In our study, PI3K/AKT and p53 pathways are inextricably linked with the same 4 miRNAs (miR-146a-5p, miR-143-3p, miR-10a-5p and miR-151a-3p) targeting the same 2 genes (MDM2 and CDK6) in these 2 pathways. MDM2 is an ubiquitin ligase that regulates the stability of p53a. AKT facilitates the functions of MDM2 to promote p53 ubiquitination. Conversely, inhibition of MDM2 leads to activation of p53 in response to cellular stresses. CDK6 is a cell cycle regulator and its overexpression influences the accumulation of pro-apoptotic p53 proteins, postulating a role in DNA damage response. Both PTGS2 and REV3L genes were commonly targeted by 3 miRNAs, within the NF-kB and FA/BRCA pathways respectively. PTGS2 overexpression has been shown in gastrointestinal adenocarcinomas; it induces carcinoma cell migration and invasion via activation of PI3K/AKT signaling. REV3L is the catalytic subunit of DNA polymerase ζ and plays a central role in DNA damage tolerance and chemoresistance towards DNA damaging agents. Notably, the NF-kB and FA/BRCA pathways were juxtaposed to the PI3K/AKT/p53 system via shared miRNA regulators.This provokes interesting questions regarding IM-resistance: are anti-apoptotic signaling and DNA damage response mechanisms driven simultaneously by these miRNAs? Can these miRNAs be potential biomarkers and therapeutic targets in IM-resistant CML? More studies are needed to further validate our findings. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Brief Research Report: Novel Compound BCR-ABL Mutations in Late Chronic Phase Imatinib Sensitive CML Patients Are Associated with Progression to Advance Disease Phase

Introduction: BCR-ABL kinase domain (KD) mutations have well established role in tyrosine kinase inhibitors (TKIs) resistance and disease progression in chronic myeloid leukemia (CML)1. In recent years, compound BCR-ABL mutations have emerged as a new threat to CML patients by causing higher degrees of resistance involving multiple TKIs, including ponatinib2. However, there are limited reports about association of compound BCR-ABL mutations with disease progression in imatinib sensitive CML patients3. Furthermore, BCR-ABL mutation detection is currently recommended only in case of drug resistance and disease progression and clinical significance of BCR-ABL mutation detection in TKI responder chronic phase CML is not well documented4. Therefore, we investigated presence of ABL-KD mutations in chronic phase (CP) and advanced phase imatinib sensitive CML to find out association of BCR-ABL mutations with progression to advanced disease phases in CML patients. .Patients and Methods: Imatinib sensitive CML patients in CP and advanced phases of the disease were included in the study.All CP patients were incomplete hematological, complete cytogenetic and major molecular responses. Due to specific study objectives, patients manifesting drug resistance during follow-up studies were excluded from the study. A total of 90 imatinib sensitive CML patients (CP=41, late CP=33, and accelerated phase=16) were finally available for analysis. All patients as well as 10 healthy controls were investigated for BCR-ABL mutations using Sanger sequencing. All response criteria were per European LeukemiaNet guidelines4. Data was analyzed using SPSS software (version 19).Results: Mean age of the patients was 33 years (Table 1). Eleven out of 33 (33.3%) patients in late-CP CML harbored 24 types of point mutations, out of which eight (72.72%) harbored compound mutated sites (Figure 1, Table 2). E355G (3.33%) was the most prevalent mutant. Five patients (45.45%), all of which had compound mutations, progressed to advanced phases of disease during follow up studies. No BCR-ABL mutation was detected in healthy subjects and in early CP CML patients. Therefore, early CP CML patients served as additional control in this study. BCR-ABL mutations were found in 3 accelerated phase patients as well. Late-CP mutations were associated with elevated platelet count (p= 0.037) and male gender (p= 0.049). The median overall survival and event free survival of CML patients (n=90) was 6.98 and 5.8 years respectively. Seven year survival was found to be 94.2%.Discussion and conclusions: Compound BCR-ABL mutations were associated with progression to advanced disease in imatinib sensitive late-CP CML patients. Although single BCR-ABL mutations have previously been found to cause CML progression5, this is first report of association of compound BCR-ABL mutations with disease progression in stable imatinib responders at late CP. Detection of new mutations can help in defining mechanism of CML progression6. Moreover, BCR-ABL mutation detection in late CP CML patients sensitive to TKI treatment can help in early assessment of risk for disease progression and/or drug resistance and subsequent clinical intervention to delay disease progression which is a major challenge of CML therapy in TKI era7.

Molecular monitoring in chronic myeloid leukemia-how low can you go?

Molecular monitoring of BCR-ABL1 transcripts for patients with chronic myeloid leukemia (CML) is now used to assess response to tyrosine kinase inhibitors (TKIs), including treatment failure that mandates a change of therapy. Therefore, many centers have adopted the molecular technique for measuring BCR-ABL1 and rely on conversion of values to the international reporting scale for appropriate clinical interpretation. However, the technique has a degree of inherent variability despite standardized procedures, which means care should be taken by the clinician when assessing response based on BCR-ABL1 cutoff limits. The last few years have witnessed the emergence of a new molecular response target, which is the achievement and maintenance of a deep molecular response. The ability to achieve treatment-free remission for some patients has shifted the relevant boundary for molecular response. However, the definitive safe BCR-ABL1 transcript level and length of the maintenance phase after which treatment cessation can be attempted has not yet been determined. For patients with TKI resistance, BCR-ABL1 kinase domain mutation analysis remains an essential assessment to guide therapy. Furthermore, low-level mutation detection is clinically relevant for response prediction to subsequent TKI therapy for some patients. Multiple low-level mutations may be a biomarker of a clonally diverse disease with the propensity for resistance evolution. Overall, molecular monitoring, including low-level monitoring is a fundamental component of management for patients with CML.

Characterization of the Genomic Landscape of BCR-ABL1 Kinase-Independent Mechanisms of Resistance to ABL1 Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia

Despite the well-established success of ABL1 tyrosine kinase inhibitors (TKIs) in the treatment of patients with chronic myeloid leukemia (CML), approximately 20% of patients treated with frontline imatinib develop resistance by 5 years on therapy. The majority (~60%) of such resistant cases are explained by acquired mutations within the BCR-ABL1 kinase domain that compromise inhibitor binding, and nearly all of these mutations are effectively targeted by one or more of the 2nd and 3rd generation ABL1 kinase inhibitors. In contrast, the remaining ~40% of imatinib-resistant cases harbor no explanatory BCR-ABL1 kinase domain mutation, presumably attributable to BCR-ABL1 kinase-independent mechanisms. We hypothesized that resistance in these patients results from acquired auxiliary molecular aberrations which persistently activate signaling pathways downstream despite inhibition of BCR-ABL1 kinase activity. To identify such mechanisms, we performed whole exome sequencing and RNA sequencing on a cohort of 135 CML patients comprising the following subgroups: newly diagnosed/TKI naïve (n=28), BCR-ABL1 kinase-dependent resistance (n=31), and BCR-ABL1 kinase-independent resistance (n=65), and TKI-induced remission (n=7). Resistant patients were required to have demonstrated clinical resistance to one or more ABL1 kinase inhibitors in the form of suboptimal response or loss of cytogenetic response; the subtype of resistance was defined based on the presence or not of an explanatory BCR-ABL1 kinase domain mutation at the time of resistance. The majority of samples collected were from patients with chronic phase CML (n=97), although smaller cohorts of accelerated phase CML, blast crisis CML, and Ph+ ALL were also profiled (n=20, 19, and 9, respectively). Among the 44,413 protein-altering and 902 splice site variants detected across the 120 WES samples, there were on average 908 missense, 146 truncation and 69 splice variants per sample. Genes with truncation and missense variants were compared between BCR-ABL1 kinase-independent and -dependent resistant chronic phase samples. A total of 44 genes were seen with a frequency difference of at least 10%, including PLEKHG5 and NKD2 (30% and 28% difference, respectively), which are involved in regulation of NF-kB and Wnt signaling. Consistent with previous reports, we also detected EZH2 and TET2 as exclusively mutated in the BCR-ABL1 kinase-independent resistance patients (6% and 3%, respectively). Further analyses stratifying variants among resistant patients according to specific ABL1 kinase inhibitor therapy failed and comparing, where available, serial samples from pre- and post-treatment for clonal expansion are underway. Additionally, sufficient material was available to perform ex vivo small-molecule inhibitor screening for 48 patient specimens, the resultant data of which was used to generate putative effective drug target profiles and integrated with exome sequencing variants to prioritize variants of functional relevance (HitWalker; Bottomly et al., Bioinformatics 2013). Among 23 patient samples exhibiting BCR-ABL1 kinase-independent resistance, the mutated genes most frequently ranked in the top 10 functional-prioritized variants were: ABL1 (which included non-kinase domain variants; 34.7%), MAP3K1, MUC4, FGF20 (each 17.4%), ARHGEF15, MEF2A, EPHA8, TYRO3, BMP2K, and IRS1 (each 13.0%). Notably, the top six candidates are members of the neutrophin (ABL1, MAP3K1, and IRS1), EPHA forward (EPHA8, ARHGEF15), and p38 MAPK signaling pathways (MAP3K1 and MEF2A). Taken together, these findings suggest that several of the same pathogenic molecular abnormalities seen in other myeloid malignancies are also present in CML patients with BCR-ABL1 kinase-independent resistance, including a subset which align to persistent re-activation of signaling pathways involved in CML disease pathogenesis and progression. As such, genetic and/or functional profiling of these patients in the clinic may translate to actionable candidates for combination therapy to maximize disease control and improve patient outcomes. DisclosuresAgarwal:CTI BioPharma Corp: Research Funding. Radich:Novartis: Consultancy, Research Funding; BMS: Consultancy; Ariad: Consultancy. Deininger:Novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Incyte: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Gilead: Research Funding; CTI BioPharma Corp.: Membership on an entity’s Board of Directors or advisory committees; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Druker:Pfizer: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Curis: Patents & Royalties; Array: Patents & Royalties; CTI: Consultancy, Equity Ownership; Pfizer: Patents & Royalties; Curis: Patents & Royalties; Array: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Oncotide Pharmaceuticals: Research Funding; Novartis: Research Funding; BMS: Research Funding; ARIAD: Patents & Royalties: inventor royalties paid by Oregon Health & Science University for licenses, Research Funding; Roche: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses; D3 Oncology Solutions: Consultancy; AstraZeneca: Consultancy; Ambit BioSciences: Consultancy; Agios: Honoraria; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Lorus: Consultancy, Equity Ownership; Cylene: Consultancy, Equity Ownership.

Dasatinib for the treatment of chronic myeloid leukemia: patient selection and special considerations.

Dasatinib is one of the second-generation tyrosine kinase inhibitors used in imatinib resistance and/or intolerance, as well as in the frontline setting in patients with chronic myeloid leukemia-chronic phase, and also in patients with advanced disease. It is also utilized in Philadelphia chromosome-positive acute lymphocytic leukemia. While choosing the appropriate tyrosine kinase inhibitor (ie, dasatinib) for each individual patient, comorbidities and BCR-ABL1 kinase domain mutations should always be taken into consideration, among other things. This review mainly focuses on patient selection prior to dasatinib administration in the treatment of chronic myeloid leukemia.

Detection of BCR-ABL Kinase Domain Mutations in Chronic Myeloid Leukemia Patients

The patient with chronic myeloid leukemia (CML) is treated successfully with tirosin kinase inhibitors (TKIs). Worldwide there are a 20 to 30% of patients, who have resistance to the treatment. The first cause of TKIs resistance is the presence of BCR-ABL kinase domain mutations. In Guatemalan population of around 35% of CML, patients are resistant to TKIs.

In chronic myeloid leukemia patients on second-line tyrosine kinase inhibitor therapy, deep sequencing of BCR-ABL1 at the time of warning may allow sensitive detection of emerging drug-resistant mutants.

BackgroundImatinib-resistant chronic myeloid leukemia (CML) patients receiving second-line tyrosine kinase inhibitor (TKI) therapy with dasatinib or nilotinib have a higher risk of disease relapse and progression and not infrequently BCR-ABL1 kinase domain (KD) mutations are implicated in therapeutic failure. In this setting, earlier detection of emerging BCR-ABL1 KD mutations would offer greater chances of efficacy for subsequent salvage therapy and limit the biological consequences of full BCR-ABL1 kinase reactivation. Taking advantage of an already set up and validated next-generation deep amplicon sequencing (DS) assay, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse.Methodsa total of 125 longitudinal samples from 51 CML patients who had acquired dasatinib- or nilotinib-resistant mutations during second-line therapy were analyzed by DS from the time of failure and mutation detection by conventional sequencing backwards. BCR-ABL1/ABL1%IS transcript levels were used to define whether the patient had ‘optimal response’, ‘warning’ or ‘failure’ at the time of first mutation detection by DS.ResultsDS was able to backtrack dasatinib- or nilotinib-resistant mutations to the previous sample(s) in 23/51 (45 %) pts. Median mutation burden at the time of first detection by DS was 5.5 % (range, 1.5–17.5 %); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 1–9 months). In 5 cases, the mutations were detectable at baseline. In the remaining cases, response level at the time mutations were first detected by DS could be defined as ‘Warning’ (according to the 2013 ELN definitions of response to 2nd-line therapy) in 13 cases, as ‘Optimal response’ in one case, as ‘Failure’ in 4 cases. No dasatinib- or nilotinib-resistant mutations were detected by DS in 15 randomly selected patients with ‘warning’ at various timepoints, that later turned into optimal responders with no treatment changes.ConclusionsDS enables a larger window of detection of emerging BCR-ABL1 KD mutations predicting for an impending relapse. A ‘Warning’ response may represent a rational trigger, besides ‘Failure’, for DS-based mutation screening in CML patients undergoing second-line TKI therapy.

BCR-ABL mutation testing in patients with CML occurrence prior to a 10X rise in BCR-ABL test results.

e18546 Background: The NCCN guidelines recommend BCR-ABL kinase domain mutation testing when CML patients show a loss of response to treatment. About 20% of patients with CML started on a first-line TKI will develop treatment resistance due to mutation in the BCR-ABL kinase domain. A “rule of thumb” is to conduct mutation testing when BCR-ABL results rise 10X or higher. We studied a group of CML patients who had mutation testing after treatment started to determine if the 10X rule of thumb is being followed. Methods: We studied data from 183 CML patients in the nationwide Medivo lab test database who were tested for BCR-ABL between 2011 - 2014. This group of patients had at least 1 mutation test after a positive slope in their previous BCR-ABL tests. We performed a simple descriptive analysis to identify the proportion of these patients who were tested for mutation when previous BCR-ABL test results had risen < 10X. Results: We found 33 instances (18%) where patients were tested for BCR-ABL mutation soon after a 10X or higher rise in BCR-ABL results, and 150 instances (82%) where patients were tested for BCR-ABL mutation when previous BCR-ABL results had risen < 10X. We are 95% confident that the true proportion of patients tested for the BCR-ABL mutation when previous BCR-ABL results had risen < 10X is between 76% and 88%. The median value for the ratio of current BCR-ABL/previous BCR-ABL for all 183 patients before mutation testing is 2.4 [IQR: 1.4 - 6.2]. Conclusions: Current NCCN guidelines recommend mutation testing in patients with CML showing lack of response, loss of response or disease progression. Our results show that mutation testing is occurring more often prior to a 10X rise in BCR-ABL test results than after a 10X rise, indicating that the “rule of thumb” is not being followed in most cases. Further study is needed to determine the underlying reasons for the differences between the groups of patients tested prior to a 10X rise and the group tested after a 10X rise in BCR-ABL test results.

A role for FOXO1 in BCR-ABL1-independent tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) patients who relapse on imatinib due to acquired ABL1 kinase domain mutations are successfully treated with second-generation ABL1-tyrosine kinase inhibitors (ABL-TKIs) such as dasatinib, nilotinib or ponatinib. However, ~40% of relapsed patients have uncharacterized BCR–ABL1 kinase-independent mechanisms of resistance. To identify these mechanisms of resistance and potential treatment options, we generated ABL-TKI-resistant K562 cells through prolonged sequential exposure to imatinib and dasatinib. Dual-resistant K562 cells lacked BCR–ABL1 kinase domain mutations, but acquired other genomic aberrations that were characterized by next-generation sequencing and copy number analyses. Proteomics showed that dual-resistant cells had elevated levels of FOXO1, phospho-ERK and BCL-2, and that dasatinib no longer inhibited substrates of the PI3K/AKT pathway. In contrast to parental cells, resistant cells were sensitive to growth inhibition and apoptosis induced by the class I PI3K inhibitor, GDC-0941 (pictilisib), which also induced FOXO1 nuclear translocation. FOXO1 was elevated in a subset of primary specimens from relapsed CML patients lacking BCR–ABL1 kinase domain mutations, and these samples were responsive to GDC-0941 treatment ex vivo. We conclude that elevated FOXO1 contributes to BCR–ABL1 kinase-independent resistance experienced by these CML patients and that PI3K inhibition coupled with BCR–ABL1 inhibition may represent a novel therapeutic approach.

Present and future of molecular monitoring in chronic myeloid leukaemia.

Currently, physicians treating chronic myeloid leukaemia (CML) patients can rely on a wide spectrum of therapeutic options: the best use of such options is essential to achieve excellent clinical outcomes and, possibly, treatment-free remission (TFR). To accomplish this, proper integration of expert clinical and laboratory monitoring of CML patients is fundamental. Molecular response (MR) monitoring of patients at defined time points has emerged as an important success factor for optimal disease management and BCR-ABL1 kinase domain mutation screening is useful to guide therapeutic reassessment in patients who do not achieve optimal responses to tyrosine kinase inhibitor therapy. Deeper MRs might be associated with improved long-term survival outcomes. More importantly, they are considered a gateway to TFR. In molecular biology, novel procedures and technologies are continually being developed. More sophisticated molecular tools and automated analytical solutions are emerging as CML treatment endpoints and expectations become more and more ambitious. Here we provide a critical overview of current and novel methodologies, present their strengths and pitfalls and discuss what their present and future role might be.

New Developments in Chronic Myeloid Leukemia: Implications for Therapy.

Context:Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by overproduction of immature and matured myeloid cells in the peripheral blood, bone marrow and spleen.Evidence Acquisition:A hallmark of CML is the presence of (9; 22) (q34; q11) reciprocal translocation, which is cytogenetically visible as Philadelphia chromosome (Ph) and results in the formation of BCR-ABL1 fusion protein. This fusion protein is a constitutively active tyrosine kinase which is necessary and sufficient for malignant transformation. The introduction of imatinib, a BCR-ABL1- targeting tyrosine kinase inhibitor (TKI) has revolutionized CML therapy. Subsequently, two other TKIs with increased activity against BCR-ABL1, dasatinib and nilotinib, were developed and approved for CML patients. Nevertheless, CML therapy faces major challenges.Results:The first is the development of resistance to BCR-ABL1 inhibitors in some patients, which can be due to BCR-ABL1 overexpression, differences in cellular drug influx and efflux, activation of alternative signaling pathways, or emergence of BCR-ABL1 kinase domain mutations during TKI treatment. The second is the limited efficiency of BCR-ABL1-TKIs in blast crisis (BC) CML. The third is the insensitivity of CML stem cells to BCR-ABL1 inhibitors. Conventional chemotherapeutics and BCR-ABL1 inhibitors which act by inhibiting cell proliferation and inducing apoptosis, are ineffective against quiescent CML stem cells.Conclusions:A better understanding of the mechanisms that underlie TKI resistance, progression to BC, genomic instability and stem cell quiescence is essential to develop curative strategies for patients with CML.

Combination of low-dose imatinib plus nilotinib for the treatment of chronic-phase chronic myeloid leukaemia after imatinib failure

Objectives: This is a feasibility study to evaluate whether simultaneous administration of low doses of imatinib and nilotinib in chronic-phase chronic myeloid leukaemia (CP-CML) patients has the potential for transcript elimination after failure to imatinib.Methods: Ten patients were enrolled; eight had cytogenetic relapse and two had confirmed loss of major molecular response (MMR). At baseline, BCR-ABL kinase domain mutation was detected in four patients.Results: After 6 months of therapy, major cytogenetic response, complete cytogenetic response, and MMR were achieved in seven, four, and four patients, respectively. Grade 4 thrombocytopenia developed in one patient, and grade 1 skin rash in four.Discussion and conclusion: These results suggest that imatinib might have inhibitory effects on the clearance of nilotinib, increasing its efficacy. This dual therapy was well tolerated and resulted in improvement of cytogenetic and molecular responses in patients with CP-CML after failure to imatinib. ClinicalTrials.gov registration number: NCT01819389.

Leucine replaced by methionine at 273 position in chronic myeloid leukemia: Knowns and unknowns…

Chronic myeloid leukemia is a clonal bone marrow stem cell disorder characterized by the presence of Philadelphia chromosome t(9;22)(q34;q11) leading to fusion oncogene BCR-ABL. Tyrosine kinase inhibitors (TKIs) act by competitively inhibiting BCR-ABL oncoprotein with significant response rates. However, up to 30% of patients fail to achieve complete cytogenetic remission on 1 st line TKI imatinib, one of the reasons being mutations in BCR-ABL kinase domain leading to imatinib resistance. Over 80 such mutations have been documented in the literature; however, some of the rare mutations still remain to be studied for their impact in development of resistance and their responsiveness to currently available therapeutic options. Here, we report one such case of a rare mutation leucine replaced by methionine at 273 position and its clinical implications.

Dasatinib treatment based on BCR- ABL mutation detection in imatinib- resistant patients with chronic myeloid leukemia

目的探讨在BCR-ABL突变检测指导下选用达沙替尼治疗伊马替尼耐药慢性髓性白血病（CML）患者的有效性。方法83例伊马替尼耐药和39例伊马替尼与尼洛替尼均耐药的CML患者，持续口服达沙替尼，其中慢性期（CP) 55例，加速期（AP) 21例，急变期（BP) 46例。达沙替尼治疗前，经BCR-ABL突变筛查，除外具有对达沙替尼高度耐药突变类型（T315I/A、F317L/V/C和V299L）的患者。定期监测血液学、细胞遗传学和分子学反应，评估疾病进展和生存情况。当发生达沙替尼治疗失败时检测BCR-ABL突变。结果CP患者中，完全血液学反应（CHR）率为92.7%，完全细胞遗传学反应（CCyR）率为53.7%，主要分子学反应（MMR）率为29.6%，MR4.5率为14.8%，4年疾病无进展生存（PFS）率和总生存（OS）率分别为84.4％和89.5%。AP患者中，血液学反应（HR）率和CCyR率分别为81.0％和35.0%，3年PFS率和OS率分别为56.1％和59.3%。BP患者中，HR率和CCyR率分别为63.0%和21.4%，1年PFS率和OS率分别为43.6％和61.8%。达沙替尼作为二线和三线酪氨酸激酶抑制剂时，治疗反应相似。当发生达沙替尼治疗失败时，75例患者中37例（48.7%）检出新突变，T315I最为多见（59.5%）。达沙替尼治疗前存在突变的患者与无突变患者相比，治疗反应、PFS和OS差异均无统计学意义，但当出现达沙替尼耐药时，发生新突变的可能性显著增高（65.7％对34.1%，P=0.006）。结论达沙替尼可以有效治疗伊马替尼耐药或伊马替尼与尼洛替尼均耐药的CML各期患者，CP和AP患者疗效持久。达沙替尼治疗前BCR-ABL突变筛查及治疗中突变监测非常重要。

The Role of New Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia.

Imatinib mesylate was the first tyrosine kinase inhibitor (TKI) approved for the management of chronic myeloid leukemia. Imatinib produces acceptable responses in approximately 60% of patients, with approximately 20% discontinuing therapy because of intolerance and approximately 20% developing drug resistance. The advent of newer TKIs, such as nilotinib, dasatinib, bosutinib, and ponatinib, has provided multiple options for patients. These agents are more potent, have unique adverse effect profiles, and are more likely to achieve relevant milestones, such as early molecular responses (3-6 months) and optimal molecular responses (12 months). The acquisition of BCR-ABL kinase domain mutations is also reportedly lower with these drugs. Thus far, none of the randomized phase III clinical trials have shown a clinically significant survival difference between frontline imatinib versus newer TKIs. Cost and safety issues with the newer TKIs, such as vascular disease with nilotinib and ponatinib and pulmonary hypertension with dasatinib, have dampened the enthusiasm of using these drugs as frontline options. While the utility of new TKIs in the setting of imatinib failure or intolerance is clear, their use as frontline agents should factor in the age of the patient, additional comorbidities, risk stratification (Sokal score), and cost. Combination therapies and newer agents with potential to eradicate quiescent chronic myeloid leukemia stem cells offers future hope.

Clinical Relevance of Low Burden BCR-ABL1 Mutations Detectable By Amplicon Deep Sequencing (DS) in Philadelphia-Positive (Ph+) Acute Lymphoblastic Leukemia (ALL) Patients (pts): The Type of Mutation Matters

Background and Aims- In Ph+ ALL pts treated with tyrosine kinase inhibitors (TKIs), the likelihood of acquiring TKI-insensitive mutations and the striking incidence of highly resistant T315I and compound mutants underscore the importance of BCR-ABL1 kinase domain (KD) sequence surveillance for timely and rational therapeutic reassessment.We used an amplicon DS strategy of the BCR-ABL1 KD to assess the following issues:i) whether DS allows earlier detection of emerging TKI-insensitive mutations in pts undergoing BCR-ABL1 KD mutation screening for minimal residual disease (MRD) persistence;ii) whether TKI-insensitive low burden mutations can be identified in relapsed pts with negative conventional sequencing results;iii) whether TKI-insensitive low burden mutations are necessary and sufficient to predict for treatment failure in all cases.Methods- This study was conducted in a total of 56 Ph+ ALL pts who received TKI-based therapies at our or collaborating institutions and were referred to our laboratory for MRD follow-up monitoring by RQ-PCR and for BCR-ABL1 KD mutation analysis in case of MRD positivity. These pts were divided into three different cohorts:i) 10 de novo and 24 advanced Ph+ ALL pts who relapsed and developed BCR-ABL1 KD mutations on TKI-based therapy administered 1st-line or for recurrent disease, respectively. To reconstruct the dynamics of mutation emergence, longitudinal re-analysis of monthly-collected samples from the time of hematologic relapse backwards was performed by DS. Whenever samples were available, the analysis was done back to the time of diagnosis (n=10/10) or back to the time of first or former relapse (n=15/24), respectively. Two to 6 samples were analyzed for each pt, for a total of 109 samples.ii) 14 Ph+ ALL pts who were known to be negative for mutations at the time of hematologic relapse as assessed by conventional sequencing. Relapse samples were reanalyzed by DS.iii) 8 Ph+ ALL pts with long-term relapse-free survival despite persistent or intermittent MRD positivity at multiple timepoints. Up to 5 samples were analyzed for each pt, for a total of 28 samples.DS was performed on a Roche GS Junior. Lower mutation detection limit of DS was 1%.Results- In the 34 de novo or advanced Ph+ ALL pts who were known to have acquired TKI-insensitive mutations at the time of relapse on tyrosine kinase inhibitor (TKI) therapy, longitudinal retrospective reanalysis by DS allowed mutation backtracking in 13 (41%) cases. One patient was found to harbour a low burden Y253H at diagnosis. In 3 imatinib (IM)-resistant pts who switched to dasatinib (DAS), a low burden T315I mutation was already detectable at baseline.In the 14 pts with no mutations detectable by conventional sequencing at the time of relapse on IM or DAS, low burden TKI-insensitive mutations were detected by DS in 6 (43%) cases. In 2 cases who had relapsed on DAS, a T315I and an F317L mutation, respectively, were present just below the lower detection limit of conventional sequencing (15.9% and 12.4%, respectively); in the remaining 4 pts, DS identified multiple (2 to 3) low burden mutations, all of which known to confer a moderate to high degree of insensitivity to the ongoing TKI.In the 8 pts with persistently or transiently detectable BCR-ABL1 transcripts at multiple timepoints despite stable hematologic remission, DS detected low burden mutations in 9 samples from 4 pts. However, no mutation known to be truly insensitive to the ongoing TKI could be recognized.Conclusions•MRD persistence in Ph+ ALL pts may hide emerging TKI-insensitive BCR-ABL1 KD mutations that DS may identify earlier than conventional sequencing - allowing a greater leeway before overt hematologic relapse occurs;•polyclonal resistance sustained by multiple TKI-insensitive low burden mutations may explain relapse in a proportion of cases with unmutated BCR-ABL1 KD sequences as assessed by conventional sequencing;•the type of mutation matters: detection of low burden mutations insensitive to the ongoing TKI was always found to predict/correlate with treatment failure. Detection of low burden mutations with low/unknown IC50 might explain low level MRD but does not predict for an impending relapse;•MRD-triggered, BCR-ABL1 KD mutation screening by DS may be precious for earlier and more effective use of preemptive rescue therapies.Supported by ELN, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi) DisclosuresSoverini:Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Abruzzese:BMS, Novartis, Pfizer, Ariad: Consultancy. Baccarani:ARIAD Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cavo:Onyx: Honoraria; BMS: Honoraria; Novartis: Consultancy, Honoraria; Millenium Pharmaceuticals: Honoraria; Celgene: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Jansenn: Consultancy, Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; ROCHE: Consultancy; AMGEN: Consultancy; Ariad: Consultancy; MSD: Consultancy.

Patients with Chronic Myeloid Leukemia Show Different Modulation of MYB-Dependent Oncogenic Pathway in the Course of Hematopoietic Differentiation upon Sensitivity to the TKI Treatment

Introduction: Inhibition of the BCR-ABL protein activity by tyrosine kinase inhibitors (TKIs) plays crucial role in leukemic transformation from the chronic phase (CP) of the chronic myeloid leukemia (CML) into fatal blast crisis (BC). However 20 - 30 % of CML patients develop resistance to the TKIs. Beside mutations in BCR-ABL kinase domain, BCR-ABL independent mechanisms of acquired TKIs resistance including microRNAs (miR) may be also involved. A significant reduction of miR-150 levels associates with CML progression. MiR-150 is an inhibitor of the Myeloblastoma oncogene c-MYB, which is required for BCR-ABL-dependent leukemogenesis in a mouse model of CML-BC. Recently MYB was found to positively regulating another miR, miR-155, that inhibits tumor suppressor and pro-differentiation factor PU.1 in AML/MPS mouse model. PU.1 is a major regulator of myelopoiesis whose levels require precise guidance. We recently reported increased levels of miR-155 in CML-BC.Objectives: We hypothesize that BCR-ABL activity together with low levels of miR-150 activates putative oncogenic signaling pathway MYB/miR-155/PU.1 in CML leading to a progressive blockade of cell differentiation and possibly also of resistance to TKIs.Material and Method: Bone marrow (BM) cells of CML-CP patients at the time of diagnosis (n=10), and of treated patients either sensitive (n=8) or resistant (n=11) to TKIs, were FACS sorted for CD34 and CD38 into subpopulations representing the stages of cell differentiation. CD34+ and CD34- leukocytes from peripheral blood of healthy donors (n=10) were isolated by MACS and used as control. Levels of miR-150, BCR-ABL, MYB, miR-155 and PU.1 were determined by qRT-PCR. Expression of surface differentiation markers on BM cells was determined using flow cytometry at the time of diagnosis (n=10) and upon TKIs resistance (n=5). The effect of miR-150 overexpression and BCR-ABL inhibition was studied in CML-BC cell line K562 using nucleofection of the synthetic miR-150 and siRNA BCR-ABL (and by cell cultivation with TKI imatinib). mRNA and protein expression of BCR-ABL, MYB and miR-150 levels were determined by qRT-PCR and immunoblotting.Results: In the groups of healthy donors and de novo diagnosed CML patients we observed significant positive or negative correlations of expression levels between studied molecules among the sorted cell subpopulations, which outlined the activity of the putative pathway BCR-ABL/miR-150/MYB/miR-155/PU.1 in CML-CP. Interestingly, in the group of patients resistant to TKI, we found that correlation between MYB and miR-155 was not observed suggesting this pathway is deregulated upon TKI resistance. Conversely, we found gain of negative correlation between expression of MYB and PU.1 in the group of patients responding to TKI suggesting that MYB and PU.1 oppose each other upon normal cell differentiation. We also observed significant differences of the surface marker phenotype profiles between BM cells of de novo diagnosed CML patients and patients resistant to TKIs treatment. While relatively differentiated (granulocyte) CD34- CD38- CD11b+ CD14- population presents about 7 % of BM cells for the group of patients at diagnosis, it comprises only 0.3 % of BM cells for resistant group (P= 0.0027) indicating that TKI resistance associates with significant blockade of myeloid differentiation.We next utilized K562 cells showing that levels of miR-150 significantly increased after BCR-ABL inhibition by imatinib (P=0.0017) and also upon BCR-ABL knockdown with siRNA (P=0.002). This suggests that miR-150 is regulated by BCR-ABL. Similarly, MYB mRNA and protein levels were markedly decreased upon miR-150 overexpression and this was enhanced by inhibition of BCR-ABL activity with imatinib.Conclusions: Dysregulation of BCR-ABL/miR-150/MYB/miR-155/PU.1 pathway was observed in CML stages and stage-specific cell populations in relation to TKI-sensitivity and disease progression. Our model consists of the BCR-ABL upregulating MYB both through miR-150 inhibition and by other downstream mechanisms by additive effect, thus the cooperation of miR-150 and imatinib through inhibiting MYB may represent an important barrier to CML progression. Even more our results show that regulation of miR-155 and PU.1 may play role in resistance to TKI treatment.Supported by GAUK 178215 and by project 00023736 from the Czech Ministry of Health DisclosuresKlamova:Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Machova Polakova:Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

The Allosteric Inhibitor ABL001 Is Susceptible to Resistance in Vitro Mediated By Overexpression of the Drug Efflux Transporters ABCB1 and ABCG2

Tyrosine Kinase Inhibitors (TKIs) result in excellent responses in Chronic Myeloid Leukaemia (CML) patients. However, secondary resistance is observed in ~35% of patients, mostly due to Bcr-Abl kinase domain (KD) mutations. Overexpression of the efflux transporter ABCB1 is a known mediator of primary resistance to imatinib (IM) and nilotinib (NIL). ABL001, a potent allosteric inhibitor, binds to the myristate pocket of the Bcr-Abl KD. In this study, we modelled ABL001 resistance in vitro with particular focus on ABCB1 and ABCG2.ABL001 sensitivity was evaluated in BCR -ABL1 + cell lines K562, K562-Dox (ABCB1 overexpressing) and K562-ABCG2 (transfected with ABCG2 overexpression vector). Also, K562, K562-Dox and KU812 cells were made resistant to ABL001 by culturing long term in increasing concentrations of ABL001 (increased once >80% survival in culture for >10 days). Parental ABL001 naive cells were maintained in parallel culture. Initial onset of resistance was characterised by significantly increased IC50ABL001 based on AnnexinV/7-AAD cytotoxicity assays performed at each ABL001 dose escalation. Resistance mechanisms were interrogated during escalation and once 10 μM was reached (based on clinically achievable doses). Protein expression levels of efflux transporters ABCB1/ABCG2 were examined by flow cytometry; KD mutation sequencing and Bcr-Abl protein quantitation were performed.K562-Dox and K562-ABCG2 cells demonstrated significantly increased IC50ABL001 compared with parental K562 control cells: 256 and 299 nM respectively vs 23 nM, p<0.001 suggesting both ABCB1 and ABCG2 are important in ABL001 transport. Furthermore, resistance was reversible through use of specific inhibitors cyclosporin (ABCB1) and Ko143 (ABCG2). IC50ABL001 +inhibitors was comparable to that of control K562 cells: K562-Dox +cyclosporin=11 nM; K562-ABCG2 +Ko143=15 nM.A prior study identified ABCB1 overexpression as an initiator of resistance to IM and NIL (expression increased up to 7- and 5-fold in IM and NIL resistant cells compared with respective control cells p<0.002). In this study, expression levels of ABCB1 and ABCG2 were interrogated in 3 ABL001 resistant cell lines; results indicated overexpression of both transporters was integral in development of resistance. Up to 17- and 60-fold greater levels of ABCB1 and ABCG2 respectively was observed in resistant vs control cells (Table 1). No KD mutations were detected in early resistance intermediates; however, a myristate pocket mutation was detected in later stage KU812 ABL001 resistant cells (percentage mutation correlated with IC50ABL001).Table 1ABL001 resistance characteristics in selected resistant intermediatesIC50ABL001 (nM)Fold change (MFI)Bcr-Abl over expressionKD mutationABCB1ABCG2K562 Control23NNK562 500 nM ABL001>2500 p <0.001-1.4 p =0.00348.3 p =0.007YNK562 10 μM ABL00127, 800p <0.001-2.3 p =0.53560.3 p <0.001YNK562 10 μM ABL001 +Ko14389 p <0.001K562-Dox Control256NNK562-Dox 500 nM ABL0012393 p <0.0012.3 p <0.001-2.9 p <0.001NNK562-Dox 500 nM ABL001 +cyclosporin17 p <0.001K562-Dox 10 μM ABL001>50, 000 p <0.001-1.6 p <0.001-0.1 p <0.001NNKU812 Control2.7NNKU812 5 nM ABL0016.4 p <0.0012.2 p =0.0084.0 p =0.002YNKU812 10 μM ABL00133, 300 p <0.0018.1 p =0.00311.0 p =0.010YY (90%)p-value: resistant vs respective control; n>3; N=No; Y=YesWhile further in vitro and in vivo studies will determine the clinical relevance of efflux-mediated resistance to ABL001 mono- and combination therapy, our preclinical data provide evidence that kinase domain mutations may not be the predominant cause of ABL001 resistance; drug transporters likely play an important role as well. Susceptibility to ABCB1 overexpression is well recognised for IM and NIL resistance and we now show it is also relevant for ABL001. ABCG2-mediated resistance has not been observed with NIL or IM but is clearly important for ABL001. ABL001 treatment induced ABCG2 overexpression in two cell lines; both have negligible basal expression. ABCG2 overexpression preceded Bcr-Abl overexpression and mutation emergence. Importantly, ABL001 resistance was completely reversible in the presence of the ABCG2 inhibitor Ko143 (K562 10 μM ABL001 cells). Given the lack of strong evidence for ABCG2-mediated transport of NIL or IM at clinically relevant concentrations, our data provide a strong rationale for the use of ABL001 in combination with either of these TKIs. DisclosuresBranford:Ariad: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Hughes:ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Aurora Kinase a: A New Component of Imatinib Resistance in Chronic Myeloid Leukemia

Imatinib (IM) targets the constitutively active BCR-ABL1 tyrosine kinase (TK) in chronic myeloid leukaemia (CML) and has become standard treatment based on good responses achieved in clinical trials. However, IM resistance can occur through several mechanisms including BCR-ABL1 kinase domain mutations, amplification, overexpression and clonal evolution.The discovery that Aurora kinases (AK) were abnormally expressed in malignancies including leukaemia prompted the development of agents that inhibit their activity. Aurora kinase A (AKA) is a central mitotic regulator necessary for mitotic entry, mitotic spindle assembly and accurate chromosome separation. The therapeutic potential of specifically targeting AKA activity as an anticancer strategy has not been rigorously investigated because all of the agents previously designed to target AKs have significant off-target effects on other family members and/or BCR-ABL1 kinase activity. However, the role of AKA in chemoresistance of CML is not well understood.In this study we investigated the mechanism of action of AKA in CML in vitro and ex vivo models (i.e. K562 cell lines and CD34+ cells derived from bone marrow aspirates of both CML at the clinical diagnosis and in mononuclear cell fraction of IM-resistant patients). Moreover, the role of two major players (i.e. FOXM1 and Polo-like kinase 1 (PLK1) a ser-thr kinase involved in G2/M progression) in the AKA signalling pathway has been investigated.First, we investigated AKA expression and activity by Western Blot and immunoprecipitation analyses. CD34+ cells from peripheral blood aphereses of hematologically healthy donors, pooled to avoid individual differences, were used as normal controls. Informed consent was obtained by all participants before their admittance to the study. Our data showed an over-expression and hyper-phosphorylation of AKA, FOXM1 and PLK1 in CD34+ cells of CML patients at clinical diagnosis compared to mononuclear cell fractions.Our results also showed a FOXM1 increment associated with IM resistance. An IM-resistant K562 cell line (LD50 0.25 mM vs 0.026 mM of parental cells) generated in our lab exhibited FOXM1 over-expression and hyper-phosphorylation contingent upon the upstream activation of AKA and PLK1. AKA, FOXM1 and PLK1 involvement in IM resistance was observed also in clonal progenitors from bone marrow samples of CML patients who developed IM resistance independent from BCR-ABL1 point mutations. Interestingly, the putative BCR-ABL1+/CD34+ LSC compartment, which is neither dependent on BCR-ABL1 TK for proliferation and survival nor killed by IM or second generation inhibitors, showed a hyper-phosphorylation of AKA and a consequent overexpression and hyper-activation of FOXM1 and PLK1.Taken together, these evidences may pave the way to the set up a new strategy to overcome drug resistance in CML. Moreover, our data identify a new signaling pathway involved both in drug resistance and in CD34+ cells survival, suggesting that AKA, FOXM1 and PLK1 are three very interesting druggable targets to eradicate the TKIs resistant CD34+ progenitors.Acknowledgments: Manuela Mancini was supported by a Fondazione Umberto Veronesi Fellowship. University of Bologna (RFO funds), Ministero della Pubblica Istruzione e della Ricerca (PRIN funds), Umberto Veronesi Foundation, AIRC, FP7 NCS-PLT project, Progetto Regione-Università 2010-12 (L. Bolondi) and BolognaAIL are acknowledged for financial support. DisclosuresSoverini:Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Rosti:Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Martinelli:BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; AMGEN: Consultancy; MSD: Consultancy; Novartis: Consultancy, Speakers Bureau.