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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5158-5158
Author(s):  
Qi Shen ◽  
Sichu Liu ◽  
Yu Chen ◽  
Lijian Yang ◽  
Shaohua Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Primary Cells ◽  
Healthy Individuals ◽  
Hoechst 33258 ◽  
Imatinib Resistance ◽  
Imatinib Resistant

Abstract 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.

scholarly journals Impact of Baseline BCR-ABL Mutations on Response to Nilotinib in Patients With Chronic Myeloid Leukemia in Chronic Phase

2009 ◽  
Vol 27 (25) ◽  
pp. 4204-4210 ◽  
Author(s):  
Timothy Hughes ◽  
Giuseppe Saglio ◽  
Susan Branford ◽  
Simona Soverini ◽  
Dong-Wook Kim ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Phase Ii ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Cytogenetic Response ◽  
Molecular Response ◽  
Imatinib Resistance ◽  
Mutational Status ◽  
Imatinib Resistant

Purpose Nilotinib is a second-generation tyrosine kinase inhibitor indicated for the treatment of patients with chronic myeloid leukemia (CML) in chronic phase (CP; CML-CP) and accelerated phase (AP; CML-AP) who are resistant to or intolerant of prior imatinib therapy. In this subanalysis of a phase II study of nilotinib in patients with imatinib-resistant or imatinib-intolerant CML-CP, the occurrence and impact of baseline and newly detectable BCR-ABL mutations were assessed. Patients and Methods Baseline mutation data were assessed in 281 (88%) of 321 patients with CML-CP in the phase II nilotinib registration trial. Results Among imatinib-resistant patients, the frequency of mutations at baseline was 55%. After 12 months of therapy, major cytogenetic response (MCyR) was achieved in 60%, complete cytogenetic response (CCyR) in 40%, and major molecular response (MMR) in 29% of patients without baseline mutations versus 49% (P = .145), 32% (P = .285), and 22% (P = .366), respectively, of patients with mutations. Responses in patients who harbored mutations with high in vitro sensitivity to nilotinib (50% inhibitory concentration [IC50] ≤ 150 nM) or mutations with unknown nilotinib sensitivity were equivalent to those responses for patients without mutations (not significant). Patients with mutations that were less sensitive to nilotinib in vitro (IC50 > 150 nM; Y253H, E255V/K, F359V/C) had less favorable responses, as 13%, 43%, and 9% of patients with each of these mutations, respectively, achieved MCyR; none achieved CCyR. Conclusion For most patients with imatinib resistance and with mutations, nilotinib offers a substantial probability of response. However, mutational status at baseline may influence response. Less sensitive mutations that occurred at three residues defined in this study, as well as the T315I mutation, may be associated with less favorable responses to nilotinib.

scholarly journals Overexpression of miR-202 resensitizes imatinib resistant chronic myeloid leukemia cells through targetting Hexokinase 2

2018 ◽  
Vol 38 (3) ◽  
Author(s):  
Yingjun Deng ◽  
Xin Li ◽  
Jinxin Feng ◽  
Xiangliang Zhang
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Specific Inhibitor ◽  
Imatinib Resistance ◽  
Hexokinase 2 ◽  
Imatinib Resistant ◽  
First Choice ◽  
Long Term Treatment

Chronic myeloid leukemia (CML) is a myeloproliferative disease which uniquely expresses a constitutively active tyrosine kinase, BCR/ABL. As a specific inhibitor of the BCR-ABL tyrosine kinase, imatinib becomes the first choice for the treatment of CML due to its high efficacy and low toxicity. However, the development of imatinib resistance limits the long-term treatment benefits of it in CML patients. In the present study, we aimed to investigate the roles of miR-202 in the regulation of imatinib sensitivity in CML cell lines and the possible mechanisms involved in this process. We found miR-202 was down-regulated in seven CML cell lines by quantitative reverse-transcription PCR (qRT-PCR) analysis. Overexpression of miR-202 significantly suppressed proliferation rates of CML cells. By establishing imatinib resistant cell lines originating from K562 and KU812 cells, we observed expressions of miR-202 were down-regulated by imatinib treatments and imatinib resistant CML cell lines exhibited lower level of miR-202. On the contrary, imatinib resistant CML cell lines displayed up-regulated glycolysis rate than sensitive cells with the evidence that glucose uptake, lactate production, and key glycolysis enzymes were elevated in imatinib resistant cells. Importantly, the imatinib resistant CML cell lines were more sensitive to glucose starvation and glycolysis inhibitors. In addition, we identified Hexokinase 2 (HK2) as a direct target of miR-202 in CML cell lines. Overexpression of miR-202 sensitized imatinib resistant CML through the miR-202-mediated glycolysis inhibition by targetting HK2. Finally, we provided the clinical relevance that miR-202 was down-regulated in CML patients and patients with lower miR-202 expression displayed higher HK2 expression. The present study will provide new aspects on the miRNA-modulated tyrosine kinase inhibitor (TKI) sensitivity in CML, contributing to the development of new therapeutic anticancer drugs.

scholarly journals Dasatinib treatment of chronic-phase chronic myeloid leukemia: analysis of responses according to preexisting BCR-ABL mutations

Blood ◽  
2009 ◽  
Vol 114 (24) ◽  
pp. 4944-4953 ◽  
Author(s):  
Martin C. Müller ◽  
Jorge E. Cortes ◽  
Dong-Wook Kim ◽  
Brian J. Druker ◽  
Philipp Erben ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Progression Free Survival ◽  
Response Rates ◽  
Imatinib Resistance ◽  
Loop Region ◽  
Imatinib Resistant ◽  
Dasatinib Treatment

Abstract Dasatinib is a BCR-ABL inhibitor with 325-fold higher potency than imatinib against unmutated BCR-ABL in vitro. Imatinib failure is commonly caused by BCR-ABL mutations. Here, dasatinib efficacy was analyzed in patients recruited to phase 2/3 trials with chronic-phase chronic myeloid leukemia with or without BCR-ABL mutations after prior imatinib. Among 1043 patients, 39% had a preexisting BCR-ABL mutation, including 48% of 805 patients with imatinib resistance or suboptimal response. Sixty-threedifferent BCR-ABL mutations affecting 49 amino acids were detected at baseline, with G250, M351, M244, and F359 most frequently affected. After 2 years of follow-up, dasatinib treatment of imatinib-resistant patients with or without a mutation resulted in notable response rates (complete cytogenetic response: 43% vs 47%) and durable progression-free survival (70% vs 80%). High response rates were achieved with different mutations except T315I, including highly imatinib-resistant mutations in the P-loop region. Impaired responses were observed with some mutations with a dasatinib median inhibitory concentration (IC50) greater than 3nM; among patients with mutations with lower or unknown IC50, efficacy was comparable with those with no mutation. Overall, dasatinib has durable efficacy in patients with or without BCR-ABL mutations. All trials were registered at http://www.clinicaltrials.gov as NCT00123474, NCT00101660, and NCT00103844.

Preclinical Investigations Demonstrate Effectiveness of Aplidin in Acute Leukemias and Lymphomas and Provide the Basis for Further Clinical Studies.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4496-4496
Author(s):  
Debabrata Banerjee ◽  
Guray Saydam ◽  
Lata G. Menon ◽  
Giuseppe S.A. Longo ◽  
Daniel Medina ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Phase Ii ◽  
Myeloid Leukemia ◽  
Xenograft Model ◽  
Vascular Endothelial ◽  
Endothelial Growth Factor ◽  
Leukemias And Lymphomas

Abstract Aplidin (dehydrodidemnin B, C57H89N7O15) (APLD) is a novel antitumor agent isolated from the Mediterranean tunicate (seasquirt) Aplidium albicans. APLD has shown impressive in vitro and in vivo activity against different human cancer cells and has recently entered Phase II clinical trials in a variety of solid tumors following promising toxicity and pharmacological properties seen in Phase I studies. Fatigue and muscular pain were the most prevalent toxicities at 5 mg/m2 iv 3 h every other week or 3.4 mg/m2/wk with little or no bone marrow toxicity. APLD inhibits protein synthesis via GTP-dependent elongation factors 1-alpha and ornithine decarboxylase (ODC) activity, induces rapid p53-independent apoptosis in vitro, cell cycle perturbation and alteration of gene expression at early times after treatment. APLD inhibits vascular endothelial growth factor (VEGF) secretion and vascular endothelial growth factor-receptor 1 (VEGF-R1/flt-1), preventing autocrine stimulation in the human lymphoid leukemic cell line MOLT-4 cells and in AML blasts. APLD is a potent inhibitor of human myeloid leukemia cell lines (K-562, HEL and HL60), as well as fresh blast cells obtained from patients with both ALL and AML and is more potent than Idarubicin. Cytototoxic doses effective against multiple myeloma cells and fresh pediatric and adult ALL/AML blasts are achievable in plasma and are well below the recommended dose, thus a positive therapeutic index is anticipated. Moreover, the lack of cross resistance with conventional agents against fresh pediatric and adult AML/ALL blasts except fludarabine and Gemcitabine makes APLD an attractive therapeutic choice. Characterization of gene expression profile is currently underway in an attempt to generate a molecular fingerprint of sensitivity/resistance to APLD that will be validated in phase II clinical studies. Based on in vitro antileukemic effect of APLD as well as early results of clinical trials, a systematic study of drug combinations with Aplidin (APLD), for use possible in hematologic malignancies was undertaken. Three cell lines viz. K562 (acute myeloid leukemia), CCRF-CEM (acute lymphocytic leukemia), and SKI-DLCL (diffuse large cell lymphoma) were used for combination studies. Cytarabine and mitoxantrone were found to be synergistic in combination with APLD in all 3 cell lines as assessed by the Chou-Talalay combination index analysis. Since cytarabine and APLD produced impressive synergistic cell kill in all three cell culture models, the combination was further tested in the CCRF-CEM ALL xenograft model in SCID mice. APLD (0.7 mg/Kg) potentiated the antitumoral effect of cytarabine (50mg/Kg) in vivo. Addition of APLD to cytarabine treatment in xenograft model resulted in greater than 50% reduction in tumor size as compared to the untreated group. T/C ratios indicated that the effect of the combination was maximal at day 5 but was still maintained on day 8 (T/C on day 3 = 0.614; day 5= 0.403 and day 8= 0.703). The preclinical results with APLD in leukemias and lymphomas, as a single agent and in combination with cytarabine provide the basis for implementation of a phase II program in resistant relapsed leukemias and lymphomas.

Dasatinib (BMS-354825) Overcomes Multiple Mechanisms of Imatinib Resistance in Chronic Myeloid Leukemia (CML).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1994-1994 ◽  
Author(s):  
Francis Y. Lee ◽  
Mei-Li Wen ◽  
Rajeev Bhide ◽  
Amy Camuso ◽  
Stephen Castenada ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitor ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Src Family Kinases ◽  
Imatinib Resistance ◽  
Over Expression ◽  
Imatinib Resistant

Abstract Resistance to imatinib is a growing concern in CML, particularly in advanced disease. The most common cause of resistance is mutations in BCR-ABL, but other mechanisms have also been identified, including over-expression of BCR-ABL, activation of SRC family kinases and the P-glycoprotein (PGP) efflux pump (via MDR1 over-expression). Dasatinib (BMS-354825) is a novel, oral, multi-targeted tyrosine kinase inhibitor that targets BCR-ABL and SRC kinases. Dasatinib has 325-fold greater potency versus imatinib in cell lines transduced with wild-type BCR-ABL and is active against 18 out of 19 BCR-ABL mutations tested that confer imatinib resistance (Shah et al, Science305:399, 2004; O’Hare et al, Cancer Res65:4500–5, 2005), and preliminary results from a Phase I study show that it is well tolerated and has significant activity in imatinib-resistant patients in all phases of CML (Sawyers et al, J Clin Oncol23:565s, 2005; Talpaz et al, J Clin Oncol23:564s, 2005). We assessed the ability of dasatinib to overcome a variety of mechanisms of imatinib resistance. First, the leukemic-cell killing activity of dasatinib was tested in vitro in three human imatinib-resistant CML cell lines (K562/IM, MEG-01/IM and SUP-B15/IM). Based on IC50 values, dasatinib had >1000-fold more potent leukemic-cell killing activity compared with imatinib versus all three cell lines. Furthermore, in mice bearing K562/IM xenografts, dasatinib was curative at doses >5 mg/kg, while imatinib had little or no impact at doses as high as 150 mg/kg, its maximum tolerated dose. We determined that the MEG-01/IM and SUP-B15/IM cell lines carried BCR-ABL mutations known to confer imatinib resistance to imatinib clinically (Q252H and F359V, respectively). In K562/IM cells, BCR-ABL mutations or BCR-ABL over-expression were not detected, but the SRC family member FYN was over-expressed. PP2, a known inhibitor of SRC family kinases but not BCR-ABL, could reverse the imatinib resistance in these cells. Together, these data suggest that activation of FYN may be a cause of imatinib resistance in K562/IM. Based on cell proliferation IC50, we found that the anti-leukemic activity of dasatinib in K562/IM cells was 29-fold more potent compared with AMN107 (a tyrosine kinase inhibitor that inhibits BCR-ABL but not SRC family kinases). Given that the human serum protein binding of dasatinib, imatinib and AMN107 were 93, 92 and >99% respectively, the difference in potency between dasatinib and AMN107 in vivo may be far greater than the simple fold-difference in the in vitro IC50 values. Finally, in K562 cells over-expressing PGP (K562/ADM), we found that dasatinib was only 6-fold less active than in parental K562 cells. Because of the extreme potency of dasatinib in K562 cells, this reduced potency still afforded an IC50 of 3 nM, which is readily achievable in vivo. Indeed, in mice bearing K562/ADM xenografts, dasatinib was curative at 30 mg/kg, with significant anti-leukemic activity at 15 mg/kg. In conclusion, the rational design of dasatinib as a multi-targeted kinase inhibitor allows this agent to overcome a variety of mechanisms of resistance to imatinib in CML, including mechanisms that are not overcome by agents with a narrower spectrum of inhibition, such as AMN107. Dasatinib is currently in Phase II evaluation in imatinib-resistant/-intolerant patients in the ‘START’ program, and in Phase I evaluation in solid tumors.

In Vitro and In Vivo Monitoring of Valproic Acid Effects on Gene Expression Signatures in Adult Acute Myeloid Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2605-2605
Author(s):  
Lars Bullinger ◽  
Konstanze Dohner ◽  
Richard F. Schlenk ◽  
Frank G. Rucker ◽  
Jonathan R. Pollack ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Serum Levels ◽  
Leukemia Cell Lines ◽  
Acute Myeloid ◽  
Myeloid Leukemia Cell

Abstract Inhibitors of histone deacetylases (HDACIs) like valproic acid (VPA) display activity in murine leukemia models, and induce tumor-selective cytoxicity against blasts from patients with acute myeloid leukemia (AML). However, despite of the existing knowledge of the potential function of HDACIs, there remain many unsolved questions especially regarding the factors that determine whether a cancer cell undergoes cell cycle arrest, differentiation, or death in response to HDACIs. Furthermore, there is still limited data on HDACIs effects in vivo, as well as HDACIs function in combination with standard induction chemotherapy, as most studies evaluated HDACIs as single agent in vitro. Thus, our first goal was to determine a VPA response signature in different myeloid leukemia cell lines in vitro, followed by an in vivo analysis of VPA effects in blasts from adult de novo AML patients entered within two randomized multicenter treatment trials of the German-Austrian AML Study Group. To define an VPA in vitro “response signature” we profiled gene expression in myeloid leukemia cell lines (HL-60, NB-4, HEL-1, CMK and K-562) following 48 hours of VPA treatment by using DNA Microarray technology. In accordance with previous studies in vitro VPA treatment of myeloid cell lines induced the expression of the cyclin-dependent kinase inhibitors CDKN1A and CDKN2D coding for p21 and p19, respectively. Supervised analyses revealed many genes known to be associated with a G1 arrest. In all cell lines except for CMK we examined an up-regulation of TNFSF10 coding for TRAIL, as well as differential regulation of other genes involved in apoptosis. Furthermore, gene set enrichment analyses showed a significant down-regulation of genes involved in DNA metabolism and DNA repair. Next, we evaluated the VPA effects on gene expression in AML samples collected within the AMLSG 07-04 trial for younger (age<60yrs) and within the AMLSG 06-04 trial for older adults (age>60yrs), in which patients are randomized to receive standard induction chemotherapy (idarubicine, cytarabine, and etoposide = ICE) with or without concomitant VPA. We profiled gene expression in diagnostic AML blasts and following 48 hours of treatment with ICE or ICE/VPA. First results from our ongoing analysis of in vivo VPA treated samples are in accordance with our cell line experiments as e.g. we also see an induction of CDKN1A expression. However, the picture observed is less homogenous as concomitant administration of ICE, as well as other factors, like e.g. VPA serum levels, might substantially influence the in vivo VPA response. Nevertheless, our data are likely to provide new insights into the VPA effect in vivo, and this study may proof to be useful to predict AML patients likely to benefit from VPA treatment. To achieve this goal, we are currently analyzing additional samples, and we are planning to correlate gene expression findings with histone acetylation status, VPA serum levels, cytogenetic, and molecular genetic data.

Deregulated Apoptotic Pathways Point to Effectiveness of IAP Inhibitor Therapy in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1275-1275
Author(s):  
Sonja C Lück ◽  
Annika C Russ ◽  
Konstanze Döhner ◽  
Ursula Botzenhardt ◽  
Domagoj Vucic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Gene Set Enrichment Analysis ◽  
Core Binding Factor ◽  
Binding Factor ◽  
Acute Myeloid

Abstract Abstract 1275 Poster Board I-297 Core binding factor (CBF) leukemias, characterized by translocations t(8;21) or inv(16)/t(16;16) targeting the core binding factor, constitute acute myeloid leukemia (AML) subgroups with favorable prognosis. However, 40-50% of patients relapse, and the current classification system does not fully reflect the heterogeneity existing within the cytogenetic subgroups. Therefore, illuminating the biological mechanisms underlying these differences is important for an optimization of therapy. Previously, gene expression profiling (GEP) revealed two distinct CBF leukemia subgroups displaying significant outcome differences (Bullinger et al., Blood 2007). In order to further characterize these GEP defined CBF subgroups, we again used gene expression profiles to identify cell line models similar to the respective CBF cohorts. Treatment of these cell lines with cytarabine (araC) revealed a differential response to the drug as expected based on the expression patterns reflecting the CBF subgroups. In accordance, the cell lines resembling the inferior outcome CBF cohort (ME-1, MONO-MAC-1, OCI-AML2) were less sensitive to araC than those modeling the good prognostic subgroup (Kasumi-1, HEL, MV4-11). A previous gene set enrichment analysis had identified the pathways Caspase cascade in apoptosis and Role of mitochondria in apoptotic signaling among the most significant differentially regulated BioCarta pathways distinguishing the two CBF leukemia subgroups. Thus, we concluded that those pathways might be interesting targets for specific intervention, as deregulated apoptosis underlying the distinct subgroups should also result in a subgroup specific sensitivity to apoptotic stimuli. Therefore, we treated our model cell lines with the Smac mimetic BV6, which antagonizes inhibitor of apoptosis (IAP) proteins that are differentially expressed among our CBF cohorts. In general, sensitivity to BV6 treatment was higher in the cell lines corresponding to the subgroup with good outcome. Time-course experiments with the CBF leukemia cell line Kasumi-1 suggested a role for caspases in this response. Interestingly, combination treatment of araC and BV6 in Kasumi-1 showed a synergistic effect of these drugs, with the underlying mechanisms being currently further investigated. Based on the promising sensitivity to BV6 treatment in some cell lines, we next treated mononuclear cells (mostly leukemic blasts) derived from newly diagnosed AML patients with BV6 in vitro to evaluate BV6 potency in primary leukemia samples. Interestingly, in vitro BV6 treatment also discriminated AML cases into two distinct populations. Most patient samples were sensitive to BV6 monotherapy, but about one-third of cases were resistant even at higher BV6 dosage. GEP of BV6 sensitive patients (at 24h following either BV6 or DMSO treatment) provided insights into BV6-induced pathway alterations in the primary AML patient samples, which included apoptosis-related pathways. In contrast to the BV6 sensitive patients, GEP analyses of BV6 resistant cases revealed no differential regulation of apoptosis-related pathways in this cohort. These results provide evidence that targeting deregulated apoptosis pathways by Smac mimetics might represent a promising new therapeutic approach in AML and that GEP might be used to predict response to therapy, thereby enabling novel individual risk-adapted therapeutic approaches. Disclosures Vucic: Genentech, Inc.: Employment. Deshayes:Genentech, Inc.: Employment.

Targeting Isoprenylcysteine Carboxyl Methyltransferase to Overcome Resistance and Improve Responses in Chronic Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3273-3273
Author(s):  
Wen Tian Sun ◽  
Bee Ling Ng ◽  
Li Yi Yang ◽  
Mei Wang ◽  
William Hwang ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Cell Lines ◽  
Rho Gtpases ◽  
Myeloid Leukemia ◽  
Small Gtpases ◽  
Synergistic Activity ◽  
Carboxyl Methyltransferase ◽  
Imatinib Resistant ◽  
Cd34 Cells ◽  
Isoprenylcysteine Carboxyl Methyltransferase

Abstract Abstract 3273 Poster Board III-1 A strategy to overcome resistance of chronic myeloid leukemia (CML) to Bcr-Abl tyrosine kinase inhibitors (TKI) is by targeting alternative pathways. Increasing evidence suggests that activation of the Ras family of small GTPases is important in leukaemogenic transformation by Bcr-Abl. Ras and Rho GTPases play important roles in the initiation and progression of cancer by disrupting the normal regulation of specific intracellular signalling pathways, promoting cell cycle progression and resistance to apoptosis. The biological function of Ras and Rho GTPases is, to a large extent, dependent on prenylation, a three-step post-translational modification process. The first step is catalysed by farnesyl transferase or geranylgeranyl transferase and the final step is catalysed by isoprenylcysteine carboxyl methyltransferase (Icmt). We have found inhibition of Icmt by a novel selective inhibitor, cysmethynil, enhanced the anti-proliferative effect of imatinib in CML cell lines. The combination of cysmethynil and imatinib was synergistic in inducing apoptosis in the imatinib-sensitive K562 and Bafp210 CML cell lines and apoptosis was further increased when the drugs were given sequentially, with cysmethynil added after imatinib. This combination was also effective in the imatinib-resistant Bcr-Abl- and P-glycoprotein-overexpressing LAMA84 cell line. The synergy was also observed when cysmethynil was combined with dasatinib in both the imatinib-sensitive Bafp210 and imatinib-resistant Bafp210 Y253F mutant but not in the Bafp210 T315I mutant cell lines. The combination therapy was more effective than single agent in inducing apoptosis in treatment-naive primary CML CD34+ cells and was not toxic to cord blood CD34+ cells (Figure 1). Figure 1 Effect of imatinib and cysmethynil on apoptosis in CD34+ cells. Abbreviations: IM + Cys, imatinib and cysmethynil added concurrently; IM −> Cys, cysmethynil added 9 hours after imatinib; Cys −> IM, imatinib added 9 hours after cysmethynil Figure 1. Effect of imatinib and cysmethynil on apoptosis in CD34+ cells. Abbreviations: IM + Cys, imatinib and cysmethynil added concurrently; IM −> Cys, cysmethynil added 9 hours after imatinib; Cys −> IM, imatinib added 9 hours after cysmethynil Imatinib decreases p-CrkL through its inhibition of Bcr-Abl kinase. Interestingly, in both K562 and Bafp210, cysmethynil also decreases p-CrkL and when combined with imatinib, p-CrkL levels are reduced further (Figure 2). CrkL activation is dependent on a multimeric protein complex containing PI-3 kinase, docking protein 2, CrkL, Vav and the Rho GTPase, Rac. We hypothesise that inhibition of CrkL phosphorylation in the CML cell lines by cysmethynil is through the inhibition of Rac prenylation. This may be responsible for the synergistic activity observed when cysmethynil is combined with imatinib. In conclusion, our preliminary results suggest that the combination of a Bcr-Abl TKI and an Icmt inhibitor exerts a selective apoptotic effect in CML cells and this combination should be explored further as a useful tool to overcome resistance and improve responses in CML. Figure 2 Effect of imatinib and cysmethynil on p-CrkL in Bafp210. Figure 2. Effect of imatinib and cysmethynil on p-CrkL in Bafp210. Disclosures: Druker: MolecularMD: Equity Ownership; Novartis Pharmaceuticals: ; Bristol-Myers Squibb:.

In Vitro Sensitivity to Tyrosine Kinase Inhibitors Is One of Possible Predictive Parameters for Therapy Switch In Patients with Chronic Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2747-2747
Author(s):  
Marketa Zackova ◽  
Tereza Lopotova ◽  
Zuzana Ondrackova ◽  
Hana Klamova ◽  
Jana Moravcova
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Transcript Level ◽  
Molecular Response ◽  
Newly Diagnosed ◽  
Therapy Switch

Abstract Abstract 2747 Backround: Tyrosine kinase inhibitors (TKI) are very effective in chronic myeloid leukemia (CML) suppression, however, the problem with development of resistance in some patients exists. It is necessary to find optimal methods for therapy response prediction and for detection of resistance. Many studies on the resistance to imatinib therapy were performed on cell lines or model systems. However, these systems are not fully consistent with CML situation in vivo. Sensitivity to imatinib and its predictivity to molecular response in patients with de novo CML were tested in vitro on patients′ leukocytes by White et al. [Blood 2005; 106: 2520]. They found that IC50 values could be predictive mainly in patients with low Sokal score. Aims: To optimize in vitro method for evaluation of patients′ sensitivity to various TKIs and to test its predictivity for molecular response in therapy and/or after therapy change. Methods: The sensitivity to TKIs: imatinib, nilotinib and dasatinib were studied on leukocytes isolated from CML patients at diagnosis and various responses to treatment. Cell lines were used as controls. Isolated leukocytes/cell lines were cultivated with/without TKIs. Optimization of cultivation was performed on cell lines (ML-2, K562, CML-T2, JURL-MK1) and on leukocytes from CML newly diagnosed patients (15) and healthy donors (6). Various incubation times (4, 24, 48 and 72h) were tested. Concentrations of TKI were used in values near to physiological levels: 2 –3 concentrations for each inhibitor (1uM, 10uM imatinib, 0,5uM and 2uM nilotinib and 1nM, 10nM and 100nM dasatinib). In given time-points the cells were harvested and lysed for protein and mRNA analyses. Sensitivity to TKIs was tested by BCR-ABL kinase inhibition – via Crkl phosphorylation (western blots) and also by WT1 transcript level kinetics [Cilloni et al, Cancer 2004; 101: 979]. Quality of cultivation was tested by apoptosis level (RNA degradation, Annexin staining – Agilent Bioanalyzer 2100). Results: We found 48 h to be the optimal time for in vitro cultivation. This time was long enough to see TKIs dependent changes on protein as well as mRNA level. At this time the intensity of apoptosis was relatively low and did not influence results. The predictive ability of cultivation with TKIs was tested on patients at diagnosis (15), with optimal (5) and suboptimal response (5) and patient with therapy failure (13). The disease state of all patients was further monitored in range from 6 to 21 months (median 12 months) after cultivation. Mostly all of newly diagnosed patients were in vitro sensitive to all three TKIs, 10 of them achieved MMR (median 7 months, range 5 – 16) on imatinib. In patients with resistance to imanitib therapy the good sensitivity to one of 2nd generation TKI on in vitro tests represented the good response to this inhibitor, 4 patients from 10 on dasatinib achieved MMR (within 4 months), the other responded to therapy with continual decrease of BCR-ABL transcript level. Thus, the cultivation test can help with the therapy switch. However, the prognosis of patients with additive chromosomal aberration was poor even if they were sensitive to TKIs in vitro. Only one of 3 patients with 8 trisomy sensitive to dasatinib in vitro achieved MMR at 4th month after starting of dasatinib. Two patients with T315I were not sensitive to any of TKIs in vitro and in vivo, as it was expected. We continue to follow up of all patients. In conclusion, the results from in vitro cultivations of patients′ leukocytes with TKIs can help with the choice of efficient inhibitor for individual patient′s therapy, however, it is necessary to take into consideration the results of cytogenetic analyses of patients and other factors influencing CML. Supported by MZOUHKT2005. Disclosures: No relevant conflicts of interest to declare.

Altered Regulation of Imprinted Non-Coding RNA Genes in Acute Myeloid Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3456-3456 ◽  
Author(s):  
Ming-Yu Yang ◽  
Jan-Gowth Chang ◽  
Pai-Mei Lin ◽  
Jui-Feng Hsu ◽  
Cheng-Han Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Large Scale ◽  
Conflicts Of Interest ◽  
Imprinted Genes ◽  
Healthy Individuals ◽  
Regulatory Functions ◽  
Acute Myeloid

Abstract Abstract 3456 Studies in large-scale genome sequencing have shown that only 2% of the mammalian genome encodes mRNAs, but the most part is transcribed as long and short non-coding RNAs (ncRNAs). The ncRNAs with gene regulatory functions are starting to be seen as a common feature of mammalian gene regulation. Genomic imprinting is a form of epigenetic regulation and imprinted genes are silenced in a parental-specific manner. Imprinted genes tend to occur in clusters and ncRNAs have been found at all well-characterized imprinted clusters. Although the exact mechanism how imprinted ncRNA regulates gene expression remains largely unknown, it is general accepted that imprinted ncRNAs binds to chromatin modifying complexes, such as PRC2, TRX, and G9a, and generates specific silencing of genomic loci both in cis and trans. Imprinting is associated with many human diseases or syndromes (e.g. Prader-Willi, Angelman, Beckwith-Wiedemann, Retts, and Silver-Russell syndromes) and various cancers (e.g. breast, prostate, and colorectal cancers), but its role in leukemogenesis remain elusive. In this present study, a panel of 24 human imprinted ncRNAs genes, including ampd3, cpa4, snuf, rasgrf1, slc22a3, lgf2, treb3c, gabrb3, c15orf2, sfmbt2, rtl1, copg2, h19, l3mbtl, ppp1r9a, tspan32, lnpp5f, impact, nr3251, nr3252, znf215, prim2, peg3as and znf264, has been mined using Bioinformatics approach. We investigated the expression of these imprinted ncRNA genes using real-time quantitative RT-PCR in 67 newly-diagnosed acute myeloid leukemia patients with normal karyotypes (AML-NK), 22 AML patients with abnormal karyotypes (AML-AK), and 39 healthy individuals. In AML-NK patients, the expression of lgf2, h19, slc22a3, copg2, and impact were significantly upregulated than in healthy individuals (p < 0.0001). In AML-AK patients, besides lgf2, h19 and impact genes, ampd3 and gabrb3 were also significantly upregulated than in healthy individuals (p < 0.0001). Expression of igf2 was almost undetectable in healthy individuals but drastically increased in all AML patients. Both lgf2 and h19 were significantly increased in both AML-NK and AML-AK patients. From our preliminary results, it is reasonable to hypothesize that loss imprinting of lgf2/h19 is critical for the leukemogenesis of AML and under NK or AK conditions different additional ncRNAs are activated and affect different imprinted gene expression and thus leading to different clinical outcomes. Based on our findings, we will further perform methylation analysis of promoter CpG sites in AML patients to investigate if hypomethylation is responsible for the upregulation of these imprinted ncRNAs. We will also carry out in vitro functional analysis to elucidate the functions and mechanisms of these imprinted ncRNAs in AML tumorigenesis. Updated results of these analysis will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

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