Imatinib-resistant Patients Research Articles

A comparative proteomic study of Homoharringtonine-induced apoptosis in leukemia K562 cells

The objective of this study was to determine the changes in protein profiles of K562 chronic myeloid leukemia (CML) cells in response to Homoharringtonine (HHT). HHT treatment significantly increased apoptosis of K562 cells. Proteomic analyses indicated 32 differentially expressed proteins, 13 of which were identified by mass spectrometry (nine down-regulated and four up-regulated). Aside from alterations in apoptotic proteins and proteins associated with transcription and translation, our data also revealed changes in oxidative stress response and redox reaction-related proteins, such as heat shock proteins (Hsps), DJ-1 and thioredoxin. Specifically, these proteins were validated to decrease after HHT treatment in K562 cells and in primary CML cells by immunoblot analysis. Additionally, Hsps, DJ-1 and thioredoxin, which were also shown to decrease in primary cells from imatinib-resistant patients, may be promising potential targets for mechanistic research and new clinical treatments.

Sensitive allele specific oligonucleotide-polymerase chain reaction in detection of preexisting mutations in imatinib-resistant chronic myelogenous leukemia patients: A retrospective analysis

Introduction: Pre-existing BCR-ABL kinase domain mutation leads to Imatinib resistance. Methods: Retrospective analysis of 50 patients of Imatinib resistance was done in GCRI, from January 2014 till May 2014. Allelle Specific Oligonucleotide–Polymerase Chain Reaction (ASO-PCR) was performed on Genomic DNA, of peripheral blood mononuclear cells (PBMCs). Results: 47 (94%) were in Chronic phase, 2 (4%) in accelerated phase, 1 (2%) in blastic crisis. Median duration of Imatinib was 48 months. 43/50 had one or more than 1 mutation, T315I mutation in 5 (10%) patients, M351T in 32% (16/50) and F311L in 8. Conclusion: We report low cytogenetic response (25%) and durability of response to 600 mg of Imatinib, even in M351T mutation.

Glutathionylation of NF-Kb and Procaspase-3 Regulates Inducible Nitric Oxide Synthase Expression and Apoptosis of Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia (CML), a myeloproliferative disorder, characterized by sustained neutrophilia and constitutive BCR-ABL tyrosine kinase activity. Constitutive expression of the BCR-ABL kinase and elevated reactive oxygen species (ROS) levels through mitochondria and NADPH oxidase 4 (NOX4) activation leads to genomic instability and enhanced cell survival. Nitric oxide (NO), a signaling molecule has been associated with hematopoesis and suppression of NOS activity may induce profound changes in hematopoietic stem cells/progenitor cells. NO addition or iNOS transfection in K562 cells (BCR-ABL+) altered genes expression involved in the iron metabolism, inhibited cell proliferation and enhanced apoptosis, which were reversed by addition of exogenous iron. Moreover, anti-cancer effect of farnesyltransferase inhibitor in these cells was also mediated by NO production/iNOS induction. The present study investigates status and regulation of NO/iNOS in neutrophils from CML patients.The present study was undertaken to explore NO generation/iNOS expression and its regulation in circulating neutrophils so as to access the role NO/iNOS in CML pathology.All CML patients (Drug/treatment naïve, n=70; imatinib responders, n=62; imatinib resistant, n=25) included in this study were diagnosed in chronic phase. The study protocol was approved by the ethical committees of CSIR-CDRI and KGMU, Lucknow and was conducted in accordance with the declaration of Helsinki. Total nitrite level in neutrophils (PMNs) was assessed by Griess reagent. Nitric oxide, Superoxide, ROS/RNS and mitochondrial ROS generation was assessed by DAF-2DA, DHE, DCF-DA and Mitosox Red respectively. H2O2was measured by Amplex red assay kit. Expression of iNOS gene was evaluated by a SYBR green real-time RT-PCR and Western blot. Binding of NF-kB (p50 and p65 subunit) to iNOS promoter was analysed by CHIP assay. NF-kB (p50 and p65 subunit) and procaspase-3 glutathionylation was assessed by Immunoprecipitation followed by Western blot. Findings in CML patients were further validated using in vitro experiments on K562 cells. Statistical analysis were performed by one way ANOVA test followed by Newman-Keul’s post hoc analysis using the Graph Pad prism software.PMNs total nitrite, NO level and iNOS expression in drug naïve and imatinib resistant patients were significantly less as compared to healthy subjects. However, significant recovery in all the parameters was observed in imatinib responsive patients. Superoxide, ROS/RNS, mitochondrial ROS generation as well as H2O2 level was significantly more in drug naïve and imatinib resistant patients and it was attenuated significantly in imatinib responsive patient’s PMNs. In vitro treatment of K562 cells with Imatinib (2µM) also showed augmented NO generation and iNOS expression, while superoxide, ROS/RNS and mitochondrial ROS generation was decreased. To decipher the molecular mechanisms underlying the modulation of iNOS in BCR-ABL+ cells, we examined binding of NF-κB to iNOS promoter/enhancer and protein S-glutathionylation. Binding of NF-κB (p50 and p65 subunits) to iNOS promoter/enhancer was less in BCR-ABL positive cells, while it was augmented following treatment with imatinib. Moreover, glutathionylation of p50, p65 and procaspase-3 was more in drug naïve as well as in imatinib resistant CML patients PMNs, while it was comparable to healthy subjects in imatinib responders CML patients PMNs. Glutathionylation of NF-κB (p50 and p65 subunit) and procaspase-3 was also attenuated in imatinib treated K562 cells.The results obtained suggest that reduced NO generation/iNOS expression in BCR-ABL positive cells was due to the S-glutathionylation of NF-κB, which decrease it’s binding to iNOS promoter. S-glutathionylation of procaspase-3 in CML however, inhibited apoptosis of BCR-ABL positive cells. The study thus highlights importance of S-glutathionylation as key regulators involved in the proliferation and apoptosis of BCR-ABL positive cells. DisclosuresNo relevant conflicts of interest to declare.

Clinical Outcome Following Change of Tyrosine Kinase Inhibitor (TKI) According to the Detection of an ABL Kinase Mutation

IntroductionPoint mutations in the kinase domain of BCR-ABL are the most frequent mechanism of acquired imatinib resistance in patients with chronic myeloid leukemia (CML). Mutation analysis is recommended to guide the selection of appropriate second line therapy in patients with imatinib failure, since some frequently occurring mutations confer clinical resistance to nilotinib and/or dasatinib. To date, more than 80 point mutations have been described following imatinib exposure, but mutations at 7 sites (G250, Y253, E255, T315, M351, F359, and H396) comprise approximately 60% of mutations reported in large series. We retrospectively analysed the impact of 83 ABL kinase mutations (P-loop mutations = 28, T315I mutation = 12 and other mutations = 43) arising in 65 chronic phase (CP) CML patients with imatinib failure. The aim of this study was to define the clinical characteristics of these patients, and to assess their outcome following introduction of second line agents.MethodsBetween July 2002 and August 2013, 123 CML patients were found to have ABL kinase mutations in our centre. Patients presenting in blast crisis (BC) or accelerated phase (AP), and those who did not require change in therapy (including patients who required imatinib dose escalation) following detection of an ABL kinase mutation were excluded from the analysis. Sixty-five patients in CP who had imatinib failure and detectable ABL kinase mutation, and who required change in therapy were evaluated. Definitions of CML phases, treatment responses and failures were as per definitions of the European LeukaemiaNet. Direct sequencing method was used to detect a range of mutations within the tyrosine kinase domain at the level of ~20% sensitivity and pyrosequencing to detect specific mutations with a sensitivity of ~5%.ResultsEighty-three ABL kinase mutations were detected in 65 CP patients at the time of imatinib failure with 35% of patients (23 of 65) harbouring P-loop mutations (including M244V), 18% (12 of 65) with T315I mutation and 46% (30 of 65) with other mutations (catalytic domain, imatinib binding site, activation loop and C-terminal). Composite mutations were present in 10 patients (15%), with 2 patients harbouring both P-loop and T315I mutations. Median time on imatinib therapy was 29.5 months (range, 2-144 months). At the time of mutation detection, 20% of patients (13 of 65) were in CCyR, 54% (35 of 65) in CP, 17% (11 of 65) in AP and 9% (6 of 65) had progressed to BC. Median time from CML diagnosis to mutation detection was 21 months for patients with T315I mutation, 45.5 months for P-loop mutations and 48 months for other mutations. Following mutation detection, patients in CP and AP were treated on second-line agents with dasatinib, nilotinib, bosutinib or ponatinib (based on sensitivity of ABL kinase mutants to ABL kinase inhibitors), and those in BC were treated with chemotherapy +/- TKI. Thirteen patients (20%) underwent allogeneic stem cell transplantation (SCT) for disease control (P-loop = 7, T315I = 5, other = 1).Following the change in TKI therapy, the best response was CMR or MMR in 54% of patients (35 of 65), CCyR in 12% (8 of 65), MCyR in 3% (2 of 65) and CHR in 26% (17 of 65). 5% (3 of 65) progressed to BC. In those who only achieved CHR, MCyR or developed progressive disease despite change in TKI, 6 out of 22 patients were found to have additional mutations during their treatment course, including 5 patients with re-emergence of their original mutation. After a median follow-up of 60 months (range, 3-137 months) from detection of mutation, 12 of the 65 patients (18%) have died, including 6 of 23 (26%) with P-loop mutations, 3 of 12 (25%) with T315I mutation, and 3 of 30 (10%) with other mutations. One patient who died had composite P-loop and T315I mutations. Median overall survival was 250 months for patients harbouring P-loop mutations (P=0.37) and not reached for T315I and other mutations.ConclusionWith longer follow-up and the availability of second and third generation TKIs, we have demonstrated that most clinically relevant ABL kinase mutations respond to change in TKI therapy following imatinib failure, with the majority of patients achieving durable cytogenetic and molecular response. This study emphasizes the importance of early detection and characterization of ABL kinase mutations in imatinib resistant patients in order to identify those patients who may benefit from alternative TKI therapy or stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Second Report of Arab Leukemia Net (ALN) Registry for Chronic Myeloid Leukemia (CML) in the Middle East &North Africa Region (AFME). Epidemiology of CML and Additional Chromosomal Abnormalities (ACAs) in Egypt, Multicenter Results

Little is known about burden of CML in Arab countries. The first ALN report demonstrated that age-specific rates for CML in Egypt and Arab nations are lower by at least two decades compared to western populations (Azzazi and Mattar ELN newsletter 2013). Lack of reliable data concerning geographic and ethnic variations and response rates to therapy contribute to the variability of incidences among CML registries. Studies underestimate the true age of CML patients as access to medical services show great diversity in AFME region.ACAs were reported in 5% of CML patients, considered at diagnosis by ELN as a “warning” requiring careful patient monitoring.(Baccarani et al Blood 2013), ACAs emerging during treatment are considered by WHO classification as accelerated phase (AP). There occurrence indicates that the leukemia has become BCR-ABL1 independent by secondary genetic acquisitions. Some ACAs are poor prognostic factors such as the acquisition of additional Ph‏ chromosome (ch) that increases the kinase activity of BCR-ABL1; the isochromosome 17q i(17q) which leads to inactivation of the tumor suppressor gene p53 and impedes the response to Imatinib; the extra 8 which lead to c-Myc over expression that enhances the transformation of leukemic cells and the extra 19 which hinder Imatinib activity via silencing gene promoters.Objectives 1) To Release 5 year follow-up data of second ALN report of CML epidemiology in Egypt. 2) To investigate the low age of CML in Egypt. 3) To evaluate ACAs role in disease and clonal evolution. 4) To build a reliable data base -the ALN- as information / support services.Method We analyzed data of 578 (302 male and 276 female) CML patients (followed-up for 5 years). Data collected according to ELN (Baccarani et al Blood 2013) GIMEMA and EUTOS recommendations via a multicenter web based data registry portal, the ALN. (www.aln-afme.com). To insure lack of bias an independent server for data storage and processing was established in Masaryk University, Institute of Biostatistics and Analyses, in Prague, Czech Republic, other logistics are managed by DMC Healthcare Inc. HQ in Toronto, Canada.Chromosome banding analysis and FISH were performed, for Y-ch, trisomy 8, duplication of Ph, i(17q), trisomy 19, and Deletion of der(9) ch (Luatti et al Blood 2012).Results Patients Median age was 43y, (40y for males, 41y for females), The age specific rates were highest for the age group of 30-35 years. Female patients presented with lower hemoglobin, higher platelet counts and smaller spleen size (P<0.0001). 98% of patients achieved CH respone, 89% PCYR, 87% CCYR, and 83% MMR. At diagnosis 87% patients were in chronic phase (CP) CML, 8.1% in accelerated, and 4.9% in blastic phase. Sokal score: Low risk 57.8% Intermediate 24.5% and High in 17.7%. EURO (Hasford) score (59% Low risk, 28.4%Intermediate risk and 12.6%High risk). 42% of patients received imatinib, 34% nilotinib 20% dasatinib and 4% needed therapies plus TKI). Transplantation rate was 19%, PFS and OS were equal in female and male patients. ACAs were found in 62 (11%) patients, they had lower cytogenetic and molecular response rates and longer response time to TKI and inferior outcome. ACAs were more frequent in younger, imatinib resistant patients, and in blast phase. We identified loss of Y ch in 18 patients (29%), trisomy 8 in 7 (11%), trisomy 19 in12 (19%), i(17q) in 12 (19%), other different single abnormalities in 8 patients (13%), complex karyotype with double ACAs in only 5 patient (8%). Four patient showed variant Ph ch: t(9;22;22)(q34;q11;q11). Deletion of der(9) ch in 17 cases (27%): (10 cases with loss of Y ch, 4 case with del(20)(q11q13), and 3 case with t(X;13) (q13;q32)). The cytogenetic and molecular response rates were uniformly lower in patients with ACAs, overall CCgR and MMR rates were significantly lower in patients with ACAs (68% vs 89% and 55% vs 86% respectively), responses were significantly slower in patients with ACAs, 54 patients presented with ACAs at diagnosis while 8 patients developed ACAs while on treatment.Conclusions: The importance of ethnicity and gender differences in relation to disease incidence, and prognosis are major health policy focus. To investigate the low mean age of CML in Egypt and evaluate role of ACAs on disease and clonal evolution, Data from 10 Centers in Egypt showed a much lower mean age of CML and more frequent (11%) ACAs, they adversely affected time and response rates to Imatinib treatment. DisclosuresNo relevant conflicts of interest to declare.

DJ-1 Plays an Important Role in Homoharringtonine-Induced Apoptosis of Imatinib-Resistant Chronic Myeloid Leukemia Cells

Background and Objective: Although the treatment of chronic myeloid leukemia (CML) has improved since the introduction of tyrosine kinase inhibitors (TKI), cases of resistance have been reported and resulted in challenges to the treatment. Recent studies have suggested that Homoharringtonine (HHT), a cephalotaxine ester, has demonstrated a clinical activity in imatinib-resistant CML patients, however, the molecular mechanisms underlying this phenomenon are unknown. Our previous study found that treatment with HHT significantly increased apoptosis of K562 cells. Moreover, the protein DJ-1, identified by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, was demonstrated to decrease after HHT treatment. Therefore, we performed the experiment to address the hypothesis that DJ-1 might play an important role in Homoharringtonine-induced apoptosis of Imatinib-resistant chronic myeloid leukemia cellsMethods and Results: To find the pivotal protein by HHT, Imatinib-resistant K562 cells were treated with HHT (10 ug/ml) for 5 h, 12 h, 24 h respectively and the control group without HHT were harvested to assess apoptosis with Annexin V-FITC and propidium iodide per the manufacturer’s protocol and analyzed by flow cytometry. The data indicated a time dependent induction of apoptosis by HHT, with the number of apoptotic cells (FITC-Annexin-V and PI double-positive cells) significantly increasing from 2.2± 1.5 % in control to 35.9 ± 6.7% in cells treated with HHT for 24 h (P<0.01). The protein DJ-1 expression change upon HHT treatment which were analyzed with western blot, found that the protein level of DJ-1 had significantly decreased after the treatment of HHT for 24 h.Furthermore, primary cells from six CML patients and three healthy donors were obtained with informed consent and divided into three groups: the CML-CP group (three newly diagnosed patients in the chronic phase), the imatinib-resistant CML group(three imatinib-resistant patients in the blastic phase) and the control group. Mononuclear cells were all cultured in vitro in the absence and presence of 10 mg/ml HHT for 5 h and 24 h. The results showed that DJ-1 expression in primary leukemia cells (both CML-CP group and imatinib-resistant CML group) were found to be decreased after HHT treatment and the expression level of DJ-1 seemed lower in the healthy donor as compared to primary CML cells, moreover, the protein changes induced by HHT were significantly different among three groups and the protein changes were not as significant in CML-CP cells as in imatinib-resistant CML cells (P<0.05).Conclusions: These findings indicated that DJ-1 might play an important role in Homoharringtonine-induced apoptosis of Imatinib-resistant chronic myeloid leukemia cells. Further study may help to assess a promising potential of this protein to be used as a target for a molecular therapy. DisclosuresNo relevant conflicts of interest to declare.

Prognostic Value of Multi-Drug Resistance 1 Gene (MDR1) Expression in Newly Diagnosed Patients with Chronic Myeloid Leukemia on Nilotinib Treatment—a Subanalysis of the ENEST1st Study

Introduction MDR1 gene expression has recently been reported to be associated with clinical outcome in imatinib-resistant patients with chronic myeloid leukemia (CML) on second-line nilotinib treatment (Agrawal et al., Leukemia 2014). High MDR1 expression was predictive for achieving deeper cytogenetic and molecular responses as well as duration of chronic phase. We thus prospectively evaluated the value of MDR1 expression analysis in the first-line situation within a large European, multicenter trial of CML patients under treatment with nilotinib.Methods 305 patients enrolled on the ENEST1st study (Evaluating Nilotinib Efficacy and Safety in clinical Trials as First-Line Treatment, NCT01061177) consented to participate in this substudy. Of these, 225 patients were evaluable due to sample availability and presence of typical e14a2 and/or e13a2 BCR-ABL1 transcripts. The median age was 52 (range 18-83), 39% were female. Prior to therapy, the expression of MDR1, BCR-ABL1, and GUSB were determined using a serial dilution of a plasmid constructs harboring MDR1 and GUSB and BCR-ABL1 and GUSB sequences. Ratios MDR1/GUSB and BCR-ABL1/GUSB were calculated. ROC curves were established using initial MDR1 and BCR-ABL1 expression levels aiming to achieve the primary endpoint of MR4 (defined as BCR-ABL1 ≤ 0.01% on the International Scale [BCR-ABL1IS] or undetectable BCR-ABL1 in cDNA with ≥ 10,000 ABL1 transcripts) at 18 months or to achieve a MMR (≤ 0.1% BCR-ABL1IS) at 9 months. Mann-Whitney tests have been applied using a significance level of 0.05.Results At 9 months, 161 patients (72%) achieved MMR and at 18 months 111 patients (49%) a MR4. The median ratio MDR1/GUSB among all evaluable patients was 11.4% (range, 0.6-318.1), the median ratio BCR-ABL1/GUSB was 22.2% (range 0.09-198.7). Initial BCR-ABL1/GUSB levels did not predict MMR or MR4 at 9 or 18 months. However, initial ratios MDR1/GUSB were predictive for the achievement of MMR at 9 months (best cut-off 3.8%, sensitivity 88.8%, specificity 26.6%, AUC 0.598, p=0.022) and for reaching MR4 at 18 months (best cut-off 7.90%, sensitivity 72.1%, specificity 51.8%, AUC 0.624, p=0.001).Conclusions High MDR1 expression levels in newly diagnosed CML patients appear to be associated with deeper molecular responses within the first 18 months of nilotinib treatment. These data merit further exploration and if validated would justify the investigation of frontline CML therapy guided by baseline MDR1 expression levels. DisclosuresDietz:Novartis: Research Funding. Hanfstein:Bristol-Myers Squibb: Honoraria; Novartis: Research Funding. Rousselot:Novartis: Research Funding. Lange:Novartis: Consultancy, Honoraria, Research Funding. Maier:Novartis: Research Funding. Foroni:Novartis: Research Funding. Gerrard:Novartis: Research Funding. Talmaci:Novartis: Research Funding. Janssen:Novartis: Research Funding. Frank:Novartis: Employment. Saussele:Pfizer: Honoraria, Travel, Travel Other; Bristol-Myers Squibb: Honoraria, Research Funding, Travel, Travel Other; Novartis: Honoraria, Research Funding, Travel Other. Giles:Novartis: Honoraria, Research Funding. Hochhaus:Novartis: Research Funding. Müller:Novartis: Honoraria, Research Funding.

Effectiveness of First and Second Line Nilotinib in Chronic Myeloid Leukemia (CML) Patients with Atypical e1a2 BCR-ABL Transcripts

▪IntroductionChronic myeloid leukemia (CML) is characterized by a reciprocal translocation between chromosome 9 and 22, resulting in a BCR-ABL fusion gene. In CML, over 95% of the breakpoints involve the major breakpoint cluster region (M-bcr) of BCR introns downstream of either exon 13 or 14 and introns downstream of ABL exon 2, resulting in fusion transcripts e13a2 (b2a2) and e14a2 (b3a2). The minor breakpoint cluster region (m-bcr) in BCR gene is a less common breakpoint involved the exon 1, fused to ABL a2, resulting in e1a2 fusion transcript. Other transcripts such as e19a2, e1a3, e6a2, e13a3, and e14a3 occur less frequently.The e1a2 transcripts exist in the most of acute lymphoblastic leukemia, and CML expressing only e1a2 transcripts is rare. Although anecdotal reports of patients with the e1a2 transcripts had a good prognosis, to our knowledge, the outcome of most of patients with this transcript alone in the era of imatinib is very poor. We performed this study to investigate the frequency of e1a2BCR-ABL CML and mutations in the kinase domain of BCR-ABL after imatinib failure, and to evaluate their treatment prognosis.ObjectiveTo investigate the frequency of e1a2BCR-ABL CML and mutations in the kinase domain of BCR-ABL after imatinib failure, and to evaluate their treatment prognosis.Patients and MethodsThe records of all CML patients at our hospital from January 2004 to March 2014 were reviewed to identify patients with only e1a2BCR-ABL fusion transcripts consequent to breakpoints in minor BCR. Patients with e1a2 coexisting with e13a2 and/or e14a2 were excluded from this analysis. BCR-ABL transcripts were revealed at diagnosis by quantitative PCR followed by conventional agarose electrophoresis of PCR products. Molecular follow-up of BCR-ABL transcripts throughout treatment was performed by quantitative PCR and cytogenetic follow-up was performed by karyotype analysis following the guidelines of the European Leukemia Net. BCR-ABL kinase domain point mutations have been assessed using Sanger sequencing in patients with imatinib failure and progression.Event-free survival (EFS) was measured from the start of each therapy until progression to the BP or death from any cause during treatment. Overall survival was defined from date of CML diagnosis to date of death or last follow-up.ResultsNineteen (0.6%) of the 3172 CML patients, during the study period had e1a2BCR-ABL CML. At the time of diagnosis, 16 patients were in CP, 2 in BP, and 1 in AP. We only retrospectively study 12 CML patients. The median follow-up since the diagnosis of CML is 35 months (range, 5-149 months).9 of 12 patients received TKI as their initial therapy (7 imatinib, 1 nilotinib;Table 1). Among patients in CP, 6 received imatinib as frontline therapy and 3 received imatinib after Hu or in combination with IFN, and after a median follow-up of 28 months (range, 9-145 months), 2 patients had no response to imatinib, and the best response for the others was complete hematologic response (CHR) in 5, mCyR in 1 and MR4.5 in 1. Notably, only 1 of 9 patients receiving imatinib achieved MR4.5. 2 patients who was not found imatinib-resistant mutation was changed to nilotinib as second line TKI and 2 patients was changed to alloSCT. Only 1 who underwent alloSCT achieved MR4.5 and the others retained a hematologic response. 2 of the patients in CP progressed to BP (including 1 with H396R mutation) after a median of 27 months (range, 12-41 months) and died at 61 and 48 months after diagnosis, respectively.2 patients in BP (including 1 after CDOP+IA failure) received imatinib . The two patients had no response to imatinib and 1 died after 8 months. 1 in AP received nolitinib as frontline therapy. The patient achieved MR4.5 after 3 months and returned to CP.Overall, 8 patients (7 CP, 1 AP) were alive at a median of 26 months (range, 5-149 months) after diagnosis: 2 with ＞MR4.5 on TKI (1 on imatinib, 1 nilotinib) and 1 with ＞MR4.5 after alloSCT. Median survival was 28 months for patients in CP at the start of therapy, and 5 months for the patient in AP.ConclusionWe conclude that the frequency of e1a2BCR-ABL CML is only 0.6%. It suggests an inferior outcome to imatinib, and the mutations in the kinase domain of BCR-ABL have been detected in 20% of imatinib-resistant patients. Perhaps nilotinib as first line treatment has a high rate of MR4.5 in newly diagnosed CML and should be considered firstly, but the role of alloSCT for these patients is irreplaceable. DisclosuresNo relevant conflicts of interest to declare.

Molecular responses at 3 and 6 months after switching to a second-generation tyrosine kinase inhibitor are complementary and predictive of long-term outcomes in patients with chronic myeloid leukemia who fail imatinib

Early molecular response (MR) defined by BCR–ABLIS levels has prognostic impact in chronic myeloid leukemia (CML). MR was evaluated at 3 and 6 months after switching to nilotinib or dasatinib in 115 patients with resistance to imatinib. Three groups were delineated at 3 months (< 1%, 1–10% or > 10% BCR–ABLIS levels) with different outcomes at 3 years regarding major molecular response (MMR, 91%, 47%, 22%, p < 0.001), failure-free survival (FFS), progression-free survival (PFS, 96%, 89% and 78%, p = 0.05) and overall survival (OS). After 6 months, patients with MR < 1% had higher 3-year MMR (83% vs. 16%, p < 0.001), FFS, PFS (94% vs. 84%, p = 0.05) and OS. Four patients had 3-month and 6-month MR > 10% and < 1%, respectively (3-year FFS 50%). Thirteen had 3-month and 6-month MR < 10% and ≥ 1%, respectively (3-year FFS 38%). These findings confirm the strong predictive value of 3-month and 6-month BCR–ABLIS levels in imatinib-resistant patients.

Abstract 4186: An integrative genomic pipeline to target the NCOR1 cistrome with precision

Abstract NCOR1 is a co-regulator of multiple transcription factors (TFs) and is mutated in major cancers. We mined publically available data-sets to reveal the diversity, function and druggability of the NCOR1 cistrome. From ENCODE, in K562 chronic myelogenous leukemia cells, NCOR1 ChIP-Seq data were integrated with H3K9me3 (a functional consequence), CpG methylation levels and open and closed chromatin states in a matrix of 45 interactions that were combined with RNA-Seq data. Boot-strapping approaches then revealed significantly affected gene networks. Surprisingly, only the combination of NCOR1, H3K9me3 and an intermediate level of CpG methylation, in open chromatin, significantly associated with a network of 212 repressed genes (lo_gene). By contrast, 15 different NCOR1 combinatorial binding patterns associated with significantly elevated gene expression. For example, NCOR1 and low CpG methylation associated with a network of 908 significantly elevated genes (hi_gene). A simple linear regression model was built on the correlation of NCOR1 and 11 different histone modifications with RNA-seq values, across 81 bins spanning gene loci, to reveal the optimal bin predictor of gene expression. NCOR1 binding at the TSS significantly and positively correlated with gene expression, and mirrored the patterns of the activating histone mark H3K4me3. Transcription factor and GO enrichment in the lo_gene and hi_gene networks revealed these significant associations with different TFs and GO terms. In the lo_genes network the most significantly associated TF was regulatory factor X (RFX1), (FDR=1e-4), whereas in the hi_gene network it was Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2), (FDR=2.7e-23). GO analyses revealed these cohorts were most associated with skeletal development (FDR, 2e-3) and transcription regulation (FDR 1.8e-2) terms respectively, underscoring their diverse functions. Mining the NCI60 database, combined with bootstrap approaches, identified that the NCOR1 hi_gene (upregulated) network was most significantly enriched for genes associated with sensitivity towards HDAC inhibitors, including vorinostat, compared to other drug classes including RTKi. Finally, mining a GEO microarray data set of 59 CML patients revealed that significantly up-regulated genes in Imatinib resistant patients were significantly enriched for targets associated with vorinostat sensitivity in the NCI60. Together this integration pipeline in R, from ENCODE, GEO and NCI60, forms a highly flexible pipeline that can be applied to any DNA binding protein or epigenetic state as a prelude to drug redeployment and/or precision medicine approaches. Specifically, these data suggest that cancer related functions of NCOR1 are surprisingly most associated with sustained gene expression, and these targets, both in CML cell lines and patients, associate with sensitivity to clinically approved HDAC inhibitors. Citation Format: Mark D. Long, Patrick van den Berg, Moray J. Campbell, Prashant K. Singh, Sebastiano Battaglia. An integrative genomic pipeline to target the NCOR1 cistrome with precision. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4186. doi:10.1158/1538-7445.AM2014-4186

Bcr-Abl Kinase Domain Mutations and Other Mutations on the Response to Nilotinib in Bcr Abl –Positive Chronic Myeloid Leukemia - a Study from Eastern India

ABSTRACT Aim: The emergence of ABL point mutations is the most frequent cause for imatinib resistance in CML patients and can occur during any phase of the disease. The treatment of CML has changed with the introduction of tyrosine kinase inhibitors (TKI).The aim of the study is to investigate two potential resistance mechanisms i.e., mutations of the BCR-ABL tyrosine kinase domain (TKI) and additional chromosomal abnormalities (ACA), during 1st and 2nd generation TKI treatment in CML. Methods: Between September 2002 and February 2012, 66 patients with Ph-positive CML and imatinib resistance or intolerance were treated with nilotinib and were analyzed. Karotyping and BCR-ABL TKD mutation screening are performed in 66 imatinib-resistant CML patients who were on imatinib at the time of loss of hematologic response (HR), cytogenetic (CyR)or molecular response (MR).Imatinib-resistance mutation analysis (Qualitative) were detected by nested RT-PCR and Sanger sequencing. Out of 66 patients, 34 received escalated imatinib, 32 received nilotinib. Results: In 66 BCR-ABL-positive imatinib-resistant patients,11 different BCR-ABL TKD mutations were detected. Lack of HR was noted in 20 patients where CyR was not evaluated. Imatinib resistance mutation analysis was promoted by no HR (n = 12); loss of HR after achieving CyR (n = 27); failure to achieve CyR milestones (n = 19); loss of CyR (n = 6); development of extra medullary BC (n = 2),The analysis revealed no mutations in 33 patients, M351T in10 patients, G250E in 7 patients, F317L in 7 patients, M244V in 4 patients, E355G in 3 patient, and T315I in 2 patients. The seven novel mutations (F317L, G250E, M244V, M351T, F359V, T315I, E355G) were seen in our patient population. T315I was found in 2/66 patients who demonstrate loss of response. Conclusions: Compared with other western studies the incidence of the T315I mutation is very low in our study population. For patients with TKI resistance, mutation and ACA screening may play a role in identifying patients with poorer prognosis.If nilotinib-resistant mutation was detected, we preferred dasatinib .We plan for bosutinib, panotinib omacetaxine (SC route) in third line therapy in nilotinib-resistant, different mutation-positive CML. Disclosure: All authors have declared no conflicts of interest.

HS-438, a new inhibitor of Imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia

Imatinib is a selective breakpoint cluster region-Abelson (BCR-ABL) tyrosine kinase inhibitor (TKI) that has significantly improved the prognosis of patients with chronic myeloid leukemia (CML). However, T315I gene mutations of the BCR-ABL kinase domain have been shown to confer resistance to imatinib. In the present study, we synthesized a novel BCR-ABL inhibitor, HS-438, and identified its anti-leukemic effects in vitro and in vivo. We found that HS-438 strongly inhibited the expression of BCR-ABL signaling pathways in wild-type BCR-ABL (BaF3/WT) cells as well as T315I-mutated BCR-ABL (BaF3/T315I) cells with resistance to imatinib. HS-438 induced cell cycle arrest, particularly during the G0/G1 cell cycle phase, and induced apoptosis. In BaF3/T315I xenograft models, HS-438 significantly delayed tumor growth, unlike imatinib. In summary, we suggest that HS-438 may be a novel drug candidate with the therapeutic potential to target BCR-ABL and overcome imatinib resistance in patients with CML.

ApoptomiRs expression modulated by BCR–ABL is linked to CML progression and imatinib resistance

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the presence of Philadelphia chromosome (Ph) leading to expression of a BCR-ABL1 fusion oncogene. The BCR-ABL protein has a constitutive tyrosine kinase activity which is responsible for CML pathogenesis by promoting cell apoptosis resistance; however, the cellular and molecular mechanisms associated with BCR-ABL expression and apoptosis impairment in CML leukemic cells have not been fully elucidated. This study evaluated apoptomiRs and their predicted apoptotic genes in BCR-ABL(+) cells from patients in different phases of CML treated with tyrosine kinase inhibitor (TKI) according to their imatinib (IM) response by qPCR. Phosphotyrosine and c-ABL expressions in HL-60.BCR-ABL cells treated with TKI were done by Western blot. We found that dasatinib (DAS) modulated miR-let-7d, miR-let-7e, miR-15a, miR-16, miR-21, miR-130a and miR-142-3p expressions while IM modulated miR-15a and miR-130a levels. miR-16, miR-130a and miR-145 expressions were modulated by nilotinib (NIL). We observed higher miR-15a, miR-130b and miR-145; and lower miR-16, miR-26a and miR-146a expressions in CML-CP in comparison with controls. CML-AP patients showed low miR-let-7d, miR-15a, miR-16, miR-29c, miR-142-3p, miR-145, and miR-146a levels in comparison with CML-CP. We noted that the miR-26a, miR-29c, miR-130b and miR-146a expressions were downregulated in IM resistant patients in comparison with IM responsive patients. This study showed the modulation of apoptomiRs by BCR-ABL kinase activity and the deregulation of apoptomiRs and their predicted apoptotic target genes in different CML phases and after treatment with TK inhibitors. ApoptomiRs may be involved in the BCR-ABL(+) cell apoptosis regulation.

Co-operating STAT5 and AKT signaling pathways in chronic myeloid leukemia and mastocytosis: possible new targets of therapy

Chronic myeloid leukemia and systemic mastocytosis are myeloid neoplasms sharing a number of pathogenetic and clinical features. In both conditions, an aberrantly activated oncoprotein with tyrosine kinase activity, namely BCR-ABL1 in chronic myeloid leukemia, and mutant KIT, mostly KIT D816V, in systemic mastocytosis, is key to disease evolution. The appreciation of the role of such tyrosine kinases in these diseases has led to the development of improved therapies with tyrosine kinase-targeted inhibitors. However, most drugs, including new KIT D816V-blocking agents, have failed to achieve long-lasting remissions in advanced systemic mastocytosis, and there is a similar problem in chronic myeloid leukemia, where imatinib-resistant patients sometimes fail to achieve remission, even with second- or third-line BCR-ABL1 specific tyrosine kinase inhibitors. During disease progression, additional signaling pathways become activated in neoplastic cells, but most converge into major downstream networks. Among these, the AKT and STAT5 pathways appear most critical and may result in drug-resistant chronic myeloid leukemia and systemic mastocytosis. Inhibition of phosphorylation of these targets has proven their crucial role in disease-evolution in both malignancies. Together, these observations suggest that STAT5 and AKT are key drivers of oncogenesis in drug-resistant forms of the diseases, and that targeting STAT5 and AKT might be an interesting approach in these malignancies. The present article provides an overview of our current knowledge about the critical role of AKT and STAT5 in the pathophysiology of chronic myeloid leukemia and systemic mastocytosis and on their potential value as therapeutic targets in these neoplasms.

Cellular Uptake of Imatinib into Leukemic Cells Is Independent of Human Organic Cation Transporter 1 (OCT1)

In addition to mutated BCR-ABL1 kinase, the organic cation transporter 1 (OCT1, encoded by SLC22A1) has been considered to contribute to imatinib resistance in patients with chronic myeloid leukemia (CML). As data are conflicting as to whether OCT1 transports imatinib and may serve as a clinical biomarker, we used a combination of different approaches including animal experiments to elucidate comprehensively the impact of OCT1 on cellular imatinib uptake. Transport of imatinib was studied using OCT1-expressing Xenopus oocytes, mammalian cell lines (HEK293, MDCK, V79) stably expressing OCT1, human leukemic cells, and Oct1-knockout mice. OCT1 mRNA and protein expression were analyzed in leukemic cells from patients with imatinib-naïve CML as well as in cell lines. Transport and inhibition studies showed that overexpression of functional OCT1 protein in Xenopus oocytes or mammalian cell lines did not lead to an increased cellular accumulation of imatinib. The CML cell lines (K562, Meg-01, LAMA84) and leukemic cells from patients expressed neither OCT1 mRNA nor protein as demonstrated by immunoblotting and immunofluorescence microscopy, yet they showed a considerable imatinib uptake. Oct1 deficiency in mice had no influence on plasma and hepatic imatinib concentrations. These data clearly demonstrate that cellular uptake of imatinib is independent of OCT1, and therefore OCT1 is apparently not a valid biomarker for imatinib resistance.

BCR-ABL kinase domain mutations, including 2 novel mutations in imatinib resistant Malaysian chronic myeloid leukemia patients—Frequency and clinical outcome

Discovery of imatinib mesylate (IM) as the targeted BCR-ABL protein tyrosine kinase inhibitor (TKI) has resulted in its use as the frontline therapy for chronic myeloid leukemia (CML) across the world. Although high response rates are observed in CML patients who receive IM treatment, a significant number of patients develop resistance to IM. Resistance to IM in patients has been associated with a heterogeneous array of mechanisms of which point mutations within the ABL tyrosine kinase domain (TKD) are the frequently documented. The types and frequencies of mutations reported in different population studies have shown wide variability. We screened 125 Malaysian CML patients on IM therapy who showed either TKI refractory or resistance to IM to investigate the frequency and pattern of BCR-ABL kinase domain mutations among Malaysian CML patients undergoing IM therapy and to determine the clinical significance. Mutational screening using denaturing high performance liquid chromatography (dHPLC) followed by DNA sequencing was performed on 125 IM resistant Malaysian CML patients. Mutations were detected in 28 patients (22.4%). Fifteen different types of mutations (T315I, E255K, G250E, M351T, F359C, G251E, Y253H, V289F, E355G, N368S, L387M, H369R, A397P, E355A, D276G), including 2 novel mutations were identified, with T315I as the predominant type of mutation. The data generated from clinical and molecular parameters studied were correlated with the survival of CML patients. Patients with Y253H, M351T and E355G TKD mutations showed poorer prognosis compared to those without mutation. Interestingly, when the prognostic impact of the observed mutations was compared inter-individually, E355G and Y253H mutations were associated with more adverse prognosis and shorter survival (P=0.025 and 0.005 respectively) than T315I mutation. Results suggest that apart from those mutations occurring in the three crucial regions (catalytic domain, P-loop and activation-loop), other rare mutations also may have high impact in the development of resistance and adverse prognosis. Presence of mutations in different regions of BCR-ABL TKD leads to different levels of resistance and early detection of emerging mutant clones may help in decision making for alternative treatment. Serial monitoring of BCR-ABL1 transcripts in CML patients allows appropriate selection of CML patients for BCR-ABL1 KD mutation analysis associated with acquired TKI resistance. Identification of these KD mutations is essential in order to direct alternative treatments in such CML patients.

Gambogic Acid Induces Apoptosis in Imatinib-Resistant Chronic Myeloid Leukemia Cells via Inducing Proteasome Inhibition and Caspase-Dependent Bcr-Abl Downregulation

Chronic myelogenous leukemia (CML) is characterized by the constitutive activation of Bcr-Abl tyrosine kinase. Bcr-Abl-T315I is the predominant mutation that causes resistance to imatinib, cytotoxic drugs, and the second-generation tyrosine kinase inhibitors. The emergence of imatinib resistance in patients with CML leads to searching for novel approaches to the treatment of CML. Gambogic acid, a small molecule derived from Chinese herb gamboges, has been approved for phase II clinical trial for cancer therapy by the Chinese Food and Drug Administration (FDA). In this study, we investigated the effect of gambogic acid on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl. CML cell lines (KBM5, KBM5-T315I, and K562), primary cells from patients with CML with clinical resistance to imatinib, and normal monocytes from healthy volunteers were treated with gambogic acid, imatinib, or their combination, followed by measuring the effects on cell growth, apoptosis, and signal pathways. The in vivo antitumor activity of gambogic acid and its combination with imatinib was also assessed with nude xenografts. Gambogic acid induced apoptosis and cell proliferation inhibition in CML cells and inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Our data suggest that GA-induced proteasome inhibition is required for caspase activation in both imatinib-resistant and -sensitive CML cells, and caspase activation is required for gambogic acid-induced Bcr-Abl downregulation and apoptotic cell death. These findings suggest an alternative strategy to overcome imatinib resistance by enhancing Bcr-Abl downregulation with the medicinal compound gambogic acid, which may have great clinical significance in imatinib-resistant cancer therapy.

&amp;lt;italic&amp;gt;MiR-320a&amp;lt;/italic&amp;gt; downregulation is associated with imatinib resistance in gastrointestinal stromal tumors

Gastrointestinal stromal tumor (GIST) is one of the most common mesenchymal tumors of the gastrointestinal tract. Though imatinib improves the outcome, drug resistance remains the major problem for extending patient survival. Genetic mutation of the drug targets is the known mechanism for imatinib resistance. However, it cannot explain all of the phenomena of imatinib resistance, and numerous additional mechanisms have been proposed to account for imatinib resistance in various model systems. In this study, we applied the SYBR-green quantitative polymerase chain reaction-based array approach to screen the differentially expressed miRNAs between primary GIST patients and imatinib-resistant patients. The selected candidate miRNAs were validated in a cohort of 12 GIST patients. We found that low expression of miR-320a was correlated with short time to imatinib resistance, and proposed the potential mechanism of miR-320a for imatinib resistance.

Drug resistance and BCR‐ABL kinase domain mutations in Philadelphia chromosome–positive acute lymphoblastic leukemia from the imatinib to the second‐generation tyrosine kinase inhibitor era: The main changes are in the type of mutations, but not in the frequency of mutation involvement

Patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) frequently relapse on imatinib with acquisition of BCR-ABL kinase domain (KD) mutations. To analyze the changes that second-generation tyrosine kinase inhibitors (TKIs) have brought in mutation frequency and type, a database review was undertaken of the results of all the BCR-ABL KD mutation analyses performed in the authors' laboratory from January 2004 to January 2013. Interrogation of the database retrieved 450 mutation analyses in 272 patients with Ph+ ALL. Prescreening of samples was performed with denaturing high-performance liquid chromatography (D-HPLC), followed by direct sequencing of D-HPLC-positive cases. BCR-ABL KD mutations were detected in 70% of imatinib-resistant patients, with T315I, E255K, and Y253H mutations accounting for 75% of cases. Seventy-eight percent of the patients reported to be resistant to second-generation TKIs after imatinib failure were positive for mutations, and 58% of them had multiple mutations. Analysis of patients relapsing on dasatinib revealed a newly acquired T315I mutation in almost two-thirds of the cases. Direct sequencing detected no mutations at diagnosis, even in patients who relapsed after a few months. Second-generation TKIs ensure a more rapid debulking of the leukemic clone and have much fewer insensitive mutations, but long-term disease control remains a problem, and the T315I mutation is revealed to be an even more frequent enemy. BCR-ABL KD mutation screening of patients with Ph+ ALL who are receiving imatinib or second-generation TKIs would be a precious ally for timely treatment optimization. In contrast, the clinical usefulness of conventional direct sequencing at diagnosis seems to be very low. American Cancer Society.

Protein-coding genes and long noncoding RNAs are differentially expressed in dasatinib-treated chronic myeloid leukemia patients with resistance to imatinib

Dasatinib has demonstrated efficacy in patients with chronic-phase chronic myeloid leukemia (CML) who had resistance or intolerance to imatinib. However, some patients also develop resistance or intolerance to dasatinib. To identify potential molecular pathways involved in primary resistance to dasatinib in CML, we analyzed gene expression profiles of mononuclear cells of 7 imatinib-resistant patients, collected before and after 1-year dasatinib treatment. Large-scale gene expression was measured with Agilent microarrays covering protein-coding genes and long (>200 nt) noncoding RNAs (lncRNAs). Sets of genes and lncRNAs significantly differentially expressed (>1.5 fold-change; q value ≤10%) were identified. Ingenuity Pathway Analysis pointed to a number of functions, canonical pathways and gene networks that were significantly enriched with differentially expressed genes. In addition to protein-coding genes, lncRNAs have been recently implicated in pathways leading to tumorigenesis. Our data point to new possible regulatory elements involved in dasatinib resistance in CML.