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

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.

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

Blood ◽

10.1182/blood.v128.22.311.311 ◽

2016 ◽

Vol 128 (22) ◽

pp. 311-311 ◽

Cited By ~ 4

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.

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

Blood ◽

10.1182/blood.v118.21.2424.2424 ◽

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.

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Value of Minimal Residual Disease (MRD) Negative Status at Response Evaluation in Chronic Lymphocytic Leukemia (CLL): Combined Analysis of Two Phase III Studies of the German CLL Study Group (GCLLSG)

Blood ◽

10.1182/blood.v124.21.23.23 ◽

2014 ◽

Vol 124 (21) ◽

pp. 23-23 ◽

Cited By ~ 2

Author(s):

Gabor Kovacs ◽

Sebastian Boettcher ◽

Jasmin Bahlo ◽

Sandra Kluth ◽

Matthias Ritgen ◽

...

Keyword(s):

Bone Marrow ◽

Clinical Response ◽

Board Of Directors ◽

Research Funding ◽

Bone Marrow Involvement ◽

Phase Iii ◽

Two Phase ◽

Advisory Committees ◽

Significant Difference ◽

Travel Grant

Abstract Introduction: Within clinical trials in CLL, response assessment is based on the NCI 1996 guidelines or its update, the iwCLL 2008 guidelines, respectively. Whereas detailed response criteria with clear cut-off values are provided for the assessment of most parameters, a few parameters such as splenomegaly are not defined quantitatively in the guidelines. In addition, the detection of MRD, which was recommended for clinical trials but not formally included in the definition of response, is gaining increasing importance. Both MRD negativity (with a threshold of <10-4 leukemic cells per leukocytes) and the occurrence of a complete response (CR) predict long progression free survival (PFS). In order to investigate the value of MRD with respect to clinical response, the MRD status was explored in patients (pts) with CR and partial remission (PR) in two phase III trials of the GCLLSG. Furthermore, we evaluated the relevance of residual splenomegaly, lymphadenopathy or bone marrow involvement in MRD negative (-) pts with clinical PR. Patients and Methods: 542 pts from two prospective phase III trials of the GCLLSG (CLL8 trial: fludarabine and cyclophosphamide without (FC) or with rituximab (FCR); CLL10 trial: FCR vs bendamustine and rituximab (BR)) were included in the analysis (Figure 1). The comprehensive dataset included MRD results from peripheral blood at final restaging (RE) (2 months after the end of last treatment cycle), bone marrow (BM), clinical and radiological assessment for organomegaly and lymphadenopathy. Clinical response was defined according to the iwCLL 2008 guidelines. Splenomegaly was determined by physical and radiological examination. Moreover, different cut-off values defining splenomegaly by CT or ultrasound (longest diameter >12 cm and >14 cm) were investigated. PFS was analyzed using Kaplan-Meier methodology, and survival curves were compared using two-sided log-rank tests. Additionally hazard ratios (HR) and 95% confidence intervals (CI) were calculated. Significance was set at a p value<0.05. No adjustments for multiple testing were performed. Results: 542 pts were included in the analysis. Their median age was 61 years, median CIRS score 2 (range 0-6), median follow up time 45.9 months (mo) (range 5.5-96.1). 121 pts (22.3%) received FC, 283 (52.2%) FCR and 138 (25.5%) BR. 13.3% of pts had Binet stage A, 52.3 % stage B and 34.4% stage C disease. Among 514 pts with IGHV results, 63.0% had an unmutated status. In 524 pts FISH was available, del(17p) was only found in 1.3% (exclusion criterion in CLL10 trial), del(11q) in 25.0%, 12+ in 10.1%, normal in 25.4%, and del(13q) in 38.2% of the pts. MRD negativity was achieved in 81.8% (175/214) of pts with CR and in 47.9% (157/328) of the pts with PR, respectively. There was a statistically significant difference in PFS between MRD- CRs and MRD positive (+) CRs (69.2 mo vs 40.4 mo; HR 0.445, 95% CI=0.282-0.703, p=0.001). Additionally, there was a statistically significant difference between MRD- PRs and MRD+ CRs (61.7 mo vs 40.4 mo; HR 0.537, 95% CI=0.340-0.847, p=0.008). No statistically significant difference between MRD- CRs and MRD- PRs was detected (69.2 mo vs. 61.7 mo; HR 0.822, 95% CI=0.572-1.182, p=0.29) (Figure 2). Of the 157 pts presenting with an MRD- PR, 106 pts were evaluable for remaining CLL involvement: 48 pts (45.3%) had a splenomegaly, 12 (11.3%) lymphadenopathy, 19 (17.9%) bone marrow involvement as the sole abnormality. Only 25.5% (27) of the pts had more than one abnormality. There was no statistically significant difference in PFS between MRD- PRs with single splenomegaly and MRD- CRs (not reached (NR) vs 69.2 mo; HR 0.737, 95% CI=0.387-1.404, p=0.4). Moreover, patients with MRD- PRs and single splenomegaly had a statistically significant longer PFS than MRD+ CR pts (NR vs 40.4 mo; HR 0.348, 95% CI=0.172-0.701, p=0.003). (Figure 3) The difference was independent of the cut-off values used for splenomegaly (12 cm or 14 cm) (p=0.001 and p=0.03). Conclusion: MRD negativity determined in the peripheral blood after end of treatment is a potent predictor of treatment efficacy regardless of the clinical response assessment. The persistence of splenomegaly as sole abnormality post treatment in MRD- patients has no negative influence on PFS. More data are needed to prove the relevance of residual BM involvement and lymphadenopathy in MRD- PR pts. These results support the use of MRD for response evaluation. Figure 1 Figure 1. Disclosures Boettcher: Roche: Honoraria, Research Funding, Travel grant Other. Ritgen:Roche: Honoraria, Research Funding, Travel grant Other. Cramer:Mundipharma: Travel grant, Travel grant Other; Roche: Travel grant Other. Maurer:Mundipharma: Travel grant Other. Doehner:Roche: Research Funding. Stilgenbauer:Roche: Consultancy, Honoraria, Research Funding. Kneba:Mundipharma: Consultancy, Research Funding; Roche: Consultancy, Research Funding. Fischer:Roche: Travel grant Other. Hallek:Mundipharma: Consultancy, Research Funding; Roche: Consultancy, Research Funding. Eichhorst:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel grant Other; Mundipharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel grant, Travel grant Other; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy. Off Label Use: The Combination of Bendamustine and Rituximab is not approved for frontline chemoimmunotherapy of CLL.

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Role of Selectins in the Pathogenesis of Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.951.951 ◽

2009 ◽

Vol 114 (22) ◽

pp. 951-951 ◽

Cited By ~ 1

Author(s):

Abdel Kareem Azab ◽

Phong Quang ◽

Feda Azab ◽

Costas M Pitsillides ◽

John T Patton ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Endothelial Cells ◽

Cell Adhesion ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Advisory Committees

Abstract Abstract 951 INTRODUCTION: Multiple Myeloma (MM) is characterized by widespread disease at diagnosis with the presence of multiple lytic lesions and disseminated involvement of the bone marrow (BM), implying that the progression of MM involves a continuous re-circulation of the MM cells in the peripheral blood and re-entrance into the BM. Selectins are adhesion molecules expressed by activated endothelium of venules and leukocytes, and are involved in the primary interaction of lymphocytes with the endothelium of blood vessels. The binding of selectins serves as a biologic brake, making leukocyte quickly decelerate by rolling on endothelial cells, as the first step of extravasation. In this study, we have investigated the role of selectins and their ligands in the regulation of homing of MM Cells to the BM and the therapeutic implications of this role. METHODS AND RESULTS: We have used flow cytometry to characterize the expression of E, L and P-selectins and their ligands on MM cell lines, patient samples and on plasma cells from normal subjects. We found that all MM cell lines and patient samples showed high expression of L and P, but little of no E-selectin. While normal plasma cells showed low expression of all selectins and ligands.(give numbers) A pan-selectin inhibitor GMI-1070 (GlycoMimetics Inc., Gaithersburg, MD) inhibited the interaction of recombinant selectins with the selectin-ligands on the MM cells in a dose response manner. We have tested the role of the selectins and their ligands on the adhesion of MM cells to endothelial cells and found that MM cells adhered preferentially to endothelial cells expressing P-selectin compared to control endothelial cells and endothelial cells expressing E-selectin (p<0.05). Moreover, we found that blockade of P-selectin on endothelial cells reduced their interaction with MM cells (p<0.01), while blockade of E and L-selectin did not show any effect. Treating endothelial cells with GMI-1070 mimicked the effect of blocking P-selectin. Moreover, we found that treating endothelial cells with the chemokine stroma cell-derived factor-1-alpha (SDF1) increased their expression of P but not E or L-selectin detected by flow cytometry. Neither the blockade of each of the selectins and their ligands nor the GMI-1070 inhibited the trans-well chemotaxis of MM cells towards SDF1-alpha. However, blockade of P-selectin (p<0.001) on endothelial cells by GMI-1070 inhibited the trans-endothelial chemotaxis of MM cells towards SDF1-alpha. Both adhesion to endothelial cells and activation with recombinant P-selectin induced phosphorylation of cell adhesion related molecules including FAK, SRC, Cadherins, Cofilin, AKT and GSK3. GMI-1070 decreased the activation of cell adhesion molecules induced by both recombinant P-selectin and endothelial cells. Using in vivo flow cytometry we found that both anti P-selectin antibody and GMI-1070 prevented the extravasation of MM cells out of blood vessels into the bone marrow in mice. Moreover, we found that, in a co-culture system, endothelial cells protected MM cells from bortezomib induced apoptosis, an effect which was reversed by using GMI-1070, showing synergistic effect with bortezomib. CONCLUSION: In summary, we showed that P-selectin ligand is highly expressed in MM cells compared to normal plasma cells, and that it plays a major role in homing of MM cells to the BM, an effect which was inhibited by the pan-selectin inhibitor GMI-1070. This provides a basis for testing the effect of selectin inhibition on tumor initiation and tumor response to therapeutic agents such as bortezomib. Moreover, it provides a basis for future clinical trials for prevention of MM metastasis and increasing efficacy of existing therapies by using selectin inhibitors for the treatment of myeloma. Disclosures: Patton: GlycoMimetics, Inc: Employment. Smith:GlycoMimetics, Inc: Employment. Sarkar:GlycoMimetics, Inc: Employment. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Magnani:GlycoMimetics, Inc.: Employment. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

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Profound Impact of Sample Processing Delay on Gene Expression of Multiple Myeloma Plasma Cells

Blood ◽

10.1182/blood.v126.23.2996.2996 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2996-2996

Author(s):

Tobias Meissner ◽

Anja Seckinger ◽

Kari Hemminki ◽

Uta Bertsch ◽

Asta Foersti ◽

...

Keyword(s):

Gene Expression ◽

Risk Stratification ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

University Of Arkansas ◽

Medical Sciences ◽

Advisory Committees ◽

Travel Grant ◽

The Uk

Abstract Introduction: Gene expression profiling (GEP) has significantly contributed to the elucidation of the molecular heterogeneity of multiple myeloma plasma cells (MMPC) and only recently it has been recommended for risk stratification. Prior to GEP MMPC need to be enriched resulting in an inability to immediately freeze bone marrow aspirates or use RNA stabilization reagents. As a result in multi-center MM trials sample processing delay due to shipping may be an important confounder of molecular analyses and risk stratification based on GEP data. In order to determine the impact of "shipping delay" on MMPC gene expression we analyzed a set of 573 newly diagnosed German MM patients including 230 in-house and 343 shipped samples. Materials and Methods: We included publicly available GEP data of newly diagnosed MM patients treated in the GMMG HD4 and MM5 trials. All samples had been processed in a central laboratory in Heidelberg and include 85 HD4 and 145 MM5 in-house and 97 HD4 and 246 MM5 shipped samples. Prediction of sample status was done on publicly available GEP, including data from the UK, UAMS and MMRC. Differential gene expression was assessed using empirical Bayes statistics in linear models for microarray data. Predictor for shipment status was generated on the MM5 cohort using prediction analysis for microarrays. Pathway enrichment analysis was done using WebGestalt. Risk signatures and molecular subgroups were obtained as previously described. Fisher's exact test was used to compare the subgroup distribution between cohorts. If applicable, results were corrected for multiple testing using the Benjamini-Hochberg method. In all statistical tests, an effect was considered statistically significant if the P-value of its corresponding statistical test was not greater than 5%. Results: Applying the Goeman's global teston the MM5 set showed that "shipping delay" significantly impacted global gene expression (P <0.001). Compared to 145 in-house samples, we detected 3301 down-regulated and 3501 up-regulated genes in 246 shipped samples. For 4280 genes we confirmed differential expression in an independent set of 85 in-house and 97 shipped samples. Of these genes 2040 had a >1.5-fold and 826 a >2-fold difference in expression level. Differentially expressed genes were enriched in processes like ribosome biogenesis, cell cycle, and apoptosis. We observed significantly lower proliferation rates in shipped samples (P <0.001). We did not detect significant differences in the distribution of molecular subgroups between in-house and shipped samples in the combined set of HD4 and MM5. Among GEP based risk predictors the IFM-15 seemed to underestimate high risk in shipped samples, whereas the GEP70 and the EMC-92 gene signatures were more robust. In order to provide a tool to assess the "shipping effect" in public repositories, we generated a 17-gene predictor for shipped samples with a 10-fold cross validation error rate of 0.06 for the training set and an error rate of 0.15 for the validation set. Applying the predictor to further publicly available data sets we detected the "shipping effect" signature in 11% of cases of the UAMS set, 94% of the UK set and 57% of the MMRC set. Conclusion: Our study shows that "shipping delay" widely influences gene expression of MMPC with different impact on molecular classification and risk stratification. Based on available data, currently no clear circumvention of the shipping impact on MMPC can be recommended. It should be avoided if possible or at least be taken into account. Disclosures Seckinger: Takeda: Other: Travel grant. Salwender:Celgene: Honoraria; Janssen Cilag: Honoraria; Bristol Meyer Sqibb: Honoraria; Amgen: Honoraria; Novartis: Honoraria. Goldschmidt:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; 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; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen-Cilag: 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; Chugai: Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. Morgan:MMRF: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; University of Arkansas for Medical Sciences: Employment; CancerNet: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Weismann Institute: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hose:Takeda: Other: Travel grant; EngMab AG: Research Funding. Weinhold:Janssen Cilag: Other: Advisory Board; University of Arkansas for Medical Sciences: Employment.

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

Blood ◽

10.1182/blood-2019-123205 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3116-3116 ◽

Cited By ~ 5

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

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

Blood ◽

10.1182/blood-2020-140673 ◽

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.

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Transcriptional Landscape of the Microenvironment in Bone Marrow Fibrosis at Single Cell Level

Blood ◽

10.1182/blood-2019-126897 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1675-1675

Author(s):

Nils B. Leimkühler ◽

Ronghui Li ◽

Helene Gleitz ◽

Inge Snoeren ◽

Stijn Fuchs ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Cell Level ◽

Bone Marrow Fibrosis ◽

Advisory Committees ◽

Molecular Alterations ◽

Ecm Proteins ◽

Travel Grant ◽

Marrow Fibrosis

Although the molecular alterations in hematopoietic cells which drive the development of myeloproliferative neoplasms (MPN) have been largely defined, reactive cellular alterations in the non-hematopoietic compartment remain rather obscure and have not been studied at single cell level. We therefore profiled enriched non-hematopoietic bone marrow cells by scRNAseq in bone marrow (BM) fibrosis compared to healthy marrow. BM fibrosis was induced by transplantation of hematopoietic stem and progenitor cells (HSPCs) with overexpression of Thrombopoietin (ThPO) into lethally irradiated mice. As ThPO-overexpression robustly leads to reticulin fibrosis in all mice (100%), we were able to study 1) pre-fibrosis (5 weeks after transplantation; reticulin fibrosis grade 0) and 2) manifest bone marrow fibrosis (10 weeks after transplantation, reticulin grade 2-3). The analysis revealed a total of 8 distinct clusters: 1-4) subpopulations of mesenchymal stromal cells (MSC-1: adipogenic, MSC-2: osteogenic, MSC-3: transition, MSC-4: interferonhigh), 5) osteoblastic lineage cells (OLCs), 6) arterial endothelial cells (ECs) and 7-8) Schwann cell precursors (SCP-1: non-myelinating SCPs; SCP-2: myelinating SCPs). Exposure to ThPO overexpressing HSPCs resulted in an overrepresentation of adipogenic MSCs at the expense of all other MSC subclusters. Differential gene expression analysis revealed a functional reprogramming of the "adipogenic" expanding MSCs with down-regulation of hematopoiesis-support and induction of a secretory phenotype including upregulation of various extracellular matrix (ECM) proteins driving fibrosis. Interestingly, only two MSC subclusters gained significant ECM expression indicating myofibroblast differentiation. Expansion of OLCs in BM fibrosis suggested a differentiation of the underrepresented MSC subpopulations into osteolineage cells which was confirmed by pseudotime analysis. Myelinating SCPs, highly expressing interleukin-33 (IL-33), showed the largest expansion in fibrosis. IL-33 is described to play a significant role in solid organ fibrosis by having both pro- and anti-fibrotic effects. Nerve injury triggers the expansion of myelinating and non-myelinating Schwann cells to promote repair, suggesting that mSCPs increase as compensatory and regenerative mechanism for the previously described MPN-induced sympathetic neuropathy. Dissection of cellular and molecular alterations in pre-fibrosis and manifest fibrosis demonstrated that only one MSC subpopulation was already significantly expanded in the pre-fibrotic phase, but only showed minor transcriptional changes. The upregulation of ECM proteins, osteogenesis as well as proinflammatory genes were hallmark features of manifest fibrosis. Interestingly, the overrepresentation of IL-33 expressing mSCPs was more pronounced in the pre-fibrotic phase, indicating that the expansion is a regenerative phenomenon failing in the stages of manifest fibrosis. Our findings were validated in the clinically relevant JAK2(V617F)-induced model of myelofibrosis. In conclusion, we here identified two distinct MSC subsets that are pro-fibrotic and contribute to osteosclerosis in PMF. The functional reprogramming of these MSCs in the bone marrow niche was accompanied by expansion of mSCPs with regenerative capacities, most likely caused by neural damage and Schwann cell death triggered by mutant HSCs. Disclosures Crysandt: Amgem: Other: travel grant; Pfizer: Other: travel grant; Gilead: Other: travel grant; Incyte: Membership on an entity's Board of Directors or advisory committees; celgene: Other: travel grant. Koschmieder:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers-Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AOP Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis Foundation: Research Funding.

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Real-World Data on Incidence, Clinical Characteristics and Outcome of Patients with Macrofocal Multiple Myeloma (MFMM) in the Era of Novel Therapies: A Study of the Greco-Israeli Collaborative Myeloma Working Group

Blood ◽

10.1182/blood-2018-99-114106 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3295-3295 ◽

Cited By ~ 1

Author(s):

Eirini Katodritou ◽

Efstathios Kastritis ◽

Moshe E. Gatt ◽

Yael C Cohen ◽

Irit Avivi ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Proteasome Inhibitor ◽

Research Funding ◽

Steering Committee ◽

Novel Therapies ◽

Advisory Committees ◽

Lytic Lesions ◽

Travel Grant

Abstract Macrofocal Multiple Myeloma (MFMM) has been described as a distinct entity of Multiple Myeloma (MM) characterized by young age, lytic lesions and limited bone marrow infiltration by clonal plasma cells (BMPCs), in the absence of other features of symptomatic MM (i.e. anemia, renal insufficiency and hypercalcemia). Few case studies have indicated a possible favorable prognosis of MFMM patients compared to patients with typical symptomatic MM. Our aim was to investigate the incidence, characteristics and outcome of patients with MFMM, under the light of modern therapeutic approach of MM. MFMM definition required: clonal BMPCs <20%, multiple lytic lesions, absence of anemia, renal insufficiency and hypercalcemia and among 4650 MM patients (3%) registered in the MM databases of Greek and Israeli centers during 2001-2017, we identified 140 patients with MFMM (M/F: 93/47, median age: 61, range: 26-89, IgG: 86, IgA: 12, light chain: 21, IgD: 4, non-secretory: 16, IgM: 1). Most of patients with MFMM (60%) were <65 years; 68% had performance status 0-2 according to Eastern Cooperative group (ECOG) scale; 70% had advanced bone disease (>3 lytic lesions). In 20/140 (14%) patients bone plasmacytomas preceded MM diagnosis. In 95/140 (68%) patients bone, soft tissue or mixed plasmacytomas in multiple locations, were present at diagnosis or during MFMM course and this was significantly more frequent compared with standard MM. Median BMPCs infiltration was 14% (range 0-19%); immunoparesis was less common in MFMM (55% vs. 90% in standard MM). Elevated lactate dehydrogenase (LDH) and β2 microglobulin (β2Μ) ≥ 3.5mg/L were found in 9% and 20% of patients, respectively. Cytogenetics by fluorescence in situ hybridization (FISH) were available in 60% of patients and high-risk features were found in 11%; overall, adverse prognostic parameters (i.e. high LDH, advanced age, high β2Μ, high risk cytogenetics) were less common in patients with MFMM compared with others (p<0.05). According to the International staging system (ISS) patients were stratified as follows: ISS1:71%, ISS2: 25% and ISS3: 4%. Per Revised ISS the distribution was R-ISS1: 54%, RISS2: 46%, no R-ISS3). Induction therapy included novel agents in 90% of patients (bortezomib-based: 61%, thalidomide-based: 14%, bortezomib-lenalidomide-dexamethasone: 4%, lenalidomide-based: 11%); 47% underwent autologous transplantation (ASCT) upfront and 13% at 1st relapse. An objective response (ORR) was achieved in 90%: 70% had at least very good partial response (vgPR), 21% partial response, 6% stable disease and 3% had progressive disease; ORR and achievement of ≥vgPR were significantly higher compared with typical MM (p<0.05). After a median follow up of 52 months (95% CI: 40-64), 33 patients have died (MM progression: 19, lung infection: 8, other causes: 6). Early deaths (<12 months) observed in 5% of patients; 53 patients received 2nd line therapy (proteasome inhibitor-based or lenalidomide-dexamethasone: 79%) and 5 patients received only radiotherapy for plasmacytomas; early relapse (<12 months) was less common in MFMM compared with standard MM (p<0.05). Progression-free survival (PFS) and overall survival (OS) were 46 months (95% CI: 40-52) and 129 months (95% CI: 79-178) respectively, both significantly longer compared with typical MM treated during the same period (p<0.001). In the univariate analysis age <65, early stage disease (ISS1, R-ISS1), 1st line treatment with proteasome inhibitor (PI)-based regimens, ASCT, and standard risk cytogenetics predicted positively for OS in MFMM patients; treatment with PI-based therapies was the only independent predictor for OS in the multivariate analysis (HR: 3.9; p<0.001). In conclusion, MFMM is a rare entity of MM characterized by limited bone marrow infiltration, extended bone lesions and frequent presence of plasmacytomas, prior or during the diagnosis or the course of the disease. MFMM patients are younger have less often adverse prognostic features compared with standard MM and achieve high quality responses when treated with novel therapies. Treatment with PI-based regimens was the strongest predictor for OS in MFMM indicating that it is probably the best therapeutic option for these patients. Disclosures Kastritis: Janssen: 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; Celgene: 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; Prothena: Honoraria, Membership on an entity's Board of Directors or advisory committees. Cohen:Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding. Aviv:ABBVIE: Consultancy; ROCHE: Research Funding. Terpos:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: member of steering committee, Research Funding; Genesis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: member of DMC, Research Funding; Amgen Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grant, steering committee member, Research Funding; BMS: Consultancy; Novartis: Consultancy. Dimopoulos:Amgen: Honoraria; Janssen: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Celgene: Honoraria.

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Role of TORC1 and TORC2 in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.1815.1815 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1815-1815

Author(s):

Patricia Maiso ◽

Yi Liu ◽

Abdel Kareem Azab ◽

Brittany Morgan ◽

Feda Azab ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Board Of Directors ◽

Cell Lines ◽

Stromal Cells ◽

Research Funding ◽

Plasma Cells ◽

Advisory Committees ◽

Cycle Arrest

Abstract Abstract 1815 Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 (Raptor) and TORC2 (Rictor). TORC1 leads to the phosphorylation of p70S6 kinase and 4E- BP1, while TORC2 regulates phosphorylation of Akt and other kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin and its analogues have not shown significant activity in MM, likely due to the lack of inhibition of TORC2. In this study, we dissected the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. Methods: Eight different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, immunochemistry, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. In vivo homing was checked by in vivo flow. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: Raptor (TORC1) and Rictor (TORC2) knockdowns led to significant inhibition of proliferation of MM cells even in the presence of bone marrow stromal cells, this effect was also accompanied by inactivation of p-Akt, p-rS6 and p-4EBP1. We used INK128, a dual and selective TORC1/2 kinase inhibitor with similar effects to Raptor plus Rictor knockdown. We examined the protein expression levels of both mTOR complex and their downstream effectors in MM plasma cells from patients and cell lines. mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all samples. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin) even in the presence of cytokines or stromal cells. INK128 induced cell cycle arrest, autophagy and apoptosis in cell lines and primary plasma cells even in the presence of bone marrow stromal cells (BMSCs). INK128 also showed a significant effect inhibiting cell adhesion in our in vivo homing model. Oral daily treatment with INK128 highly decreased the percentage of CD138+ tumor plasma cells in mice implanted with MM cells and reduced the levels of p-Akt and p-4EBP. These results suggest that potent and complete blockade of mTOR as part of TORC1 and TORC2 is potential therapeutic strategy to induce cell cycle arrest, apoptosis and disruption of MM cells interaction with the BM microenvironment. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Liu: Intellikine: Employment. Roccaro:Roche: Research Funding. Rommel:Intellikine: Employment. Ghobrial:Celgene: Consultancy; 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; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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