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

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 <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 < 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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Dual Targeting of -TORC1 and -TORC2 as a New Strategy In the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.133.133 ◽

2010 ◽

Vol 116 (21) ◽

pp. 133-133 ◽

Cited By ~ 2

Author(s):

Patricia Maiso ◽

AbdelKareem Azab ◽

Yang Liu ◽

Yong Zhang ◽

Feda Azab ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Cell Lines ◽

Mechanism Of Action ◽

Plasma Cells ◽

Bone Marrow Microenvironment ◽

Advisory Committees

Abstract Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC 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 analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine 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, 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. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. 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: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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CYC065, a Potent Derivative of Seliciclib Is Active In Multiple Myeloma In Preclinical Studies

Blood ◽

10.1182/blood.v116.21.2999.2999 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2999-2999 ◽

Cited By ~ 1

Author(s):

Samantha Pozzi ◽

Diana Cirstea ◽

Loredana Santo ◽

Doris M Nabikejje ◽

Kishan Patel ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Preliminary Data ◽

Research Funding ◽

Preclinical Studies ◽

Dependent Manner ◽

Advisory Committees

Abstract Abstract 2999 Multiple myeloma (MM) is a treatable but incurable hematological malignancy and novel targeted therapies are under investigation. MM is characterized by dysregulation of the cell cycle, consequent to the overexpression of cyclins and their related kinases, the cyclins dependent kinases (CDK), a group of Ser/Thr proteine kinases. CDKs represent a promising therapeutic target, and inhibitors have been developed for anticancer treatment. We have previously studied seliciclib in the context of MM. CYC065, a second generation CDK inhibitor is the more potent derivative of seliciclib. It is mainly active on CDK 2, 5 and 9, involved in progression of the cell cycle and protein transcription. It has already shown promising results in preclinical studies in breast cancer and acute leukemia. We tested CYC065 in in vitro experiments in MM. Our preliminary data in 7 MM cell lines showed cytotoxicity of CYC065, both in MM cell lines sensitive as well as resistant to conventional chemotherapy, with an IC50 ranging between 0.06 and 2μ M, at 24 and 48h. Tritiated thymidine uptake assay confirmed the antiproliferative effects of CYC065 in MM, and its ability to overcome the growth advantage conferred by co-culture with bone marrow stromal cells derived from MM patients, and cytokines like interleukin 6 (10ng/ml) and insulin like growth factor-1 (50ng/ml). The anti-proliferative effect was evident both at 24 and 48h, starting at concentrations as low as 0.015μ M. The AnnexinV/PI assay in the MM1.s cell line confirmed CYC065's ability to induce apoptosis in a time dependent manner starting at 9 hours of treatment, at a concentration of 0.125 μ M, inducing 82% of apoptosis after 48h of exposure. Cell cycle analysis in the same MM1.s cell line showed an increase of subG1 phase, starting at 9 hours of treatment, at 0.125 μ M of CYC065. Preliminary results of western blot analysis confirmed the apoptotic effect of CYC065 in the MM1s cell line, highlighted by the cleavage of caspase 3, 8, 9 and PARP. The compound was tested in primary CD138+ cells isolated from three refractory MM patients, confirming its efficacy at 0.125 μ M, both at 24 and 48h. Comparative analysis in PBMCs from normal donors, for the evaluation of the drug toxicity is ongoing and will be presented. In conclusion our preliminary data confirm the efficacy of CYC065 in MM cell lines and primary MM cells, at nanomolar concentrations. Ongoing mechanistic and in vivo studies will delineate its role in the now increasing spectrum of CDK inhibitors in MM and better define its potential for clinical development in MM. Disclosures: Green: Cyclacel: Employment. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Acetylon: Research Funding.

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

Blood ◽

10.1182/blood-2018-99-117067 ◽

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.

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Baseline and on-Treatment Bone Marrow Microenvironments Predict Myeloma Patient Outcomes and Inform Potential Intervention Strategies

Blood ◽

10.1182/blood-2018-99-113169 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1882-1882 ◽

Cited By ~ 1

Author(s):

Samuel A Danziger ◽

Mark McConnell ◽

Jake Gockley ◽

Mary Young ◽

Adam Rosenthal ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Expression Profiles ◽

Cell Types ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Abstract Introduction The multiple myeloma (MM) tumor microenvironment (TME) strongly influences patient outcomes as evidenced by the success of immunomodulatory therapies. To develop precision immunotherapeutic approaches, it is essential to identify and enumerate TME cell types and understand their dynamics. Methods We estimated the population of immune and other non-tumor cell types during the course of MM treatment at a single institution using gene expression of paired CD138-selected bone marrow aspirates and whole bone marrow (WBM) core biopsies from 867 samples of 436 newly diagnosed MM patients collected at 5 time points: pre-treatment (N=354), post-induction (N=245), post-transplant (N=83), post-consolidation (N=51), and post-maintenance (N=134). Expression profiles from the aspirates were used to infer the transcriptome contribution of immune and stromal cells in the WBM array data. Unsupervised clustering of these non-tumor gene expression profiles across all time points was performed using the R package ConsensusClusterPlus with Bayesian Information Criterion (BIC) to select the number of clusters. Individual cell types in these TMEs were estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) augmented with 5 bone marrow- and myeloma-specific cell types. Results Our deconvolution approach accurately estimated percent tumor cells in the paired samples compared to estimates from microscopy and flow cytometry (PCC = 0.63, RMSE = 9.99%). TME clusters built on gene expression data from all 867 samples resulted in 5 unsupervised clusters covering 91% of samples. While the fraction of patients in each cluster changed during treatment, no new TME clusters emerged as treatment progressed. These clusters were associated with progression free survival (PFS) (p-Val = 0.020) and overall survival (OS) (p-Val = 0.067) when measured in pre-transplant samples. The most striking outcomes were represented by Cluster 5 (N = 106) characterized by a low innate to adaptive cell ratio and shortened patient survival (Figure 1, 2). This cluster had worse outcomes than others (estimated mean PFS = 58 months compared to 71+ months for other clusters, p-Val = 0.002; estimate mean OS = 105 months compared with 113+ months for other clusters, p-Val = 0.040). Compared to other immune clusters, the adaptive-skewed TME of Cluster 5 is characterized by low granulocyte populations and high antigen-presenting, CD8 T, and B cell populations. As might be expected, this cluster was also significantly enriched for ISS3 and GEP70 high risk patients, as well as Del1p, Del1q, t12;14, and t14:16. Importantly, this TME persisted even when the induction therapy significantly reduced the tumor load (Table 1). At post-induction, outcomes for the 69 / 245 patients in Cluster 5 remain significantly worse (estimate mean PFS = 56 months compared to 71+ months for other clusters, p-Val = 0.004; estimate mean OS = 100 months compared to 121+ months for other clusters, p-Val = 0.002). The analysis of on-treatment samples showed that the number of patients in Cluster 5 decreases from 30% before treatment to 12% after transplant, and of the 63 patients for whom we have both pre-treatment and post-transplant samples, 18/20 of the Cluster 5 patients moved into other immune clusters; 13 into Cluster 4. The non-5 clusters (with better PFS and OS overall) had higher amounts of granulocytes and lower amounts of CD8 T cells. Some clusters (1 and 4) had increased natural killer (NK) cells and decreased dendritic cells, while other clusters (2 and 3) had increased adipocytes and increases in M2 macrophages (Cluster 2) or NK cells (Cluster 3). Taken together, the gain of granulocytes and adipocytes was associated with improved outcome, while increases in the adaptive immune compartment was associated with poorer outcome. Conclusions We identified distinct clusters of patient TMEs from bulk transcriptome profiles by computationally estimating the CD138- fraction of TMEs. Our findings identified differential immune and stromal compositions in patient clusters with opposing clinical outcomes and tracked membership in those clusters during treatment. Adding this layer of TME to the analysis of myeloma patient baseline and on-treatment samples enables us to formulate biological hypotheses and may eventually guide therapeutic interventions to improve outcomes for patients. Disclosures Danziger: Celgene Corporation: Employment, Equity Ownership. McConnell:Celgene Corporation: Employment. Gockley:Celgene Corporation: Employment. Young:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Reiss:Celgene Corporation: Employment, Equity Ownership. Davies:MMRF: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; TRM Oncology: Honoraria; Abbvie: Consultancy; ASH: Honoraria; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria. Copeland:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Fitch:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Barlogie:Celgene: Consultancy, Research Funding; Dana Farber Cancer Institute: Other: travel stipend; Multiple Myeloma Research Foundation: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Millenium: Consultancy, Research Funding; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC. Trotter:Celgene Research SL (Spain), part of Celgene Corporation: Employment, Equity Ownership. Hershberg:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Dervan:Celgene Corporation: Employment, Equity Ownership. Ratushny:Celgene Corporation: Employment, Equity Ownership. Morgan:Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding.

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

Blood ◽

10.1182/blood-2019-127332 ◽

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.

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

Blood ◽

10.1182/blood-2020-140526 ◽

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 (>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.

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Methyljasmonate, An Inhibitor of Glycolytic Energy Production, Displays Pre-Clinical Activity against Multiple Myeloma Cells.

Blood ◽

10.1182/blood.v114.22.1741.1741 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1741-1741

Author(s):

Steffen Klippel ◽

Jana Jakubikova ◽

Jake Delmore ◽

Melissa G. Ooi ◽

Douglas McMillin ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Death ◽

Cancer Cells ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Energy Production ◽

Advisory Committees ◽

Normal Cells

Abstract Abstract 1741 Poster Board I-767 Background In contrast to most normal cells, cancer cells typically produce energy predominantly by glycolysis as demonstrated by O. Warburg more than 50 years ago. Methyljasmonate (MJ), a hormone produced by plants in response to biotic & abiotic stresses such as herbivory and wounding, has been shown to prevent the interaction of hexokinase (Hxk) and voltage dependent anion channels (VDACs), thereby significantly impacting the onset of glycolytic energy production. This may explain promising preclinical results observed with MJ against a variety of cancer cells, including myeloid leukemia and B-cell lymphoma cell lines. Methods and Results We tested the potential of MJ against Multiple Myeloma (MM) cells. We first evaluated the response of 16 different MM cell lines to 24 h of exposure to MJ concentrations of 0.5 – 3.5 mM using MTT assays. 15/16 of the MM cell lines tested displayed an IC50 of < 1.5 mM. In contrast, HS-5 stroma cells and peripheral blood mononuclear cells (PBMCs) did not respond to that MJ concentration, and even at a concentration of 2.5 mM MJ showed a maximal reduction of cell viability of 40%. Similarly to MM cell lines, purified CD138+ primary tumor cells of 3 MM patients displayed an IC50 of < 1.5 mM, suggesting that the differential sensitivity of MM vs. normal cells to MJ is not restricted to cell lines, but is also observed with primary tumor cells. Importantly, neither co-culture with HS-5 stroma nor IL-6 protected MM cells against MJ. Cell death commitment assays revealed that 1h exposure of 1.5 mM MJ induced cell death. Annexin V/PI FACS analysis of MJ-exposed MM cells showed that the cell death is mainly driven by apoptosis, evidenced by cleavage of caspases 3, 8 and 9 as well as of PARP. However, pre-incubation of MM cells with specific caspase inhibitors such as 10 mM of AC-DEVD-CHO, Z-IETD-fmk, Z-LEHD-fmk or 50 mM of Z-VAD only minimally protects the cancer cells from MJ exposure. Therefore, the impact of the MJ is not solely due to caspase triggered proteolytic cascades. Measurements of cellular ATP content by cell titer glow (CTG; Promega, Madison, WI) assay showed rapid depletion of ATP triggered by MJ action in sensitive MM cell lines. Additionally, we observed that 1 h exposure to 2 mM MJ modulated signaling pathways including IRS1/PI3K/AKT, MEK1/2, as well as Stat3 and JNK. FACS-based cell cycle analysis after propidium iodide staining did not show cell cycle arrest, but rather a rapid transition of cells to G0/G1 No correlation of sensitivity of MM cell lines and the number of mitochondria per cancer cell, as determined by Mitotracker Green (Invitrogen, Carlsbad, CA) -based flow analysis, was observed. We next examined if MJ exhibits either significant antagonism or synergy with established or novel anti-MM agents, including Bortezomib, Lenalidomide, Doxorubicin, Rapamycin or Dexamethasone, but discovered neither. However, MJ displayed synergy when combined with 2-Deoxyglucose. Finally, MJ was tested in vivo in scid/nod mice irradiated with 150 rads, injected with 1× 106 MM1S cells, and then, treated at 500 mg/kg by IP administration on a 5 days on / 2 days off schedule starting two weeks after tumor cell injection, There was an overall survival advantage of MJ-treated animals over the respective controls, with all treated mice (n=10) still alive but 6/10 control mice dead after 27 d. Conclusions Based on its rapidity of anti-MM action, favorable safety profile in preclinical models, distinct pattern of molecular sequelae, and compatibility with established anti-MM agents, MJ represents a promising investigational anti-MM agent. Disclosures Laubach: Novartis: Consultancy, Honoraria. Richardson:Millennium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Novartis Pharmaceuticals: Consultancy, Honoraria; Milllennium: Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

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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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Phase II Study Of The c-MET Inhibitor ARQ 197 (Tivantinib) In Patients With Relapsed Or Relapsed/Refractory Multiple Myeloma (RRMM)

Blood ◽

10.1182/blood.v122.21.1953.1953 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1953-1953

Author(s):

Robert Z. Orlowski ◽

Shadia Zaman ◽

Sheeba K. Thomas ◽

Raymond Alexanian ◽

Jatin J. Shah ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Phase Ii Study ◽

Single Agent ◽

Advisory Committees ◽

Arq 197 ◽

Met Inhibitor ◽

Grade 3

Abstract Background c-MET receptor tyrosine kinase (RTK) activity has been implicated in establishing the oncogenic phenotype across several human cancers with high levels of the activating c-MET ligand, hepatocyte growth factor (HGF). Malignant plasma cells secrete HGF-activator (HGFA), which converts HGF to its active form, and high HGF levels are correlated with a poor prognosis in multiple myeloma. Syndecan 1 (CD138) on malignant plasma cells binds HGF and potentiates interleukin-6-induced growth and migration. HGF stimulation of myeloma cells also activates autophosphorylation of c-MET and other critical downstream signaling pathways promoting oncogenesis. Finally, pre-clinical studies have shown that suppression of c-MET signaling with a number of small molecules, including ARQ 197, induced myeloma cell apoptosis. Tivantinib-mediated cytotoxic response was observed at concentrations of less than 5 µM, which are achievable in the clinic. These findings supported the hypothesis that suppression of the HGF/c-MET signaling axis could be a rational strategy against RRMM. Methods In this phase II study, the efficacy and safety of ARQ 197, a non-competitive and highly selective inhibitor of the c-MET RTK, was studied in patients with RRMM. Primary objectives were to determine the overall response rate (ORR) to single-agent tivantinib in patients who had received one to four prior lines of therapy, and to define the toxicities in this population. A Simon’s Minimax 2-stage design was used for the study. ARQ 197 was administered at a starting oral dose of 360 mg twice daily with meals for each day of every 4-week treatment cycle. This dose was selected from prior phase I investigations in solid tumors, and at this dose level, steady-state plasma levels of ARQ 197 were 7 µM. Treatment could continue providing that patients did not experience undue toxicities, or disease progression. Tivantinib is provided through the Cancer Therapy Evaluation Program (CTEP), and this study was supported by CTEP, as well as the MD Anderson Cancer Center SPORE in Multiple Myeloma. Results A total of 16 patients were enrolled and treated to date, including 9 men and 7 women, who had received a median of 1 prior line of therapy (range 1-3), including stem cell transplant in ten. The mean patient age was 66 (range 49-76), with ethnicity including 13 Caucasian Americans, 2 African Americans, and 1 Asian American. Patients have received a median of 3 cycles of therapy to date (1-11) with one patient continuing on study, and all were evaluable for toxicity, while 11 were evaluable for response based on having completed two treatment cycles. The most common adverse events (AEs) of any grade seen in at least 25% of patients and felt to be at least possibly drug related included fatigue or decreased neutrophils (94% each), pain (81%), myalgias (56%), diarrhea (38%), memory impairment, respiratory disorders, and rash (31% each), and hypertension (25%), and these were predominantly grade 1 or 2. Grade 3 or 4 AEs included neutropenia (31% and 25%, respectively), syncope, infection, pain (13% of each, all grade 3), and anal fissure, cough, fatigue, hypertension, and pulmonary embolism (6% each, all grade 3). Stable disease (SD) has been seen as the best response in 4/11 (36%) evaluable patients, which was maintained for up to 11 cycles, while the remaining patients showed evidence of disease progression. Conclusion Single-agent tivantinib has been well tolerated in patients with RRMM, and the ability to achieve stable disease in patients with previously progressing myeloma does support the possibility that targeting c-MET has some promise. Future studies with rationally designed combination regimens incorporating a c-MET inhibitor and other novel agents may better define the role of this class of drugs in our armamentarium against myeloma. Disclosures: Orlowski: Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene Corporation: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees. Thomas:Millenium: Research Funding; Novartis: Research Funding; Celgene: Research Funding; Immunomedics: Research Funding; Pharmacyclics: Membership on an entity’s Board of Directors or advisory committees; Onyx: Membership on an entity’s Board of Directors or advisory committees. Shah:Celgene: Consultancy, Research Funding; Array: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Millenium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

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