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.

Targeting EZH2 in Multiple Myeloma Could be Promising for a Subgroup of MM Patients in Combination with IMiDs

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 311-311 ◽  
Author(s):  
Laurie Herviou ◽  
Alboukadel Kassambara ◽  
Stephanie Boireau ◽  
Nicolas Robert ◽  
Guilhem Requirand ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Newly Diagnosed ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Ezh2 Expression ◽  
Bone Marrow Plasma

Abstract Multiple Myeloma is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow.Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes. We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio > 2 and FDR (false discovery rate) < 5%) were identified, including the EZH2 histone methyltransferase. EZH2, the enzymatic subunit of Polycomb Repressive Complex 2, is a histone methyltransferases able to repress gene expression by catalyzing H3K27me3 histone mark. EZH2 overexpression has been associated with numerous hematological malignancies, including MM. We thus studied EZH2 role in MM physiopathology and drug resistance. EZH2 expression was analyzed in normal bone marrow plasma cells (BMPCs; N=5), primary myeloma cells from newly diagnosed patients (MMCs; N=206) and human myeloma cell lines (HMCLs; N=40) using Affymetrix microarrays. EZH2 gene is significantly overexpressed in MMCs of patients (median 574, range 105 - 4562) compared to normal BMPCs (median = 432; range: 314 - 563) (P < 0.01). The expression is even higher in HMCLs (median 4481, range 581 - 8455) compared to primary MMCs or BMPCs (P < 0.001). High EZH2 expression is associated with a poor prognosis in 3 independent cohorts of newly diagnosed patients (Heidelberg-Montpellier cohort - N=206, UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N =158). Furthermore, GSEA analysis of patients with high EZH2 expression highlighted a significant enrichment of genes involved in cell cycle, downregulated in mature plasma cells vs plasmablasts, and EZH2 targets. Specific EZH2 inhibition by EPZ-6438 EZH2 inhibitor induced a significant decrease of global H3K27me3 in all the HMCLs tested (P < 0.01) and inhibited MM cell growth in 5 out of the 6 HMCLs tested. The inhibitory effect of EZH2 inhibitor on MM cell growth appeared at day 6 suggesting that it is mediated by epigenetic reprogramming. To confirm that EZH2 is also required for the survival of primary MMCs from patients, primary MM cells (n = 17 patients) co-cultured with their bone marrow microenvironment and recombinant IL-6 were treated with EPZ-6438. As identified in HMCLs, EZH2 inhibition significantly reduced the median number of viable myeloma cells by 35% (P = 0.004) from a subset of patients (n=9) while the other group (n=8) was resistant. Of interest, EPZ-6438 induced a significant global H3K27me3 decrease in both groups of patient. RNA sequencing of 6 HMCLs treated with EPZ-6438 combined with H3K27me3 ChIP analyses allowed us to create an EZ GEP-based score able to predict HMCLs and primary MM cells sensitivity to EZH2 inhibitors. We also observed a synergy between EPZ-6438 and Lenalidomide, a conventional drug used for MM treatment. More interestingly, pretreatment of myeloma cells with EPZ-6438 significantly re-sensitize drug-resistant MM cells to Lenalidomide. Investigating the effect of EPZ-6438/Lenalidomide combination in MMC, we identified that IKZF1, IRF4 and MYC protein levels were significantly more inhibited by the combination treatment (65.5%, 63.9% and 14.8% respectively) compared with Lenalidomide (51.5%, 43% and 2.2%) or EPZ-6438 (45.2%, 38.7% and 6.2%) alone. Clinical trials are ongoing with EZH2 inhibitors in lymphoma and could be promising for a subgroup of MM patients in combination with IMiDs. Furthermore, the EZ score enables identification of MM patients with an adverse prognosis and who could benefit from treatment with EZH2 inhibitors. Disclosures Goldschmidt: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

scholarly journals A Multicenter Phase II Single Arm Trial of Isatuximab in Patients with High Risk Smoldering Multiple Myeloma (HRSMM)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3116-3116 ◽  
Author(s):  
Elisabet E. Manasanch ◽  
Sundar Jagannath ◽  
Hans C. Lee ◽  
Krina K. Patel ◽  
Connor Graham ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
High Risk ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Infusion Reaction ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
History Of ◽  
Smoldering Myeloma

Background High risk smoldering multiple myeloma (HRSMM), defined as having immunoparesis and at least 95% abnormal plasma cells/all plasma cells by advanced flow cytometry, has a risk of progression to multiple myeloma of about 75% after 5 years of diagnosis. These patient have no symptoms and current standard is to follow them without treatment. Isatuximab is an IgG1 monoclonal antibody that binds to CD38 highly expressed in myeloma cells. Isatuximab has activity as monotherapy (overall response rate (ORR) 35%), with lenalidomide/dexamethasone (ORR 56%) and pomalidomide/dexamethasone (ORR 62%) in relapsed MM. We designed a phase II study to test the efficacy of isatuximab in high risk smoldering myeloma. Our study is registered in clinicaltrials.gov as NCT02960555. Methods The primary endpoint of the study is the ORR of isatuximab 20 mg/kg IV days 1, 8, 15, 22 cycle 1; days 1, 15 cycles 2-6 and day 1 cycles 7-30 in high risk smoldering myeloma. 24 patients were accrued in the first stage (of maximum 61 patients). Secondary endpoints are PFS, OS, clinical benefit rate (CBR). Exploratory endpoints are quality of life analysis (QoL), MRD, molecular/immune characterization using DNA/RNA sequencing of myeloma cells and the microenvironment before and after treatment. Results 24 patients with HRSMM were accrued from 02/08/2017 until 12/21/2018 (Table 1). All patients are evaluable for response. Best responses: ORR (≥PR) 15(62.5%), CR MRD- flow at 10-5 1 (5%), VGPR 4 (17%), PR 10 (42%), minor response (MR) 4 (18%), stable disease 5 (21%); CBR (≥MR) 79%. Median number of cycles received were 11.5 (range 6-30). Five patients have stopped treatment (one has completed the study, one with heavy history of smoking was diagnosed with squamous cell cancer of the tongue, one could no longer travel to treatments due to relocation, two progressed to active multiple myeloma after 16 and 6 cycles of treatment, respectively). There have been no deaths. DNA/RNA seq is ongoing for biomarkers of response. There were 5 grade 3 severe treatment-related adverse events (RAE) which resolved to baseline: dyspnea -related to infusion reaction (n=2), headache (n=1), ANC decrease (n=1), urinary tract infection (n=1). Most common grade 1-2 related adverse events (n): nausea (7), vomit (5), WBC decrease (3), diarrhea (3), fatigue (6), headache (4), mucositis (4), myalgia (4) and infusion reaction (3). In patients with available QoL functional scores (n=9 at baseline and n=7 after 6 months of therapy), isatuximab was effective in reducing their anxiety and worry of progression to multiple myeloma. Isatuximab also improved general QoL scores by the end of cycle 6 of treatment which were now comparable to those in the general population (Figure 1). Conclusion Isatuximab is very well tolerated, results in high response rates in HRSMM and has the potential to change the natural history of this disease. In ongoing QoL analysis, initial data shows improvement in QoL and decreased cancer worry after isatuximab treatment. Immune-genomic analysis is ongoing and may identify patients that benefit the most from treatment. Disclosures Manasanch: celgene: Honoraria; merck: Research Funding; quest diagnostics: Research Funding; sanofi: Research Funding; BMS: Honoraria; Sanofi: Honoraria. Jagannath:Multiple Myeloma Research Foundation: Speakers Bureau; BMS: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Medicom: Speakers Bureau; Merck: Consultancy. Lee:Daiichi Sankyo: Research Funding; Celgene: Consultancy, Research Funding; GlaxoSmithKline plc: Research Funding; Sanofi: Consultancy; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Patel:Poseida Therapeutics, Cellectis, Abbvie: Research Funding; Oncopeptides, Nektar, Precision Biosciences, BMS: Consultancy; Takeda, Celgene, Janssen: Consultancy, Research Funding. Kaufman:Janssen: Other: travel/lodging, Research Funding. Thomas:Xencor: Research Funding; BMS: Research Funding; Celgene: Research Funding; Amgen: Research Funding. Mailankody:Takeda Oncology: Research Funding; Juno: Research Funding; Celgene: Research Funding; Janssen: Research Funding; CME activity by Physician Education Resource: Honoraria. Lendvai:Janssen: Employment. Neelapu:Acerta: Research Funding; Celgene: Consultancy, Research Funding; BMS: Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Incyte: Consultancy; Merck: Consultancy, Research Funding; Allogene: Consultancy; Cellectis: Research Funding; Poseida: Research Funding; Karus: Research Funding; Pfizer: Consultancy; Unum Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; Precision Biosciences: Consultancy; Cell Medica: Consultancy. Orlowski:Poseida Therapeutics, Inc.: Research Funding. Landgren:Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Theradex: Other: IDMC; Abbvie: Membership on an entity's Board of Directors or advisory committees; Merck: Other: IDMC. OffLabel Disclosure: Isatuximab for the treatment of smoldering myeloma

scholarly journals Defining the Differentiation States of Multiple Myeloma at Single Cell Resolution Reveals Opportunities for Immunotherapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3091-3091
Author(s):  
Julia Frede ◽  
Praveen Anand ◽  
Andrew J. Yee ◽  
Tushara Vijaykumar ◽  
Monica S. Nair ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Single Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Advisory Board ◽  
Therapeutic Resistance ◽  
Cellular Plasticity ◽  
Advisory Committees ◽  
Myeloma Cells

Introduction: Despite recent advances in the treatment of multiple myeloma, responses may be short-lived and therapeutic resistance develops almost invariably. Non-genetic cellular plasticity and dedifferentiation have recently emerged as a basis for therapeutic resistance in cancer as cells acquire transcriptional states which no longer depend on the drug target. Therefore, a better understanding of plasticity and adaptive state changes in myeloma cells is critical to develop effective therapeutic approaches that can overcome drug resistance. Here we show that cellular plasticity, though frequently invoked as a basis for therapeutic resistance in cancer, can also lead to new therapeutic opportunities. Methods: To define transcriptional states in myeloma at a single cell level, we performed fluorescence activated cell sorting and full-length single-cell RNA sequencing. We assayed a total 6000 CD38+CD138+ plasma cells and CD45+ immune cells from the bone marrow of 8 patients with relapsed and refractory multiple myeloma (RRMM) before and after immuno-modulatory treatment on a clinical trial with elotuzumab, pomalidomide, bortezomib and dexamethasone (Elo-PVD; NCT02718833) and 2 healthy donors. Surface expression of selected markers was validated by flow cytometry. Results: Assessing pre-treatment samples, we discovered that the transcriptional states of single myeloma cells are highly distinct between individual patients, despite the presence of the same established genomic classifiers, such as t(11;14). Furthermore, distinct transcriptional states co-exist within individual patients, indicating there is substantial inter- and intra-individual heterogeneity. Transcriptional states diverge from normal plasma cells towards more immature cells, of the B lymphoid lineage, suggesting a substantial cellular plasticity. Notably, we detected co-expression of myeloid and lymphoid developmental programs in the same single cells. Interestingly, these altered differentiation states were associated with up-regulation of potential immunotherapeutic targets, such as CD20, CD19, and CD33, indicating that this plasticity may result in novel therapeutic vulnerabilities. To define gene-regulatory relationships, we identified a shared core regulatory network present in malignant and normal plasma cells with the active transcription factors XBP1, ATF4, and CREB3, suggesting that myeloma cells retain lineage-specific regulons. However, we further identified patient-specific regulons not detected in any of the mature immune cell populations assayed, such as TEAD4, ELF3 and SNAI1, illustrating an aberrant and promiscuous activation of transcriptional regulators in myeloma cells. Consistent with this finding, we observed an increased number of expressed genes in myeloma cells compared to normal plasma cells as well as an increase in single cell transcriptional entropy, measures that have been linked to cell potency in normal development and cancer. Comparison of pre- and post-treatment samples interestingly revealed a further increase in transcriptional diversity and signatures associated with stemness and developmental potential following treatment. Conclusions: In conclusion, we find that higher transcriptional diversity and activation of alternate gene regulatory programs facilitate the emergence of altered transcriptional states. Interestingly, these altered states are associated with up-regulation of putative immune-therapeutic targets in myeloma cells, thus providing novel therapeutic vulnerabilities. Disclosures Lipe: amgen: Research Funding; Celgene: Consultancy; amgen: Consultancy. O'Donnell:Celgene: Consultancy; Takeda: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Amgen: Consultancy. Munshi:Celgene: Consultancy; Amgen: Consultancy; Oncopep: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Oncopep: Consultancy; Takeda: Consultancy. Richardson:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Anderson:Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board; OncoPep: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder . Lohr:T2 Biosystems: Honoraria; Celgene: Research Funding. OffLabel Disclosure: Samples for ancillary research were obtained in the context of a phase II clinical trial evaluating Elotuzumab, pomalidomide, bortezomib, dexamethasone The combination of elo-PVD is off label.

Investigational Agent MLN9708 Target Tumor Suppressor MicroRNA-33b in Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 136-136
Author(s):  
Ze Tian ◽  
Jian-Jun Zhao ◽  
Jianhong Lin ◽  
Dharminder Chauhan ◽  
Kenneth C. Anderson
Keyword(s):  
Multiple Myeloma ◽  
Tumor Suppressor ◽  
Board Of Directors ◽  
Expression Profiling ◽  
Research Funding ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Investigational Agent ◽  
Reporter Activity

Abstract Abstract 136 Investigational Agent MLN9708 Target Tumor Suppressor MicroRNA-33b in Multiple Myeloma Cells Ze Tian, Jianjun Zhao, Jianhong Lin, Dharminder Chauhan, Kenneth C. Anderson Medical Oncology, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA, 02115 MicroRNAs (miRNAs) are 19–25 nucleotide-long noncoding RNA molecules that regulate gene expression both at the level of messenger RNA degradation and translation. Emerging evidence shows that miRNAs play a critical role in tumor pathogenesis by functioning as either oncogene or tumor suppressor genes. The role of miRNA and their regulation in response to proteasome inhibitors treatment in Multiple Myeloma (MM) is unclear. Here, we utilized MLN9708, a selective orally bio-available proteasome inhibitor to examine its effects on miRNA alterations in MM.1S MM cells. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to its biologically active form MLN2238. Our previous study using both in vitro and in vivo models showed that MLN2238 inhibits tumor growth and triggers apoptosis via activation of caspases. Moreover, MLN2238 triggered apoptosis in bortezomib-resistant MM cells, and induced synergistic anti-MM activity when combined with HDAC inhibitor SAHA, dexamethasone, and lenalidomide. In the current study, we treated MM.1S cells with MLN2238 (12 nM) for 3 hours and harvested; total RNA was subjected to miRNA profiling using TaqMan® Array Human miRNA A-Card Set v3.0 and the data was analyzed using dChip analysis. Results showed that MLN2238 modulates miRNA expression with a total of 36 miRNA changing their expression profiling (δδCT>1.5 or δδCT <-1.5; 19 were upregulated and 17 showed a downregulation). Among all miRNA, miR-33b was highly (δδCT>7) upregulated in response to MLN2238 treatment. We therefore hypothesized that miR-33b may play a role in MM pathogenesis as well as during MLN2238-induced proteasome inhibition in MM cells. We first utilized quantitative polymerase chain reaction (q-PCR) to validate the changes in miRNA expression profiling. Results confirmed that MLN2238 treatment triggers significant increase in the miR-33b expression in MM.1S cells (2.1 and 2.2 folds at 3h and 6h, respectively; P<0.001). Examination of normal PBMCs and plasma cells showed higher expression of miR-33b than patient MM cells (P<0.001). We further investigated the functional role of miR-33b in MM cells at baseline and during MLN2238 treatment. Drug sensitivity, cell viability, apoptosis, colony formation, and migration assays were performed using cell TilTer-Glo, Annexin V-FITC/PI staining, MTT staining, and Transwell assays, respectively. Signaling pathways modulated post miR-33b overexpression were evaluated by q-PCR, immunoblot, and reporter assays. Our findings show that overexpression of miR-33b significantly decreased cell viability, cell migration, colony formation, as well as increased apoptosis and sensitivity of MM cells to MLN2238 treatment. Targetscan analysis predicted pim-1 as a putative downstream target of miR-33b. Overexpression of miR-33b downregulated pim-1 mRNA and protein expression. To further corroborate these data, we co-tranfected miR-33b and Pim-1-wt or Pim-1-mt in 293T and MM.1S cell lines. In concert with our earlier findings, miR-33b decreases pim-1-wt, but not pim-1-mt reporter activity in both cell lines. Reflecting the overexpression study results, MLN2238 treatment also decreases pim-1-wt, but not pim1-mt reporter activity. Moreover, a biochemical inhibitor of pim1/2 triggered apoptosis in MM cells. Finally, overexpression of miR-33b inhibits tumor growth (P<0.001) and prolongs survival (P<0.001) in both subcutaneous and disseminated human MM xenograft models. In summary, our study suggests that miR-33b is a tumor suppressor, which plays a role during MLN2238-induced apoptotic signaling in MM cells, and provide the basis for novel therapeutic strategies targeting miR-33b in MM. Disclosures: Anderson: Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership.

Inhibition of SUV39H Methyltransferase As a Potent Therapeutic Target in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1771-1771 ◽  
Author(s):  
Julie Devin ◽  
Elena Viziteu ◽  
Laurie Herviou ◽  
Anja Seckinger ◽  
Grandmougin Camille ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Normal Bone ◽  
Significant Toxicity ◽  
Wide Range ◽  
Travel Grant

Abstract Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes. We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio > 2 and FDR (false discovery rate) < 5%) were identified, including the SUV39H1 histone methyltransferase. SUV39H1 and SUV39H2 are regulators of chromatin organization. SUV39H1-dependent trimethylation of H3K9 is essential for maintenance of both pericentromeric and telomeric heterochromatin. SUV39H1 deficiency reduced cell viability severely and is associated to heterochromatin decompaction, loss of silencing, genome instability, and a wide range of defects in cell cycle, cell growth, and meiosis. SUV39H1-mediated H3K9me has been linked to gene silencing of the tumor suppressor genes, such as p15INK4B and E-cadherin, in acute myeloid leukemia (AML). Therefore, it is highly possible that the default function of SUV39H1 is to maintain genome stability by limiting the acute activation of oncogenes while its dysregulation could cause tumor formation. We reported that high SUV39H1 expression, in MM cells, is associated with a poor prognosis in two independent cohorts of patients (Heidelberg-Montpellier cohort - N=206 and UAMS-TT2 cohort - N=345). SUV39H1 expression was downregulated by conditional shRNA expression through lentiviral delivery. SUV39H1 knock down significantly inhibits H3K9me3, growth of myeloma cells, induces apoptosis, cell cycle deregulation, reactive oxygen species production and spontaneous accumulation of DNA double strand breaks. According to these results, SUV39H1 depletion sensitizes myeloma cells to melphalan. Chaetocin is a selective inhibitor of SUV39H1. We identified that chaetocin has anti-myeloma effects at low nanomolar doses (range: 4 to 17 nM), on 11 different human myeloma cell lines, that are representative of the molecular heterogeneity of the patients, in association with H3K9 trimethylation inhibition. Furthermore, this significant toxicity of chaetocin in MM was confirmed on primary myeloma cells of 5 patients cocultured with their bone marrow microenvironment without significant toxicity on normal bone marrow cells and hematopoietic stem cells. Interestingly, the IC50 doses of chaetocin in MM were 50 fold lower compared to results published in AML, suggesting H3K9 histone methyltransferases could be a potent therapeutic target in MM. Disclosures Seckinger: EngMab AG: Research Funding; Takeda: Other: Travel grant. Goldschmidt:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millenium: Honoraria, Research Funding, Speakers Bureau; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai: Honoraria, Research Funding, Speakers Bureau. Hose:EngMab AG: Research Funding; Takeda: Other: Travel grant.

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 High Levels of APOBEC3B Gene Expression Contribute to Poor Prognosis in Multiple Myeloma Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3897-3897
Author(s):  
Valeriy V Lyzogubov ◽  
Pingping Qu ◽  
Cody Ashby ◽  
Adam Rosenthal ◽  
Antje Hoering ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Board Of Directors ◽  
Research Funding ◽  
Poor Prognosis ◽  
Target Genes ◽  
Plasma Cells ◽  
Nuclear Fraction ◽  
Advisory Committees ◽  
Significant Difference

Abstract Introduction: Poor prognosis and drug resistance in multiple myeloma (MM) is associated with increased mutational load. APOBEC3B is a major contributor to mutagenesis, especially in myeloma patients with t(14;16) MAF subgroup. It was shown recently that presence of the APOBEC signature at diagnosis is an independent prognostic factor for progression free survival (PFS) and overall survival (OS). We hypothesized that high levels of APOBEC3B gene expression at diagnosis may also have a prognostic impact in myeloma. To consider APOBEC3B as a potential target for therapy more studies are necessary to understand how APOBEC3B expression is regulated and how APOBEC3B generates mutations. Methods: Gene expression profiling (GEP, U133 Plus 2.0) of MM patients was performed. APOBEC3B gene expression levels were investigated in plasma cells of healthy donors (HD; n=34), MGUS (n=154), smoldering myeloma (SMM; n=219), MM low risk (LR; n=739), MM high risk (HR; n=129), relapsed MM (RMM; n=74), and primary plasma cell leukemia (pPCL; n=19) samples. The samples from relapse were taken on or after the progression/relapse date but within 30 days after progression/relapse from Total Therapy trials 3, 4, 5 & 6. GEP70 score was used to separate samples into LR and HR groups. We also investigated APOBEC3B expression in different MM molecular subgroups and used logrank statistics with covariate frequency distribution to determine an optimal cut off APOBEC3B expression value. Gene expression was compared in cases with low expression of APOBEC3B (log2<7.5) and high expression of APOBEC3B (log2>10), and an optimal cut-point in APOBEC3B expression was identified with respect to PFS. To explore the role of MAF and the non-canonical NF-ĸB pathway we performed functional studies using a cellular model of MAF downregulation. TRIPZ lentiviral shRNA MAF knockdown in the RPMI8226 cell lines was used to explore MAF-dependent genes. NF-ĸB proteins, p52 and RelB, were investigated in the nuclear fraction by immunoblot analysis. Results: Expression of APOBEC3B in HD control samples (log2=10.9) was surprisingly higher than in MGUS (log2=9.51), SMM (log2=9.09), and LR (log2=9.40) and was comparable to HR (log2=10.4) and RMM (log2=10.6) groups. Expression levels of APOBEC3B were gradually increased as disease progressed from SMM to pPCL. The high expression of APOBEC3B in HD places plasma cells at risk of APOBEC induced mutagenesis where the regulation of APOBEC3B function is compromised. The correlation between APOBEC3B expression and GEP70 score in MM was 0.37, and there was a significant difference in APOBEC3B expression between GEP70 high and low risk groups (p=0.0003). An optimal cut-point in APOBEC3B expression of log2=10.2 resulted in a significant difference in PFS (median 5.7 yr vs.7.4 yr; p=0.0086) and OS (median 9.1 yr vs. not reached; p<0.0001), between high and low expression. The highest APOBEC3B expression was detected in cases with a t(14;16). We analyzed t(14;16) cases with the APOBEC mutational signature and compared them to t(14;16) cases without the APOBEC signature and found elevated MAF (2-fold) and APOBEC3B (2.7-fold) gene expression in samples with the APOBEC signature. No APOBEC signature was detected in SMM cases, including those with a t(14;16). High APOBEC3B levels in myeloma patients was associated with overexpression of genes related to response to DNA damage and cell cycle control. Significant (p<0.05) increases of NF-κB target genes was seen in high APOBEC3B cases: TNFAIP3 (4.4-fold), NFKB2 (1.7-fold), NFKBIE (1.9-fold), RELB (1.4-fold), NFKBIA (2.0-fold), PLEK (2.5-fold), MALT1 (2.5-fold), WNT10A (2.4-fold). However, in t(14;16) cases there was no significant increase of NF-κB target genes except BIRC3 (2.5-fold) and MALT1 (2.0-fold). MAF downregulation in RPMI8226 cells did not lead to changes in NF-κB target gene expression but MAF-dependent genes were identified, including ETS1, SPP1, RUNX2, HGF, IGFBP2 and IGFBP3. Analysis of nuclear fraction of NF-ĸB proteins did not show significant changes in expression of p52 and RelB in RPMI8226 cells after MAF downregulation. Conclusions: Increased expression of APOBEC3B is a negative prognostic factor in multiple myeloma. MAF is a major factor regulating expression of APOBEC3B in the t(14;16) subgroup. NF-ĸB pathway activation is most likely involved in upregulation of APOBEC3B in non-t(14;16) subgroups. Disclosures Davies: TRM Oncology: Honoraria; MMRF: Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy. Morgan:Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Takeda: Consultancy, Honoraria.

scholarly journals First Description of B Cell Maturation Antigen Expression in Light Chain Amyloidosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5452-5452
Author(s):  
Susan Bal ◽  
Allison Sigler ◽  
Alexander Chan ◽  
David J. Chung ◽  
Ahmet Dogan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Board Of Directors ◽  
Light Chain ◽  
Research Funding ◽  
Plasma Cells ◽  
Staining Intensity ◽  
Advisory Committees ◽  
Light Chain Amyloidosis ◽  
B Cell Maturation

Background B-cell maturation antigen (BCMA) is a transmembrane protein belonging to the tumor necrosis factor (TNF) superfamily involved in the regulation of B cell proliferation and survival as well as maturation/differentiation into plasma cells. In multiple myeloma cells, overexpression of BCMA has been shown to activate mitogen activated protein kinase pathways (AKT, ERK1/2, and NF-κB) and upregulates anti-apoptotic proteins (MCL1, BCL2, BCL-xL) resulting in cellular proliferation. Immunotherapeutic strategies targeting BCMA are showing great promise in heavily pre-treated refractory multiple myeloma. Light Chain Amyloidosis (AL) is a multisystem disorder of clonal plasma cells that results in the production of an abnormal light chain which misfolds and deposits in the organs leading to disruption of tissue architecture, cellular stress, dysfunction and eventually, death. The smaller burden and lower proliferative potential of the offending clonal plasma cells in amyloidosis may potentially lend itself favorably to immunotherapeutic strategies targeting BCMA. Given the efficacy of this approach in MM, the evaluation of BCMA expression on the surface of amyloidogenic plasma cells is warranted. Methods All patients diagnosed with Light chain Amyloidosis at Memorial Sloan Kettering Cancer Center, NY between January 1, 2012, and December 31, 2018, who had unstained bone marrow samples were identified. These unstained BM biopsy samples were prospectively stained for BCMA expression using Immunohistochemistry (IHC). We utilized a clinical-grade assay (clone D6; catalog sc-390147; company Santa-Cruz; monoclonal antibody; dilution 1:400) in a CLIA compliant setting. We scored the biopsies for BCMA expression, intensity, and site of staining. We also obtained their demographic details, staging, and cytogenetic information for the patients with available samples. Results During the queried period, 28 unstained samples were available for testing from the time of disease diagnosis. The median age of the population was 63 years (range 41-73). 64% of patients were male and consistent with the literature; a majority of patients (75%) had lambda-typic clonal plasma cells. Cytogenetic abnormalities using fluorescence in situ hybridization (FISH) were reviewed, t(11;14) was seen in 36% patients, and chromosome 1q and del 13q were each seen in 32% of patients. No patient had t(4;14) or del 17p. The median clonal PC burden in BM at diagnosis was 10% (range2-80%) and 36% had > 10% plasma cells. In clonal PCs, the median BCMA expression was 80% (range 20-100%). Only one patient had a staining intensity under 50% (20%). Membranous staining was noted in 82% of patients and a Golgi pattern in 11%. The median staining intensity was 2 (range 1-3). Of the patients with baseline diagnostic samples available for testing, six patients had additional unstained bone marrow samples for staining at the time of relapse. The majority of patients (83%) who relapsed had >10% plasma cells with a higher median plasma cell burden of 35% (range 10-80). The median BCMA expression was 65% (range 50-80) with no patient having <50% expression. The staining pattern was membranous in 50%, Golgi in 17%, and Golgi-membranous in 33%. At the time of relapse, the median clonal PC burden was 13% (range 5-30). BCMA expression continued to be present at the time of relapse with a median 75% (range 50-100) with predominantly membranous staining (83%). The median staining intensity in both diagnostic and relapsed tissue within the six samples studied was 1. Conclusions Our study represents the first description of BCMA expression on the surface of amyloidogenic plasma cells to our knowledge. BCMA is uniformly expressed by pathologic PCs in AL amyloidosis both at the time of diagnosis and relapse. Given the efficacy of BCMA directed therapy in multiple myeloma, further investigation of these agents in light-chain amyloidosis are warranted and may provide an effective therapeutic strategy in this devastating disease. Figure Disclosures Dogan: Corvus Pharmaceuticals: Consultancy; Celgene: Consultancy; Seattle Genetics: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Roche: Consultancy, Research Funding. Giralt:Takeda: Consultancy, Research Funding; Johnson & Johnson: Consultancy, Research Funding; Kite: Consultancy; Novartis: Consultancy; Actinium: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Miltenyi: Research Funding; Spectrum Pharmaceuticals: Consultancy. Hassoun:Novartis: Consultancy; Janssen: Research Funding; Celgene: Research Funding. Landau:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees; Caelum: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Descriptive Analysis of Isatuximab Use Following Prior Daratumumab in Patients with Relapsed/Refractory Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-21
Author(s):  
Melody R Becnel ◽  
Sandra B. Horowitz ◽  
Sheeba K. Thomas ◽  
Swami P. Iyer ◽  
Krina K. Patel ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
High Risk ◽  
North America ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Laboratory Research ◽  
Private Company ◽  
Advisory Committees ◽  
Advisory Boards

Background: Anti-CD38 monoclonal antibodies (mAb) like daratumumab (dara) have become integral in managing relapsed/refractory (RR) and newly diagnosed (ND) multiple myeloma (MM). Isatuximab (isa), a newer CD38 mAb, induces direct rather than indirect apoptosis of MM cells. However, little is known about whether the use of one prior CD38 mAb will alter the efficacy of another in subsequent lines of therapy. Methods: All patients (pts) with MM treated at MD Anderson with isa after receiving dara in prior lines of therapy were identified. We conducted a retrospective analysis with data points including patient and disease characteristics, responses to dara, response to isa, the presence of high risk features, and the presence of t(11,14). Results: 9 pts were identified, ages 56-72. 5 pts (55%) were male. 5 pts (55%) were alive at the time of data cutoff. 5 pts were Hispanic, 3 White, and 1 Black. 8 pts (89%) had high risk features as represented by the presence of del17p, t(4,14), t(14,16), t(14,20), p53 mutations, gain 1q, extramedullary disease (EMD), CNS disease, early relapse (within 1 year) after autologous transplant, or an increased (&gt;5%) peripheral blood plasma cells (PBPC). 2 (22%) had t(11,14). 4 (44%) had IgG MM. 2 (22%) with light chain disease, 2 (22%) with IgA MM, and 1 (11%) with IgD MM. Dara was initially used in lines 2-7. Dara combinations with pomalidomide (pom), bortezomib (bor), thalidomide (thal), lenalidomide (len), or carfilzomib (car); and pom combinations that also included elotuzumab (elo) or Cytoxan (cytox) are noted in table 1. Dara was discontinued (dc'd) in 8 pts due to progressive disease (PD) and in 1 pt due to toxicity. 8 pts (89%) experienced a best overall response (ORR) of partial response (PR) to dara; 1 pt had stable disease (SD). All pts received prior len and 8 pts received prior pom at some time during the treatment of MM. All pts received isa in combination with pom/dexamethasone (dex). Best ORR to isa/pom/dex: 5 pt (55%) had PR, 2 pt with minimal response (MR), 1 SD, 1 PD. Median treatment duration of isa/pom/dex was 5 weeks (2-14 weeks) at data cutoff. 3 pts dc'd isa/pom/dex due to infections, and 2 due to later progression. 2 pts remain on therapy. 1 pt chose to dc all MM therapy for quality of life purposes despite PR with isa/pom/dex. 1 pt died from cardiac disease unrelated to MM or treatment. Conclusions: Our current study of heavily pretreated pts with RRMM demonstrates that despite prior anti-CD38 therapy with dara, most patients (77%) experienced a response of MR or better with treatment with another anti-CD38 therapy isa. To our knowledge, this is the first report of outcomes to isa in patients with prior dara therapy. Further long term follow up will be needed to determine the length of response. Additional studies are planned to further evaluate this patient population. Table 1 Disclosures Thomas: Pharmacyclics: Other: Advisory Boards; BMS: Research Funding; Ascentage: Membership on an entity's Board of Directors or advisory committees, Research Funding; X4 Pharma: Research Funding; Xencor: Research Funding; Genentech: Research Funding. Iyer:Rhizen: Research Funding; CRISPR: Research Funding; Spectrum: Research Funding; Merck: Research Funding; Curio Biosciences: Honoraria; Target Oncology: Honoraria; Afffimed: Research Funding; Daiichi Sankyo: Consultancy; Legend Biotech: Consultancy; Trillium: Research Funding; Seattle Genetics, Inc.: Research Funding. Patel:Celgene: Consultancy, Research Funding; Cellectis: Research Funding; Nektar: Consultancy, Research Funding; Oncopeptides: Consultancy; Poseida: Research Funding; Precision Biosciences: Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding. Manasanch:Adaptive Biotechnologies: Honoraria; GSK: Honoraria; Sanofi: Honoraria; BMS: Honoraria; Takeda: Honoraria; Quest Diagnostics: Research Funding; Merck: Research Funding; JW Pharma: Research Funding; Novartis: Research Funding; Sanofi: Research Funding. Kaufman:Janssen: Research Funding; Bristol Myers Squibb: Research Funding; Karyopharm: Honoraria. Lee:Genentech: Consultancy; GlaxoSmithKline: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Sanofi: Consultancy; Daiichi Sankyo: Research Funding; Regeneron: Research Funding; Genentech: Consultancy. Orlowski:Sanofi-Aventis, Servier, Takeda Pharmaceuticals North America, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen, Inc., AstraZeneca, BMS, Celgene, EcoR1 Capital LLC, Forma Therapeutics, Genzyme, GSK Biologicals, Ionis Pharmaceuticals, Inc., Janssen Biotech, Juno Therapeutics, Kite Pharma, Legend Biotech USA, Molecular Partners, Regeneron Pharmaceuticals, Inc.,: Honoraria, Membership on an entity's Board of Directors or advisory committees; STATinMED Research: Consultancy; Founder of Asylia Therapeutics, Inc., with associated patents and an equity interest, though this technology does not bear on the current submission.: Current equity holder in private company, Patents & Royalties; Laboratory research funding from BioTheryX, and clinical research funding from CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Research Funding.

scholarly journals Single-Cell Pathway Enrichment and Regulatory Profiling of Multiple Myeloma across Disease Stages

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 364-364
Author(s):  
Tianjiao Wang ◽  
Hua Sun ◽  
Daniel Cui Zhou ◽  
Ruiyang Liu ◽  
Lijun Yao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Cell Types ◽  
Differentially Expressed ◽  
P Value ◽  
Advisory Committees ◽  
Pathway Enrichment

Multiple myeloma (MM) is a hematological malignancy, defined by aberrant monoclonal proliferation of plasma cells in the bone marrow, that to date remains an incurable disease despite advances in treatment. Key genetic and epigenetic alterations that drive MM pathogenesis have been identified, but a comprehensive profile of affected cellular pathways has yet to be fully characterized. In this study, we integrate whole-genome and whole-exome sequencing data with single-cell RNA sequencing (scRNA-seq) data from 13 patients across multiple treatment stages to 1) assess differential pathway enrichment between tumor subpopulations, 2) trace the clonal evolution of dominant disease mechanisms, and 3) investigate signaling interactions between surrounding cell types. We also analyzed bulk genomic and transcriptomic data from 662 additional Multiple Myeloma Research Foundation (MMRF) tumor samples as a large reference cohort for highly prevalent pathway disturbances. To assess whether tumor subpopulations rely on different oncogenic programs for proliferation, we analyzed the differential expression of key genes (FDR-adjusted p-value &lt;0.05) in 12 canonical oncogenic pathways. Cell cycle, Hippo, RTK/RAS, and NFkB pathways contain the highest numbers of differentially expressed genes, with certain subclusters upregulating as many as 25% of annotated cell cycle genes and over 90% of annotated Hippo genes, whereas p53, Notch, Nrf2, and DNA repair genes tend to be uniformly expressed across subpopulations. Next, we evaluated changes in pathway enrichment across different disease timepoints, with the goal of capturing the reorganization of functional profiles through successive treatment and relapse cycles. We assessed statistical enrichment of pathways containing differentially expressed genes (DEGs) unique to Smoldering Multiple Myeloma (SMM), primary, and relapse stages using the KEGG pathway database (n = 2, 17, and 7 pathways, respectively; FDR-adjusted p-value of enrichment &lt; 0.05). SMM is the only stage where hematopoietic differentiation and the PI3K-Akt pathways are variably expressed between plasma cell subpopulations, suggesting that these pathways may play a role in initiating events. Only primary tumor samples show significant intra-tumor variability of p53 regulation, which is lost in the relapsed tumor and may reflect selection due to treatment. Relative to SMM, primary and relapse samples are enriched for changes in the MAPK, NFkB, RAP1, and cell cycle pathways, indicating potential sources of tumor resistance. We then analyzed pathway enrichment within the tumor microenvironment to enhance our understanding of tumor development in the context of surrounding tissues. We see frequent changes in many immune cell types in TLR signaling as the disease progresses, driven by differential expression of NFkB1A, JUN, and FOS, all of which are key upstream regulators of the AP-1 pathway and responders to the MAPK and PI3K signaling cascades. These microenvironment changes may be complementary to the PI3K and MAPK dysregulation observed in tumor plasma cells. Proteasome and ubiquitin genes, which affect secretion, autophagy, and apoptosis pathways that may be relevant to MM pathogenesis are also frequently differentially expressed in immune cells between disease stages. Finally, we integrate bulk whole-exome and whole-genome sequencing analysis (from both the 13-patient cohort and MMRF) to obtain a more complete understanding of how pathways become dysregulated in MM. Our findings advance the understanding of how MM tumor subpopulations differentially regulate cellular pathways and interact within the tumor microenvironment. Disclosures O'Neal: Wugen: Patents & Royalties: Patent Pending; WashU: Patents & Royalties: Patent Pending. Rettig:WashU: Patents & Royalties: Patent Application 16/401,950. Oh:Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Vij:Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Honoraria; Karyopharm: Honoraria; Sanofi: Honoraria; Takeda: Honoraria, Research Funding. DiPersio:Amphivena Therapeutics: Consultancy, Research Funding; Magenta Therapeutics: Equity Ownership; Karyopharm Therapeutics: Consultancy; Incyte: Consultancy, Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; NeoImmune Tech: Research Funding; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees.

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