Agents In Myeloma Research Articles

Testing Novel Anti - Multiple Myeloma (MM) Agents in a Suitable Three-Dimensional (3D) Co-Culture Platform

Introduction: In the past several years significant progress has been made in the understanding of MM pathogenesis, drug resistance and the related role of the bone marrow microenvironment (BMM). Hence various treatment strategies are currently in (pre-) clinical use, targeting both malignant plasma cells and the BM niche. In February 2015 the first epigenetic modifying pan histone deacetylase inhibitor (HDACi), panobinostat, granted approval for relapsed or refractory MM. However there is insufficient data so far to conclusively unravel the complex interactions between "epi-drugs" and the rapid increasing number of antimyeloma agents. Thus, we assess the impact of novel drug combinations (NOX-A12, auranofin, class selective HDACis, Sirt-2 inhibitors) on the BMM in a suitable matrix-based 3D co-culture platform to best mimic in vivo conditions whilst ensuring easy handling.Methods: The innovative 3D co-culture platform is based on transparent agarose matrices with 2 mm deep conical micro-cavities, wherein semi-adherent cell populations can be either seeded in contact or distance co-culture (Fig. 1). Due to the uniquely defined conical shape of the cavities, proliferation as well as treatment response could be monitored up to several weeks by determining the circular area of the cell clusters via transmitted light scanner. To further compare common flat 2D versus 3D culturing with regard to treatment response and growing behavior, FACS analysis and viability assays were performed. In addition, we examined altered expression levels of adhesion molecules (CXCR4, CD138, CD38, Slam-F7) in terms of culture conditions and epigenetic modulations (class-selective HDACis) using multi-panel flow cytometry. Qualitative and quantitative distribution of fluorescence dyes (apoptosis, proliferation) inside the cell agglomerations was visualized using mCherry transduced RPMI-8226 cells combined with confocal imaging and ScanR-analysis.Results: Human MM cell lines (MMCLs; U266 and RPMI-8226) exhibited substantial differences concerning growth and therapeutic responses under 2D versus agarose-based 3D culture conditions. MMCLs and especially primary MM patient samples could be cultured without reconditioning steps for more than three weeks under moderate logarithmic growth conditions inside the agarose matrices. Due to simple media and/or stromal exchange a loss or dying of cells could be consistently avoided. In contrast semi-adherent MMCLs (initial concentration 105 cells/ml) showed exponential proliferation in common flat culture until nutrient starvation induced apoptosis after approx. six days. Furthermore, malignant plasma cells became significantly resistant to proteasome inhibitors (PI) bortezomib (BTZ; 6nM, 48h) and auranofin (AUR; 3 µM, 48h) by forming solid 3D cell-agglomerations and almost remained unaffected if co-cultured additionally with HS-5 BM stromal cells (BMSCs). To exclude uneven distribution of agents, we determined the equal penetration of fluorescent dyes (CellTrace Violet/CFSE) into cell-clusters via confocal imaging.Further data suggest a great impact of 3D co-culture conditions on the expression pattern of modern target structures per se, leading e.g. to a decrease in CXCR4 but persistence of CD138 levels if compared to 2D mono-culture. Moreover we demonstrated a significant decrease of CD138 in low toxic concentrations of panobinostat (8nM, 48h) indicating a intriguing modulation of the BMM by epigenetic modifying agents. For subsequent steps we already verified synergistic antimyeloma effects of the novel HDAC6-selctive inhibitor JS28 in combination with BTZ and aim to further elucidate the influences of various selective deacetylase inhibitors on the BM niche.Conclusions and Ouline: Our recent data underline the importance of accurate 3D co-culture models to mimic in vivo proliferation and drug resistance and to predict later clinical treatment success more precisely. Based on this suitable platform, we are currently assessing the impact of cluster formation, stromal support and "epi-drugs" on the expression pattern of adhesion molecules to derive most rational drug combinations with modern target structures, such as CXCR4, CD138, CD38 and SlamF7 and to allow more effective targeting of the BMM. [Display omitted] DisclosuresEngelhardt:Janssen: Research Funding; MSD: Research Funding; Amgen: Research Funding; Celgene: Research Funding.

A GRP78-Directed Monoclonal Antibody Recaptures Response in Refractory Multiple Myeloma with Extramedullary Involvement.

Glucose-regulated protein (GRP) 78 is overexpressed in multiple myeloma, and both its surface expression and its biologic significance as key sensor of the unfolded protein response make GRP78 an ideal candidate for immunotherapeutic intervention. The monoclonal antibody PAT-SM6 targets surface GRP78 and leads to disease stabilization when used as single agent in a clinical trial. In this article, we evaluated expression of GRP78 in relapsed-refractory disease and explored PAT-SM6 therapy in combination regimens. GRP78 expression was immunohistochemically analyzed during disease progression and development of drug resistance throughout different stages of multiple myeloma. Activity of PAT-SM6 was evaluated in combination with anti-multiple myeloma agents lenalidomide, bortezomib, and dexamethasone in vitro Finally, we report on a multiple myeloma patient with relapsed-refractory disease treated with PAT-SM6 in combination with bortezomib and lenalidomide. Although sGRP78 expression was present at all stages, it increased with disease progression and was even strongly elevated in patients with drug-resistant and extramedullary disease. Pretreatment with dexamethasone as well as dual combination of PAT-SM6/lenalidomide further increased sGRP78 expression and consecutively showed synergistic anti-multiple myeloma effects with PAT-SM6 in proliferation assays. As proof of concept, a 62-year-old male with triple resistant multiple myeloma treated with PAT-SM6, bortezomib, and lenalidomide experienced partial remission of both intra- and extramedullary lesions. PAT-SM6 therapy in combination regimens showed efficacy in relapsed-refractory multiple myeloma. Clin Cancer Res; 22(17); 4341-9. ©2016 AACR.

Abstract 3558: Predicting response to Mcl-1 targeting agents in NSCLC and multiple myeloma

Abstract Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins and is frequently amplified or over-expressed in both solid tumors and hematological malignancies, suggesting that its activity may be important for the survival of cancer cells. CDK9 inhibition results in the down regulation of Mcl-1 mRNA and subsequent protein levels by inhibiting transcription and represents an indirect approach to targeting Mcl-1. Mcl-1 can also be targeted directly using an inhibitor that disrupts the Mcl-1 complexes to induce apoptosis. Using both molecular and pharmacological approaches, we sought to identify predictive biomarkers of Mcl1 dependency in sensitive NSCLC and multiple myeloma cell lines. Here we demonstrate that NSCLC cell lines lacking MCL1 gene copy number gains are not sensitive to siRNA mediated knockdown of Mcl-1 or Mcl-1 inhibition (cell line sensitivity to CDK9 or Mcl-1 inhibition is defined by potency and extent of caspase activation). However, the presence of a copy number alteration does not predict sensitivity to Mcl-1 inhibition. To better understand what the drivers of sensitivity are, we developed quantitative assays on the Peggy platform (a capillary based immunoassay platform by Protein Simple) to measure both Mcl-1 and Bcl-xL protein levels. Using these assays, we show a correlation between sensitivity to a CDK9 or Mcl1 inhibitor and Mcl-1 levels, as well as to the ratio of Mcl-1 to Bcl-xL protein in a NSCLC cell line panel. These findings were then extended into a panel of multiple myeloma cell lines. While somewhat broad activity for CDK9 or Mcl-1 inhibition is seen across the cell lines tested, a subset of the sensitive lines have MCL1 amplification and express high levels of Mcl-1 protein. Mcl-1 levels alone, however, do not predict for sensitivity across the panel and, similar to NSCLC, the ratio of Mcl1 to Bcl-xL expression has greater positive predictive value. These results provide the rationale for exploring Mcl-1 copy number alterations and Mcl-1 and Bcl-xL protein levels as predictive biomarkers for tumor response when treating with a CDK9 or Mcl-1 inhibitor in both NSCLC and multiple myeloma. Citation Format: Kristen McEachern, Greg O’Connor, Justin Cidado, Matthew Belmonte, Evan Barry, Hannah Dry, Paul Secrist, Lisa Drew. Predicting response to Mcl-1 targeting agents in NSCLC and multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3558.

Mechanisms of drug resistance and sensitivity concerning novel agents for multiple myeloma.

Multiple myeloma (MM) is a neoplastic plasma-cell disorder characterized by clonal proliferation of malignant plasma cells in the bone marrow microenvironment. Novel treatments such as proteasome inhibitors and immunomodulatory agents significantly prolong both progression free survival and overall survival (OS) in patients with MM. However, recent clinical data have indicated that it is difficult to eradicate all MM clones even with the administration of such novel agents. To overcome drug resistance (DR), further investigation of the mechanism underlying DR is required, and strategies to up-regulate, maintain, and restore drug sensitivity will become increasingly important in the clinical setting. The findings from basic research focusing on the microenvironment, genetic abnormalities, and signal transduction disturbances related to MM pathogenesis are excellent strategies for developing new agents. In addition, based on the improved OS over 10 years and the low continuation rate of standard therapy in frail patients, a stratified strategy has the potential to provide total therapy.

Systematic review of the economic evaluations of novel therapeutic agents in multiple myeloma: what is the reporting quality?

Given the increasing healthcare costs and the recent introduction of novel agents in the treatment for multiple myeloma (MM), an incurable haematologic malignancy, more efficient use of existing resources is fundamental. The objective of this study was to systematically review economic evaluations of the use of novel agents in MM and assess their reporting quality. A literature search was performed in PubMed/Medline, Latin American and Caribbean Health Sciences Literature, Cost-Effectiveness Analysis Registry and the National Health Services Economic Evaluation Database for economic evaluations up to June 2015. The search strategy included Medical Subject Headings terms or text words related to MM, economic evaluations and drugs. Full economic evaluations of bortezomib, thalidomide or lenalidomide in patients with MM that were published in English, Portuguese or Spanish were included. Two independent authors performed study selection, data extraction and quality assessment using 24 items from the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) statement. Of the 132 potentially relevant records identified, eight satisfied the inclusion criteria. Most studies were cost-effectiveness analyses combined with cost-utility analyses (n = 6) from the public payer perspective (n = 4) and were performed in Europe (n = 6) on patients with refractory or relapsed MM (n = 5). All studies were based on economic models, with four of them using discrete event simulation. We found bortezomib-based therapies to be one of the more commonly selected treatment strategies for comparison (n = 7). Overall, the intervention was more effective and costlier than the alternative strategy (average of $54 630 per life year; $68 261 per quality-adjusted life year-QALY). The CHEERS' total score was 14·6 (SD = 2·6) with the most frequent problems being the lack of precision measures for all model parameters, no evaluation of heterogeneity of the results by subgroup analyses and no description of the role the funder in the identification, design, conduct and reporting of the analysis. Most analyses of the novel therapeutic agents determined that they were cost-effective in MM at a threshold of up to $100 000/QALY. Nevertheless, the poor reporting quality of the economic studies requires improvement to ensure greater transparency.

Renal impairment in multiple myeloma: A single center experience.

To determine the features of the different forms of kidney diseases associated with multiple myeloma (MM), we retrospectively studied 144 patients with kidney injury and MM at our institute from 1974 to 2014. The mean age of the patients was 60.1 years and the male:female ratio was 1.25. Renal disease was concomitant with the discovery of MM in 92% of cases. The mean follow-up of our patients was 2.1 years. Initial renal insufficiency was found in 131 (91%) patients. Cast nephropathy of the distal tubule was found in 110 (26%) patients, renal amyloidosis in 16 (11.1%), and light chain deposition disease in five (3.47%). Twelve (8.3%) patients had chronic glomerular nephropathy. Twenty-six patients reached end-stage renal failure within 13.4 months. Renal survival was 30.45 months. Predictive factors for improvement of renal function undergoing chemotherapy included serum creatinine <250 µmol/L, proteinuria <1 g/24 h, and the non-use of renal replacement therapy. Multiple pathogenic mechanisms can contribute to kidney injury in myeloma patients. Novel myeloma agents have shown considerable promise in reversing renal failure in some patients and improving outcomes.

Two first-in-class agents for multiple myeloma

Two first-in-class agents for multiple myeloma

Dual DNA Damage Repair Inhibition Synergizes with Alkylator Chemotherapy for Myeloma and Acute Leukemia

Alkylator chemotherapy remains important in pretransplant conditioning for multiple hematologic malignancies. Resistance to alkylators such as busulfan and melphalan results in part from the ability to repair DNA damage. DNA repair inhibition has therefore become an attractive treatment target. Here we tested the hypothesis that inhibitors of DNA repair overcome resistance to alkylator therapy.We initially treated REH, K562, HL60, NB4, RPMI8226 and U266 cells with the PARP inhibitor ABT-888. We noted widely varying GI50 values from 8.7 - 11x104 μM. Acute leukemia cells were overall insensitive to ABT-888 alone. However myeloma cells appeared relatively sensitive to ABT-888 with lower GI50 values (8.7μM for RPMI8226 cells, 49μM for U266 cells) and increased γH2AX foci at 24 hours after treatment. Synergy between ABT-888 and alkylator chemotherapy was observed in proliferative assays in all cell lines when treated with a fixed dose of ABT-888 (4μM) and increasing doses of either melphalan or busulfan. Combination index for RPMI8226 and HL60 cells was 0.5 at 50% proliferation. These findings were confirmed in a clonogenic assay. Increased cytotoxicity did not appear to be a result of apoptotic cell death as shown by Annexin V/PI co-staining. Instead combination treated RPMI8226 cells underwent accelerated senescence compared to melphalan treated cells at 24 hours as shown by βGal staining (27% vs. 51%, p=0.02). This was confirmed by upregulation of senescence related genes p16 (1.6 fold increase) and p21 (1.5 fold increase). Combination treated RPMI8226 cells were also noted to arrest in G2/M phase (43% vs. 50%). We hypothesized that cells may acquire the senescence associated secretory phenotype. We noted a 1.5-fold upregulation in IL6 gene expression in combination treatments compared to melphalan alone. CD86 gene expression increased by 1.6-fold and mean fluorescence intensity also increase by a factor of 1.3 compared to melphalan treatment. In mixed lymphocyte culture of treated RPMI8226 cells and CD3+ T cells, the stimulation index was 1.2 for melphalan treated cells and 3.2 for combination treated cells (p=0.003) showing that potentially RPMI8226 cells were able to directly present antigen to T-cells following combination treatment.As combination treatment resulted in upregulation of double strand repair genes such as BRCA1, BRCA2, FANCD2 and DNA-PK in HL60 cells we hypothesized that inhibitors of double strand repair may be synergistic with the alkylator chemotherapy and ABT-888. Treatment with NU7026 at fixed concentrations (10μM) to inhibit DNA-PK in addition to ABT-888 (4μM) resulted in 46% reduction in proliferation in RPMI8226 cells and 52% in U266 cells. No effect was seen in HL60 cells. However when used in combination with alkylator chemotherapy we were able to show marked synergy with alkylator and PARP inhibition in HL60 and RPMI8226 cells. Combination index in melphalan, ABT888 and NU7026 treated RPMI 8226 cells was 0.6.Finally we hypothesized that efficacy of DNA repair inhibition combination treatments was reliant on abnormal DNA repair in malignant cells in comparison to normal cells. To illustrate this we tested the impact of treatment on CD34+ cell hematopoietic colony formation. We show that NU7026, ABT-888 or a combination of both drugs with alkylator chemotherapy did not decrease colony formation over alkylator chemotherapy alone.Here we show that PARP and DNA-PK inhibition synergizes with alkylator chemotherapy in AML, ALL and myeloma cells lines, providing a rationale for the addition of these agents to conditioning chemotherapy. In addition we show the activity of these agents in myeloma without the use of alkylating agents. DisclosuresNo relevant conflicts of interest to declare.

Appropriate Dose Adjustment of Dexamethasone Does Not Compromise Outcomes in Relapsed Refractory Multiple Myeloma

Background: Dexamethasone (dex) is an integral part of various anti-myeloma regimens both in the frontline and relapsed/refractory (RR) setting. Several clinical trials have shown that dex adds to the efficacy of novel therapeutic agents for multiple myeloma (MM). Yet, a substantial number of patients are intolerant to even the standard of care low-dose dex (40 mg weekly), especially in heavily pre-treated RRMM, possibly due to cumulative steroid toxicities and thus, require dex dose adjustments. There is a concern of compromising the efficacy of anti-MM regimens by adjusting the dex dose lower. We describe the frequency of dex dose adjustment, characteristics of patients who required it and its effect on patient outcomes in a large prospective clinical trial of pomalidomide (pom) and dex in RRMM (MC0789).Methods: We utilized data from the Pom-dex trial (MC0789), which treated RRMM patients in different cohorts of pom (2 vs. 4 mg; 21 vs 28 days cycle) with fixed dose dex (40 mg orally weekly). Patients were divided into 2 subgroups, with any dose adjustment/elimination of dex vs. not. Continuous and categorical variables were compared between the two groups using two-sample t-test/Wilcoxon rank sum test or Chi-squared test, respectively. Logistic regression and Cox regression models were used to find the univariate and stepwise multivariate predictors of response to treatment and overall mortality, respectively. Kaplan-Meier method was used to estimate overall survival (OS) at 1, 3, and 5 years. All statistical tests were two-sided with the alpha level set at 0.05 for statistical significance.Results: Results from the MC0789 clinical trial have been reported previously. Almost half of all treated patients in this trial had dex dose adjusted (n=175/343; 51%). The median number of cycles for the first dose adjustment in this group was 3 (range 1-62), with 46.5% of patients (n=82) having multiple dose adjustments and majority (58.3%, n=102) within the first 3 cycles of treatment. Patients in the dose adjustment group were significantly older (mean 65 vs 60 years; p=0.0001). Males, Whites and patients diagnosed before 2006 or with normal cytogenetics also had a higher incidence of dex dose adjustment, though this was not statistically significant. Patients with dex dose adjustment had a longer duration of treatment (mean 339 days) as compared to the non-adjusted cohort (mean 123.65 days) (95% CI 160.13, 270.58; p <0.0001). Dex dose adjustment was associated with a higher probability of responding in the clinical trial after a univariate (p<0.001) as well as multivariate (p<0.001) analysis. Patients with dex dose adjustment also had a significantly better OS at 1, 3 and 5 years by univariate (p<0.001) and multivariate (p<0.001) analysis. (Figure 1) The only other significant factor was lesser number of prior regimens (3 or less), associated with a higher probability of response (p=0.002) and survival (p<0.001).Conclusion: Our analysis established that in these heavily pre-treated RRMM patients, appropriate dose adjustment of dex did not compromise their response rates; patients remained on treatment significantly longer and had an improved survival. Majority of patients (58.3%) requiring dex dose adjustment had it instituted early during treatment (<3 cycles), suggesting that early disease progression was less likely to determine dex dose continuation or adjustment. Appropriate dex adjustments should be judiciously employed as they may ensure longer treatment duration with novel anti-MM therapeutic agents, conferring superior response and survival in this difficult to treat MM subgroup. This also paves the path to further optimize dex dose in various anti-MM regimens, especially in these patients who are more likely to be intolerant to it. [Display omitted] DisclosuresKumar:Skyline, Noxxon: Honoraria; Celgene, Millennium, Onyx, Janssen, Noxxon, Sanofi, BMS, Skyline: Consultancy; Celgene, Millennium, Onyx, Novartis, Janssen, Sanofi: Research Funding.

99m technetium-Tocilizumab Fragments As Molecular Imaging Agent for Multiple Myeloma

Introduction: Multiple myeloma (MM) is a neoplasm of B lymphoid line that is characterized by clonal proliferation of malignant plasma cells in the bone marrow, producing monoclonal paraprotein (M) in blood and/or serum. Interleukin-6 (IL-6) is one of the key molecules related to growth, survival and proliferation of MM cells. Tocilizumab (TCZ) is a humanized monoclonal antibody directed against IL-6 receptor (IL-6R). When radiolabeled and used for tumor imaging, intact IgG exhibits high liver uptake. Antibody fragments (Fab´s) are quickly eliminated from blood and normal tissues (except kidneys), achieving high tumor-to-blood and tumor-to-normal tissue ratios with renal clearance. The aim of our work was to develop a 99mTc radiolabeled TCZ Fab´s fragment and to perform its chemical and biological evaluation in order to be used as a potential MM imaging agent for staging and restaging.Methods: Antibody fragmentation was carried out with papain and, once purified, Fab´s(TCZ) fragments were identified and derivatized with NHS-HYNIC-Tfa as bifunctional coupling agent. MALDITOF/TOF was used to confirm all procedures. A mixture of Tricine/SnCl2.2H2O was added to Fab´s(TCZ)-Tfa-HYNIC and radiolabeled with 99mTcO4-. Radiochemical purity and in-vitro stability in saline, serum and different concentration of L-cysteine up to 4 h were analyzed by ITLC and HPLC. In-vitro binding assays were performed using U266 and MM1S cell lines up to 120 min. Biodistribution and SPECT/CT images were evaluated on healthy Balb/c mice and MM1S tumor-bearing Balb/c nude mice at 0.5, 2 and 4 h.Results: Radiolabeling of HYNIC-Tfa-Fab´s(TCZ) was carried out in a fast, reproducible, easy, stable way showing high radiochemical purity and high specific activity. In vitro binding assays confirm that after its derivatization and radiolabeleing, Tfa-HYNIC-Fab`s(TCZ) does not interfere with the epitope recognition. In vivo biodistribution studies on healthy Balb/c mice and MM1S tumor-bearing Balb/c mice showed that 99mTc-HYNIC-Fab´s (TCZ) has significant renal uptake with neglectable uptake in other organs, indicating renal clearance. Tumor uptake was 12.84±1.80 %ID/g followed by 8.94±0.61 %ID/g and 3.05±1.49 %ID/g at 2 and 4 h, respectively. U266 tumor-to-muscle ratios were 5.79, 8.61 and 2.71 at 0.5, 2 and 4 h, respectively.Tumor uptake for MM1S tumor-bearing Balb/c nude mice was 10.05±1.32 %ID/g, 8.59±2.36 %ID/g and 3.88±0.68 %ID/g at 0.5, 2 and 4 h, respectively. MM1S tumor-to-muscle ratios were 6.32, 4.61 and 3.08 at 0.5, 2 and 4 h, respectively. Biodistribution data of 99mTc-HYNIC-Fab´s(TCZ) on U266 tumor-bearing Balb/c nude mice showed good tumor uptake and retention 0.5 h after its injection SPECT/CT images on healthy Balb/c mice and MM1S tumor-bearing Bal/c nude mice of 99mTc-HYNIC-Fab´s(TCZ) showed renal uptake and a discrete tumor uptake at 4 h p.i (Figure 1).Conclusions: Labeling Fab´s(TCZ) with 99mTc using HYNIC was performed in an easy, fast, stable and reproducible way preserving its biological activity. Biodistribution and SPECT/CT imaging assays allowed us to observe and evaluate its potential role as a diagnostic molecular imaging agent for MM. DisclosuresNo relevant conflicts of interest to declare.

A Striking Reduction of Monoclonal Protein in a Patient with Concurrent Plasma Cell Dyscrasia and CMML-2, after Treatment with Rigosertib (01910.Na)

Rigosertib (ON 01910.Na) is a member of a broader class of unsaturated sulfone kinase inhibitors capable of inducing profound mitotic spindle abnormalities, abnormal centrosome localization, G2-M cell cycle phase arrest and mitotic catastrophe, culminating in apoptosis. Rigosertib is a Ras mimetic that interferes with phosphoinositide 3-kinase (PI-3K)/Akt, reactive oxygen species and Ras/Raf/polo-like kinase (PLK) signaling pathways. Although broadly cytotoxic against malignant cells, it is remarkably non-toxic for non-neoplastic cells. For this reason, this is a particularly attractive compound to test against neoplastic diseases of the bone marrow such as MDS and acute leukemia.This is a report of an unexpected reduction in monoclonal IgG, during a subject participation in a Phase III, randomized study of rigosertib, in patients with MDS who have either failed to respond, or progressed after receiving hypomethylating agents (ONTIME Trial).A 75-year-old man with CMML-2 had a CBC on day 1 of the trial that demonstrated leukocytosis, with absolute monocytosis, 7% blasts in the peripheral blood, Hgb of 9.4 gm/dl, and platelets of 7 K. He was transfusion dependent for both pRBCs and platelets. His chemistry panel demonstrated a high total protein of 9.9 (NL: 6.0 - 8.2 G/DL) with low albumin at 2.4 (NL: 3.5 - 5.0 G/DL); therefore, an SPEP/IPEP was performed, reporting the presence of monoclonal IgG kappa. Quantitative immunoglobulins showed an elevated IgG of 3594 mg/dl (NL: 596 - 1584 MG/DL). Serum free light chains were remarkable for an elevated Kappa fraction at 38.94 (NL: 0.33 - 1.94 MG/DL).On day 1 of cycle 5 of rigosertib, he was started on pulse decadron for 2 months, after which his disease progressed to AML, and he died shortly thereafter. Neither his bone marrow biopsies, nor his hematological parameters demonstrated a response to treatment with rigosertib.In contrast and interestingly, his total protein, serum kappa light chain load, and total IgG, all were drastically reduced shortly after initiation of rigosertib, as can be seen in the graph below depicting a substantial drop in his kappa light chain as well as the kappa/light chain ratio. Importantly, reduction in the monoclonal protein was noted prior to initiation of pulse decadron.Even though his initial bone marrow biopsy did not note a monoclonal plasma cell population, a subsequent bone marrow reported a low-level involvement with a plasma cell dyscrasia, with kappa light chain restriction. His final bone marrow biopsy confirmed progression to AML, but the previously seen plasma cell dyscrasia was no longer present.Conclusion: We are not aware of prior reports describing a similar effect of rigosertib on M-proteins. However, in vitro studies with rigosertib have demonstrated antitumor effects and induction of apoptosis in myeloma cell lines1. This observation merits further exploration of this agent in multiple myeloma.

Patterns of Relapse and Progression in Multiple Myeloma Patients Treated with Conventional and Novel Agent-Based Therapy: A Single Centre Experience

BackgroundThe incorporation of novel agents (NA) for multiple myeloma (MM) has improved the response rates (RR), overall survival (OS), and progression free survival (PFS) when compared to conventional agents (CA). Unfortunately, relapse is inevitable and few studies focus on patterns of relapse, especially in non-transplant patients (pts). We aim to describe the different patterns of relapse in non-transplant MM pts and determine if any pre-treatment clinical or disease characteristics can predict the patterns relapse. We will evaluate whether NA treated pts have higher rates of aggressive relapse with plasmacytomas or plasma cell leukemia (Leuk Res. 2009 Aug;33(8):1137-40). Secondly, RR and PFS for pts treated with CA vs NA will be described.MethodsA retrospective evaluation of 156 consecutive newly diagnosed non-transplant eligible MM pts at Princess Margaret Cancer Centre receiving at least two consecutive cycles of CA or NA from 1999 to 2015. CA included steroids and alkylators while NA had immunomodulatory (IMiD) drugs (thalidomide, lenalidomide) and proteasome inhibitors (PI) (bortezomib). Response type was defined by the revised International Myeloma Working Group criteria (Leukemia. 2006 Sep;20(9):1467-73. Epub 2006 Jul 20); relapse patterns as defined in the Spanish Registry (Haematologica. 2002 Jun;87(6):609-14)ResultsFor 156 non-transplant MM pts: 81 (52%) male, average age 76 yrs, 87 (56%) treated with NA (thalidomide=15; PI=52). Baseline characteristics were not significantly different between groups (Table 1). Sixty three (52%) pts had a clinical relapse, 37 (30%) pts had a biochemical relapse, and 22 (18%) were switched immediately to second line therapy given suboptimal response (lack of clinical benefit or PD). Six pts relapsed with isolated plasmacytomas (4 CA vs 2 NA). There was one case of plasma cell leukemia relapse in an IMiD-treated pt.Twenty seven (17.3%) pts had not relapsed at the time of analysis and had ongoing follow-up. There was no significant difference in the types of relapse patterns for pts treated with CA versus NA (p=0.26) or for CA versus IMiD versus PI therapy (p=0.22). Pts with insufficient response to first line chemotherapy were more likely to have a 17p deletion (p=0.07). All pts with a biochemical relapse did not have a 17p deletion. The median follow-up time was 16.4 (range 0.6 to 99) months (mo) for CA vs. 19.6 (range 0.4 to 107) mo for NA.Table 1Patient CharacteristicsRelapse Pattern - Mean (sd)Clinicaln =63Biochemicaln =37Insufficient n=22p -valueHgb108 (17)99 (21)110 (18)0.08WBC6.1 (2.4)6.4 (3.3)5.6 (1.9)0.72Plt231 (107)224 (117)237 (103)0.64Ca2.5 (0.3)2.5 (0.4)2.4 (0.3)0.58Cr125 (92)130 (87)136 (133)0.92B2M492 (537)596 (448)618 (618)0.19Alb37 (7)36 (6)36 (5)0.29CRP6.7 (7.4)9.3 (19.1)13.0 (17.5)0.56Relapse Pattern - Count (%)ConventionalNovel35 (56)28 (44)15 (41)22 (59)13 (59)9 (41)0.26IgGIgAFLCOther34 (54)17 (27)10 (16) 2 (3)23 (62) 6 (16) 8 (22) 0 (0)14 (63) 4 (18) 4 (18) 0 (0)0.78KappaLambda32 (58)23 (42)19 (59)13 (41)12 (57)9 (43)0.99Chr 13 Del8/29 (28)7/10 (41)3/9 (33)0.64t(4,14)2/29 (7)3/15 (20)0/8 (0)0.3417p Del4/28 (14)0/15 (0)3/9 (33)0.07Extramed. Inv.4 (6)1 (3)1 (5)0.85Sixty (38%) pts achieved VGPR/CR/sCR, 53 (34%) PR, 35 (22%) SD, and 8 (5%) PD with upfront therapy. VGPR/CR/sCR was seen in 13 (21%) pts with CA vs 47 (78%) with NA (p<0.01). For NA, 28 (47%) pts in the PI-based group achieved VGPR/CR/sCR compared to 19 (32%) in IMiD-based (p<0.01).The median PFS for all pts was 21 (95% CI 17-23) mo, with 17 (95% CI 13-23) mo for CA vs 23 (95% CI 17-29) mo in NA. There is a statistically significant difference between CA and NA in PFS (p=0.0045; Figure 1).DiscussionIn non-transplant MM pts, we did not find a significant difference in the patterns of disease relapse between those treated with CA versus NA. Baseline characteristics such as renal failure or type of treatment do not seem to predict for the pattern of relapse; except the presence of 17p deletion trended toward more treatment failure. We note that the number of pts with aggressive relapses (plasmacytomas or plasma cell leukemia) was low and this limits our ability to detect differences in outcomes and baseline factors. Pts treated with NA continue to have better RR and PFS than those treated with CA. Future work with longer follow-up intervals is needed in order to capture late relapses, better describe relapse patterns with NA as well as understanding disease biology. [Display omitted] DisclosuresChen:Celgene: Consultancy, Honoraria, Research Funding. Prica:Janssen: Honoraria; Celgene: Honoraria. Reece:Lundbeck: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Research Funding; Merck: Research Funding; Millennium Takeda: Research Funding; Bristol-Myers Squibb: Research Funding; Otsuka: Research Funding; Novartis: Honoraria, Research Funding; Onyx: Consultancy; Amgen: Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Tiedemann:Janssen Ortho: Honoraria; Celgene: Honoraria; Amgen: Honoraria. Kukreti:Celgene: Honoraria; Amgen: Honoraria; Lundbeck: Honoraria; Roche: Honoraria; Janssen Ortho: Honoraria.

Voriconazole Increase the Risk of PN in Multiple Myeloma Patients Treated with Bortezomib-Based Chemotherapy

Background Bortezomib (btz) is the major and potent agent in multiple myeloma (MM) treatment and intravenous (IV) injection is its standard administration route. Our previous study showed that Subcutaneous (SC) administration of btz was an important alternative with comparable efficacy but better safety profile, significantly decreased and delayed the development of peripheral neuropathy (PN) in patients with MM. However, PN still always limited the use of btz. The primary objective is try to identify some correlative factors for PN, then to decrease the risk of PN and the grade of PN.Methods This retrospective study was undertaken at a single centre in China. Patients with NDMM were assigned to receive up to nine 21-day cycles of Btz based regimens, including PAd/VDd/BCd (btz 1.3mg/m2, on days 1, 4, 8 and 11; adriamycin 9mg/m2 intravenously on days 1 - 4; or PLD 30mg/m2, intravenously on days 1, or CTX 500mg/m2, orally on days 1, 8, 15, and dexamethasone 20mg/day, orally or intravenously on days 1, 2, 4, 5, 8, 9, 11 and 12). Btz was administered by SC injection or IV infusion. The basic characteristics, drug combination (especially drugs for fungi), the effect and adverse events were recorded. Then the correlation were analysed.Results 252 (male, n=159) NDMM patients were received Btz based treatment from Oct. 2008 to Nov. 2014. The median age were 56 (26-77). In this group, patients were treated with a median 4 cycles of btz-based chemotherapy. 98, 101 and 53 patients received PAd, BCd and VDd regimen, respectively. 114 patients received IV btz, and 148 received SC btz. The median total Btz dosage was 20.4 (2.6-56.6) mg/m2. Patients achieved at least PR at median 1 (1-7) cycle, and achieved the best response at median 2 (1-9) cycles. The overall response rate (ORR, >=PR) was 95%, with 33.3%, 12.4%, 22.4% achieved CR, nCR and VGPR, respectively. With median follow up 24 months, the median PFS and OS were 28 months, and not arrived, respectively. There was no significant difference between IV and SC group. During their course, 49, 19, 73, and 46 cases received fluconazole, itraconazole, voriconazole, and caspofungin respectively as prophylaxis or treatment for fungi. 140 (55.56%) patients developed PN during their courses, with 17.06% were ≥ grade 3. The median dosage of btz when PN developed was 15.6 mg/m2. In the subgroup which combined voriconazole, 59 (80.82%) patients developed PN, with 39.07% were ≥ grade 3. The median dosage of btz when PN developed was 12.3mg/m2 in this subgroup. In addition, 10 patients (13.70%) developed diarrhea of grade 3/4 and 20 cases (27.4%) developed paralytic ileus of any grade, which may be related to the neuropathy toxicity of btz too. Correlation analysis showed that only voriconazole combination was significantly correlated with PN development (p<0.0001). However, any other factors, including age, sex, the percentage of plasma cells in bone marrow, the level of M-protein, IL-6, TNF-α, β2-MG, ALB, calcium, the type of M-protein, etc, were all without correlation with the development of PN.Conclusions Voriconazole combination significantly increased the risk and the grade of PN in patients treated with btz-based chemotherapy. If this combination can't be avoided, patients should be observed more closely or btz dosage should be decreased earlier. DisclosuresNo relevant conflicts of interest to declare.

Junctional adhesion molecule-A is overexpressed in advanced multiple myeloma and determines response to oncolytic reovirus.

Despite the development of several new agents for multiple myeloma (MM) therapy over the last decade, drug resistance continues to be a significant problem. Patients with relapsed/refractory disease have high mortality rates and desperately need new precision approaches that directly target specific molecular features that are prevalent in the refractory setting. Reolysin is a proprietary formulation of reovirus for cancer therapy that has demonstrated efficacy in multiple clinical trials. Its selective effects against solid tumors have been largely attributed to RAS-mediated control of reovirus replication. However, the mechanisms regulating its preferential anti-neoplastic effects in MM and other hematological malignancies have not been rigorously studied. Here we report that the reovirus receptor, junctional adhesion molecule-A (JAM-A) is highly expressed in primary cells from patients with MM and the majority of MM cell lines compared to normal controls. A series of experiments demonstrated that JAM-A expression, rather than RAS, was required for Reolysin-induced cell death in MM models. Notably, analysis of paired primary MM specimens revealed that JAM-A expression was significantly increased at relapse compared to diagnosis. Two different models of acquired resistance to bortezomib also displayed both higher JAM-A expression and elevated sensitivity to Reolysin compared to parental cells, suggesting that Reolysin may be an effective agent for patients with relapsed/refractory disease due to their high JAM-A levels. Taken together, these findings support further investigation of Reolysin for the treatment of patients with relapsed/refractory MM and of JAM-A as a predictive biomarker for sensitivity to Reolysin-induced cell death.

Subcutaneous administration of bortezomib significantly decreased and delayed the development of peripheral neuropathy in patients with newly diagnosed multiple myeloma

Bortezomib (btz) is the major and potent agent in multiple myeloma (MM) treatment and intravenous (IV) injection is its standard administration route. However, peripheral neuropathy (PN) always limited the use of btz. Subcutaneous (SC) administration is an important alternative. The primary outcome of our previous study showed that SC administration of btz significantly decreased and delayed the development of PN in patients with newly diagnosed MM (NDMM) and relapse/refractory MM (RRMM). Here, we amplified the cohorts and extended follow up to further confirm our previous primary outcome in patients with NDMM. This retrospective, historical control study was undertaken at a single centre in China. Patients with NDMM were assigned to receive up to nine 21-day cycles of Btz based regimens, including PAd/VDd/BCd (btz 1.3 mg/m2 , on days 1, 4, 8 and 11; adriamycin 9 mg/m2 on days 1-4; or PLD 30mg/m2 on days 1, or CTX 500mg/m2 on days 1, 8, 15, and dexamethasone 20 mg/day on days 1, 2, 4, 5, 8, 9, 11 and 12). Btz was administered by SC injection or IV infusion. Then the efficacy and safety of SC versus IV btz were compared. 330 NDMM patients were received Btz based treatment. 151 received a median of 5 cycles of SC btz (SC group) from 2012.3 to 2014.12. 179 cases were treated with a median 3 cycles of IV btz (historical control IV group) from 2008.5 to 2012.2. The overall response rate (ORR, >1⁄4PR) was 97.4% and 97.2% in these two groups, with 37.7% and 36.3% achieved CR respectively, indication the efficacy was very comparable. SC group had better safety profile. Most of any grade adverse events (AE) were less common with SC than with IV administration, including thrombocytopenia, neutropenia, anaemia. The most importance was PN of any grade (32.1% vs 58.1%; p1⁄40$01) was significantly decreased with SC btz. Especially severe PN (grade 3/4) was also significantly decreased (5.3% vs 18.4%, p1⁄40.013). Notably, the incidence and seriousness of diarrhea were lower (any grade, 9.8% vs 15.7%, >1⁄4grade 3, 0.9% vs 4.7%, respectively), so were constipation, and intestinal obstruction, which may be related to the neuropathy toxicity of btz. The time of developing PN was delayed in SC group. Median dosage of btz when PN developed was 15.6 mg/m2 in SC group and 10.4mg/m2 in IV group. The efficacy of SC btz in patients with NDMM was comparable with standard IV administration. SC btz was locally well tolerated. It significantly decreased and delayed the development of PN. PO-143 Melphalan 140mg/m“2” significantly reduces 5 year progression free survival in multiple myeloma patients receiving a peripheral blood autologous stem cell transplant compared with melphalan 200mg/m“2” C. Attwood, E. Nicholson, R. Chakraverty, A. Fielding, P. Kottaridis, D. Hughes, A. Mehta, W. Stewart, C. Kyriakou, S. Worthington, S. Grace, A. Wechalekar, S. MacKinnon Royal Free Hospital, London

TM-233, a novel analog of 1'-acetoxychavicol acetate, induces cell death in myeloma cells by inhibiting both JAK/STAT and proteasome activities.

Although the introduction of bortezomib and immunomodulatory drugs has led to improved outcomes in patients with multiple myeloma, the disease remains incurable. In an effort to identify more potent and well-tolerated agents for myeloma, we have previously reported that 1′-acetoxychavicol acetate (ACA), a natural condiment from South-East Asia, induces apoptotic cell death of myeloma cells in vitro and in vivo through inhibition of NF-κB-related functions. Searching for more potent NF-κB inhibitors, we developed several ACA analogs based on quantitative structure–activity relationship analysis. TM-233, one of these ACA analogs, inhibited cellular proliferation and induced cell death in various myeloma cell lines with a lower IC50 than ACA. Treatment with TM-233 inhibited constitutive activation of JAK2 and STAT3, and then downregulated the expression of anti-apoptotic Mcl-1 protein, but not Bcl-2 and Bcl-xL proteins. In addition, TM-233 rapidly decreased the nuclear expression of NF-κB and also decreased the accumulation of cytosolic NF-κB. We also examined the effects of TM-233 on bortezomib-resistant myeloma cells that we recently established, KMS-11/BTZ and OPM-2/BTZ. TM-233, but not bortezomib, inhibited cellular proliferation and induced cell death in KMS-11/BTZ and OPM-2/BTZ cells. Interestingly, the combination of TM-233 and bortezomib significantly induced cell death in these bortezomib-resistant myeloma cells through inhibition of NF-κB activity. These results indicate that TM-233 could overcome bortezomib resistance in myeloma cells mediated through different mechanisms, possibly inhibiting the JAK/STAT pathway. In conclusion, TM-233 might be a more potent NF-κB inhibitor than ACA, and could overcome bortezomib resistance in myeloma cells.

A Patient with Multiple Myeloma who Developed Severe Myoclonus after Stem Cell Transplantation

There are various neurologic manifestations of Multiple Myeloma (MM) seen either at presentation or as a complication of various anti- myeloma agents administered during the course of the disease. These neurologic complications might occasionally be challenging to diagnose and treat. Peripheral nervous system is more commonly affected and peripheral neuropathy is the most common form of neurologic complications seen in MM. Here we report a gentleman with MM on regular renal replacement therapy developed severe myoclonus 3 days status post autologous stem cell (ASCT). Besides MM, he had Hypertension and Diabetes Mellitus as to his renal failure. He was also suffering from neuropathy for which gabapentin was commenced.

Induction of Bim-Dependent and -Independent Apoptosis in Multiple Myeloma

Multiple myeloma is a plasma cell malignancy, estimated to affect 24,050 new Americans in 2014. While new treatments have emerged in recent years greatly increasing survival rates, our understanding of the mechanisms whereby these therapies kill myeloma cells is still lacking. By understanding how drugs induce cell death we can help to predict cell sensitivity, dissect resistance pathways, and design more effective combination treatment regimens.The Bcl-2 family of proteins regulates cell survival and death. We have previously reported that in myeloma cells, one can map the Bcl-2 protein that is necessary for survival by determining which anti-apoptotic protein is sequestering the pro-apoptotic protein, Bim. Bim is known as a crucial component in the activation of pro-apoptotic proteins Bak and Bax, which is necessary for cells to undergo apoptosis. While other proteins, such as Puma and tBid, are also capable of activating Bak and Bax, Bim is thought to be the primary activator in myeloma cells.Given the role of Bim in apoptosis, we were interested in determining the contribution of Bim induction in response to therapeutic agents and resistance mechanisms. RNAseq analysis of 69 myeloma cell lines revealed that only two human myeloma cell lines, LP-1 and KMS26 were devoid of Bim expression. This is due to biallelic deletion in both lines as determined by array CGH. Therefore we examined the responses of these two cell lines to ABT-737, which functions by directly inhibiting Bcl-2 and Bcl-xL as well as 3 therapeutic agents that are commonly used in myeloma therapy; dexamethasone, carfilzomib, and melphalan. We compared the responses of Bim-deficient lines to the Bim-expressing cell lines MM.1s and RPMI8226. As expected LP-1 and KMS26 were highly insensitive to ABT-737 and dexamethasone (IC50s (μM): ABT-737: 8226 – 0.65, MM.1s – 0.5, KMS26 – 3.0, LP-1 – 4.5; Dexamethasone: 8226 – 1.9, MM.1s – 1.5, KMS26 – could not be calculate due to no response at tested concentrations, LP-1 – 19.7) as we and others have previously demonstrated Bim-dependence of these agents in myeloma through silencing experiments. When treated with carfilzomib, LP-1 and KMS26 exhibited an intermediate sensitivity (IC50s: (nM): 8226 – 7.9, MM.1s – 6.0, KMS26 – 20.3, LP-1 – 16.2). Given previous data generated in our lab indicating proteasome inhibitor-induced cell death is inhibited by over-expression of Bcl-2, Bcl-xL and Mcl-1 and that the apical caspase activated following proteasome inhibition is caspase-8, the apoptotic response to carfilzomib in KMS26 and LP-1 is most consistent with the activation of Bid. Interestingly, when treated with melphalan, all four cell lines exhibited similar sensitivities (IC50s (μM): 8226 – 65, MM.1s – 19.4, KMS26 – 42, LP-1 – 26.7). Previous studies have demonstrated that alkylating agents can induce PARP1-dependent necrosis in the absence of Bax/Bak. Therefore to determine if the melphalan-induced death was caspase-dependent, we co-treated the cell lines with melphalan and the caspase-inhibitor, qVD-OPH. Upon co-treatment, the apoptotic response in all cell lines was drastically reduced, indicating a caspase-dependent death (IC50s (μM): 8226 – 131, MM.1s – 118, KMS26 – 386, LP-1 – 80.5).In order to verify that these responses are representative of loss of Bim and not unique to LP-1 and KMS26, we utilized ABT-737 resistant cell lines generated in our lab that express very low levels of Bim (KMS11-ABTR and U266-ABTR). Similar to LP-1 and KMS26, these cell lines displayed an intermediate sensitivity to carfilzomib when compared to control sensitive lines (KMS11-CR2 and U266-CR2). Additionally, both KMS11-ABTR and U266-ABTR were sensitive to melphalan in a caspase-dependent manner. We are currently determining the role of Puma and Bid as BH3-activating proteins in proteasome inhibitor- and melphalan-induced cell death.Our data indicate that active therapeutic agents in myeloma initiate the intrinsic apoptosis pathway through different BH3-activator proteins. Defining the activator proteins for therapeutics could allow for more precise therapeutic decision-making based on patient expression data. Additionally these findings suggest that rational combination therapies could be designed based on combining Bim-dependent and -independent therapies without having to understand how the specific targets of therapies interact (e.g. steroids and alkylating agents). DisclosuresBoise:Onyx: Consultancy.

Dexamethasone Synergizes with ABT-199 through the Induction of Bim and Bcl-2 Dependence in Myeloma

Multiple myeloma is a plasma cell malignancy, with a 10-year survival rate of approximately 25%. While this survival rate is largely attributed to the FDA approval of new therapies for the disease, such as IMiDs and proteasome inhibitors, eventual relapse and drug resistance is the reality facing the majority of myeloma patients. Subsequently, the quest for new drugs and combination therapies is ongoing.Novel agents targeting the Bcl-2 family of cell survival regulators are promising avenues of research. We have previously reported on a method of predicting sensitivity of myeloma cell lines and patient samples to the Bcl-2/xL inhibitor ABT-737, based on the binding pattern of pro-apoptotic protein Bim to anti-apoptotic proteins Mcl-1, Bcl-xL, and Bcl-2. In Mcl-1-dependent cells, Bim is primarily associated with Mcl-1, and insensitive to ABT-737. Alternatively, in cells that are co-dependent on Mcl-1 and Bcl-2/xL for survival, Bim is either predominantly associated with Bcl-2/xL or when it is released from Bcl-2/xL it cannot bind to Mcl-1 because of the presence of the Mcl-1 inhibitor Noxa, rendering the cells sensitive to ABT-737. Unfortunately, ABT-737, or the clinical compound Navitoclax, is not a viable treatment for myeloma, due to its potential for causing thrombocytopenia. Therefore we chose to investigate the sensitivity of myeloma cell lines and freshly isolated patient samples to ABT-199.ABT-199, a specific inhibitor of anti-apoptotic protein Bcl-2, is currently in phase I clinical trials for multiple myeloma. We have previously reported on its preclinical efficacy as a single agent in myeloma as well as in combination with other commonly used therapeutics. While ABT-199 was ineffective as a single agent in the cell lines we tested, combining it with dexamethasone significantly decreased the IC50s (Fold change: 8226 – 2.6, MM.1s – 43.5, OPM2 – 14.1, KMS11 – 23.3, KMS18 – 4.8). Here we report on the mechanism by which dexamethasone increases sensitivity to ABT-199, as well as the effectiveness of this combination in freshly isolated patient samples.We performed real-time PCR analysis to determine changes in the expression of the Bcl-2 family of proteins following treatment with ABT-199, dexamethasone, and the combination. Minimal changes in expression were seen with ABT-199 treatment; however, dexamethasone treatment greatly induced the expression of both Bcl-2 and Bim, which was also seen in the combination treatment, along with a decrease in Bcl-xL expression. Next we performed CoIP studies to examine how these changes in expression affected the binding pattern of Bim to the anti-apoptotic proteins. As expected, we found that upon treatment with ABT-199, the small amount of Bim previously bound to Bcl-2 was released. Alternatively, when cells were treated with dexamethasone alone, the amount of Bim bound to Bcl-2 increased, likely due to the significant increase in expression of both proteins. When cells were subjected to both drugs simultaneously, there was a decrease in the amount of Bim bound to Bcl-xL, as well as an absence of Bim on Bcl-2. Thus, co-treatment brings about a scenario whereby ABT-199 releases Bim bound to Bcl-2, and at the same time, prevents the Bim induced by dexamethasone from binding to Bcl-2. These changes, coupled with a decrease in amount of Bcl-xL available to bind Bim, result in the induction of apoptosis.To further verify this drug synergy, we treated ficoll-isolated buffy coat cells from 10 myeloma patients with ABT-199, either alone or in combination with dexamethasone. Samples from 4 of the patients were sensitive to ABT-199 alone, thus combination had little effect. For the other 6 patient samples, the addition of dexamethasone to ABT-199 significantly decreased the IC50 over ABT-199 alone (Fold Change: MM61 – 7.3, MM62 – 17.5, MM63 – 20.1, MM64 – 2.2, MM65 – 1.8, MM66 – 8.7).Previous studies have reported that the t(11;14) subset of multiple myeloma is highly sensitive to ABT-199. Five of the 10 patient samples we tested were positive for the t(11;14), and all but one, MM61, were sensitive to ABT-199 alone. However, MM61 was sensitive to the combination treatment. Taken together, our data demonstrate that the addition of dexamethasone expands the potential of ABT-199 to a broader set of patients who would otherwise likely be resistant to monotherapy. DisclosuresKaufman:Millennium: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Onyx: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Spectrum: Consultancy, Honoraria; Merck: Research Funding. Lonial:Millennium: The Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding.

The in vivo Transcriptional Response Towards Epigenetic Modulating Agents in Multiple Myeloma

Multiple myeloma (MM) is a hematological malignancy characterized by a plasma cell accumulation in the bone marrow (BM), for which novel treatment options are urgently needed. Epigenetic modulating agents such as histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) are under intense investigation for cancer therapy. As shown in numerous functional in vitro studies, HDACi and DNMTi affect various biological processes important for tumor control including tumor cell survival, proliferation, differentiation and DNA repair. Given the broad mechanisms of action of these agents, it remains important to continue pre-clinical evaluation to identify in vivo relevant mechanisms of action. This may lead to the identification of novel biologically relevant targets and predictive biomarkers allowing clinical trial optimization.Recently, we developed gene expression based risk scores after treating human MM cell lines with TSA (HA-score) or decitabine (DM-score). These scores were predictive for MM patient survival using two independent cohorts (Heidelberg-Montpellier (HM) and University of Arkansas for Medical Sciences-Total Therapy 2) and identified potential biomarkers predictive for drug sensitivity. However, the transcriptional response of MM cells in vivo may be influenced by the close contact with the BM-environment. Therefore, we here aimed to characterize the transcriptional response of MM cells after in vivo treatment with the HDACi JNJ-26481585 (quisinostat) or the DNMTi decitabine using the syngeneic immunocompetent murine 5T33MM model.5T33MM mice (n=4/group) with established disease were treated with quisinostat or decitabine for 5 days after which tumor cells were isolated from the BM and subjected to microarray analysis. Using Significance Analysis of Microarray, we identified 574 and 180 probesets deregulated by respectively quisinostat and decitabine (of which 111 are in common). To assess the prognostic value of the deregulated genes, we performed MaxStat analysis in the HM cohort. JNJ-585 deregulated 31 genes associated with good prognosis and 31 associated with bad prognosis. Decitabine altered expression of 20 genes linked with poor prognosis while 5 genes were linked with good prognosis. The prognostic value of these genes was then implemented in a murine (Mu)-DM and Mu-HA score. The score values were significant higher in MM patients and human myeloma cell lines compared to MGUS and healthy bone marrow plasma cells. In addition, the scores were useful to separate patients of the 2 cohorts into a low risk and high risk group. Patients from the proliferation subgroup had a higher score compared to all other subgroups. In concordance, the scores were highest in patients with a high gene-expression based proliferation index. Using gene ontology (GO) tools (DAVID) and pathway tools (Reactome, STRING and Pathway-guide), we next explored the association of in vivo deregulated genes with biological processes and pathways. GO analysis showed that quisinostat-deregulated genes were mainly involved in immune modulation. Pathway analysis revealed associations with lymphocyte activation and proliferation, immune effector mechanisms and T-helper-1 development through processes like cytokine interactions, chemokine signaling and T-cell receptor- and NK-cell-signaling. In concordance, the signature represented elevated presence and signaling of interferon, tumor necrosis factor, interleukin-1 (IL-1), IL-2 and IL-12. The second most prominent alterations were genes linked with transcriptional misregulation in cancer. Pathways predicted to be affected by these alterations are linked with differentiation, resistance and cell survival. For decitabine, the gene list was substantially smaller and for more than half shared by JNJ-585. The pathway analysis also identified genes linked to immune system, gene expression regulation and metabolism pathways.In conclusion, in vivo treatment with epigenetic modulating agents identified a prognostic gene signature. In addition, HDACi (and to a lesser extent DNMTi) deregulated immunomodulatory genes and genes involved in transcriptional regulation. This indicates that immune regulation is an important in vivo anti-tumor property of HDACi and supports the rational to combine HDACi with immunomodulatory therapies such lenalidomide or cellular/peptide vaccination strategies. DisclosuresHose:Novartis: Research Funding. Seckinger:Novartis: Research Funding.