Imatinib-resistant Chronic Myeloid Leukemia Research Articles

Dasatinib-induced Chylous Pleural Effusions

Tyrosine kinase inhibitors have revolutionized treatment of chronic myeloid leukemia through their targeted treatment of the BCR-ABL transcript. Imatinib was the first of this class and was found to induce fluid retention and volume overload resulting in pleural effusions. In cases of imatinib-resistant chronic myeloid leukemia, dasatinib provides another treatment option, as a second-generation tyrosine kinase inhibitor, which is 325 times more efficacious in vitro when compared with imatinib. However, a common adverse pulmonary effect is the development of a pleural effusion. Dasatinib-induced toxicity and the development of exudative pleural effusions are dose dependent. Chylous pleural effusions have been described with dasatinib toxicity; however, not all pleural effusions documented with dasatinib are chylous. In our patient, serial chest radiographs show the regression of her effusions with dasatinib discontinuation. Despite the regression of the patient’s effusions, chylomicrons were still present in low concentrations. We suspect the development of chylous pleural effusions from dasatinib exposure is related to lymphatic dysregulation.

Protein Kinase C-βii-Dependent Up-Regulation Of Heme Oxygenase-1 In Chronic Lymphocytic Leukemia Cells Is Crucial For Resistance Against Imatinib

ObjectiveResistance toward imatinib and other BCR/ABL tyrosine kinase inhibitors(TKI) remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia (CML). Heme oxygenase-1 (HO-1) inhibition plus TKI were found to produce growth inhibition in imatinib-resistant K562/IM cells and in Ba/F3 cells harboring the T315I mutant of BCR/ABL. In this study, we aimed to explore the molecular mechanism of HO-1 explosive expression and relationship between HO-1 and Protein Kinase C-βII(PKC-βII) in imatinib-resistant CML cells. MethodsWe explored that mRNA expression of various drug-resistant-relative gene between K562 cells and K562/IM cells via high throughout screening assay detected by real-time PCR. According to the results mentioned above, relative protein expression and phosphorylation examined by western blot. And protein translocation was observed and recorded by fluorescence microscope. Cells apoptosis, reactive oxygen species(ROS) and intracellular calcium were analyzed by flow cytometry. Cells proliferation inhibition was detected by CCK-8 assay. The peripheral blood mononuclear cells(PBMNCs) were purified from 45 patients with freshly diagnosed CML or imatinib-resistant CML(including all kinds of drug-resistance involved in various of reasons). Compared the differences of mRNA expression between HO-1 and PKC-βII in those patients. ResultsOur results indicated that PKC-βII contributed to activation of HO-1 expression through PI3K/AKT pathway. PKC-βII phosphorylation localized to membrane from cytoplasim to activate PI3K/AKT pathway, meanwhile, the 7.8 folder higher (p<0.01) expression of HO-1 following the nucleus translocation of NF-E2–related factor 2(Nrf-2) was observed after pAKT up-regulation. The accumulation of ROS and intracellular calcium overload were reduced by HO-1 to protect cells against cytotoxicity, less apoptotic imatinib-resistant K562 cells were detected at the same concentration of imatinib(the total apoptotic rate of K562/IM:42.4%±3.3% vs. K562: 72.1%±4.1%, at the concentration of 1 μM, p<0.01). In addition, the results of HO-1 and PKC-βII mRNA expression in PBMNCs from 45 patients indicated that HO-1 activation kept positively correspondent with expression of PKC-βII in primary leukemic cells obtained from those patients. In addition, blockage of PI3K/AKT, silence of PKC-βII or HO-1 by small interfere RNA( or inhibited by targeted inhibitor) could significantly increase apoptotic rate of imatinib-resistant CML cells(the apoptotic rate of regulated expression/ natural expression: when blockage of PI3K/AKT: 1.54±0.17; silence of PKC-βII: 2.09±0.23; inhibition of HO-1: 1.87±0.12, p<0.05). ConclusionSelectively targeted-PKC-βII plus imatinib produced growth inhibition in primary leukemic cells. In summary, these data showed that HO-1 expression involved in PKC-βII activation in imatinib-treated CML cells, and targeted- PKC-βII may be the same promising novel strategy as HO-1 inhibition in imatinib resistant CML. Disclosures:No relevant conflicts of interest to declare.

Proliferation Inhibition and Apoptosis Induction Of Imatinib Resistance Chronic Myeloid Leukemia Cells By Down-Regulated PPP2R5C

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder that occurs because of t(9;22)(q34;q11) translocations. The prognosis in CML improved markedly after introduction of abl tyrosine kinase inhibitors (TKI), still a lot of CML patients die due to abl mutation related drug resistance and the blast crisis, moreover, de novo or secondary TKI-resistance is a significant problem in CML. The aim of the study is to down-regulate the PPP2R5C gene expression in imatinib-sensitive or imatinib-resistant chronic myeloid leukemia (CML) cell lines: K562, K562R (imatinib resistance without abl gene mutation), 32D-Bcr-Abl WT (imatinib sensitive, murine CML cell lines with wild type abl gene) and 32D-Bcr-Abl T315I (imatinib resistance, with abl gene T315I mutation) and primary cells from CML patients by RNA interference, thereby inhibit the CML cells proliferation and induce apoptosis. PPP2R5C-siRNAs numbered 799 or 991 were obtained by chemosynthesis. Non-silencing siRNA control (SC)-treated, mock-transfected, untreated cells were used as controls. PPP2R5C expression in mRNA levels from CML cells were analyzed after siRNAs delivered by nucleofection using the real-time quantitative PCR. The PPP2R5C protein levels were analyzed by Western blotting. Cell proliferation in vitro was assayed by the cell count kit-8 method after treatment. The morphology and the percentage of apoptosis were revealed by Hoechst 33258 stain and flow cytometry (FCM). Bone marrow mononuclear cells (BM-MNCs) from healthy individuals were transferred by PPP2R5C-siRNA-991. BFU-E, CFU-Meg and CFU-GM were performed from PPP2R5C-siRNA-991 treated BM-MNCs by methyl cellulose semi-solid culturing method, to estimate the role of differentiation and proliferation in BM-MNCs after PPP2R5C-siRNA transfection. The results showed that both PPP2R5C-siRNA-799 and PPP2R5C-siRNA-991 took best silencing results after nucleofection in all of four cells and primary cells from CML patients. The reduction about 2 to 7 folds in PPP2R5C mRNA level was observed in PPP2R5C-siRNA799 or PPP2R5C-siRNA991 treated cells. And PPP2R5C protein expression inhibition rate reached 38.08%-55.26% at 48 or 72 h after treatment. The proliferation rates of PPP2R5C-siRNA-799 or 991 treated CML cells were significantly decreased at 72 h (P < 0.05). PPP2R5C-siRNA-799 or 991 treated CML cells lines showed a significantly increase in AnnexinV/PI-positive cells (apoptosis) (P < 0.05), similar results in the morphological changes of apoptosis were found by Hoechst 33258 staining test. PPP2R5C gene mRNA expression levels in BM-MNCs from healthy individuals were significantly lower than that in K562 cells (P < 0.05), and the expression level was not significant changed after PPP2R5C-siRNA-991 transfection. The formation of BFU-E, CFU-Meg and CFU-GM from BM-MNCs showed no significant difference between PPP2R5C-siRNA-991 treatment and MOCK control group (P > 0.05). In conclusions, suppression of PPP2R5C by RNA interference could inhibit the proliferation and induce the apoptosis effectively in CML cells either in imatinib sensitive or imatinib resistance cell lines, while no significant effect of PPP2R5C-siRNA on the proliferation and differentiation of BM-MNCs in vitro, suggesting that PPP2R5C-siRNA might specially target on the CML cells. Down-regulating the PPP2R5C gene expression might be considered as a new target therapeutic strategy in CML, especially in imatinib-resistant CML. Disclosures:Li:This work was supported by Grants from National Natural Science Foundation of China (30871091 and 91129720), the Collaborated grant for HK-Macao-TW of Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (2012B0506: Research Funding.

Initial Molecular Response At 3 Months Can Identify Three Groups Of Patients With Different Responses and Event-Free Survival At 36-Months Following Second-Generation Tyrosine Kinase Inhibitor Therapy In Imatinib-Resistant Chronic Myeloid Leukemia

IntroductionIn patients with imatinib-resistant chronic myeloid leukemia (CML) treated with a second generation tyrosine kinase inhibitor (2GTKI), the initial molecular response at 3 months (3m-IMR) has been shown to be predictive of long-term outcomes. However, there is no consensus regarding the best cutoff for the BCR-ABL1 transcript levels, with 1% and 10% being proposed in different studies. Also, additional prognostic factors such as baseline tyrosine kinase (TK) mutations and a prognostic scoring system (PSS) (Jabbour, 2011) have been proposed. In this study, we addressed those issues in an homogeneous group of imatinib-resistant CML patients treated in a single center for a median follow-up of 36 months (14-60). Patients and Methods134 patients with a diagnosis of CML treated either with dasatinib (N=64) or nilotinib (N=70) due to imatinib failure were included. Imatinib failure was defined as any of the following: no complete hematologic response at 3 months, no cytogenetic response at 6 months, no major cytogenetic response at 12 months, no complete cytogenetic response (CCR) at 18 months, or loss of hematologic or cytogenetic response at any time. Patients with the T315I mutation were excluded. Molecular analysis was performed in a properly standardized laboratory every 3 months from the beginning of the 2GTKI, and results are presented according to the international scale. The study's primary endpoint was the 3-year event-free survival (EFS) after 2GTKI. Secondary endpoints were the best response levels attained under 2GTKI (BMR). The factors analysed were the 3m-IMR, age, gender, Sokal and EUTOS scores at diagnosis, failure to achieve a complete cytogenetic response, TK mutations, PSS score and type of 2GTKI. A multivariate analysis with the factors significant at P<0.2 in the univariate analysis for EFS was also performed. ResultsThere were 74 males and 60 females, with a median age of 46 years (4-79). The Sokal score was available in 93 patients: it was low in 25 (27%), intermediate in 24 (26%) and high in 44 patients (47%). The EUTOS score was low in 61 and high in 32 patients. The best cytogenetic response to imatinib was complete in 32 patients, major in 18, minor in 12, and minimal or no response in 72 patients. The 62 patients who attained at least a minor cytogenetic response lost it before starting the 2GTKI. TK mutations were present in 33 patients (105 tested). The PSS score was low-risk in 59 (44%), intermediate in 61 (46%) and high-risk in 14 (10%) patients. The 3m-IMR was < 1% in 59 (44%) patients, 1% to <10% in 35 (26%) patients and ≥ 10% in 40 patients (30%). During the study period there were 16 deaths and 60 events. The adverse prognostic factors for the 3-year EFS in the univariate analysis were: 3m-IMR response 1% to <10% (HR of 6.15, <0.001), 3m-IMR ≥ 10% (HR 16.98 , P<0.001), failure to achieve a CCR (HR 4.44, P=0.001), presence of TK mutations (HR 1.76, P=0.044) and a high PSS score (HR 3.00, P<0.001). In the multivariate analysis, only the 3m-IMR remained predictive for EFS (HR 7.6 for 1% to<10% and HR 20 for ≥ 10%). Finally, the 3m-IMR was associated with the best response to 2GTKI: CCR of 100%, 49% and 15% (P<0.001); major molecular response of 88%, 31% and 10% (P<0.001), and molecular response 4.0 of 57.6%, 11.4% and 7.5% (P<0.001) for BCR-ABL1 transcript levels < 1%, 1% to < 10% and > 10%, respectively. ConclusionsBCR-ABL1 transcript levels at 3 months identified a good-risk group (BCR-ABL1< 1%) with 3y-EFS of 87%, an intermediate-risk group (BCR-ABL1 1% to <10%) with 3y-EFS of 47% and a poor-risk group (BCR-ABL1 ≥ 10%) with 3y-EFS of 11% (P<0.001) (figure1). The present results confirm, in an independent series, that the BCR-ABL1 transcript level at 3 months is the most relevant surrogate for response and long-term outcome following 2GTKI after imatinib-failure in CML patients. [Display omitted] Disclosures:Boquimpani:BMS: Consultancy; Novartis: Consultancy, Research Funding. Lobo:Novartis: Research Funding. Spector:Novartis: Honoraria, Research Funding.

The E3 Ubiquitin Ligases Siah2 Contributes To Imatinib Resistance In Chronic Myeloid Leukemia In Hypoxia Condition

Background and ObjectiveSevere hypoxia has been shown to favor the self-renewal of human hematopoietic stem cells. Recent studies demonstrate that hypoxia via hypoxia-inducible factor (Hif-1a) can modify the proliferation and differentiation of CML stem cells. The ubiquitin E3 ligase SIAH2 is an important regulator of the hypoxic response as it leads to the ubiquitin/proteasomal degradation of prolyl hydroxylases such as PHD3, which in turn increases the stability of Hif-1a. The Hif-1a has been linked to chemosensitivity while the underlying molecular mechanism remains elusive. Therefore, we comprehensively analysed SIAH2 and Hif-1a role in determining chemosensitivity via signal molecule vascular epithelial growth factor (VEGF) pathway. MethodsWe tested the level of Siah2, Hif-1a and VEGF in Imatinb-sensitive CML Patients (n=15) and insensitive CML patients(n=10) by real-time reverse transcription PCR and western blot analysis. K562-wild type (K562-W) and K562-imatinib-resistance type (K562-R) cell were cultured for 24h and 48h under the condition of normal and hypoxia concentration of oxygen (1%). Knockdown of SAIH2 by RNA interference ,Cell viability and IC50 under 1% concentration oxygen were tested by cck-8;detected cell cycle and apoptosis by flow cytometry (FCM) ; analyzed the expression levels of Siah2, Hif-1a and VEGF respectively by real-time PCR and western blot. ResultsThe level of mRNA and protein of SIAH2 ,Hif-1a and VEGF were significantly higher in IM- resistant CML patients compared to IM sensitive CML patients, respectively (P<0.05). The similar results were observed in K562-R and K562-W cells(P<0.01). Under 21%,1% oxygen concentration cultured for 24h ,the IC50 of K562-W and K562-R cells was no significant difference (P<0.05),but there was significant difference after cultured for 48h Cell cycle analysis showed that more G0/G1 cells in K562-R than K562-W after cultured for 48h under hypoxia condition(P<0.05). After being cultured in 1% oxygen concentration for 48 hours, we confirmed SIAH2, Hif-1a and VEGF were up-regulated in both cell lines, moreover, it was more obvious in K562-R cells. In SIAH2-sh K562-R cells, the apoptosis was higher than K562-R cells obviously under 1% oxygen concentration for 48 hours(P<0.05);the level of Hif-1a Hardly monitored, and the level of VEGF was also lower. ConclusionsThere were higher level of SIAH2, Hif-1a and VEGF in IM-resistant CML patients. Under hypoxia condition, K562 cells were likely to improve their resistance to Imatinib .After we Knockdown SAIH2, K562-R Cell apoptosis rate increased significantly, along with low level Hif-1a and VEGF. It indicated that in hypoxia micro-environment, Siah-2 might be one of the critical molecules that induce Imatinib-resistance in CML in the way of maintaining leukemic cell survival and stimulating them into quiescence phase. Disclosures:No relevant conflicts of interest to declare.

The Prospective Analysis Of Clinical Efficacy and Adverse Events In Chronic Myeloid Leukemia In Chronic Phase (CML-CP) Patients With Imatinib Resistance Or Intolerance, Evaluated By European Leukemia Net (ELN) 2013 Criteria

BackgroundDasatinib is a highly potent BCR-ABL inhibitor, with a 325-fold higher potency than imatinib and a 16-fold higher potency than nilotinib in vitro. Across a series of phase II and III trials with more than 2 years of follow-up, dasatinib has demonstrated durable efficacy in patients with CML following resistance or intolerance to imatinib. We conducted a phase II study to evaluate the efficacy and safety of dasatinib in Japanese patients with imatinib resistance or intolerance CML. MethodsFifty- five CML-CP patients from 2009 to 2011 with resistance (n=40) or intolerance (n=26) to imatinib were registered to dasatinib administration (100mg once daily). Eleven among 26 patients with intolerance also showed resistance to imatinib at the registration. Imatinib resistance was defined as a lack of partial cytogenetical response at 3 months, a lack of complete cytogenetic response at 6 months or a lack of a major molecular response at 12 months of imatinib treatment. This criteria was identical to the criteria in ELN 2013 recommendation for first line. In another words, the resistance in this study means non-optimal criteria (warning and failure) of definition of the response to TKIs, first line, in ELN 2013 recommendation. Efficacy and safety were assessed using rates of major molecular response (MMR)/ MR4.5 (either (i) detectable disease with<0.0032% BCR-ABL1 IS or (ii) undetectable disease in cDNA with >32,000 ABL1 transcripts in the same volume of cDNA used to test for BCR-ABL1) at 12 months and drug-related adverse events (AEs) respectively. All analyses were based on the modified to intension-to treat. ResultsOne patient was withdrawal before dasatinib administration. The median duration of imatinib therapy was 545 days. The overall incidence of MMR (primary endopoint) and MR4.5 at 12 months was 67.2% (95% confidence interval, 53.3-81.1%), and 20.2 % (95% CI, 8.4–32.0 %), respectively. Forty patients with resistance to imatinib, who were warning and failure patients judged by ELN 2013 criteria, were reassessed. Cumulative MMR and MR4.5 rate were 63.4% (95% CI: 46.3-80.6) and 18.0% (95% CI: 4.9-31.1) respectively at 12 months. Eleven patients, who showed more than 1% IS at 3 months of dasatinib treatment, did not reach to MMR at 12 months or discontinued dasatinib due to insufficient efficacy in this resistance cohort. However, progression to the accelerated or blastic phase had not been observed. When imatinib were changed to dasatinib, 5 patients showed the mutations, which were effective in dasatinib therapy. New mutations have not been observed during the treatment of dasatinib.Among 54 patients, most non-hematological AEs were in grade 1/2. including diarrhea (12.7%), rash (7.3%), myalgia (5.5%) and vomiting (3.6%). Grade 3/4 non-hematological AEs were infrequent, including decreased potassium (3.6%), and increased creatinine (3.6%). Grade 1/2 fluid retention AEs were shown in 9.1% of patients, including edema (3.6%). Pleural effusion, which was only in Grade 1/2, was shown in 32.7% of patients. Grade 3/4 hematological toxicities included anemia (7.3%) and thrombocytopenia (3.6 %). Only 3 patients have permanently discontinued dasatinib treatment due to AEs. ConclusionsThe patients with non-optimal responses (warning and failure) judged by ELN 2013 criteria should have early intervention to dasatinib, which is less toxicity in CML-CP patients. This intervention might induce good prognosis. The BCR-ABL1 IS less than 1% at 3 months of dasatinib administration will be the valuable landmark for outcome. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Imatinib-induced apoptosis could be enhanced by Triptolide in Imatinib-resistant chronic myeloid leukemia cell line through down-regulation of HIF-1αand Nrf2

BackgroundThe emergence of Imatinib has brought a new era for the treatment of chronic myeloid leukemia. However, resistant to Imatinib might lead to the treatment failure and death of patients. Thus, finding a drug which could enhance the Imatinib-induced apoptosis in vitro could provide the experimental base for the treatment of chronic myeloid leukemia. K562 cell line is a common cell line used in the study of chronic myeloid leukemia while K562/G cell line which is resistant to Imatinib is derived from K562 cell line. AimThis study aims to explore whether low-dose Triptolide(TPL) could enhance the Imatinib-induced apoptosis in K562/G cells and related mechanism. MethodsK562/G cells were subjected to different treatments and thereafter MTT assay, flow cytometry and Western blot or RT-PCR were used to determine IC50, apoptotic status and expression of Nrf2, HIF-1α and their target genes. ResultsTriptolide is highly cytotoxic to K562/G cells in a concentration-dependent manner. To determine the combination effect of TPL and anticancer agents, K562/G cells were exposed to Imatinib(50μM) with or without TPL (25nM) for 24h. The apoptotic cells were determined by PI/Annexin V staining and flow cytometric analysis. Apoptotic ratio of cells treated by Imatinib together with TPL is significantly increased compared to cells treated by Imatinib alone (24.78±1.12 vs. 77.52±7.75, P<0.01). Next, the underlying mechanism was investigated. High expression of Hypoxia-inducible factor-1α (HIF-1α) has been shown to be related with bad clinical outcome in patients while Nrf2 is responsible for chemo-resistanct in cancer cells. Imatinib in combination with TPL could decrease Nrf2 and HIF-1α expression at protein and mRNA levels. Down-stream genes of Nrf2 e.g NQO1, GSR and HO-1 as well as target genes of HIF-1α e.g BNIP3, VEGF and CAIX are also down-regulated at mRNA level. ConclusionTPL could significantly increase the Imatinib-induced apoptotic ratio in K562/G cells through down-regulation of HIF-1α and Nrf2. Disclosures:No relevant conflicts of interest to declare.

A T315I Gate Keeper Mutation Responding To Pegylated Interferon Alfa-2a (Peg-IFN) Monotherapy With Major Molecular Response (MMR4) At 12 Months In An Imatinib-Resistant Chronic Myeloid Leukemia (CML) Patient

Since the IRIS Study in 2000, tyrosine kinase inhibitors (TKI) have become a standard therapy for CML patients in lieu of interferon based therapy. Nevertheless, resistance occurs in roughly 30% of imatinib-treated patients. This resistance is attributable to ABL kinase domain mutations in 50% of cases, in which T315I mutation accounts for 10 to 15%. This latter is resistant to all TKIs available except for ponatinib which enables 75% of complete cytogenetic response (CCyR) in this setting (NEJM November 29, 2012). Before Ponatinib, various strategies were proposed to overcome the T315I mutation including more importantly ‘non-targeted' therapy such as Omacetaxine. IFN is also a ‘non-targeted therapy' known to have a well-established immunological effect. There are also clues about its cellular effects: microarray analyses have shown that IFN alfa can upregulate genes that encode for apoptotic proteins (i.e TRAIL, Fas, caspases 4 and 8 and XAF-1…) conferring the anti-growth effect different from ATP competition specific to TKIs.We report here an 80 years-old imatinib-resistant CML patient harboring the T315I mutation who has been successfully treated with Peg-IFN (Pegasys).His past medical history involved hypertension, asthma and chronic obstructive pulmonary disease. He was diagnosed in July 2008 with a chronic phase CML when he presented with weight loss, splenomegaly, leukocytosis (WBC 80G/l), slight anemia (Hemoglobin 112G/l) and normal Platelet count 240G/L. Karyotyping of 26 metaphase cells in the bone marrow showed Philadelphia (Ph1) chromosome without any additional abnormality. The molecular analysis by real time PCR (Light Cycler 480) detected a BCR-ABL p210 transcript. The Sokal score was high (at 1.24) while the Eutos score was low (31).He started Imatinib 400mg daily in July 2008 and achieved 5% IS BCR-ABL ratio at 3 months and complete cytogenetic response (CCyR) at 12 months. But the patient had never reached the major molecular response (MMR) and ended up losing the cytogenetic response in February 2012 (at 40 months) with an increase of 1.5log in the BCR-ABL ratio. Sequencing analysis for ABL mutation was carried out using the applied biosystems 3500 gentic analyzer. It revealed the T315I mutation at 20%. After a request for Ponatinib was dismissed by our health authorities, we decided to treat the patient with Peg-IFN in June 2012 at the dose of 90 µg per week. At this point, the imatinib had been stopped for 3 months, 8 metaphase cells out of 20 showed Ph1 chromosome without additional abnormality, BCR-ABL ratio was 3% IS, the mutational analysis showed 50% of T315I and 20% of another acquired E255K mutation. But the patient was still in complete haematologic response. figure 1 highlights the decline in the BCR-ABL ratio with MMR reached for the first time at 9 months (from Peg-IFN treatment). Of note, the CCyR was obtained again at this landmark. At 12 months from the Peg-IFN the CCyR was confirmed, the BCR-ABL ratio came very close to MMR4 (0.02% IS) while both T315I and E255K mutations disappeared on mutational analysis. [Display omitted] No side effect was reported except for weight loss when the dose of Peg-IFN was increased to 180 µg per week which was the dose suggested by J.H. Lipton (Leukemia and Lymphoma, March 2007). The average dose in the past 12 months was 90 µg per week. The latter dose is well tolerated and is still ongoing.This observation shows that, though we are in the ponatinib era, there still is a room for interferon, in the management of CP CML patients even in BCR-ABL domain mutated patients because of its different mechanism of action. In this case the Pegylated Interferon a-2-a at the dose of 90µg per week has allowed clearance of both T315I and E255K mutations with a response close to MMR4. Taking into account the slope of the BCR-ABL decline, deeper molecular response is expected before the ASH meeting in December 2013. Disclosures:No relevant conflicts of interest to declare.

Simultaneous manifestation of pleural effusion and acute renal failure associated with dasatinib: a case report

Dasatinib is a novel second-generation inhibitor of multiple tyrosine kinases, indicated for the treatment for Philadelphia chromosome-positive (Ph+) chronic myeloid leukaemia (CML), acute lymphoblastic leukaemia (ALL) and lymphoid blast CML with resistance or intolerance to prior therapy. Although dasatinib is a potent, efficacious and generally well-tolerated drug, patients are also subject to various adverse effects. The most common pulmonary-related side effect is pleural effusion (PE). Renal failure has been reported rarely as a side effect of dasatinib treatment. We report the first case of a patient with imatinib-resistant CML who developed PE and acute renal failure (ARF) simultaneously, after being placed on dasatinib therapy. We report a 58-year-old female dasatinib-treated patient with Ph+ chronic phase CML who was admitted to our hospital due to persisted dyspnoea and fever. After reviewing the laboratory and clinical findings, we determined our patient as having simultaneously ARF and PE related to dasatinib therapy. Dasatinib was discontinued, and after 10days of treatment with ampicillin-sulbactam, allopurinol, amlodipine, furosemide and methylprednisolone, she was discharged home effusion free and with ameliorated renal function. PE is the most common extra-haematological toxicity observed during dasatinib treatment whose pathogenesis is still unclear. A possible role of cytokines, such as platelet-derived growth factor receptor (PDGFR)-β and vascular endothelial growth factor (VEGF), in causing endothelial permeability has been suggested. The aetiology of renal failure is also unclear in these patients, but two different possible mechanisms have been suggested such as tumour lysis syndrome and toxic tubular damage. In conclusion, here we describe the first case of simultaneous manifestation of PE and ARF associated with dasatinib. Thus, in patients treated with tyrosine kinase inhibitors, especially those with predisposing nephrological or haematological factors, serum creatinine levels should be monitored routinely.

Phosphorylated Crkl reduction levels are associated with the lowest P-glycoprotein activity levels in cells from chronic myeloid leukemia patients

ABCB1/P-glycoprotein (Pgp) and ABCG2/BCRP overexpression have been described as related to imatinib resistance in chronic myeloid leukemia (CML). We showed in CML cells from 55 patients that Pgp activity was more frequently detected than BCRP activity (p=0.0074). Imatinib-induced Crkl phosphorylated protein (pCrkl) reduction was more pronounced in K562 (Pgp-negative) than in K562-Lucena (Pgp-positive) CML cell line. Expressive pCrkl reduction levels after in vitro imatinib treatment was observed in samples from patients exhibiting lower Pgp activity levels compared with patients exhibiting higher Pgp activity levels (p=0.0045). Pgp activity in association with pCrkl reduction levels might help to distinguish between imatinib-resistant and imatinib-sensitive CML cells.

Proliferation inhibition and apoptosis induction of imatinib-resistant chronic myeloid leukemia cells via PPP2R5C down-regulation

Despite the success of imatinib and other tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable, and a number of CML patients die due to Abl mutation-related drug resistance and blast crisis. The aim of this study was to evaluate proliferation inhibition and apoptosis induction by down-regulating PPP2R5C gene expression in the imatinib-sensitive and imatinib-resistant CML cell lines K562, K562R (imatinib resistant without an Abl gene mutation), 32D-Bcr-Abl WT (imatinib-sensitive murine CML cell line with a wild type Abl gene) and 32D-Bcr-Abl T315I (imatinib resistant with a T315I Abl gene mutation) and primary cells from CML patients by RNA interference. PPP2R5C siRNAs numbered 799 and 991 were obtained by chemosynthesis. Non-silencing siRNA scrambled control (SC)-treated, mock-transfected, and untreated cells were used as controls. The PPP2R5C mRNA and protein expression levels in treated CML cells were analyzed by quantitative real-time PCR and Western blotting, and in vitro cell proliferation was assayed with the cell counting kit-8 method. The morphology and percentage of apoptosis were revealed by Hoechst 33258 staining and flow cytometry (FCM). The results demonstrated that both siRNAs had the best silencing results after nucleofection in all four cell lines and primary cells. A reduction in PPP2R5C mRNA and protein levels was observed in the treated cells. The proliferation rate of the PPP2R5C-siRNA-treated CML cell lines was significantly decreased at 72 h, and apoptosis was significantly increased. Significantly higher proliferation inhibition and apoptosis induction were found in K562R cells treated with PPP2R5C-siRNA799 than K562 cells. In conclusion, the suppression of PPP2R5C by RNA interference could inhibit proliferation and effectively induce apoptosis in CML cells that were either imatinib sensitive or resistant. Down-regulating PPP2R5C gene expression might be considered as a new therapeutic target strategy for CML, particularly for imatinib-resistant CML.

Aberrant signalling by protein kinase CK2 in imatinib-resistant chronic myeloid leukaemia cells: Biochemical evidence and therapeutic perspectives

Chronic myeloid leukaemia (CML) is driven by the fusion protein Bcr-Abl, a constitutively active tyrosine kinase playing a crucial role in initiation and maintenance of CML phenotype. Despite the great efficacy of the Bcr-Abl-specific inhibitor imatinib, resistance to this drug is recognized as a major problem in CML treatment. We found that in LAMA84 cells, characterized by imatinib-resistance caused by BCR-ABL1 gene amplification, the pro-survival protein kinase CK2 is up-regulated as compared to the sensitive cells. CK2 exhibits a higher protein-level and a parallel enhancement of catalytic activity. Consistently, CK2-catalysed phosphorylation of Akt-Ser129 is increased. CK2 co-localizes with Bcr-Abl in the cytoplasmic fraction as judged by subcellular fractionation and fluorescence immunolocalization. CK2 and Bcr-Abl are members of the same multi-protein complex(es) in imatinib-resistant cells as demonstrated by co-immunoprecipitation and co-sedimentation in glycerol gradients. Cell treatment with CX-4945, a CK2 inhibitor currently in clinical trials, counteracts CK2/Bcr-Abl interaction and causes cell death by apoptosis. Interestingly, combination of CX-4945 with imatinib displays a synergistic effect in reducing cell viability. Consistently, knockdown of CK2α expression by siRNA restores the sensitivity of resistant LAMA84 cells to low imatinib concentrations. Remarkably, the CK2/Bcr-Abl interaction and the sensitization towards imatinib obtained by CK2-inhibition in LAMA84 is observable also in other imatinib-resistant CML cell lines.These results demonstrate that CK2 contributes to strengthen the imatinib-resistance phenotype of CML cells conferring survival advantage against imatinib. We suggest that CK2 inhibition might be a promising tool for combined strategies in CML therapy.

Identification of nilotinib-altered microRNA expression patterns in imatinib-resistant chronic myeloid leukemia cells

Drug resistance is a key contributor for treatment failure in hematologic and other malignancies. Nilotinib has been observed with significant activity in patients with chronic phase chronic myeloid leukemia (CML) who have failed or are intolerant of imatinib therapy. Recent reports have suggested that microRNA (miRNA) expression changes are involved in the drug response of human cancer. Several miRNAs have been previously found to be consistently deregulated in imatinib resistance; however, very limited information is available for nilotinib-treated patients who have failed imatinib therapy. Many reports have discovered circulating miRNAs as noninvasive biomarkers of disease and therapy response. The aim of this study was to explore miRNA regulation and find candidate miRNA markers for CML that can be used for drug response prediction. Here we demonstrate the circulating miRNA profile in the culture supernatant of imatinib-resistant K562 CML cells (K562-R) by microarray chip analysis. We have identified specific miRNAs that are associated with nilotinib sensitivity by comparison of miRNA expression patterns from the culture supernatant of nilotinib-treated K562-R cells with the culture supernatant of untreated K562-R cells. The information obtained from this study may have the potential to become a novel biomarker to predict drug response in the future and can also be applied to developing novel therapeutic treatment for CML.

Endoplasmic reticulum stress-mediated apoptosis in imatinib-resistant leukemic K562-r cells triggered by AMN107 combined with arsenic trioxide

The first tyrosine kinase inhibitor (TKI) imatinib mesylate (imatinib) targets the kinase domain of BCR-ABL and induces apoptosis in newly diagnosed chronic myeloid leukaemia (CML). However, resistant and relapse are common problems in imatinib-treated patients. Although second-generation TKI such as AMN107 appears to improve the treatment of CML, TKI resistance and relapse are also frequently occurred in the patients. To test whether arsenic trioxide (ATO) could potentiate the efficacy of AMN107 in imatinib-resistant cells, we conducted a series of assays in TKI-resistant K562-r cells treated with AMN107 and ATO. Based on a time-course cDNA microarray analysis, we found many genes typically involved in the endoplasmic reticulum (ER) stress signalling were significantly up-regulated, implicating the occurrence of ER stress-mediated apoptosis in K562-r cells treated with the combination of ATO and AMN107. Such implication was also supported by the data showing the activation of members in the JNK pathway, which are known to be characteristic markers bridging ER-stress and apoptosis. Partial knock-down of the JNK activities alleviated the effects of apoptosis (p < 0.05) triggered by combining AMN107 with ATO. In conclusion, this study for the first time demonstrates a synergistic effect of AMN107 with ATO, allowing insights into the possible mechanisms underlying imatinib-induced resistance in CML. Our data also suggest that combination of AMN107 with ATO may represent a new strategy for the treatment of imatinib-resistant CML patients.

Down-regulation of miR-181c in imatinib-resistant chronic myeloid leukemia

The association of microRNA alterations with progression and treatment outcome has been revealed in different types of cancers. To find miRNAs involved in imatinib response we performed miRNA microarray followed by RT-qPCR verification of 9 available diagnostic bone marrow core biopsies from 9 CML patients including 4 imatinib-resistant and 5 imatinib-responder patients. Only one differentially expressed miRNA, miR-181c, was found when the imatinib-resistant group was compared with imatinib-responders. Significant down-regulation of miR-181c in imatinib-resistant versus imatinib-responders was confirmed by qRT-PCR. Some miR-181c target genes such as PBX3, HSP90B1, NMT2 and RAD21 have been associated with drug response.

The roles of epigenetic modifications of proapoptotic BID and BIM genes in imatinib-resistant chronic myeloid leukemia cells

In chronic myeloid leukemia (CML), epigenetic modifications such as promoter hypermethylation and inactive histone modification are known mechanisms of drug resistance. In our study, we investigated the roles of promoter hypermethylation of BIM and BID genes and H3K27me3 histone modification on imatinib resistance.We detected higher expression levels of BIM and BID genes and lower expression levels of EZH2, EED2, SIRT1, and SUZ12 genes in imatinib-resistant K562/IMA-3 cells compared to imatinib-non-resistant K562 cells. While we determined the EZH2 and DNMT enzymes as bounded to the promoter of the BIM gene, we did not detect hypermethylation of this promoter. We also found the H3K27me3 histone modification promoter of BIM and BID genes in both cell lines. In conclusion, our results support the notion that DNA promoter methylation may be formed independently from EZH2-H3K27me3 and pro-apoptotic BIM and BID genes are not methyllated in the imatinib resistance of CML cells.

Phase I study of NRC-an-019, a tyrosine kinase inhibitor, in imatinib-resistant chronic myeloid leukemia (CML) in an Indian tertiary care hospital.

7080 Background: NRC-AN-019 is an orally administered tyrosine kinase inhibitor (TKI) of the Bcr-Abl protein-tyrosine kinase. The drug was given to patients as a ready-to-use liquid formulation. Methods: The primary objectives of the Phase-I study were to estimate the Dose Limiting Toxicity (DLT) and Maximum Tolerated Dose (MTD) of NRC-AN-019 and also to establish the safe dose for Phase II clinical trial. The secondary objectives were to study the pharmacokinetic properties and antileukemic activity of NRC-AN-019. Philadelphia positive CML patients in all phases, in the age group of 18-65 years were eligible. They had to be resistant or intolerant to Imatinib, with ECOG ≤ 2. Dosing was initiated at 50 mg/day and dose escalation was done in increments of 50 mg per each additional cohort. The maximum dose administered was 450 mg/day. The protocol-specific study duration was 30 days; patients continued to receive the study drug subsequently based on Investigator’s decision. Results: A total of 30 patients were enrolled (3 at 50 mg, 4 at 100 mg, 3 at 150 mg, 3 at 200 mg, 4 at 250 mg, 4 at 300 mg, 3 at 350 mg, 3 at 400 mg, and 3 at 450 mg). DLT was not observed and MTD was not reached. The recommended Phase II-A dose is 300 mg/day, with a scope for escalation up to 450 mg/day. The maximum plasma concentration of NRC-AN-019 was 9,342 ng/mL and AUC was 1,262,191 ng.hr/mL. Common adverse events were skin rash, nausea, and vomiting. Hematological response was observed in 11 patients, cytogenetic response in 7 patients and molecular response in 5 patients. The maximum duration a patient has been on NRC-AN-019 is 863 days. Conclusions: Based on the Phase-I data, NRC-AN-019 showed considerable safety and response; it could be a potential treatment option for imatinib-resistant CML. Clinical trial information: CTRI/2009/091/000204.

Study of different mutations in chronic myeloid leukemia in India and their co-relation with drug resistance.

7083 Background: Emergence of ABL point mutations is the most frequent cause for imatinib resistance in CML. Aim of our study is to investigate two potential resistance mechanisms i.e.,mutations of BCR-ABL tyrosine kinase domain (TKD) and Additional Chromosomal Abnormalities during TKI treatment in CML. Methods: Karyotyping and BCR-ABL TKD mutation screening are performed in 100 imatinib resistant CML patients who were on imatinib at the time of loss of hematologic response, cytogenetic or molecular response. Imatinib–Resistance Mutation Analysis (Qualitative) were detected by Nested RTPCR and Sanger’s Sequencing. In 100 cases, 34 received escalated imatinib, 34 nilotinib and another 32 dasatinib. Results: In 100 BCR-ABL positive imatinib, nilotinib and dasatinib resistant cases, 11 different BCR-ABL TKD mutations were detected. Analysis revealed no mutations-43 cases, M351T-12 cases, G250E-10 cases, F317L-8 cases, M244V-5 cases, E255K-4 cases, V379I-4 cases, F359V-3 cases, H396R-3 cases, Y253F-3 cases, E355G-3 cases, T315I-2 cases. 11 novel mutations (F317L, G250E, M244V, Y253F, E255K, M351T, F359V, H396R, V379I, E355G, T315I) conferring imatinib resistance, 10 nilotinib–resistant mutations (M244V, F359V, T315I, E355G, G250E) and 8 dasatinib-resistant mutations (H396R, F317L, H396R, T315I, M351T) were seen in our patient population. T315I was found more frequently in cases on dasatinib than on imatinib therapy. Conclusions: T315I which confers resistance to all TKIs was detected only in 2/100 patients who demonstrated loss of response in our population. As compared with other western studies, incidence of T315I mutation was very low in our study. In addition analysis of mutation patterns at baseline may help in stratifying patients for treatment. For cases with TKI resistance, mutation and ACA screening may play role in identifying patients with poorer prognosis. In our practice if nilotinib–resistant mutation was detected, dasatinib was preferred and for dasatinib-resistant mutation, nilotinib was preferred. We are planning for using bosutinib, panotinib and omacetaxine (SC route) in third line therapy in imatinib resistant different mutation positive chronic myeloid leukemia.

Evaluation of T315I mutation frequency in chronic myeloid leukemia patients after imatinib resistance

The occurrence of resistance mutations in the Abl kinase domain plays a central role in drug treatment failure in chronic myeloid leukemia (CML) patients. Among them, the T315I mutation at the gatekeeper position affects a common Abl kinase contact residue and confers complete resistance to all known ATP-competitive BCR-ABL inhibitors. In the present study, an allele-specific oligonucleotide reverse transcriptase polymerase chain reaction assay was used to detect T315I mutation in a cohort of 60 imatinib-resistant CML patients. In terms of disease phase, 43 patients (71%) were in late chronic phase, 4 (7%) in accelerated phase, and 13 (22%) in blastic phase. The prevalence of the T315I mutation was found to be 7% (4/60). All four patients with mutation were in advance phases and had previously lost all their responses. The results of the study confirmed that this method is low cost and easy tool to operate for T315I mutation screening and direct sequencing should be performed in positive cases for confirmation.

Abstract 996: Receptor tyrosine kinase AXL is upregulated in metformin resistant prostate cancer cells.

Abstract Metformin is a biguanine derivative, widely used as an antidiabetic drug. Epidemiological studies on several types of cancers including breast, prostate, ovarian and pancreatic cancer show that patients treated with metformin have a lower incidence of cancer. Mechanistic studies have shown that metformin inhibits cancer cell viability by activating the AMP activated protein kinase (AMPK) pathway and inhibiting mTOR. In this study we developed a metformin resistance LnCap prostate cancer cells by exposing the cells continuously increasing concentrations of metformin. Micro-array data analysis comparing the resistant cells (4-fold) to parental cells showed that genes associated with receptor binding and with cell proliferation are primarily affected. EDIL3 (EGF-like repeats and discoidin I-like domains 3), EREG (eEpiregulin) and AXL (Axl receptor tyrosine kinase) are top three up regulated genes (∼ 35 fold). CALB2 (cCalbindin 2) and TPTE (transmembrane phosphatase with tensin homology) were the top down regulated genes (10 fold down regulated). AXL overexpression has earlier been demonstrated in Imatinib resistant CML cell lines and in CML cells from patients. We are currently investigating the role of Axl receptor tyrosine kinase in metformin resistant prostate cancer cells. Citation Format: Nitu Bansal, Prasun Mishra, Kathleen Cruz, Joseph Bertino. Receptor tyrosine kinase AXL is upregulated in metformin resistant prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 996. doi:10.1158/1538-7445.AM2013-996