scholarly journals The Gain of the Short Arm of Chromosome 2 (2p+) Induces XPO1 Overexpression and Drug Resistance in Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 492-492 ◽  
Author(s):  
Adrien Cosson ◽  
Elise Chapiro ◽  
Jérome Lambert ◽  
Hong-Anh Cung ◽  
Caroline Algrin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Drug Resistance ◽  
Cell Death ◽  
Prognostic Factors ◽  
Functional Characterization ◽  
Snp Array ◽  
Genetic Alterations ◽  
Lymphocytic Leukemia ◽  
Bax Protein

Abstract Introduction: CLL is a heterogeneous disease in terms of response to treatment, with some patients reaching complete and prolonged remissions, while others relapsing early and requiring several lines of treatments. This highly variable course is partly explained by the existence of a heterogenic panel of genetic alterations (mutations, chromosomal abnormalities) that allow the development of drug-resistant aggressive CLL subclones. Therefore, a functional characterization of the cytogenetic alterations associated to CLL drug resistance may provide new means of improving the current therapeutic strategies. We and others have already reported that the gain of 2p (2p+) is recurrent in CLL. However, the candidate gained gene(s) on the 2p remain to be identified. Previously data: we have observed that the 2p gain is frequent in previously untreated CLL Binet stages B/C (21/132, 15.9%), and is associated with bad prognostic factors, such as 11q deletion (p=0.0008) and unmutated IGHV (p=0.02). Using a SNP-array approach, we have identified a minimally gained region of 1.28Mb on 2p16.1-15. This region included the gene CRM1/XPO1 (Chromosome Region Maintenance 1/Exportin-1), a gene also recurrently mutated in CLL. A qPCR assessment confirmed that XPO1 was overexpressed in the 2p+/CLL patients (1.4-fold increase compared to 2p-/CLL; p=0.02). The objective of our work was to identify the potential role of XPO1 in CLL drug resistance by using the selective XPO1 inhibitor Selinexor (KPT-330, provided by Karyopharm Therapeutics), which is currently in Phase II human clinical trials in hematological and solid cancers. Methods: We have analyzed 36 2p+/CLL and we have searched for XPO1 mutations in 436 CLL samples. CLL drug resistance associated to XPO1 overexpression/mutation was assessed by measuring the rate of programmed cell death (PCD) on cells from 2p- and wildtype (wt) XPO1/CLL (n=20), 2p+/XPO1 wt/CLL (n=8) and on XPO1 mut/CLL (n=6). After 24 hours treatment with Fludarabin + Cyclophosphamid + Rituximab (FCR), Ibrutinib (Ibru), Idelalisib + Rituximab (Ide+R) and Selinexor, cells were stained with Annexin-V and propidium iodide and PCD was assessed by flow cytometry. KPT-301 was used as a negative control. For the inhibition assay, the inhibitor Q-VD-Oph was added 30 min before inducing cell death. Mitochondrial membrane depolarisation was assessed using tetramethyllrhodamine ethyl ester probe and flow cytometry analysis. Results: (i) Using a FISH approach, we fully confirmed the gain of XPO1 in 2p+/CLL samples. Additionally, we found that the XPO1 gain was often subclonal, suggesting that it tends to arise late in leukemic development. Longitudinal FISH analyses, performed on 8 2p+/CLL-treated patients, showed a similar or increasing percentage of cells carrying XPO1 gain at relapse, when compared to diagnosis; (ii) XPO1 was mutated in 23/436 (5.3%) CLL and in 2/30 (6.7%) 2p+/CLL; (iii) Selinexor induced PCD in 2p-/XPO1 wt/CLL (35% of PCD). The results were similar in all tested CLL, independently of prognostic factors (del13q, tri12, del11q, del17p, IGHV status), while sparing the non leukemic cells from patients or B cells from healthy donors; (iv) Selinexor induced CLL PCD through a caspase-dependant apoptotic pathway, as evidenced by inhibition of cell death by Q-VD-Oph, and cleavage of the caspase-3. Selinexor also induced mitochondrial depolarization and was associated with upregulation and activation of the pro-apopototic Bax protein; (v) XPO1 mut/CLL were significantly resistant to PCD induced by Selinexor (p=0.003). In contrast, the mutations in XPO1 had no effect in FCR and Ibru PCD induction; (vi) 2p+/CLL cells were resistant to PCD induced by all tested drugs: FCR (p=0.01), Ibru (p=0.003), Ide+R (p=0.004) and Selinexor (p=0.0001). Conclusion: Our data show that 2p+/CLL is associated to FCR, Ibru and Ide+R drug resistance. Strikingly, Selinexor, a new XPO1 inhibitor, is unable to induce PCD in 2p+ and/or XPO1 mut CLL, which strongly suggests a key role for XPO1 in the CLL drug resistance associated to the 2p gain. Altogether, our work provide substantial progress in the understanding of the role of XPO1 in CLL drug resistance and suggests that the assessment of the 2p gain and the mutations in XPO1 will be considered before to decide a CLL therapy. As 2p gain could be observed in other B malignancies, it is tempting to extend these recommendations to all Selinexor treatments. Disclosures Choquet: Janssen: Consultancy; Roche: Consultancy. Leblond:Janssen: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Mundipharma: Honoraria.

Overexpression of TEL-MN1 Fusion Enhances Resistance of HL-60 Cells to Idarubicin

Chemotherapy ◽  
2018 ◽  
Vol 63 (6) ◽  
pp. 308-314 ◽  
Author(s):  
Shenglan Gong ◽  
Mengqiao Guo ◽  
Gusheng Tang ◽  
Jianmin Yang ◽  
Huiying Qiu
Keyword(s):  
Flow Cytometry ◽  
Drug Resistance ◽  
Cell Viability ◽  
Cell Apoptosis ◽  
Chromosome Abnormality ◽  
Chemotherapy Resistance ◽  
Retroviral Vector ◽  
Promote Cell Proliferation ◽  
Enhanced Resistance

Background: The translocation t(12; 22) (p13;q12) is a recurrent but infrequent chromosome abnormality in human myeloid malignancies. To date, the role of TEL-MN1 fusion in leukemogenic process and drug resistance is still largely unknown. Methods: In the present study, the TEL-MN1 fusion was transfected into HL-60 cells to upregulate TEL-MN1 expression via a retroviral vector. MTT assay was employed to examine cell viability and flow cytometry was performed to evaluate cell apoptosis. Idarubicin was used to treat HL-60 cells for estimating the effect of TEL-MN1 fusion on the chemotherapy resistance. Results: The results showed that overexpression of TEL-MN1 in HL-60 cells could promote cell proliferation, suggesting that TEL-MN1 may be involved in the leukemogenesis process. HL-60 cells treated with idarubicin showed a weakened cell viability, whereas TEL-MN1 overexpression attenuated the idarubicin-induced inhibition of cell viability and acceleration of cell apoptosis of HL-60 cells. Conclusion: Taken together, our results indicated that TEL-MN1 fusion is an oncogene involved in the leukemogenesis process and TEL-MN1 overexpression enhanced resistance of HL-60 cells to idarubicin, which may provide a useful tool for studying the mechanism of leukemogenesis and drug resistance.

scholarly journals Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells

Cancers ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 291 ◽  
Author(s):  
Simon Latour ◽  
Marion Zanese ◽  
Valérie Le Morvan ◽  
Anne-Marie Vacher ◽  
Nelly Menard ◽  
...  
Keyword(s):  
Cell Death ◽  
Calcium Signaling ◽  
Lymphocytic Leukemia ◽  
Non Hodgkin Lymphoma ◽  
Human B Cells ◽  
Rational Drug ◽  
Intracellular Compartments ◽  
Hodgkin Lymphoma Cell ◽  
Anti Cd20

GA101/obinutuzumab is a novel type II anti-CD20 monoclonal antibody (mAb), which is more effective than rituximab (RTX) in preclinical and clinical studies when used in combination with chemotherapy. Ca2+ signaling was shown to play a role in RTX-induced cell death. This report concerns the effect of GA101 on Ca2+ signaling and its involvement in the direct cell death induced by GA101. We reveal that GA101 triggered an intracellular Ca2+ increase by mobilizing intracellular Ca2+ stores and activating Orai1-dependent Ca2+ influx in non-Hodgkin lymphoma cell lines and primary B-Cell Chronic Lymphocytic Leukemia (B-CLL) cells. According to the cell type, Ca2+ was mobilized from two distinct intracellular compartments. In Raji, BL2, and B-CLL cells, GA101 induced a Ca2+ release from lysosomes, leading to the subsequent lysosomal membrane permeabilization and cell death. Inhibition of this calcium signaling reduced GA101-induced cell death in these cells. In SU-DHL-4 cells, GA101 mobilized Ca2+ from the endoplasmic reticulum (ER). Inhibition of ER replenishment, by blocking Orai1-dependent Ca2+ influx, led to an ER stress and unfolded protein response (UPR) which sensitized these cells to GA101-induced cell death. These results revealed the central role of Ca2+ signaling in GA101’s action mechanism, which may contribute to designing new rational drug combinations improving its clinical efficacy.

Potential Role of Dual NF-κB and Oxidative Stress Pathway Inhibitor, OT-304 as a Novel Drug Resistance-Reversing Agent.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4211-4211
Author(s):  
Shaker A. Mousa ◽  
Ghanshyam Patil ◽  
Abdelhadi Rebbaa
Keyword(s):  
Oxidative Stress ◽  
Drug Resistance ◽  
Tumor Cells ◽  
Genetic Alterations ◽  
Potential Candidate ◽  
Drug Resistant ◽  
P Glycoprotein ◽  
And Oxidative Stress

Abstract The development of resistance to chemotherapy represents an adaptive biological response by tumor cells that leads to treatment failure and patient relapse. During the course of their evolution (intrinsic resistance) or in response to chemotherapy (acquired resistance), tumor cells may undergo genetic alterations to possess a drug resistant phenotype. Dysregulation of membrane transport proteins and cellular enzymes, as well as altered susceptibility to commit to apoptosis are among the mechanisms that contribute to the genesis of acquired drug resistance. Recently, the development of approaches to prevent and/or to reverse this phenomenon has attracted special interest and a number of drug candidates have been identified. Despite strong effects observed for these candidates in vitro, however, most of them fail in vivo. In the present study, we have identified a novel small molecule inhibitor of dual NF-κB and oxidative stress pathways, OT-304, as a potential candidate to reverse drug resistance. Initial investigations indicate that this compound effectively inhibits proliferation of doxorubicin-sensitive and doxorubicin-resistant cells to the same extent, suggesting that it is capable of bypassing the development of drug resistance. Additional experiments reveal that OT-304 enhances cancer cell sensitivity to doxorubicin and to etoposide, particularly in cells characterized by the over-expression of the drug transporter P-glycoprotein. These findings suggest that either the expression/and or the function of P-glycoprotein could be affected by OT-304. In vivo studies using tumor xenografts in nude mice showed that OT-304 is also capable of preventing the growth of drug resistant cancer cells. This later finding further confirms the role of OT-304 as a drug resistance-reversing agent and warrants further pre-clinical and clinical investigation to determine its efficacy in treating aggressive tumors.

ROS-Mediated Upregulation of Noxa Overcomes Drug-Resistance Due to P53-Dysfunction in Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2382-2382
Author(s):  
Sanne H. Tonino ◽  
Jacoline M van Laar ◽  
Marinus H. J. van Oers ◽  
Jean Y.J. Wang ◽  
Eric Eldering ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Combination Treatment ◽  
Conflicts Of Interest ◽  
Alkylating Agents ◽  
Single Agent ◽  
Treatment Strategies ◽  
Cd40 Ligand ◽  
Lymphocytic Leukemia

Abstract Abstract 2382 Poster Board II-359 Although recent advances in treatment-strategies for chronic lymphocytic leukemia (CLL) have resulted in increased remission rates and response duration, the disease eventually relapses, which necessitates repeated cycles of therapy. Eventually most patients develop chemo-resistant disease which infers a very poor prognosis. The activity of purine-analogs and alkylating agents, the backbone of current treatment regimens, depends on functional p53 and chemo-resistance is highly associated with a dysfunctional p53-response. P53-independent sensitization of CLL cells to these compounds could represent a novel strategy to overcome chemo-resistance. Platinum-based compounds have been successfully applied in relapsed lymphoma and recently also in high-risk CLL. In various cancer-types, the activity of such compounds has been found to be p53-independent and in part mediated by p73. In this study we investigated the efficacy and mechanism of action of platinum-based compounds in chemo-refractory CLL. Neither cisplatinum nor oxaliplatin as a single agent induced cell death in clinically relevant doses. However, independent of p53-functional status, platinum-based compounds acted synergistically with fludarabine, which was found to be caspase-dependent. Combination-treatment resulted in strong upregulation of the pro-apoptotic BH3-only protein Noxa. We did not find evidence for a role of p73; however, the observed synergy was found to involve generation of reactive oxygen species (ROS). Co-treatment with ROS-scavengers completely abrogated Noxa-upregulation and cell-death upon combination treatment in p53-dysfunctional CLL. Noxa RNA-interference markedly decreased sensitivity to combination treatment, supporting a key role for Noxa as mediator between ROS signaling and apoptosis induction. In addition to these findings, we tested the effects of platinum-based compounds and fludarabine on drug-resistance resulting from CD40-ligand stimulation of CLL cells, which represents a model for CLL cells in the protective micro-environment of the secondary lymph node-tissue (Hallaert et al Blood 2008 112(13):5141). Combination treatment could overcome CD40-ligand induced chemo-resistance and was, at least in part, mediated by the generation of ROS and marked induction of expression of Noxa. Our data indicate that interference with the cellular redox-balance represents an interesting target to overcome drug resistance due to both p53-dysfunction as well as micro-environmental protective stimuli in CLL. Disclosures: No relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia-Associated Macrophages: Not Just Nurse Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 46-46
Author(s):  
Loic Ysebaert ◽  
Mary Poupot ◽  
Yovan Sanchez-Ruiz ◽  
Camille Laurent ◽  
Guy Laurent ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Conditioned Medium ◽  
Apoptotic Cell ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Lymphocytic Leukemia ◽  
Functional Interpretation

Abstract Abstract 46 Introduction: CLL cells interact with many accessory cells in an environment mimicking that of normal mature B cells. Role of antigen, cytokines, adhesion pathways are critical for many aspects in the disease course (proliferation/survival, migration or homing, drug resistance, and presumably relapse). Nurse-like cells (NLC) belong to a monocytic-derived, bystander population among CLL lymph node and spleen stromal cells. Aim: To investigate the nature, functions, and location of NLC within CLL microenvironment. Methods: Gene expression profiles (GEP) from in vitro expanded NLC from patients (n=10) were produced and compared to those from normal CD14+ monocytes, M1-polarized macrophages, M2-polarized macrophages and tumor-associated macrophages (produced in the lab or downloaded from GEO datasets). Principal Component Analysis was used to categorize these five populations of cells and in-house-built GSEA software was used for functional interpretation of their relevant gene lists. Protein expression patterns were validated with multi-analyte ELISArray kits, proteome profiler arrays, flow cytometry (FC) or immunohistochemistry (IHC). Results: New insights into the physiopathological role of NLC in CLL are suggested from five lines of evidence: 1/a Òmonocytic gene signatureÓ (i.e. a set of 549 genes) is shared by the NLC and the monocyte subtypes. The genes over-represented in NLC vs normal monocytes pinpointed positive modulation of apoptotic cell clearance (scavenger, mannose and complement receptors, LXRalpha), lipid metabolism (Apolipoprotein E, PPAR signaling), extracellular matrix-receptor interactions (integrins, SPARC, Matrix MetalloProteinases) and actin cytoskeleton remodeling. 2/unsupervised clustering show that NLC represent an M2-skewed, TAM-like cell population. They down-regulate mRNA and proteins for classic M1 inflammatory markers (e.g. IL-1, IL-6, IL-12, COX2) while increase secretion of TGFbeta, IL-10, CCL17 and CCL22 soluble factors. 3/these and previously published observations suggest that B-CLL-to-NLC interactions may orchestrate immunosuppression in this disease. PBMCs from Òwatch and waitÓ CLL patients (all stage A/Rai 0, mutated IgVH, low risk cytogenetics profile) or healthy donors were stimulated with anti-CD3/CD28 beads + IL-2, either in standard RPMI+10% FCS or in conditioned medium (CM, after 14d CLL-NLC co-culture in vitro) and their proliferation/phenotype were compared after 2 weeks. Significant expansion of T cells with Treg (CD4+CD25+FoxP3+) phenotype was observed only from CLL PBMCs grown in conditioned medium (mean % Treg: 2.85 vs 3.05 in CM for normal PBMCs, and 1.54 vs 15.9 in CM for CLL PBMCs, P< 0.05). 4/although NLC make immune synapses with live B-CLL, they do not phagocytose them. Over-expression of CD47 (ÒdonÕt eat meÓ signal) by B-CLL cells (mfi= 3490 vs 2581 on normal cells, P< 0.05, n=18) may provide them with a protective signal against NLC. 5/from our GEP, flow cytometric and IHC analyses, we propose CD163 (classic M2 marker) as a reliable tool to identify NLC in vivo. Although in vitro, CLL cells can pervert healthy donor monocytes into NLC, only CLL-derived NLC are truly CD14+ CD163+. In vivo, CD163 staining reveals putative NLC in CLL lymph nodes(LN)/spleen sections but not in bone marrow. In LN from all patients, NLC reside in the subcapsular areas and line vessel structures, suggesting a role in CLL cells trafficking. Most interestingly, NLC infiltrate pseudofollicles structures only in a subset of cases. We will present updated IHC and clinical presentation correlation studies. Conclusions: Our results suggest that the role of NLC in CLL might be broader than initially thought. Beside of nursing and conferring drug resistance, NLC may also be crucial in the setting of immunosuppression, of CLL cells recruitment, and should thus be considered as therapeutic targets. Disclosures: Off Label Use: GA101 is not currently approved for CLL treatment.

HSP70 Inhibitor, YK5, Synergizes with Chemotherapeutic Agents and Prevents Chemoresistance in Acute Myelogenous Leukemia (AML).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2476-2476
Author(s):  
Krishan K. Sharma ◽  
Juan Felipe Rico ◽  
Michael W. Becker ◽  
Gail J. Roboz ◽  
Gabriela Chiosis ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Drug Resistance ◽  
Cell Death ◽  
Heat Shock ◽  
Quantitative Pcr ◽  
Caspase 3 ◽  
Chemotherapeutic Agents ◽  
Fold Change ◽  
Myelogenous Leukemia ◽  
Intracellular Hsp70

Abstract Abstract 2476 Stress-inducible heat shock protein 70 (HSP70) is a major cytoprotective factor and a molecular chaperone that interacts with HSP90 to form a multi-chaperone complex. Cancer cells are highly dependent on this complex due to their increased demand for protein synthesis. HSP70 overexpression inhibits apoptosis and has been associated with drug resistance and poor prognosis. YK5, a novel inhibitor of tumor-HSP70, has been shown to induce potent cell death in AML blast, progenitor, and stem cell populations with minimal effects in normal hematopoietic cells. Due to the role of HSP70 in drug resistance, we examined the effect of combining YK5 with other chemotherapeutic agents, including arsenic trioxide, cytarabine, suberoylanilide hydroxamic acid (SAHA) and PU-H71, a novel tumor-specific HSP90 inhibitor. We tested the ability of YK5 to synergize with either AsO3, AraC, SAHA, or PU-H71 in primary AML samples. Using multiparameter flow cytometry to measure viability after 48 hours of treatment, we found that combining 1μM YK5 with either 500nM AsO3 or PU-H71 resulted in a significant increase in cell death when compared to either agent alone (n=9, mean viability: 51.8, 67.2, and 13.4% for AsO3, YK5, and AsO3/YK5, respectively, P = 0.0018; mean viability: 57.1 and 20.8% for PU-H71 and PU-H71/YK5, respectively, P = 0.0029). A synergistic relationship between YK5 and both AsO3 and PU-H71 was found in all nine primary samples (combination indexes 0.29 – 0.76 with YK5/AsO3, 0.33 – 0.83 with YK5/PU-H71). In contrast, the combination of YK5 with either AsO3 or PU-H71 in CD34+ cord blood mononuclear cells did not result in a significant increase in cell death when compared to either agent alone (mean viability: 42.4, 72.4, and 37.2% for AsO3, YK5, and AsO3/YK5, respectively; mean viability: 61.1 and 51.1% for PU-H71 and PU-H71/YK5, respectively). YK5 in combination with either AraC or SAHA, however, did not result in a significant increase in cell death when compared to either drug alone, with an additive effect being demonstrated with a 1:1 YK5 to AraC/SAHA drug ratio (Mean CI = 0.9918). To determine the mechanism of the observed synergistic activity, intracellular HSP70 and active caspase-3, a client of HSP70, were measured using flow cytometry. Both AsO3 and PU-H71 significantly increased intracellular HSP70 and caspase-3 (Mean fold change = 18.3, 21.0 of HSP70 and 9.9, 8.3 of Caspase-3 for AsO3 and PU-H71 treatment, respectively), while treatment with AraC or SAHA resulted in no change in HSP70 levels. Furthermore, quantitative PCR revealed that treatment with either AsO3 or PU-H71 strongly upregulated HSPA1A and HSPA6, the main stress-inducible isoforms of HSP70 (Mean fold change = 15.9, 14.1 of HSPA1A, and 20.8, 23.4 of HSPA6 for AsO3 and PU-H71 treatment, respectively). AraC and SAHA had no significant upregulation of these genes. We have previously shown that increased levels of HSPA1A correlate with sensitivity to HSP70 inhibition via YK5. To further explore the mechanism of this observed synergy, flow cytometry was used to measure the levels of reactive oxygen species (ROS). Treatment with AsO3, PU-H71, AraC, or SAHA resulted in a significant increase in ROS (Mean fold change = 2.75, 1.92, 2.89, 1.67, respectively). Quantitative PCR also confirmed the activation of the oxidative stress response by the upregulation of heme oxygenase 1 (HMOX1) by treatment with these drugs (Mean fold change = 10.9, 8.7, 11.2, 7.7, respectively). YK5, however, did not induce ROS or upregulate HMOX1. Interestingly, pretreatment with NAC in primary AML samples (n=4) resulted in no protection from YK5 synergistic effect when combined with either AsO3 or PU-H71. These results suggest that YK5 synergizes with AsO3 and PU-H71 due to the increase in intracellular HSP70 caused by these drugs. This synergy is most likely due to the activation of the heat shock response and independent of the production of ROS due to drug treatment. In summary, we have found that the novel tumor-HSP70 inhibitor YK5 can synergize with AsO3 and PU-H71 in primary human AML, and that the basis of this synergism is due to the increase in intracellular HSP70 caused by these chemotherapeutic agents. HSP70 inhibition represents a novel approach in AML treatment and can be particularly significant to drug-resistant patients when combined with other chemotherapy. Disclosures: Roboz: Astex Pharmaceuticals: Research Funding.

scholarly journals The role of prognostic factors in assessing ‘high-risk’ subgroups of patients with chronic lymphocytic leukemia

Leukemia ◽  
2007 ◽  
Vol 21 (9) ◽  
pp. 1885-1891 ◽  
Author(s):  
N E Kay ◽  
S M O'Brien ◽  
A R Pettitt ◽  
S Stilgenbauer
Keyword(s):  
Prognostic Factors ◽  
High Risk ◽  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia

scholarly journals Targeting PAK1 Overcomes Resistance to Ibrutinib in Chronic Lymphocytic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1546-1546
Author(s):  
Zijuan Wu ◽  
LEI Fan ◽  
Luqiao Wang ◽  
Hanning Tang ◽  
Yi Miao ◽  
...  
Keyword(s):  
Drug Resistance ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
High Throughput ◽  
Combination Index ◽  
Lymphocytic Leukemia ◽  
Chromatin Conformation ◽  
Rna Seq ◽  
Ibrutinib Resistance

Abstract Objective: Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that mainly affects the elderly and is characterized by the expansion of small mature B-cells. New targeted drugs, such as the BTK inhibitor ibrutinib, have greatly improved patient survival but have also posed the challenge of drug resistance. The three-dimensional (3D) spatial structure of chromatin is highly dynamic and varies greatly between cell types and developmental stages, with the maintenance of chromatin homeostasis being of major significance in disease prevention. Accumulating evidence has suggested that changes in 3D genomic structures play an important role in cell development and differentiation, disease progression, as well as drug resistance. Nevertheless, the characteristics and functional significance of chromatin conformation in the resistance of CLL to ibrutinib remain unclear. In this study, we aimed to investigate the mechanism underlying ibrutinib resistance through multi-omics profiling, including the study of chromatin conformation. Thus, we would be able to demonstrate the importance of chromatin spatial organization in CLL and highlight the oncogenic factors contributing to CLL development and mediating ibrutinib resistance. Methods: An ibrutinib-resistant cell line was established by exposing cells to increasing doses of ibrutinib. High-throughput chromosome conformation capture (Hi-C), assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), bulk RNA sequencing (RNA-seq), and Tandem Mass Tag (TMT) were performed to explore differences between ibrutinib-resistant and parental cells. Peripheral blood mononuclear cells (PBMCs) from 53 CLL patients were collected for RNA-seq. Mitochondrial respiration and glycolysis were assessed via Seahorse analysis. The growth-inhibitory effects of tested drugs were evaluated via a CCK8 assay, and the combination index (CI), indicating synergy, was calculated using CompuSyn software. Apoptosis was detected via annexin V staining. Results: Between ibrutinib-resistant and parental cells changes in some chromosomes, including chr11 were observed (Figure 1A). p21-activated kinase 1 (PAK1), which is located on chr11 and frequently overexpressed or excessively activated in almost all cancer types and involved in almost every stage of cancer progression, was first explored for its role in CLL progression and drug resistance. The oncogene PAK1 was observed locate in a region where B-to-A compartment switching occurred (Figure 1B). Consistent with the results of ATAC-seq, RNA-seq, and TMT, Hi-C analysis revealed a transcriptional upregulation of PAK1 in ibrutinib-resistant CLL cells (Figure 1C). Functional analysis demonstrated that PAK1 overexpression significantly promoted cell proliferation, while knockdown markedly suppressed cell viability (Figure 1D). Cell viability assays indicated that the depletion of PAK1 increased ibrutinib sensitivity (Figure 1E). In addition, PAK1 positively regulates glycolysis and oxidative phosphorylation in CLL cells (Figure 1F and G). To verify the results of sequencing and further explore the role of PAK1 in CLL, B-cells from healthy volunteers and PBMCs from CLL patients were collected. The level of PAK1 mRNA expression was significantly higher in CLL primary cells than in B-cells from healthy volunteers (Figure 1H). Kaplan-Meier survival analysis of qRT-PCR data confirmed that patients with high PAK1 expression had a significantly lower OS (Figure 1I). IPA-3, the small molecular inhibitor of PAK1 suppressed the proliferation of ibrutinib-resistant and parental CLL cells in a dose-dependent manner. The combination of IPA-3 and ibrutinib exerted potent cell growth inhibition (Figure 1J), and the combination index (CI) calculated using the CompuSyn software confirmed the synergistic effect (CI&lt;1) of this combinatorial therapy (Figure 1K). Conclusions: In the current study, we have provided a genome-wide view of alterations in 3D chromatin organization between ibrutinib-resistant and parental CLL cells and confirmed the oncogenic role of PAK1 in CLL. Most importantly, our research provides promising therapeutic targets for overcoming ibrutinib resistance. In particular, the treatment of CLL patients with a combination of IPA-3 and ibrutinib may improve clinical outcomes. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting Nicotinamide Adenine Dinucleotide (NAD) Glycohydrase Activity of CD38 Exerts Anti-Myeloma Effect Accompanying Intracellular NAD Elevation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1810-1810
Author(s):  
Saki Kushima ◽  
Takayuki Sasano ◽  
Masao Matsuoka ◽  
Hiroyuki Hata ◽  
Yawara Kawano
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Cell Lines ◽  
Nicotinamide Adenine Dinucleotide ◽  
Research Funding ◽  
Therapeutic Strategy ◽  
Cd38 Expression ◽  
Low Concentrations ◽  
Mammalian Tissues

Introduction. The development of novel agents has improved the outcomes of multiple myeloma (MM) patients. Especially, daratumumab, an anti-CD38 monoclonal antibody which exerts therapeutic effect against MM cells through direct cell damage, antibody dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), has shown its high efficacy in clinical practice. CD38 is a transmembrane glycoprotein highly expressed in plasma cells. CD38 is also a major nicotinamide adenine dinucleotide (NAD) glycohydrase in mammalian tissues, which regulate cellular levels of NAD. However, the role of CD38 as a NAD glycohydrase (NADase) in survival of MM cells is not well understood. In the present study, we conducted CD38 enzyme activity inhibition on MM cells using a small molecule compound 78c, a specific inhibitor for NADase enzymatic activity of CD38, in order to study the role of CD38 NADase activity in MM cell survival and to examine whether CD38 enzyme inhibition could be a new therapeutic strategy of MM. Materials and methods. MM cell lines (NCI-H929, KMS-12BM, KMS-12PE, U266) were treated with CD38 NADase inhibitor, 78c, in vitro. Viability of MM cell lines and patient-derived MM cells were analyzed by flow cytometry after 7AAD staining. MM cell lines possessing CD38 positive and negative fraction were sorted according to the CD38 expression using CD38 Micro-Beads. CD38 low MM cell lines were treated with All-trans retinoic acid(ATRA)to increase surface CD38 expression. Intracellular NAD and NADH concentrations in MM cells were analyzed using NAD / NADH assay kit. Detection of apoptosis in MM cell lines were examined by Annexin V and PI staining followed by flow cytometry analysis. Caspase inhibitor, Z-VAD-FMK, was used in combination with 78c to study the mechanism of 78c induced MM cell death. Results. 78c induced cell death in MM cell lines at low concentrations (IC50 10-20 μM). Addition of 78c to patient derived bone marrow cells showed cytotoxicity to MM cells, while toxicity to non-MM cells were limited. CD38 positive fraction of MM cell lines had better sensitivity to 78c compared to CD38 negative fraction. CD38 induction by ATRA in CD38 low MM cell lines showed increased sensitivity to 78c. These results proved that 78c efficacy correlates with surface CD38 expression. Comparison of intracellular NAD and NADH concentrations between CD38 positive and negative fractions of MM cell lines demonstrated a significant increase of NAD in the CD38 negative fraction compared to their positive counterparts, indicating that CD38 is indeed controlling the intracellular NAD concentration. Marked increase of NAD / NADH ratio was observed in 78c treated MM cell lines compared to control, proving that CD38 NADase inhibition affects intracellular NAD concentration in MM cells (Fig. 1). 78c treatment of MM cell lines significantly reduced the number of viable cells in the Annexin- / PI- region, however, addition of Z-VAD-FMK did not lead to recovery of viable cell numbers, indicating non-apototic cell death induction by CD38 NADase inhibition. Conclusions. CD38 is the major NADase in mammalian tissues, and involved in catabolism of NAD. CD38 NADase inhibitor, 78c, inhibited the growth of MM cells at low concentrations. 78c induced cell death was found to be highly specific to MM cells and its cytotoxic effect was associated with surface CD38 expression of MM cells. Increased amount of NAD in MM cells by 78c treatment suggests that NAD elevation is associated with MM cell death induced by CD38 NADase inhibition. Since, daratumumab has limited effect against CD38 NADase activity, modulation of intracellular NAD levels by CD38 NADase inhibition could provide a novel therapeutic strategy for MM (Fig. 2). Disclosures Matsuoka: Kyowa Kirin Co., Ltd.: Research Funding; Bristol-Myers Squibb Corp.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria.

scholarly journals The Critical Role of HDAC1-IRF4-Pim-2 Axis in Myeloma Cell Growth and Survival: Therapeutic Impacts of Targeting the HDAC1-IRF4-Pim-2 Axis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1939-1939 ◽  
Author(s):  
Takeshi Harada ◽  
Asuka Oda ◽  
Yohann Grondin ◽  
Jumpei Teramachi ◽  
Ariunzaya Bat-Erdene ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Death ◽  
Cell Growth ◽  
Critical Role ◽  
Class I ◽  
Growth And Survival ◽  
Class I Hdacs ◽  
Rpmi 8226 ◽  
Cell Growth And Survival

Abstract Multiple myeloma (MM) is a heterogeneous clonal plasma cell proliferative disorder with CRAB features. Although survival of MM patients has been greatly prolonged by recent implementation of various combinatory treatments with novel anti-MM agents, MM still remains incurable. MM cells preferentially grow and expand in the bone marrow to elicit the alteration of gene expression and thereby drug resistance. To improve the therapeutic efficacy, we urgently need to develop novel treatment strategies targeting the BM microenvironment-mediated drug resistance. The serine/threonine kinase Pim-2 is constitutively over-expressed and acts as a pro-survival mediator in MM cells. We have reported that cocultures with bone marrow stromal cells (BMSCs) or osteoclasts (OCs) further up-regulate Pim-2 expression in MM cells to confer drug resistance (Leukemia 2011, 2015). Therefore, Pim-2 appears to be an important therapeutic target to impair the BM microenvironment-mediated drug resistance in MM. Histone deacetylases (HDACs) are generally accepted to be therapeutic targets for MM treatment. However, clinical application of currently available pan-HDAC inhibitors is limited with their adverse effects induced by a non-selective HDAC inhibition. To develop safe and effective HDAC inhibitor-based treatment, the therapeutic roles of HDAC isoform-specific inhibition should be elucidated. In this regard, we have recently reported therapeutic impacts on MM cells of inhibition of class-I HDACs, especially HDAC1 and HDAC3. HDAC3-selective inhibitor BG45 induces anti-MM activity in combination with DNA methyltransferase (DNMT) inhibitor azacytidine (Leukemia 2017). In the present study, we aimed to clarify the underlying mechanisms for impairment of MM cell growth and survival by HDAC1 inhibition. We first referenced the expression of class-I HDACs using a publicly available GSE6691 data set. Among class-I HDACs, HDAC1 and HDAC3 were highly expressed in MM cells. We then knockdowned HDAC1 gene using lentiviral shRNA system in MM cell lines. The HDAC1 gene silencing induced MM cell death with caspase-3 activation, indicating the critical role of HDAC1 in MM cell growth and survival. To determine target molecules of HDAC1, we carried out RNA-sequencing with and without the HDAC1 gene silencing in RPMI 8226 cells. Among genes whose expression significantly changed by the HDAC1 knockdown (adjusted P values < 0.05, log fold change > 0.5), we focused on IRF4 together with PIM2, because MM cell has been demonstrated to addict to aberrant IRF4-c-Myc regulatory network (Nature 2008). Downregulation of IRF4 and Pim-2 by the HDAC1 knockdown was further confirmed by quantitative PCR (Q-PCR) and immunoblotting in RPMI 8226 and MM.1S cells. Treatment with the class I HDAC-selective inhibitor MS-275 (entinostat) also induced MM cell death along with reduction of IRF4 and Pim-2 expression. Since previous study has shown that IRF4 binds to PIM2 promoter in MM cells (Nature 2008), we examined whether IRF4 regulates PIM2 expression. We found that IRF4 binds to the PIM2 promoter region by analyzing ChIP-Seq data in KMS-12 cells (GSE22901). We further confirmed the binding of IRF4 on PIM2 promoter by ChIP-Q-PCR. Indeed, the IRF4 knockdown downregulated Pim-2 expression in RPMI 8226 cells. These results suggest that HDAC1 inhibition downregulates IRF4 expression, thereby transcriptionally reducing PIM2 expression in MM cells. Pim-2 expression can also be augmented by multiple signaling pathways, including HIF-1a, JAK-STAT and NF-kB-mediated ones in MM cells through the interaction with BM microenvironment. Interestingly, the Pim inhibitor SMI-16a and MS-275 cooperatively induced apoptotic cell death in MM cell lines and CD138-positive primary MM cells even in the presence of BMSCs. Taken together, our results demonstrate the critical role of the HDAC1-IRF4-Pim-2 axis in MM cell growth and survival, and provoke the novel treatment strategy targeting the HDAC1-IRF4-Pim-2 axis in MM cells. Disclosures Anderson: Takeda Millennium: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership; Celgene: Consultancy; Bristol Myers Squibb: Consultancy.

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