scholarly journals Combining Next Generation Proteomics Platforms with Drug Sensitivity Resistance Testing Allows Identification of Physiologically Distinct Sub-Clones That Can Inform Therapeutic and Drug Development Strategies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1901-1901
Author(s):  
Despina Bazou ◽  
Muntasir M Majumder ◽  
Ciara Tierney ◽  
Sinead O'Rourke ◽  
Pekka Anttila ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Resistance Testing ◽  
Proteomics Analysis ◽  
Advisory Committees ◽  
Group I

Abstract Introduction: A hallmark of Multiple Myeloma (MM) is the sequel development of drug resistant phenotypes, which may be present initially or emerge during the course of treatment. These drug resistant phenotypes reflect the intra-tumor and inter-patient heterogeneity of this cancer. Most MM cells are sensitive to proteasome inhibitors (PIs), which have become the standard of care in the treatment of newly diagnosed and relapsed MM. However, resistance develops (intrinsic/acquired). Although several novel drugs have recently been approved or are in development for MM, there are few molecular indicators to guide treatment selection. To address this limitation we have combined mass spectrometry-based proteomics analysis together with ex vivo drug response profiles and clinical outcome to elucidate a best possible accurate phenotype of the resistant sub-clones, thus yielding a theranostic profile that will inform therapeutic and drug development strategies. Methods: We performed mass spectrometry-based proteomics analysis on plasma cells isolated from 38 adult MM patient bone marrow aspirates (CD138+). Samples were obtained at diagnosis or prior to commencing therapy. The participating subjects gave written informed consent in accordance with the Declaration of Helsinki that was approved by local ethics committees. For the proteomics analysis, peptides were purified using the filtered aided sample preparation (FASP) method. Subsequently, samples were prepared for label-free liquid chromatography mass spectrometry (LC-MS/MS) using a Thermo Scientific Q-Exactive MS mass spectrometer. Proteins were analysed using the MaxQuant and Perseus software for mass-spectrometry (MS)-based proteomics data analysis, UniProtKB-Swiss Prot database and KEGG Pathway database. In parallel, we undertook a comprehensive functional strategy to directly determine the drug dependency of myeloma plasma cells based on ex vivo drug sensitivity and resistance testing (DSRT)as previously described (1). Results: Our initial proteomic analysis was generated by examining MM patient plasma cells, grouped based on DSRT to 142 anticancer drugs including standard of care and investigational drugs. Each of the 142 drugs was tested over a 10,000-fold concentration range, allowing for the establishment of accurate dose-response curves for each drug in each patient. MM patients were stratified into four distinct subgroups as follows: highly sensitive (Group I), sensitive (Group II), resistant (Group III) or highly resistant (Group IV) to the panel of drugs tested. We then performed blinded analysis on the 4 groups of CD138+ plasma cells divided based on the ex vivo sensitivity profile, identifying a highly significant differential proteomic signature between the 4-chemosensitivity profiles, with Cell Adhesion Mediated-Drug Resistance (CAM-DR) related proteins (e.g. integrins αIIb and β3) significantly elevated in the highly resistant phenotype (Group IV). In addition our results showed that Group I patients displayed significant upregulation of cell proliferation proteins including: MCM2, FEN1, PCNA and RRM2. Furthermore, Group I patients have shorter Progression Free Survival (PFS) as well as Overall Survival (OS) compared to the other subgroups. Figure 1 shows the Heatmap summarizing the expression of proteins (log2 fold change) in the four distinct MM patient subgroups. Conclusions:Our findings suggest that combining a proteomics based study together with drug sensitivity and resistance testing allows for an iterative adjustment of therapies for patients with MM, one patient at a time, thus providing a theranostic approach. Our results suggest that the disease driving mechanisms in the patient subgroups are distinct, with highly resistant patients exhibiting cell adhesion mediated cytoprotection, while highly sensitive patients show an increased cell proliferation protein profile with shorter PFS and OS. Our study aims to guide treatment decisions for individual cancer patients coupled with monitoring of subsequent responses in patients to measure and understand the efficacy and mechanism of action of the drugs. Future work will include the establishment of flow cytometry-based screening assays to identify the different resistant phenotypes at diagnosis/relapse. References: (1) M. M. Majumder et al., Oncotarget 8(34), 56338 (2017) Disclosures Anttila: Amgen: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Silvennoinen:Amgen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Heckman:Orion Pharma: Research Funding; Celgene: Research Funding; Novartis: Research Funding.

scholarly journals Phosphoproteomic Analysis of Primary Myeloma Patient Samples Identifies Distinct Phosphorylation Signatures Correlating with Chemo-Sensitivity Profiles in an Ex Vivo Drug Sensitivity Testing Platform

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2666-2666
Author(s):  
Katie Dunphy ◽  
Paul Dowling ◽  
Juho J. Miettinen ◽  
Caroline A. Heckman ◽  
Paula Meleady ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Resistance Mechanisms ◽  
Group Iv ◽  
Group I ◽  
Resistant Group ◽  
Sensitive Group

Abstract Introduction: Multiple myeloma (MM) is characterized by the clonal expansion of plasma cells in the bone marrow resulting in end-organ damage. Despite an extensive increase in the five-year survival rate in recent years, MM is still considered an incurable disease as patients will repeatedly relapse and develop resistance to current chemotherapies. A key focus for the personalization of myeloma therapy is understanding the biological mechanisms of drug resistance and identifying clinically useful biomarkers of therapeutic response. Highly efficient techniques for the enrichment of phosphorylated peptides followed by high resolution mass spectrometry facilitates the quantitation of thousands of site-specific phosphorylation events. Here, we have performed a phosphoproteomic analysis on MM cell lysates stratified based on their ex vivo drug response profiles to advance our understanding of drug resistance mechanisms. Materials and Methods: CD138 + plasma cells were isolated from 20 adult MM patient bone marrow aspirates at diagnosis (n=7) or relapse (n=13). Samples were grouped based on ex vivo drug sensitivity and resistance testing (DSRT) as follows: highly sensitive (Group I), sensitive (Group II), resistant (Group III), highly resistant (Group IV) [1]. For the phosphoproteomic analysis, peptides were generated and purified using the filter aided sample preparation (FASP) protocol. Peptide tandem mass tag (TMT) labelling, Fe 3+ immobilized metal ion affinity chromatography (IMAC), synchronous precursor selection (SPS), and triple stage tandem mass spectrometry (MS3) was performed. Nonenriched peptides were used for proteomic analysis. Resulting data was analysed using MaxQuant, followed by normalization of phosphosite intensities using the internal reference scale (IRS) method, and statistical analysis using Perseus. Functional enrichment and kinase enrichment analyses were performed on significant phosphoproteins using g:profiler and KEA2, respectively. Results: Our quantitative MS-based phosphoproteomic analysis identified 2,945 phosphorylation sites on 2,232 phosphopeptides from 690 phosphoproteins. Of these phosphorylation sites, 176 were significantly changed between all four DSRT groups and 267 were significantly changed between Group I and Group IV (False Discovery Rate (FDR) < 0.05). Hierarchical clustering was performed to highlight the distinct phosphoproteomic profiles associated with each DSRT group, of which the very sensitive (Group I) and very resistant (Group IV) subgroups demonstrated a well-defined separation (Fig. 1A, 1B). KEGG pathway analysis and gene ontology (GO) analysis of significantly increased phosphorylated proteins in Group IV compared to Group I MM patients demonstrated an increased phosphorylation of proteins associated with tight junctions, the Rap1 signalling pathway and the phosphatidylinositol signalling system; indicating an upregulation of cell adhesion associated processes in drug resistant MM. Phosphoproteins increased in abundance in Group I compared to Group IV MM patients revealed an increased phosphorylation of proteins involved in translation and RNA processing including the spliceosome, RNA transport and RNA binding pathways (Fig. 1C). We identified filamin A serine 2152, RAS guanyl-releasing protein 2 serine 576 and proto-oncogene tyrosine-protein kinase Src serine 17 as increased in Group IV MM, and nuclease-sensitive element-binding protein 1 (YBX1) serine 165, CD44 serine 697 and Bcl2-associated agonist of cell death (BAD) serine 99 as increased in Group I MM. KEA of the upregulated phosphoproteome in Group IV revealed an enrichment of cyclin dependent kinase 1 (CDK1) and ribosomal s6 kinases (RPS6K) whereas casein kinase 2 (CK2) and the glycolysis-associated kinases were enriched in Group I (Fig. 1D). Conclusion: Our study has generated a phosphoproteomic dataset demonstrating distinct phosphorylation signatures associated with drug sensitivity in clinical MM plasma cells. The identification of phosphorylation events associated with drug resistance provides a basis for further exploration of these events and associated signalling pathways to further understand drug resistance mechanisms in MM and identify potential biomarkers of therapeutic response and targets for drug re-sensitization in MM. References: [1] M. M. Majumder et al., Oncotarget 8(34), 56338 (2017) Figure 1 Figure 1. Disclosures Heckman: Novartis: Research Funding; Orion Pharma: Research Funding; Celgene/BMS: Research Funding; Oncopeptides: Consultancy, Research Funding; Kronos Bio, Inc.: Research Funding.

scholarly journals Identification of Novel Regulatory Pathway for Immunoglobulin Production Provides Rational Treatment for Bortezomib-Resistant Multiple Myeloma Patients

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 40-42
Author(s):  
Alexander Vdovin ◽  
Michal Durech ◽  
Tomas Jelinek ◽  
Tereza Sevcikova ◽  
Juli R. Bago ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Multiple Myeloma ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Patient Survival ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Ubiquitinated Proteins ◽  
Ig Synthesis

Introduction Monoclonal immunoglobulin (Ig) is a valuable diagnostic marker in patients with multiple myeloma (MM). An inevitable consequence of extensive Ig synthesis is overload of misfolded proteins that saturate proteasome capacity making the myeloma cells highly sensitive to proteasome inhibitors (PI). Even though PI are regularly used in the clinic, resistance often emerges leaving clinicians with limited treatment options. Therefore, there is a need for a robust marker selecting MM patients for precise PI-based combination therapy. Methods We performed a multiple database search for genes associated with Ig production and MM patients' survival. Additionally, we compared gene expression profiles (RNAseq) of primary MM cells with low and high Ig levels. Next, we validated the identified hits by shRNA knockdown and overexpression studies using myeloma cell lines, primary MM samples, and mouse models. We also applied mass spectrometry-based proteomic analysis, advanced biochemical approaches, and genetic models to reveal the Ig production pathway components and function. Finally, we performed a limited rational drug screening to select suitable compounds for combination treatment. Results RNAseq and database mining revealed a strong association between the expression of plasma cell-specific deubiquitinase OTUD1, Ig production, and MM patient survival. Suppression of OTUD1 with shRNAs in RPMI8226 and MM1.S cell lines reduced Ig levels, increased proliferation, and induced bortezomib resistance. Conversely, inducible OTUD1 overexpression enhanced Ig production, slowed down proliferation, and increased bortezomib sensitivity. In the xenografts mouse models cells with high OTUD1 levels synthesized more Ig and developed smaller tumors. Intriguingly, the transcription of Ig genes was not influenced by OTUD1 expression suggesting that OTUD1 functions as a posttranslational regulator of Ig assembly. To gain mechanistic insight into the Ig pathway regulation by OTUD1, we utilized the biotin proximity labeling method (Turbo-ID) combined with mass spectrometry analysis. We found several novel OTUD1 interaction partners including the E3 ubiquitin ligase KEAP1 and endoplasmic reticulum (ER) redox protein PRDX4. We demonstrated that KEAP1 acts upstream of OTUD1 by regulating OTUD1 ubiquitination and stability. Consistently, survival analysis revealed that MM patients with high KEAP1 expression (low OTUD1) had a worse prognosis than patients with low levels of KEAP1 (high OTUD1). PRDX4 regulates disulfite bonds formation during protein folding and is uniquely expressed in fully differentiated plasma cells. Here, we revealed that OTUD1 specifically deubiquitinates and thus stabilizes PRDX4 inside the ER. Additionally, we performed rescue genetic experiments and found a direct link between the OTUD1-PRDX4 axis and Ig production. The increase in OTUD1 expression (high Ig) led to a dramatic increase in the total pool of ubiquitinated proteins formed mainly by misfolded Ig, while OTUD1 knockdown (low Ig) had an opposite effect. We showed that changes in the level of ubiquitinated proteins correlated with PI sensitivity. Of note, OTUD1 did not affect the expression of proteasome subunits, either their enzymatic activity. Our mechanistic findings prompted us to propose a novel therapeutic opportunity in PI resistant MM patients. We hypothesize that the resensitization of Ig low MM cells to PI could be achieved by enhancing ER stress leading to an increase in misfolded proteins that would ultimately saturate proteasomes. Indeed, from clinically relevant drugs tested so far, the HSP-90 inhibitor (17-AAG) reverted the PI resistance in OTUD1 low (Ig low) myeloma cells. An in vivo validation of the combination treatment and testing of Ig involvement in PI sensitivity and proliferation of MM cells is ongoing. Conclusion Here we present the discovery of a novel regulatory mechanism for Ig production in plasma cells. Based on our results and previously published studies, we conclude that Ig synthesis is a clinically significant factor related to PI response and MM patient survival. Our findings suggest that the intracellular Ig level is an important biomarker to identify patients benefiting the most from PI-based therapies. Finally, we provide a rational solution for selective, combination therapy to overcome PI resistance in MM patients with a decreased capacity to synthesize Ig. Figure Disclosures Hajek: Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria; PharmaMar: Consultancy, Honoraria; Oncopeptides: Consultancy.

scholarly journals A Phase 2 Study of Carfilzomib Plus Elotuzumab Plus Dexamethasone for Myeloma Patients Relapsed after 1-3 Prior Treatment Lines

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1975-1975 ◽  
Author(s):  
Raija Helena Silvennoinen ◽  
Dimitrios Tsallos ◽  
Hareth Nahi ◽  
Pekka Anttila ◽  
Perttu Koskenvesa ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Thrombotic Microangiopathy ◽  
Research Funding ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Prior Treatment ◽  
Phase 2 ◽  
Advisory Committees ◽  
Ras Genes ◽  
Phase 2 Study

Abstract Introduction: The median progression free survival (PFS) and overall survival (OS) of multiple myeloma (MM) patients have been prolonged due to novel agents combined with ASCT but the median OS in MM is still 7-8 years. Thus, the feasibility of new combinations and dosing of available agents must be investigated. The first proteasome inhibitor (PI), bortezomib (B), combined with elotuzumab and dexamethasone (d) showed superiority to Bd with PFS of 9.7 vs. 6.9 months, respectively, without excessive toxicity (Jakubowiak et al. Blood 2016;127:2833-40). In our study we investigate the safety, feasibility and initial efficacy of a second generation PI carfilzomib (K), SLAMF7 antibody elotuzumab (E) and dexamethasone (D) combination (KED) in relapsed or refractory MM (RRMM) patients. Patients and methods: Forty RRMM patients after 1-3 prior lines will be included in this phase 2 study after written informed consent. The primary endpoint is overall response rate (ORR). In patients achieving at least very good partial remission (VGPR) the quality of response will be assessed with high-sensitivity multicolour flow cytometry (MFC) according to the 8-colour EuroFlow protocol. Carfilzomib is given once weekly 20 mg/m2 on D1C1 and thereafter 70 mg/m2 in 28 day cycles on days 1, 8 and 15 in cycles 1-8 and on days 1 and 15 thereafter combined with weekly elotuzumab 10 mg/kg on days 1, 8 and 15 in cycles 1-2, thereafter on days 1 and 15; dexamethasone 40 mg on days 1, 8, 15 and 22 on cycles 1-8, thereafter on days 1 and 15. Treatment will continue until progression or excessive toxicity. Carfilzomib dose was 20/56 mg/m2 for the first two cycles for the first five patients to evaluate the safety. Additionally, patient samples collected prior to treatment will be comprehensively profiled by whole exome and RNA sequencing and evaluated for ex vivo response to the agents. Together, the study addresses clinical response, ex vivo-in vivo translation, identifies molecular biomarkers for the KED combination and facilitates precision guided clinical trials for RRMM. Results: By the end of July 2018, 11 patients have been enrolled. Median number of prior lines was 2 (1-3). Seven IgG-κ, two IgA-κ and one kappa and lambda light-chain patient are included. After a median of 6 (2-12) cycles ORR is 91% with 3 patients in VGPR (median MFC-MRD of 0,002%), 7 patients in PR, one in MR. Initial molecular characterization highlighted diverse subclonal backgrounds among the treated patients (Figure 1), but interestingly, the best responding VGPR patients displayed mutations to RAS genes in the dominant clones (NRAS 828, 2662; KRAS 733). At least PR was achieved after a median of 1 (1-4) cycle. Three patients have progressed and one patient withdrawn due to suspected thrombotic microangiopathy with manifestation of convulsions and pulmonary embolism. We noticed only one grade 2 infusion reaction after premedication. One patient developed autoimmune hemolytic anemia (AIHA), without red cell antibodies, suspected to be related to elotuzumab. She recovered with steroids and elotuzumab discontinuation and continued Kd without reappearance of AIHA. Another patient had grade 3 liver transaminase elevation but was able to continue treatment with dose reduction of carfilzomib and dexamethasone. Conclusion: To the best of our knowledge this is the first study evaluating the carfilzomib, elotuzumab and dexamethasone combination in RRMM with comprehensive molecular annotations. Among this small group of patients we noticed two unexpected severe adverse events; atypical AIHA and suspected thrombotic microangiopathy. AIHA should be excluded if unexpected anemia will appear during elotuzumab treatment. Preliminary results of this KED combination showed efficacy in patients with clonal RAS mutations and ORR of 91% after the median of two prior treatment lines using weekly carfilzomib dose of 70 mg/m2. Figure 1: Subclonal diversity in patient derived plasma cells. Clonal and subclonal fractions were evaluated by assessing peaks of variant allele frequencies of somatic mutations present in the myeloma plasma cells using kernel density estimation. For each plot, the fraction of cells carrying the mutation is represented on the x axis (1 = 100% of cells), and the probability density on the y axis. The mutated cell fraction (x axis) was calculated by adjusting mutant allele burden by copy number of the mutated loci. Disclosures Silvennoinen: Takeda: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Tsallos:Novartis: Research Funding. Anttila:Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. Räsänen:Amgen: Honoraria. Luoma:Amgen: Honoraria. Jantunen:Amgen: Honoraria; Genzyme/Sanofi: Honoraria; Takeda: Honoraria. Heckman:Celgene: Research Funding; Orion Pharma: Research Funding; Novartis: Research Funding.

scholarly journals Predicting Response to Dasatinib Using a Computational Model and Its Validation: A Beat AML Project Study

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1541-1541
Author(s):  
Jeffrey W. Tyner ◽  
Brian J. Druker ◽  
Cristina E. Tognon ◽  
Stephen E Kurtz ◽  
Leylah M. Drusbosky ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Computational Biology ◽  
Board Of Directors ◽  
Research Funding ◽  
Drug Response ◽  
Drug Sensitivity ◽  
Ex Vivo ◽  
Patient Specific ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Background: New prognostic factors have been recently identified in AML patient population that include frequent mutations of receptor tyrosine kinases (RTK) including KIT, PDGFR, FLT3, that are associated with higher risk of relapse. Thus, targeting RTKs could improve the therapeutic outcome in AML patients. Aim: To create a digital drug model for dasatinib and validate the predicted response in AML patient samples with ex vivo drug sensitivity testing. Methods: The Beat AML project (supported by the Leukemia & Lymphoma Society) collects clinical data and bone marrow specimens from AML patients. Bone marrow samples are analyzed by conventional cytogenetics, whole-exome sequencing, RNA-seq, and an ex vivo drug sensitivity assay. For 50 randomly chosen patients, every available genomic abnormality was inputted into a computational biology program (Cell Works Group Inc.) that uses PubMed and other online resources to generate patient-specific protein network maps of activated and inactivated pathways. Digital drug simulations with dasatinib were conducted by quantitatively measuring drug effect on a composite AML disease inhibition score (DIS) (i.e., cell proliferation, viability, and apoptosis). Drug response was determined based on a DIS threshold reduction of > 65%. Computational predictions of drug response were compared to dasatinib IC50 values from the Beat AML ex vivo testing. Results: 23/50 (46%) AML patients had somatic mutations in an RTK gene (KIT, PDGFR, FLT3 (ITD (n=15) & TKD (n=4)), while 27/50 (54%) were wild type (WT) for the RTK genes. Dasatinib showed ex vivo cytotoxicity in 9/50 (18%) AML patients and was predicted by CBM to remit AML in 9/50 AML patients with 4 true responders and 5 false positive. Ex vivo dasatinib responses were correctly matched to the CBM prediction in 40/50 (80%) of patients (Table1), with 10 mismatches due to lack of sufficient genomic information resulting in profile creation issues and absence of sensitive loops in the profile. Only 4/23 (17%) RTK-mutant patients and 5/27(19%) RTK-WT patients were sensitive to dasatinib ex vivo, indicating that presence of somatic RTK gene mutations may not be essential for leukemia regression in response to dasatinib. Co-occurrence of mutations in NRAS, KRAS and NF1 seemed to associate with resistance as seen in 10 of the 14 profiles harboring these mutations. Conclusion: Computational biology modeling can be used to simulate dasatinib drug response in AML with high accuracy to ex vivo chemosensitivity. DNA mutations in RTK genes may not be required for dasatinib response in AML. Co-occurrence of NRAS, KRAS and NF1gene mutations may be important co-factors in modulating response to dasatinib. Disclosures Tyner: Leap Oncology: Equity Ownership; Syros: Research Funding; Seattle Genetics: Research Funding; Janssen: Research Funding; Incyte: Research Funding; Gilead: Research Funding; Genentech: Research Funding; AstraZeneca: Research Funding; Aptose: Research Funding; Takeda: Research Funding; Agios: Research Funding. Druker:Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Millipore: Patents & Royalties; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Celgene: Consultancy; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Patient True Talk: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy. Sahu:Cellworks Research India Private Limited: Employment. Vidva:Cellworks Research India Private Limited: Employment. Kapoor:Cellworks Research India Private Limited: Employment. Azam:Cellworks Research India Private Limited: Employment. Kumar:Cellworks Research India Private Limited: Employment. Chickdipatti:Cellworks Research India Private Limited: Employment. Raveendaran:Cellworks Research India Private Limited: Employment. Gopi:Cellworks Research India Private Limited: Employment. Abbasi:Cell Works Group Inc.: Employment. Vali:Cell Works Group Inc.: Employment. Cogle:Celgene: Other: Steering Committee Member of Connect MDS/AML Registry.

scholarly journals Niche-like Ex Vivo High Throughput (NEXT) Drug Screening Platform in Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-13
Author(s):  
Reinaldo Dal Bello Figueiras ◽  
Justine Pasanisi ◽  
Romane Joudinaud ◽  
Matthieu Duchmann ◽  
Gaetano Sodaro ◽  
...  
Keyword(s):  
Precision Medicine ◽  
Board Of Directors ◽  
Drug Screening ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Low Dose ◽  
Ex Vivo ◽  
Advisory Committees ◽  
Ex Vivo Culture

Context. Functional precision medicine is gaining momentum in AML, notably through ex vivo drug sensitivity screening (DSS) of primary patient (pt) cells (Pemovska Cancer Discov 2013, Tyner Nature 2018). The DSS landscape differs across genetic AML subgroups (Tyner Nature 2016), of which NPM1mut is the most frequent (Papaemmanuil NEJM 2016). DSS in AML has mostly been done in standard conditions, with overall viability as unique endpoint. Niche signals, which can be partly mimicked in vitro, convey drug resistance in vivo. Drugs can induce a variety of cell fates in AML. Induction of differentiation rather than killing of blasts, can result in false negative results in global viability assays. Persistence of leukemic stem cells (LSC) represents a major cause of treatment failure. GPR56 is a ubiquitous surface marker enriching for LSCs and stable upon short-term ex vivo culture (Pabst Blood 2016). Objectives. To develop an ex vivo niche-like multiparametric DSS platform for primary AML cells. To validate its clinical relevance in NPM1mut pts treated with conventional DNR-AraC chemotherapy. To discover new sensitizers to DNR-AraC chemotherapy in NPM1mut AML. Results. We designed an MFC panel to count viable blasts and measure their differentiation (CD11b/CD14/CD15) and stemness (GPR56) after exclusion of residual lymphocytes (Figure 1A). We validated GPR56 expression as stemness marker based on increased retention of GPR56+ cells in niche-like coculture combining hypoxia (O2 3%) and MSC compared to standard conditions (p<0.0001, Figure 1B) and limit dilution assays of residual GPR56+ cells at 72h of niche-like culture in 3 NPM1mut AMLs (Figure 1C). Using a limited panel of 14 drugs or combinations at fixed concentrations, our MFC readout after 72h of coculture with MSC+hypoxia revealed the distinct mode of action of different agents or combinations including the differentiation activity of ATO-ATRA, the LSC-sparring cytotoxicity of DNR-AraC and the anti-LSC- activity of VEN (Figure 1D). To further mimic in vivo conditions, we derived a MEMa-based plasma-like medium (PLM) based on targeted metabolomics (Figure 1E) and electro-chemoluminescent cytokine assays of 29 diagnostic AML bone marrow plasma samples compared to conditioned media of primary AML cells cultured in niche-like conditions (MSC, hypoxia). This instructed the design of our custom PLM with dialyzed FBS and defined low-dose (~1 ng/mL range) cytokines (CK) and amino-acid (AA) concentrations. We next investigated the contribution of MSCs, hypoxia, plasma-like AAs and CKs on blasts viability, differentiation, stemness and drug response in 3 NPM1mut AMLs exposed to fixed concentrations of 6 core AML therapies. This analysis uncovered significant interactions between these 4 niche components in dictating blast viability and stemness upon 72h ex vivo culture (Figure 1F) and revealed the distinct contribution of these niche components to drug sensitivity. RNA-seq of primary blasts cultured in niche-like, plasma-like conditions revealed marked enrichment of stemness pathways compared to ex vivo culture in standard conditions. Finally, we explored DNR-AraC (five-point serial dilution) alone or in combination with fixed, clinically relevant concentrations of 24 drugs in 49 primary AML samples (including 34 NPM1mut). Using AUCs of DNR-AraC on lymphocytes as internal control, we first validated our NEXT assay on NPM1 MRD levels in the 34 NPM1mut pts treated frontline with conventional DNR-AraC regimens (Figure 1G). Across all 49 pts, we uncovered 11 different optimal 'third-drugs', stressing the role of our NEXT assay to deploy precision medicine in daily practice. At the population level, we could nominate 3 top combinations, two of which are currently in clinical investigation (Venetoclax and Selinexor). The unpublished sensitizing effect of low dose (0.25µM) Ruxolitinib on DNR-AraC uncovered with our NEXT assay is currently being investigated in PDX models. Conclusion. We designed the NEXT assay, a multiparametric drug screening of AML viability, differentiation and stemness in niche-like culture combining hypoxia, stromal interactions and plasma-like medium. Components of the niche-like culture interact to govern leukemic viability and stemness. Our assay could predict MRD achievement in NPM1mut AML and identifies novel sensitizers to DNR-AraC in these pts. Disclosures Clappier: Amgen: Honoraria, Research Funding. Ades:Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; novartis: Research Funding; Celgene/BMS: Research Funding. Itzykson:Amgen: Membership on an entity's Board of Directors or advisory committees; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Stemline: Membership on an entity's Board of Directors or advisory committees; Oncoethix (now Merck): Research Funding; Janssen: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria; Daiichi Sankyo: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS (Celgene): Honoraria; Sanofi: Honoraria; Astellas: Honoraria.

scholarly journals Characterization of Synergistic Selinexor Combinations of Dexamethasone, Pomalidomide, Elotuzumab and Daratumumab in Primary MM Samples Ex Vivo

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 29-30
Author(s):  
Kenneth H. Shain ◽  
Rafael Renatino-Canevarolo ◽  
Mark B. Meads ◽  
Praneeth Reddy Sudalagunta ◽  
Maria D Coelho Siqueira Silva ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Board Of Directors ◽  
Nuclear Export ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Ex Vivo ◽  
Single Agent ◽  
Gene Mutations ◽  
Drug Combinations ◽  
Advisory Committees

Introduction. Multiple myeloma (MM) is an incurable plasma cell malignancy with a growing list of anti-MM therapeutics. However, the development of predictive biomarkers has yet to be achieved for nearly all MM therapeutics. Selinexor (SELI), a nuclear export inhibitor targeting exportin 1 (XPO1), has been approved with dexamethasone (DEX) in penta-refractory MM. Clinical studies investigating promising SELI- 3 drug combinations are ongoing. Here, we have investigated potential synergistic combinations of SELI and anti-MM agents in terms of ex vivo sensitivity, as well as paired RNAseq and WES to identify companion biomarkers. Methods. MM cells isolated from fresh bone marrow aspirates were tested for drug sensitivity in an organotypic ex vivo drug sensitivity assay, consisting of co-culture with stroma, collagen matrix and patient-derived serum. Single agents were tested at 5 concentrations, while two-drug combinations were tested at fixed ratio of concentrations. LD50 and area under the curve (AUC) were assessed during 96h-exposure as metrics for drug resistance. Drug synergy was calculated as a modified BLISS model. Matching aliquots of MM cells had RNAseq and WES performed through ORIEN/AVATAR project. Geneset enrichment analysis (GSEA) was conducted using both AUC and LD50 as phenotypes for single agents and combinations. Both curated pathways (KEGG and cancer hallmarks) and unsupervised gene clustering were used as genesets. Student t-tests with multiple test correction were used to identify non-synonymous mutations in protein coding genes associated with single agent or combination AUC. Results. For this analysis, a cohort of specimens from 103 patients (48% female, 4% Hispanic, 11% African American) was tested with SELI and/or DEX. with a median of 2 lines of therapy (0-12). A smaller cohort of 37 have been examined with SELI, pomalidomide (POM), elotuzumab (ELO) and daratumumab (DARA). Within this cohort we observed synergy between SELI and DEX, POM and ELO as shown in Figure 1. The volcano plot illustrates the number of samples, maximum drug concentration, as well as magnitude (x- axis) and significance (y- axis) of synergy. Although the SELI-DARA combination trended toward synergy, statistical significance was not achieved. To identify molecular mechanisms and biomarkers associated with sensitivity to SELI and SELI- combinations, we investigated paired RNAseq and WES with ex vivo sensitivity. Initially, we conducted GSEA on two cohorts of primary MM samples tested with SELI alone at 5µM (n=53) and 10µM (n=50). Cell adhesion (KEGG CAMS), inflammatory cytokines (KEGG ASTHMA), and epithelial mesenchymal transition (HALLMARK EMT) were associated with resistance in both cohorts, while the HALLMARK MYC TARGETS was associated with sensitivity (FWER p<0.05). Mutational analysis identified 46 gene mutations associated with SELI resistance and 100 associated with sensitivity at 5µM, and 87 and 27 mutations associated with SELI resistance and sensitivity, respectively, at 10µM. Two gene mutations were identified in both cohorts: BCL7A, involved in chromatin remodeling, was associated with sensitivity and CEP290, a microtubule binding protein, associated with resistance (p<0.05). Analysis of both gene sequences (NetNES 1.1) identified nuclear export signal (NES) residues suggesting these may be XPO1 cargo. Additionally, translocation t(11;14) was associated with SELI resistance in the 5µM cohort (p=0.037). The completed set of 50 specimens ex vivo, RNAseq and WES analysis will be mature and updated for the potential presentation at ASH. Conclusions. We observed ex vivo synergy between SELI and DEX, POM and ELO. Molecular analysis of matched ex vivo drug sensitivity, transcriptome and mutational profile identified environment-mediated drug resistance pathways positively correlated with SELI single agent resistance, as well as MYC regulated genes associated with ex vivo sensitivity. We also identified a list of mutations associated with SELI drug resistance and sensitivity, with special emphasis on two novel NES-containing genes, CEP290 and BCL7A. The next step of this project is to analyze transcriptional and mutational patterns associated with ex vivo synergy in the combinations here described, as putative biomarkers for future clinical investigation. Disclosures Shain: Amgen: Speakers Bureau; Adaptive: Consultancy, Honoraria; Karyopharm: Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GlaxoSmithKline: Speakers Bureau; Janssen: Honoraria, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; AbbVie: Research Funding. Kulkarni:M2GEN: Current Employment. Zhang:M2GEN: Current Employment. Hampton:M2GEN: Current Employment. Argueta:Karyopharm: Current Employment. Landesman:Karyopharm Therapeutics Inc: Current Employment, Current equity holder in publicly-traded company. Siqueira Silva:AbbVie: Research Funding; NIH/NCI: Research Funding; Karyopharm: Research Funding.

Broad Activity of Apto-253 in AML and Other Hematologic Malignancies Correlates with KLF4 Expression Level

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1358-1358 ◽  
Author(s):  
Stephen E. Kurtz ◽  
Daniel Bottomly ◽  
Beth Wilmot ◽  
Shannon K. McWeeney ◽  
William Rice ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Board Of Directors ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Ex Vivo ◽  
Hematologic Malignancies ◽  
Employment Equity ◽  
Advisory Committees ◽  
Flt3 Inhibitor ◽  
Equity Ownership

Abstract Introduction: Aberrant expression of the homeodomain transcription factor CDX2 has recently been reported in a large proportion of AML cases. One consequence of CDX2 deregulation appears to be repressed expression of the transcription factor KLF4. Repression of KLF4 was shown to be critical for CDX2-mediated tumorigenesis, and forced genetic de-repression of KLF4 led to apoptosis of AML cells. APTO-253 is a novel small molecule that induces the expression of KLF4 and is cytotoxic to AML cell lines at low-nanomolar concentrations. We evaluated the activity of APTO-253 against a broad panel of primary specimens from patients with acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN). APTO-253 was tested both as a single agent and in combinations with 2 other emerging targeted therapies, the BET bromodomain inhibitor JQ1 and the FLT3 inhibitor quizartinib. Methods: We used an ex vivo drug sensitivity assay to determine the activity of APTO-253, JQ1, and quizartinib across increasing concentrations of each agent up to 10 μM. Combinations were tested at fixed, equimolar ratios over the same concentration range. After a 3-day ex vivo culture, cell viability was assessed using a colorimetric tetrazolium-based MTS assay, and IC50 values were calculated. RNA-Seq was performed on AML specimens to permit investigation of correlations of drug sensitivity with gene expression levels. Results: We evaluated specimens from 177 patients with a variety of hematologic malignancy diagnoses (80 AML, 72 CLL, 25 MDS/MPN). The highest frequency of APTO-253 sensitivity occurred in AML, with 43/80 (54%) samples exhibiting an IC50 <1 μM. At this cutoff, 25/72 (35%) CLL samples and 3/25 (12%) MDS/MPN samples were sensitive to APTO-253. The average expression of KLF4 mRNA was 2-fold lower among AML samples with an IC50 <1 µM compared to those with IC50 >1 µM (p=0.07). Approximately 65% (56/87) of cases tested with a combination of APTO-253 and JQ1 showed the combination IC50 to be at least 2-fold lower than the IC50 of either agent alone. This enhanced efficacy of APTO-253 with JQ1 was observed across all 3 hematologic malignancies tested, whereas quizartinib enhancement of APTO-253 sensitivity was confined to AML (14/38, or 37% showed reduced IC50). Conclusions: These results support the potential of KLF4 as an important and frequently dysregulated master transcription factor in AML and suggest that the KLF4 inducer APTO-253 is effective at killing tumor cells in a majority of AML samples. The data also indicate activity of APTO-253 in other hematologic malignancies, namely CLL. Expression level of KLF4 may be one component of a biomarker for prediction of APTO-253 efficacy; a more extensive global gene expression signature analysis is under way. Finally, these data have identified prominent interaction of APTO-253 with the BET bromodomain inhibitor JQ1, as well as AML-restricted interaction of APTO-253 with the FLT3 inhibitor quizartinib, suggesting these classes of drugs as potential combination partners for APTO-253. Disclosures Rice: Aptose Biosciences: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Howell:Aptose Biosciences: Consultancy, Equity Ownership; Angstrom: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Abeoda: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; InhibRx: Equity Ownership. Vellanki:Aptose Biosciences: Employment, Equity Ownership. Druker:Oncotide Pharmaceuticals: Research Funding; Sage Bionetworks: Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis Pharmaceuticals: Research Funding; Henry Stewart Talks: Patents & Royalties; McGraw Hill: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; AstraZeneca: Consultancy; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tyner:Incyte: Research Funding; Janssen Pharmaceuticals: Research Funding; Constellation Pharmaceuticals: Research Funding; Array Biopharma: Research Funding; Aptose Biosciences: Research Funding.

scholarly journals Associating Ex Vivo Drug Sensitivity with Differentiation Status Identifies Effective Drug Combinations for Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1301-1301
Author(s):  
Christopher A. Eide ◽  
Stephen E. Kurtz ◽  
Andy Kaempf ◽  
Nicola Long ◽  
Daniel Bottomly ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Ex Vivo ◽  
Single Agent ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Acute Myeloid

Abstract The development of molecularly-targeted therapies to improve outcomes relative to chemotherapy for acute myeloid leukemia (AML) is impeded by the heterogeneity of genetic aberrations that contribute to disease. Among the multitude of biological mechanisms that lead to AML disease initiation and progression is dysregulation of cytokine signaling pathways, a hallmark of chronic inflammation, which contribute to the growth, survival, and differentiation state of AML cells. We have previously shown that IL-1β, a pro-inflammatory cytokine expressed by many cell types including macrophages and monocytes, stimulates proliferation of leukemic blasts independent of mutational status in primary AML samples via enhanced phosphorylation of p38α MAPK, an effect that can be blocked by IL-1 receptor knockdown or by pharmacologic inhibition (Carey 2017). Additionally, recent studies have shown sensitivity to the approved BCL2 inhibitor venetoclax in AML associates with undifferentiated leukemic cells (Pei 2020; Zhang 2018; Majumder 2020). Based on these associations, we evaluated the combination of doramapimod (DORA), a p38 MAPK inhibitor, with venetoclax (VEN) for potential enhanced sensitivity on primary AML cells. Ex vivo drug screening of primary AML patient samples (n=335) revealed significantly enhanced efficacy of VEN+DORA compared to either single agent (Nemenyi test; p&lt;0.0001). This broad sensitivity of the VEN+DORA combination was not significantly associated with an array of clinical, genetic, and mutational features in the patient samples tested, in contrast to single agents, particularly VEN. Analysis of blood cell differential counts of patient samples tested identified increased monocyte levels were significantly correlated with sensitivity to DORA and resistance to VEN as single agents (Spearman r = -0.3 and 0.6; p&lt;0.0001), associations that were not apparent with the combination. For patient samples with accompanying FAB differentiation state-based designations (n=108), sensitivities of the combination were similar across classifications of undifferentiated (M0/M1) through monocytic (M4/M5) acute leukemia. In contrast, single-agent VEN was significantly more sensitive in undifferentiated compared to monocytic specimens, whereas DORA sensitivity showed the reverse trend (though to a lesser degree). These differences in sensitivity were further validated by immunophenotyping data where available (n=105), which showed surface markers associated with resistance to VEN (CD11b, CD14, CD16, CD56, CD64, HLADR; Wilcoxon Rank Sum, p&lt;0.001 to p=0.007) or sensitivity to VEN (CD117; p=0.001) or DORA (CD14; HLADR; p=0.004). By contrast, none of these associations significantly distinguished sensitivity for the VEN+DORA combination. Expression levels of MAPK14 and BCL2, the respective primary targets of DORA and VEN, were concordant with their respective drug sensitivities associated with FAB classification; that is, significantly higher levels of BCL2 in M0/M1 leukemias and MAPK14 in M4/M5 cases (Mann-Whitney test; p&lt;0.0001; n=145). Further dissection of transcriptomic and drug sensitivity data revealed strong correlation and gene set enrichment for DORA and VEN sensitivities with monocyte-like and progenitor-like signatures, respectively (n=225), for cell differentiation states previously described for AML (van Galen 2019), and these associations diminished for the combination treatment. Lastly, the VEN+DORA combination enhanced efficacy and synergistic inhibition was confirmed using human AML cell line models tested with a matrix of potential dose concentrations. Taken together, these findings suggest that exploiting distinct, complementary sensitivity profiles of targeted therapies with respect to leukemic differentiation state, such as dual targeting of p38 MAPK and BCL2, offers an opportunity for broad, enhanced efficacy across the clinically challenging heterogeneous landscape of AML. Disclosures Druker: Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Recludix Pharma, Inc.: Consultancy; EnLiven: Consultancy, Research Funding; Pfizer: Research Funding; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; Aileron: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; GRAIL: Current equity holder in publicly-traded company; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Iterion Therapeutics: Membership on an entity's Board of Directors or advisory committees; Nemucore Medical Innovations, Inc.: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharma: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees. Tyner: Genentech: Research Funding; Takeda: Research Funding; Astrazeneca: Research Funding; Constellation: Research Funding; Agios: Research Funding; Petra: Research Funding; Incyte: Research Funding; Array: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Seattle Genetics: Research Funding; Schrodinger: Research Funding.

Deregulation of TNFRSF18 (GITR) Through Promoter CpG Island Methylation Induces Tumor Proliferation in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2424-2424
Author(s):  
Yang Liu ◽  
Yong Zhang ◽  
Phong Quang ◽  
Hai T Ngo ◽  
Feda Azab ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Advisory Committees ◽  
Brdu Assay

Abstract Abstract 2424 Introduction Tumor necrosis factor receptor super families (TNFRSFs) play an important role in activation of lymphocyte and cell apoptosis. However the function of TNFRSFs in multiple myeloma (MM) remains unknown. Loss of function mutation of Fas antigen (TNFRSF6) was identified in MM cells, thus suggesting the possible role of TNFRSFs in regulating MM pathogenesis. We therefore investigated the epigenetic mechanisms that may mediate inactivation of TNFRSFs and its functional role in MM. Methods Dchip software was utilized for analyzing gene expression dataset. DNA was extracted from both primary CD138+ MM plasma cells and MM cell lines using blood & tissue DNA isolation kit (Qiagen, Inc.). Expression of GITR in primary CD138+ plasma cells was detected by Imunohistochemistry (IHC) DNA methylation was analyzed by methylated DNA immunoprecipitation (Medip) assay and bisulfate sequencing. 5'azacytidine was used to demethylate genomic DNA. Gene expression was detected by qRT-PCR and confirmed at the protein level by flow cytometry and western-blot. Over-expression of GITR was obtained in MM1.S cells by using GITR recombinant plasmid and electroporation. Apoptosis was determined using Annexin/PI staining and flow cytometry analysis. Activation of apoptotic signaling was studied by western blot. Cell survival and proliferation were analyzed by MTT and BrdU assay, respectively. Recombinant GITR-lentivirus was obtained from the supernatant of culture medium after 72 hours transfection in 293 cells. GFP positive MM cells were sorted and analyzed by flow cytometry. In vivo effect of GITR on MM tumor growth was determined by injection of GITR over-expressing MM cells in null mice. Mice skull, femur and vertebrae were isolated after 4 weeks injection. Anti-human CD138+ mAb microbead was used to detect MM cells extracted from mice tissue by flow cytometry. Results Gene-expression profiling showed down-regulation of TNFRSFs, including TNFRSF11A, TNFRSF11B, TNFRSF8, TNFRSF10C, TNFRSF9, TNFRSF21, TNFRSF1B, TNFRSF1A and TNFRSF18, compared to normal plasma cells. Moreover, Our IHC results also showed that GITR expression was positive in primary CD138+ plasma cells from 9 normal bone marrow, but negative in 9 MM samples. Importantly, we found that low GITR expression significantly correlated with MM progression. Indeed, GITR gene levels were lower in smoldering and active MM patients compared to MGUS patients and normal donors. Promoter CpG island (CGI) methylation of GITR was indentified in 5 out of 7 MM primary bone marrow (BM)-derived CD138+ cells but not in normal BM-derived plasma cells. Bisulfate sequencing and Medip assay showed that methylation of GITR was significantly associated with GITR expression in 5 MM cell lines, including MM1.S, OPM1, U266, RPMI and INA6. Promoter CGI of GITR was highly methylated leading to complete silencing of GITR in MM1.S cell line. GITR expression was significantly up-regulated in MM cells upon treatment with the 5'azacytidine. MTT and BrdU assay revealed that the proliferation and survival of MM1.S cells was disrupted in the GITR over-expressing MM1.S cells, notably with inhibition of cell proliferation compared to control vector infected cells. Moreover induction of cytotoxicity in GITR over-expressing cells was confirmed by using GFP competition assay. GITR-induced apoptosis was supported by induction of caspase 8 and 3 cleavage. The inhibition of human CD138+ plasma cell growth in the bone marrow of SCID mice using a disseminated MM xenograft model was observed in the experimental group injected with GITR expressing cells compared to the control group after 4 weeks injection. Conclusion Our findings uncovered a novel epigenetic mechanism contributing to MM pathogenesis, showing the role of GITR methylation as a key regulator of MM cell survival. Disclosures: Roccaro: Roche:. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Development of a Cancer Pharmacopeia-Wide Ex-Vivo Drug Sensitivity and Resistance Testing (DSRT) Platform: Identification of MEK and mTOR As Patient-Specific Molecular Drivers of Adult AML and Potent Therapeutic Combinations with Dasatinib

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2487-2487
Author(s):  
Mika Kontro ◽  
Caroline Heckman ◽  
Evgeny Kulesskiy ◽  
Tea Pemovska ◽  
Maxim Bespalov ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Drug Response ◽  
Drug Sensitivity ◽  
Ex Vivo ◽  
Molecular Profiling ◽  
Resistance Testing ◽  
Patient Specific ◽  
Targeted Drugs ◽  
Response Profiles

Abstract Abstract 2487 Introduction: The molecular drivers of adult AML as well as the determinants of drug response are poorly understood. While AML genomes have recently been sequenced, many cases do not harbor druggable mutations. Treatment options are particularly limited for relapsed and refractory AML. Due to the molecular heterogeneity of the disease, optimal therapy would likely consist of individualized combinations of targeted and non-targeted drugs, which poses significant challenges for the conventional paradigm of clinical drug testing. In order to better understand the molecular driver signals, identify individual variability of drug response, and to discover clinically actionable therapeutic combinations and future opportunities with emerging drugs, we established a diagnostic ex-vivo drug sensitivity and resistance testing (DSRT) platform for adult AML covering the entire cancer pharmacopeia as well as many emerging anti-cancer compounds. Methods: DSRT was implemented for primary cells from adult AML patients, focusing on relapsed and refractory cases. Fresh mononuclear cells from bone marrow aspirates (>50% blast count) were screened in a robotic high-throughput screening system using 384-well plates. The primary screening panel consisted of a comprehensive collection of FDA/EMA-approved small molecule and conventional cytotoxic drugs (n=120), as well as emerging, investigational and pre-clinical oncology compounds (currently n=90), such as major kinase (e.g. RTKs, checkpoint and mitotic kinases, Raf, MEK, JAKs, mTOR, PI3K), and non-kinase inhibitors (e.g. HSP, Bcl, activin, HDAC, PARP, Hh). The drugs are tested over a 10,000-fold concentration range resulting in a dose-response curve for each compound and with combinations of effective drugs explored in follow-up screens. The same samples also undergo deep molecular profiling including exome- and transcriptome sequencing, as well as phosphoproteomic analysis. Results: DSRT data from 11 clinical AML samples and 2 normal bone marrow controls were bioinformatically processed and resulted in several exciting observations. First, overall drug response profiles of the AML samples and the controls were distinctly different suggesting multiple leukemia-selective inhibitory effects. Second, the MEK and mTOR signaling pathways emerged as potential key molecular drivers of AML cells when analyzing targets of leukemia-specific active drugs. Third, potent new ex-vivo combinations of approved targeted drugs were uncovered, such as mTOR pathway inhibitors with dasatinib. Fourth, data from ex-vivo DSRT profiles showed excellent agreement with clinical response when serial samples were analyzed from leukemia patients developing clinical resistance to targeted agents. Summary: The rapid and comprehensive DSRT platform covering the entire cancer pharmacopeia and many emerging agents has already generated powerful insights into the molecular events underlying adult AML, with significant potential to facilitate individually optimized combinatorial therapies, particularly for recurrent leukemias. DSRT will also serve as a powerful hypothesis-generator for clinical trials, particularly for emerging drugs and drug combinations. The ability to correlate response profiles of hundreds of drugs in clinical ex vivo samples with deep molecular profiling data will yield exciting new translational and pharmacogenomic opportunities for clinical hematology. Disclosures: Mustjoki: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Porkka:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Kallioniemi:Abbot/Vysis: Patents & Royalties; Medisapiens: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bayer Schering Pharma: Research Funding; Roche: Research Funding.

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