Synergistic Anti-myeloma Effects Research Articles

Abstract 4554: Novel combination therapy for treating proteasome inhibitor-resistant multiple myeloma

Abstract Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by osteolytic bone disease and immunosuppression. Proteasome inhibitors (PIs) have remarkably improved the survival of MM patients, but dose-limiting toxicities and the development of drug resistance limit their long-term utility. Elevated TGF-β levels in MM patient sera correlate with drug resistance, disease progression, metastasis and poor prognosis. Therefore, we evaluated the anti-MM therapeutic potential of a TGF-β type I receptor kinase inhibitor, Vactosertib. In vitro treatment of Vactosertib synergistically inhibited the growth of bortezomib (BTZ)-resistant MM cells in combination with either BTZ or ixazomib (IXZ) by suppressing TGF-β activation of Smad2/3 and expression of PSMB5, which encodes the proteasome 5 catalytic subunit targeted by PIs. Oral administration of Vactosertib as a single agent decreased MM progression and mortality of the mice bearing BTZ-resistant MM. Vactosertib alone also attenuated PSMB5 expression and proteasome activity, reduced the expansion of CD11b+Gr-1+ myeloid derived suppressor cells (MDSCs) in bone marrow (BM) tumor microenvironment (TME), and diminished the population of Foxp3+ regulatory T cells (Treg) in the spleen. Combination therapy of Vactosertib plus PI (BTZ or IXZ) exhibited a synergistic anti-myeloma effect when compared to either Vactosertib or PI alone by greatly prolonged survival and significant a reduction in both MDSCs and Tregs. Furthermore, therapy of BCMA+ CAR T cells in combination with Vactosertib exhibited a synergistic anti-tumor effect, compared to either CAR T cells or Vactosertib. Taken together, our data provide the rationale for clinical evaluation of Vactosertib in MM and demonstrate proof-of-concept that combination of Vactosertib and either PI or cell therapy may overcome drug resistance and enhance durable patient responses. Citation Format: Byung-Gyu Kim, Sung Hee Choi, Huong Nguyen, Fu-Sen Liang, Seong-Jin Kim, John Letterio, Alex Huang. Novel combination therapy for treating proteasome inhibitor-resistant multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4554.

Harnessing the cytotoxic granule exocytosis to augment the efficacy of T-cell-engaging bispecific antibody therapy.

T-cell-engaging bispecific antibody (T-BsAb, also known as BiTE) therapy has emerged as a powerful therapeutic modality against multiple myeloma. Given that T-BsAb therapy redirects endogenous T cells to eliminate tumor cells, reinvigorating dysfunctional T cells may be a potential approach to improve the efficacy of T-BsAb. While various immunostimulatory cytokines can potentiate effector T-cell functions, the optimal cytokine treatment for T-BsAb therapy is yet to be established, partly due to a concern of cytokine release syndrome driven by aberrant interferon (IFN)-γ production. Here, we functionally screen immunostimulatory cytokines to determine an ideal combination partner for T-BsAb therapy. This approach reveals IL-21 as a potential immunostimulatory cytokine with the ability to augment T-BsAb-mediated release of granzyme B and perforin, without increasing IFN-γ release. Transcriptome profiling and functional characterization strongly support that IL-21 selectively targets the cytotoxic granule exocytosis pathway, but not pro-inflammatory responses. Notably, IL-21 modulates multiple steps of cytotoxic effector functions including upregulation of co-activating CD226 receptor, increasing cytotoxic granules, and promoting cytotoxic granule delivery at the immunological synapse. Indeed, T-BsAb-mediated myeloma-killing is cytotoxic granule-dependent, and IL-21 priming significantly augments cytotoxic activities. Furthermore, in vivo IL-21 treatment induces cytotoxic effector reprogramming in bone marrow T cells, showing synergistic anti-myeloma effects in combination with T-BsAb therapy. Together, harnessing the cytotoxic granule exocytosis pathway by IL-21 may be a potential approach to achieve better responses by T-BsAb therapy.

Talquetamab + Pomalidomide in Patients with Relapsed/Refractory Multiple Myeloma: Safety and Preliminary Efficacy Results from the Phase 1b MonumenTAL-2 Study

Introduction: Talquetamab (tal) is a T-cell redirecting bispecific antibody (BsAb) targeting GPRC5D and CD3. GPRC5D is a novel antigen that is highly expressed on malignant plasma cells but has low expression on B cells and normal plasma cells. Tal has demonstrated deep and durable responses, including in high-risk populations, and a clinically manageable safety profile in patients (pts) with relapsed/refractory multiple myeloma (RRMM) in the MonumenTAL-1 study (NCT03399799/NCT04634552). Pomalidomide (pom) is an established immunomodulatory drug (IMiD) that has direct on-tumor apoptotic activity and enhances immune activity. Combining pom with T-cell redirection therapy may lead to synergistic antimyeloma effects. We report initial efficacy and safety results of tal + pom from the MonumenTAL-2 study. Methods: MonumenTAL-2 (NCT05050097) is a multi-arm, phase 1b study of tal in combination with antimyeloma agents in pts with MM. Pts received the recommended phase 2 doses of subcutaneous tal 0.4 mg/kg weekly (QW) or 0.8 mg/kg every other week (Q2W), with step-up dosing, + oral pom 2 mg daily (dose escalation to 4 mg daily permitted) starting in cycle 2. Pts received ≥2 prior lines of therapy (LOT) including a proteasome inhibitor and an IMiD; prior T-cell redirection therapies including BsAbs and chimeric antigen receptor (CAR)-T along with prior pom exposure were permitted. CRS and ICANS were graded by ASTCT criteria; all other adverse events (AEs) were graded by CTCAE v5.0. Response was assessed by IMWG criteria. Efficacy endpoints are presented as individual cohorts (QW and Q2W); safety is presented across both cohorts. Results: As of June 5, 2023, 35 patients were enrolled with a median follow-up of 11.4 months (range, 1.2-14.9) in the QW cohort (N=16) and 7.7 months (range, 1.6-10.8) in the Q2W cohort (N=19). Median ages were 69.5 years (range, 49-78) and 63.0 years (range, 43-76), respectively; 41.7% and 33.3% of pts had high-risk cytogenetics (del[17p], t[4;14], or t[14;16]) and 12.5% and 10.5% of pts had extramedullary disease, respectively. Median prior LOT were 3 in both cohorts; 25.0% and 21.1% were triple-class refractory, respectively, and 6.3% were penta-drug refractory (all in QW cohort). Prior treatments included CAR-T (18.8% and 0%), BsAb (6.3% and 0% [0% refractory]), and anti-CD38 Ab (75.0% and 73.7% [56.3% and 36.8% refractory]) in the QW and Q2W cohorts, respectively; 31.3% and 15.8% had prior pom exposure (18.8% and 5.3% refractory). All pts had ≥1 AE; most common were dysgeusia (77.1%), CRS (74.3%; most grade 1/2, 2.9% grade ≥3), and neutropenia (60.0%). Grade 3/4 AEs occurred in 88.6% of pts; most common were neutropenia (48.6%), anemia (25.7%), and thrombocytopenia (20.0%). Nail, skin, and rash toxicities occurred in 65.7%, 40.0%, and 20.0% of pts (majority grade 1/2 with no discontinuations), respectively. ICANS occurred in 2 pts (both grade 1). Infections occurred in 71.4% of pts (22.9% grade 3/4); most common were pneumonia (20.0%) and COVID-19 (14.3%). AEs led to dose reduction or schedule change of tal in 34.3% of pts and dose reduction of pom in 31.4% of pts. Two pts (5.7%) in the Q2W cohort had AEs, myocardial infarction and pulmonary embolism (PE), that led to treatment discontinuation (not drug related). One death due to PE occurred (same pt who discontinued treatment). ORR was 86.7% and 83.3% in the QW and Q2W cohorts, respectively, with ≥CR in 60.0% and 44.4% and ≥VGPR in 86.7% and 77.8%, respectively. ORRs were consistent across pt subgroups (>80% independent of prior pom or CAR-T exposure). Median time to first response was 1.0 month (range, 0.9-2.1) in the QW cohort and 1.3 months (range, 0-4.8) in the Q2W cohort. At 6 months, 100% of responders were still responding in both cohorts. Median DOR and PFS were not reached, with 6-month PFS rates of 93.3% (QW) and 88.9% (Q2W). Conclusions: In this first reported combination of a GPRC5D-targeted therapy and an IMiD, tal + pom showed rapid, deep responses in pts with RRMM and ≥2 prior LOT. The safety profile of the combination, including grade 3/4 hematologic toxicity, was consistent with the individual agents, with no evidence of additive hematologic toxicities; additionally, there were low rates of treatment discontinuation due to AEs. The promising efficacy and manageable safety profile of this combination further supports tal as a versatile combination partner and warrants further evaluation of this regimen.

CUDC-101 as a dual-target inhibitor of EGFR and HDAC enhances the anti-myeloma effects of bortezomib by regulating G2/M cell cycle arrest.

CUDC-101, an effective and multi-target inhibitor of epidermal growth factor receptor (EGFR), histone deacetylase (HDAC), and human epidermal growth factor receptor 2 (HER2), has been reported to inhibit many kinds of cancers, such as acute promyelocytic leukemia and non-Hodgkin's lymphoma. However, no studies have yet investigated whether CUDC-101 is effective against myeloma. Herein, we proved that CUDC-101 effectively inhibits the proliferation of multiple myeloma (MM) cell lines and induces cell apoptosis in a time- and dose-dependent manner. Moreover, CUDC-101 markedly blocked the signaling pathway of EGFR/phosphoinositide-3-kinase (PI3K) and HDAC, and regulated the cell cycle G2/M arrest. Moreover, we revealed through in vivo experiment that CUDC-101 is a potent anti-myeloma drug. Bortezomib is one of the important drugs in MM treatment, and we investigated whether CUDC-101 has a synergistic or additive effect with bortezomib. The results showed that this drug combination had a synergistic anti-myeloma effect by inducing G2/M phase blockade. Collectively, our findings revealed that CUDC-101 could act on its own or in conjunction with bortezomib, which provides insights into exploring new strategies for MM treatment.

Chidamide and venetoclax synergistically exert cytotoxicity on multiple myeloma by upregulating BIM expression

BackgroundMultiple myeloma (MM) is the second most common hematologic malignancy with almost all patients eventually having relapse or refractory MM (RRMM), thus novel drugs or combination therapies are needed for improved prognosis. Chidamide and venetoclax, which target histone deacetylase and BCL2, respectively, are two promising agents for the treatment of RRMM.ResultsHerein, we found that chidamide and venetoclax synergistically exert an anti-myeloma effect in vitro in human myeloma cell lines (HMCLs) with a combination index (CI) < 1. Moreover, the synergistic anti-myeloma effect of these two drugs was demonstrated in primary MM cells and MM xenograft mice. Mechanistically, co-exposure to chidamide and venetoclax led to cell cycle arrest at G0/G1 and a sharp increase in DNA double-strand breaks. In addition, the combination of chidamide and venetoclax resulted in BCL-XL downregulation and BIM upregulation, and the latter protein was proved to play a critical role in sensitizing HMCLs to co-treatment.ConclusionIn conclusion, these results proved the high therapeutic potential of venetoclax and chidamide combination in curing MM, representing a potent and alternative salvage therapy for the treatment of RRMM.

Central Memory T-Cell Differentiation Correlates with Depth of Response in Relapsed/Refractory Multiple Myeloma Patients Receiving Elotuzumab in Combination with Carfilzomib, Lenalidomide and Dexamethasone (Elo-KRd)

Central Memory T-Cell Differentiation Correlates with Depth of Response in Relapsed/Refractory Multiple Myeloma Patients Receiving Elotuzumab in Combination with Carfilzomib, Lenalidomide and Dexamethasone (Elo-KRd)

Pan-PIM Kinase Inhibitors Enhance Lenalidomide's Anti-Myeloma Activity Via Cereblon-IKZF1/3 Cascade

Pan-PIM Kinase Inhibitors Enhance Lenalidomide's Anti-myeloma Activity Via Cereblon-IKZF1/3 CascadeYonggang Sha1, Jing Zheng2, Jianda Hu2+, and Yubin Kang1+1Division of Hematologic Malignancies and Cellular Therapy, Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA; 2Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, China+ Corresponding author: Yubin Kang, MD, Email: yubin.kang@duke.edu; and Jianda Hu, Email: drjiandahu@163.com.ABSTRACTPurpose: Multiple myeloma (MM) remains an incurable disease, and continuous efforts are required to develop novel therapeutic agents and novel drug combinations with more effective anti-myeloma activity. Immunomodulatory agents (IMiDs) including lenalidomide are one of the mainstays in the treatment of MM. It is important to identify and develop novel agents that can work with IMiDs for enhanced anti-myeloma effects. Here, we determined anti-myeloma effect of pan-PIM inhibitors and the synergistic anti-myeloma effects of pan-PIM inhibitors with IMiDs (lenalidomide).Experimental Design: The anti-myeloma activities of pan-PIM inhibitors (SGI1776 and CX6258) and the synergistic anti-myeloma effects of pan-PIM inhibitors with lenalidomide were determined in human myeloma cells, transplanted VK*MYC myeloma mice, and myeloma xenograft mouse models. Western blot, protein stability assay, shRNA specific gene knockdown, and ubiquitination assay were performed to determine the molecular mechanisms involved.Results: Pan-PIM inhibitors (SGI1776 and CX6258) induced the apoptosis of MM cells and exhibited significant anti-myeloma activities in human myeloma cells and in transplanted VK*MYC myeloma mice. Combination of PIM inhibitors with lenalidomide showed synergistic anti-myeloma effects without additional hematological or liver toxicities in vivo in myeloma xenograft mouse models. The treatments of pan-PIM inhibitors promoted ubiquitination and degradation of IKZF1 and IKZF3, the two transcription factors important for the survival of myeloma cells, and combining Pan-PIM inhibitors with lenalidomide leds to more effective degradation of IKZF1 and IKZF3 in vitro in human multiple myeloma cells and in vivo in myeloma xenograft mouse model. We further demonstrated that treatments with pan-PIM inhibitors resulted in an increased expression of Cereblon, and knockdown of Cereblon with shRNA abolished the effects of pan-PIM inhibitors on IKZF1 and IKZF3, demonstrating a central role of Cereblon in Pan-PIM inhibitor-mediated IKZF1/3 degradation.Conclusions: We demonstrated that pan-PIM inhibitors (SGI1776 and CX6258) exhibited significant anti-myeloma activities and combination of pan-PIM inhibitors with lenalidomide showed synergistic anti-myeloma effects without additional hematological or liver toxicities. Mechanistically, pan-PIM kinase inhibitors enhance lenalidomide's anti-myeloma activity via Cereblon-IKZF1/3 Cascade. Our study provides molecular rationale and justification for clinical trials combining pan-PIM inhibitors and lenalidomide for the treatment of myeloma. DisclosuresNo relevant conflicts of interest to declare.

The Novel Subtype-Selective Histone Deacetylase (HDAC) Inhibitor, Chidamide, Exerts Dual Anti-Myeloma and Bone Protective Effect in Vitro and In Vivo

Background and Purpose: Despite the advance in multiple myeloma treatment, drug resistance is still inevitable and the prognosis of most patients remains poor. Chidamide, a new subtype-selective histone deacetylase (HDAC) inhibitor, which is produced independently in China, is designed to inhibit the activity of HDAC 1, 2, 3 and 10. In the present work, we investigated the effect of chidamide on multiple myeloma and its associated osteolytic bone disease with different models.Experiment Design: In vitro cytotoxicity of chidamide was tested on myeloma cell lines (MM.1s, CAG, ARP-1, 8226, U266, H929, LP-1, ANBL-6, OPM2) with CCK-8 and flow cytometry. Synergistic combinations with standard drugs in 8226 and CAG cell lines were analyzed by compusyn software. The effect on osteoclast formation was assessed in osteoclastic cultures. The mechanisms of chidamide were explored by western blot and immunofluorescence. Murine models were employed for in vivo works, IVIS in vivo imaging system, ELISA (human Ig lambda, CTX-I, PINP) and micro-CT were used to surveillance the tumor burden and examine the bone destruction.Results: The cytotoxic effect of chidamide on myeloma cells is due to induction of cell apoptosis and cell cycle disruption (G1 phase arrest) in a dose dependent manner (IC50 varies from 2 to 5 uM in different cell lines) which mediated by increasing Cleaved Caspase-3, Caspase-7, Caspase-8, Caspase-9, p21 and p27, decreasing C-Myc, Mcl-1, Bcl-xL, CDK4 and CDK6. SensitiveSOCS3/JAK2/STAT3 cell signaling was involved in regulating these proteins by increasing the expression of SOCS3, reducing the expression of phospho-JAK2 and phospho-STAT3, thus inhibiting the proliferation and inducing cell cycle disruption. After treating with chidamide, the expression levels of HDAC1, 2, 3, 10 were not changed significantly, which indicated chidamide suppressed the function of these targets while not affected their expression. Notably, chidamide exerted synergistic anti-myeloma effect with dexamethasone, carfilzomib or pomalidomide (combination index < 1). Also, chidamide showed effective cytotoxicity in the co-culture of myeloma cells with bone marrow stromal cells and osteoclasts. Importantly, chidamide suppressed in vitro osteoclast formation and resorption, inhibited the expression of the key molecules (Cathepsin K, c-fos, NFATc1) and de-phosphorylation of p-AKT and p-JNK, and disrupted the F-acting ring formation. Finally, chidamide significantly reduced the tumor volume in the subcutaneous xenografts models, also the level of monoclonal protein Ig lambda secreted by myeloma cells decreased in disseminated myeloma murine models after treatment. Moreover, chidamide not only prevented tumor-associated bone loss in the disseminated murine model which was partially related with reducing the tumor burden but also had a direct bone protective effect on RANKL-induced rapid bone loss in non-tumor bearing mouse model with serum PINP (bone formation) and trabecular numbers increased while serum CTX-I (bone resorption) and trabecular space diminished compared with the vehicle group.Conclusion: Our work demonstrates dual anti-myeloma and bone protective effect of chidamide in vitro and in vivo, and shows its synergistic anti-myeloma effect with dexamethasone, carfilzomib and pomalidomide. These findings strongly suggest the potential clinical use of this drug in multiple myeloma in the near future. DisclosuresNo relevant conflicts of interest to declare.

Upregulation of CD38 Expression on Multiple Myeloma Cells By the HDAC Inhibitor Ricolinostat Augments the Efficacy of Daratumumab

Background:The anti-CD38 antibody daratumumab is effective and increasingly being utilized to treat multiple myeloma. A clinical observation with daratumumab is that expression of CD38 on pretreatment myeloma cells correlates with efficacy, and patients with higher CD38 expression have a higher chance to respond. Moreover, during daratumumab therapy, there are reduced CD38 expression levels on myeloma cells, favoring immune escape and disease progression. We have recently shown that the histone deacetylase (HDAC) inhibitor panobinostat induces CD38 upregulation and augments the antibody-dependent cellular cytotoxicity (ADCC) of daratumumab (García-Guerrero E, Blood 2017). Here, we investigated the alternative HDAC inhibitor ricolinostat and all-trans retinoic acid (ATRA) to determine whether upregulation of CD38 is a class effect, and to identify the substance that works most synergistically with daratumumab.Methods:The myeloma cell lines MM1.S and OPM2 were treated with titrated doses of ricolinostat (0, 5, 10 µM), ATRA (0, 10, 25 nM) and panobinostat (0, 10, 25 nM). Expression of CD38, B-cell maturation antigen (BCMA) and SLAMF7 was analyzed by flow cytometry after 24, 48 and 72 h of exposure. ADCC against HDAC inhibitor-treated and untreated myeloma cells was analyzed after addition of PBMC at an effector to target ratio of 25:1 in the presence of daratumumab (1, 10, 50 ug/mL) or isotype control.Results:We first treated the myeloma cell line MM1.S with each of the HDAC inhibitors and analyzed their direct cytotoxic antimyeloma effect. We found the strongest effect with ricolinostat where live MM1.S myeloma cells had decreased to 50% and 20% after 48 h of exposure to 5 and 10 µM of the drug, respectively. Consistent with previous work the direct anti-myeloma effect of panobinostat (decrease of live MM1.S myeloma cells to 85% and 50% after 48 h of exposure to 10 and 25 nM, respectively) and ATRA (no cytotoxic effect) was lower compared to ricolinostat.We then analyzed expression of CD38 on residual live i.e. 7-AAD negative MM1.S cells by flow cytometry and observed a 2-fold (5 µM) and 2,5-fold (10 µM) increase in CD38 expression by mean fluorescence intensity (MFI) after treatment with ricolinostat-treated compared to untreated MM1.S cells. The increase in CD38 expression was already detectable after 24 h and further increased with longer exposure to the drug. Off note, withdrawal of ricolinostat resulted in rapid decline of CD38 expression to baseline levels but increased again to the same magnitude within 24 h of re-exposure to the drug. Overall, the increase in CD38 expression was strongest with ricolinostat followed by panobinostat and ATRA, which we confirmed with several myeloma cell lines and primary myeloma cells. We also confirmed that ricolinostat-induced upregulation of CD38 specifically occurred in myeloma cells and not in the lymphoma cell lines Raji and JeKo-1. In contrast to CD38, expression of BCMA and SLAMF7 after treatment of myeloma cells with ricolinostat had decreased.We then treated myeloma cells with ricolinostat for 48 hours to induce upregulation of CD38 and then performed ADCC assays with daratumumab in the presence of ricolinostat. We found, that the combination treatment ricolinostat plus daratumumab eliminated 95,6% of MM1.S cells after 20h; while 87,4% were eliminated with ricolinostat alone, and only 22,6% with daratumumab alone. Preliminary data show that ricolinostat does not affect expression of the complement inhibitor CD55 and induces a lower increase in CD59 expression compared to panobinostat. Experiments to evaluate complement-dependent cytotoxicity (CDC), and to confirm the synergistic antimyeloma effect of ricolinostat and daratumumab in a murine xenograft myeloma model, are ongoing.Conclusions:Our data demonstrate that the HDAC inhibitor ricolinostat induces upregulation of CD38 expression on myeloma cells. The increase in CD38 expression after treatment with ricolinostat is stronger compared to panobinostat and ATRA. Our data suggest that upregulation of CD38 on myeloma cells is a class effect of HDAC inhibitors, and identify ricolinostat as a highly synergistic combination partner for daratumumab, due to its considerable intrinsic antimyeloma activity. Our data suggest that combination treatment with ricolinostat and daratumumab could increase response rates and extend duration of responses of daratumumab therapy. DisclosuresNo relevant conflicts of interest to declare.

The combination of a sphingosine kinase 2 inhibitor (ABC294640) and a Bcl-2 inhibitor (ABT-199) displays synergistic anti-myeloma effects in myeloma cells without a t(11;14) translocation.

Multiple myeloma (MM) remains an incurable disease in need of the development of novel therapeutic agents and drug combinations. ABT‐199 is a specific Bcl‐2 inhibitor in clinical trials for MM; however, its activity as a single agent was limited to myeloma patients with the t(11;14) translocation who acquire resistance due to co‐expression of Mcl‐1 and Bcl‐xL. These limitations preclude its use in a broader patient population. We have recently found that a sphingosine kinase 2‐specific inhibitor (ABC294640) induces apoptosis in primary human CD138+ cells and MM cell lines. ABC294640 is currently in phase I/II clinical trials for myeloma (clinicaltrials.gov: #NCT01410981). Interestingly, ABC294640 down‐regulates c‐Myc and Mcl‐1, but does not have any effects on Bcl‐2. We first evaluated the combinatorial anti‐myeloma effect of ABC294640 and ABT‐199 in vitro in 7 MM cell lines, all of which harbor no t(11;14) translocation. Combination index calculation demonstrated a synergistic anti‐myeloma effect of the combination of ABC294640 and ABT‐199. This synergistic anti‐myeloma effect was maintained even in the presence of bone marrow (BM) stromal cells. The combination of ABC294640 and ABT‐199 led to enhanced cleavage of PARP and caspase‐3/9 and increased Annexin‐V expression, consistent with the induction of apoptosis by the combination treatment. In addition, the combination of ABC294640 and ABT‐199 resulted in the down‐regulation of the anti‐apoptotic proteins Mcl‐1, Bcl‐2, and Bcl‐xL and the cleavage of Bax and Bid. The combination induced both the mitochondrial mediated‐ and caspase‐mediated apoptosis pathways. Finally, the combination of ABC294640 and ABT‐199 resulted in augmented anti‐myeloma effect in vivo in a mouse xenograft model. These findings demonstrate that the co‐administration of ABC294640 and ABT‐199 exhibits synergistic anti‐myeloma activity in vitro and in vivo, providing justification for a clinical study of this novel combination in patients with relapsed/refractory multiple myeloma.

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.

Mobilization of human immature hematopoietic progenitors through combinatory use of bortezomib and immunomodulatory drugs.

Combination use of the proteasome inhibitor bortezomib and the immunomodulatory drugs lenalidomide or thalidomide has provided superior outcomes in multiple myeloma over their single use; however, these combinations can produce significant toxicities. Unexpectedly, we found a small but significant increase in the population of immature granulocytes and erythrocytes/megakaryocytes in peripheral blood in 16 of 22 patients (73%) treated with dexamethasone in combination with bortezomib and immunomodulatory drugs (triplet), but not in any of 25 patients treated with either bortezomib or immunomodulatory drugs with dexamethasone (doublet). These immature cells gradually increased to a peak level (mean 2.6% per white blood cells) with triplet therapy, and disappeared immediately after therapy cessation. The numbers of circulating CD34+ cells and colony-forming cells derived from peripheral blood mononuclear cells increased after triplet therapy compared with those in patients treated by either bortezomib or immunomodulatory drugs plus dexamethasone. Furthermore, triplet regimen downregulated the expression of CXCR4, a chemokine receptor essential for bone marrow retention, on CD34+ cells, suggesting an unexpected effect on normal hematopoietic stem/progenitor cells through the reduced interaction with the bone marrow microenvironment. Our observations suggest that combination use should be carefully evaluated to exert synergistic anti-myeloma effects while avoiding unexpected adverse events.

Identification of a Cereblon-Independent Protein Degradation Pathway in Residual Myeloma Cells Treated with Immunomodulatory Drugs

Recent studies have shown that binding of immune modulatory drugs (IMiDs)R lenalidomide and pomalidomide (Pom) to the CRBN-CRL4 E3 ubiquitin ligase complex leads to degradation of IKZF1 and IKZF3, which in turn mediates anti-proliferative effects on myeloma cells and enhance IL2 expression by T cells. However as the drug-induced IKZF1 degradation is proteasome-mediated, this mechanism does not explain the apparent paradox that IMiDs mediate synergistic anti-myeloma effects with proteasome inhibitors in the clinic. In order to better understand how MM cells may resist potent IMiDs such as Pom, we examined the clonogenic growth properties of residual MM cells following Pom exposure. Surprisingly, residual MM cell lines and primary MM cells following Pom exposure exhibit paradoxically enhanced clonogenic growth in culture. Human MM cells persisting after Pom exposure also exhibit greater capacity for growth in vivo. Enhanced clonogenic growth was associated with increased expression of embryonal stem cell genes such as SOX2 and RNAi-mediated inhibition of SOX2 abrogated Pom-induced enrichment of clonogenic potential. We hypothesized that as with CBRN/IKZF1-mediated effects, the induction of ES/pluripotency genes in Pom-exposed cells may be related to degradation of repressors of gene transcription. Recent studies have identified MBD3 as a critical component of the pluripotency repressor complex. Pom-exposure led to ubiquitin and proteasome-mediated degradation of MBD3 and RNAi-mediated inhibition of MBD3 led to induction of pluripotency genes by MM cells. As expected, loss of CRBN abrogates the capacity of Pom to mediate degradation of IKZF1. Surprisingly, Pom-mediated degradation of MBD3, enrichment of ES genes and enhancement of clonogenic growth was independent of CRBN as it was observed in MM1s-R10R cells with genomic loss of CRBN, and was not impacted by RNAi-mediated inhibition of CRBN. RNAi-mediated inhibition of IKZF1 also does not lead to enrichment of ES genes and does not phenocopy the effects of MBD3 depletion. Residual MM cells in patients (n=2) treated with Pom also revealed marked depletion of MBD3 protein as well as enrichment of ES gene transcripts. Taken together, these data identify a CRBN/IKZF1-independent pathway for Pom-induced depletion of MBD3 and enrichment of pluripotency genes. Pre-exposure to proteasome inhibitor MG132 abrogated Pom-mediated loss of MBD3 and enrichment of ES genes. Similar results were obtained following treatment with Trichostatin A (TSA) and Romidepsin (FK228), which also inhibited Pom-induced depletion of MBD3. Therefore concurrent therapy of IMiDs with proteasome or HDAC inhibitors may mediate synergistic anti-tumor effects by abrogating adverse effects of Pom exposure on clonogenic growth of residual cells. In order to further dissect the underlying mechanism of Pom-induced degradation of MBD3 in MM cells, we hypothesized engagement of another E3 ligase complex other than CRBN. We show that Pom-induced degradation of MBD3 depends on TRIM27/29 E3 ligase complex, known to be overexpressed in MM plasma cells. Accordingly, RNAi-mediated downregulation of TRIM27/29 inhibits Pom-induced MBD3 depletion and enrichment of clonogenic growth. In summary, these data identify a novel CRBN-independent, TRIM27/29 E3 ligase-dependent pathway for degradation of MDB3 engaged by Pomalidomide, which contributes to the biology of residual MM cells via enrichment of ES genes following exposure to these drugs in vitro/in vivo and provides a novel mechanism for observed synergy with proteasome and HDAC inhibitors. Targeting these pathways may be essential to enhance the therapeutic potential of IMiDs and minimize residual disease in MM. DisclosuresNo relevant conflicts of interest to declare.

Higher Decorin Levels in Bone Marrow Plasma Are Associated with Superior Treatment Response to Novel Agent-Based Induction in Patients with Newly Diagnosed Myeloma - A Retrospective Study.

The growth of myeloma cells depends on bone marrow (BM) stroma consisting of stromal cells, secreted cytokines and the extracellular matrix (ECM). Decorin, a small leucine-rich proteoglycan in the ECM, is a signaling ligand and native anti-tumor agent. However, the role of decorin in patients with myeloma is not clear. We evaluated the correlation between the decorin levels measured by enzyme-linked immunosorbent assay in BM plasma from 121 patients with newly diagnosed myeloma based on their clinical features and treatment response. The median decorin levels in the patients and the normal control group were 12.31 ng/mL [standard deviation (SD), 7.50 ng/mL; range, 2.45 to 44.46 ng/mL] and 10.31 ng/mL (SD, 2.42 ng/mL; range, 4.85–15.14 ng/mL), respectively (P < 0.001). Using 15.15 ng/mL as a cut-off, 46 patients (38%) exhibited higher decorin levels (H-DCN), whereas the other patients exhibited normal to lower decorin levels (NL-DCN). Except for the median age, which was significantly younger in the H-DCN than in the NL-DCN group (60.6±14.0 vs. 65.8±12.2 years, respectively; P = 0.034), there were no differences between the two groups. However, in 79 patients who had received novel agent-based induction, the overall response rate was significantly better in the H-DCN than in the NL-DCN (97 vs. 63%, respectively; P < 0.001), as was the depth of responses (P = 0.008), which were not observed in those who had received chemotherapeutic agents alone. Progression-free survival (PFS) was significantly longer in H-DCN than NL-DCN (not reached vs. 19.5 mo, respectively; P = 0.0003). Multivariate analyses indicated that H-DCN, as a significantly independent factor, was associated with better treatment response (odds ratio, 20.014; 95% CI, 2.187–183.150; P = 0.008) and longer PFS (hazard ratio, 0.135; 95% CI, 0.051–0.361; P < 0.001). These findings disclose the potential role of decorin in myeloma and provide a basis for further study on possible synergistic anti-myeloma effects between decorin and the novel agents that target BM stroma.

A critical role of autocrine sonic hedgehog signaling in human CD138+ myeloma cell survival and drug resistance

AbstractHedgehog (Hh) signaling plays an important role in the oncogenesis of B-cell malignancies such as multiple myeloma (MM). However, the source of Hh ligand sonic hedgehog (SHH) and its target cells remains controversial. Previous studies showed that stromally induced Hh signaling is essential for the tumor cells and that CD19+CD138– MM stem cells are the target cells of Hh signaling. Here we demonstrate that SHH was mainly secreted by human myeloma cells but not by stromal cells in MM bone marrow. Autocrine SHH enhanced CD138+ myeloma cell proliferation and protected myeloma cells from spontaneous and stress-induced apoptosis. More importantly, autocrine SHH protected myeloma cells against chemotherapy-induced apoptosis in vitro and in vivo. Combinational treatment with chemotherapy and SHH-neutralizing antibody displayed synergistic antimyeloma effects. Mechanistic studies showed that SHH signaling activated the SHH/GLI1/BCL-2 axis, leading to the inhibition of myeloma cell apoptosis. Thus, this study identifies the myeloma autocrine Hh signaling pathway as a potential target for the treatment of MM. Targeting this pathway may improve the efficacy of chemotherapy in MM patients.

Salvage therapy with pegylated liposomal doxorubicin, bortezomib, cyclophosphamide, and dexamethasone in relapsed/refractory myeloma patients

In vitro studies have shown synergistic anti-myeloma effects of bortezomib combined with alkylating agents or anthracycline. We tested safety and efficacy of the combination of bortezomib, doxorubicin cyclophosphamide, and dexamethasone (ABCD) in the treatment of relapsed/refractory myeloma. ABCD consisted of bortezomib given intravenous (IV) at dosage 1.3 mg/m(2) , dexamethasone 40 mg IV on days 1, 4, 8, and 15, pegylated liposomal doxorubicin (PLD) 20 mg IV on days 1 and 15, plus cyclophosphamide 100 mg/d per os for 15 d. Between January 2008 and February 2009, 24 patients received a median of four 28-d ABCD cycles (range 1-6). All patients had been already treated with a median of two previous lines of treatment (range 1-6): 38% were resistant to previous therapies and 62% were relapsed. Clinical response was observed in 12 patients (50%), including 29% of very good partial remissions or better. Side effects included hematological toxicity (31% any grade), grades 3-4 thrombocytopenia (9%), grades 3-4 anemia (17%). Non-hematological toxicity affected 32% of administered cycles and included gastrointestinal disturbances (54%), peripheral neuropathy (8%), and infections (8%). After a median follow-up of 21.5 months (range 2-44 months), median of progression-free survival (PFS) was 8.7 months and median overall survival was 22.5 months. Achieving at least partial response within the second cycle was associated with a better PFS (19.5 months vs. 3.5 months), P = 0.03, HR 0.35 (CI 95% 0.13-0.90). ABCD is safe and effective for relapsed/refractory MM subjects previously treated with novel agents.

Bortezomib-Cyclophosphamide-Dexamethasone for Relapsing Multiple Myeloma

In vitro studies have shown synergistic antimyeloma effects with the combination of bortezomib and alkylating agents. Combinations of bortezomib, cyclophosphamide, and dexamethasone are rational with the prospect of superior antitumor activity with independent toxicity. Between December 2004 and April 2007, we treated 44 patients with relapsing multiple myeloma with the combination of bortezomib 1.3 mg/m intravenously on days 1, 4, 8, 11; dexamethasone 20 mg/m orally daily for 4 days beginning on days 1, 9 and 17; and cyclophosphamide 70 mg/m orally twice daily for 4 days. A second course was given 1 month later. Clinical response was observed in 32 patients (73%) including 26 with disease in partial remission (59%), and 6 with disease in complete remission (14%). Side effects were uncommon and mild, except for grade 3 thrombocytopenia in 15%, infection in 5% and constipation in 2% of patients. The median remission time of responding patients was 10 months that contributed to significantly longer median survival for patients with responsive disease (33 mo) than for those with unresponsive disease (12 mo) (P < 0.01). Bortezomib-cyclophosphamide-dexamethasone was an effective, well-tolerated combination for the treatment of relapsing multiple myeloma.

Phase I Exploratory Study of IV Formulation of Panobinostat in Combination with Bortezomib in Relapsed/Refractory Multiple Myeloma Patients: Effect On Serum PTH and Gene Expression Profiling (GEP) Studies

Abstract 4073 Background:Inhibition of histone deacetylase (HDAC) provides a novel approach for cancer treatment. Bortezomib and panobinostat are active agents in multiple myeloma (MM) and have a synergistic antimyeloma effect when used in combination. Both compounds have also shown anabolic bone effect. Methods:We conducted a phase I exploratory study IV formulation of panobinostat in combination with bortezomib in relapsed/refractory MM patients who had failed at least one line of prior therapy. Patients were assigned to their dose levels in a classical 3+3 cohort design. Primary objective was to assess the toxicity of a fixed dose of bortezomib in combination with escalating doses of panobinostat to define the maximum tolerated dose (MTD). The secondary objective was to determine the osteoblastic response. Bortezomib was administered iv at the dose of 1 mg/m2 during cycles on days 1, 4, 8, and 11 of each 21 days cycle. Panobinostat was administered iv starting at cycle 2 on days 1 and 8 of the 21 day cycle, 5 mg/m2 n=1–3, 10 mg/m2 n=4–9, 15 mg/m2 n=10–12 (one screening failure). Patients with at least stable disease after the first 2 cycles (Phase I Portion of the Study) were allowed to continue to a maximum of 12 cycles.PTH was measured during the first two cycles, every 20 minutes starting one hour before infusion for a total of seven time points on days 1 and 4, and single pre dose on days 8 and 11 during cycles 1 and 2.Bone marrow (BM) aspiration and biopsies were collected at baseline, after the completion of second cycle and at the end of the study. H&E stained core biopsy slides were scanned using Scan Scope XT system. We developed classifier algorithms using Genie pattern recognition image analysis software to calculate Trabecular Volume (TV), Bone Volume (BV). RNA obtained from separate core biopsies at baseline and after second cycle was used to complete Gene Expression Profiling (GEP) studies. The paired t test was used to identify significantly gene changes. Results:Eleven patients with a median age of 58 years (range 44 to 70) were enrolled. 45% of patients received ≥ 3 previous lines of therapy. 45% was previously exposed to Bortezomib and 63% of patients failed prior autologous stem cell transplant. All eleven patients completed two cycles of therapy, 10 patients continued treatment, and three completed a total of 12 cycles. No MTD was reached with a 15 mg/m2IV Panobinostat dose. Using IMWG criteria after the second cycle of treatment we observed 1CR, 3 PR, 5 SD, 2 patients experienced PD. Best response during the entire treatment included 2 CR, 2 VGPR, 4 SD (≥ SD 72%). Common grade 3/4 AEs included: thrombocytopenia (36%), neutropenia (45%), leukopenia (54%), lymphopenia (18%), with no treatment-related mortality.The median baseline of Trabecular Volume/Hematopietic Area (TV/HA) was 27.6% (range 8.1–46%). Comparing the baseline to the end of study samples, an increment of TV/HA was noted in 6 of 9 available patients. The average TV/HA increment was 19.4% (range 9.9–38.3%).72% of patients (n=8) showed changes in PTH level during treatment. Serial PTH samples significantly increased during treatment, from a mean of 39.4 pg/L (±22.3) at baseline, to a mean of 50.2 pg/L (±36.8) at day 4 cycle 2 (p=0.003). GEP from bone marrow baseline core biopsies were compared to post-treatment samples. A total of 3783 probe sets were found to be differentially expressed. 30% of genes were over-expressed and 70% were down regulated. Interestingly decorin, which function has been associated to antimyeloma effect of osteoblasts, and its down-stream target p21-activated kinase 4 (PAK4) were significantly increased in the post-treatment samples (p=0.04, p=0.03 respectively with 2.0 ratio) suggesting a bone anabolic and antimyeloma effect of these drugs in combination. Conclusions:The combination of IV Panobinostat and Bortezomib has predictable and manageable safety profile with significant antimyeloma activity as reflected by ≥ SD 72%, ≥ PR 36% including 18% CR. Changes in bone indices were observed by a computer assisted image analysis. Hormonal changes and genes studies also support an osteoblastic/anabolic effect of panobinostat in combination with bortezomib in relapsed/refractory MM patients. Disclosures:Zangari:Millennium, Onyx, Cephalon, Incyte: Research Funding, Speakers Bureau.

Inhibition of the HDM-2 E3 Ligase Induces Types I and II Cell Death in Wild-Type and Mutant p53 Models of Multiple Myeloma, and Acts Synergistically in Both with ABT-737.

Abstract 2940 Background:The ubiquitin-proteasome pathway has been validated as a therapeutic target for multiple myeloma by the incorporation of proteasome inhibitors such as bortezomib and carfilzomib into our standards of care against this disease. A minority of patients achieve complete remission with single-agent therapy, however, and proteasome inhibitors may induce on- and off-target anti-apoptotic effects and clinical toxicities that limit their utility. Unlike proteasome inhibitors, which have a broad impact on intracellular proteolysis, E3 ligases are responsible for ubiquitination of only a small subset of proteins. Our hypothesis proposed that inhibition of the Human double minute (HDM)-2 E3 ligase, which in part is responsible for p53 ubiquitination, could be a more rational therapeutic target for myeloma, where p53 mutations are less common than is the case in solid tumors. Methods:We used Nutlin-3a and MI-63, which bind to the p53 binding pocket of HDM-2, as prototypic agents, and studied their activity against both wild-type (wt) and mutant (mut) p53 cell line models of multiple myeloma. These studies were supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results:Inhibition of HDM-2 in wt p53 (MM1.S, H929, MOLP-8) cells with Nutlin-3a or MI-63 reduced viability, with a median inhibitory concentration (IC50) in the 0.5–2 μM range. In wt cells, genomic and proteomic studies showed that MI-63 induced transcription of downstream gene targets including p21, PUMA and Bax, and promoted accumulation of p53, p21, p27, and Bax. This was accompanied by cell cycle arrest at G1, and induction of apoptosis as demonstrated by Annexin V staining and caspase activation. Interestingly, these agents also showed activity against mut p53 cells such as RPMI 8226 and U266, though with higher IC50’s of 30–40 μM. Knockdown of HDM-2 sensitized mut p53 myeloma cells to MI-63, suggesting that this agent was inducing cell death in an on-target manner. In p53 mutant cells, MI-63 induced both type I cell death and type II, autophagic cell death, as determined by conversion of microtubule-associated protein 1 light chain 3, and activation of transcription of autophagy (ATG) genes ATG3 and 5. Since autophagy can be a mechanism of cell survival or cell death, we tested the impact of the autophagy inhibitors chloroquine and 3-methyladenine, and found that they reduced the impact of MI-63 on viability. Moreover, knockdown of ATG5 helped rescue cells as well, supporting the possibility that autophagic activation was a mechanism of cell death in these myeloma models. Synergistic anti-myeloma effects were seen when MI-63 was combined with the BH3 mimetic ABT-737, with decreased viability and enhanced apoptosis, accompanied by increased Bax activation and caspase cleavage. Notably, ABT-737 also enhanced the effects of MI-63 on mut p53 myeloma models, where increased levels of both type I and II programmed cell death were seen. Conclusions:Together, our findings support the possibility that HDM-2 is a promising therapeutic target for multiple myeloma, and also provide a rationale for translation of HDM-2 inhibitors alone, and in combination with ABT-737, to the clinic. Disclosures:Leopold:Ascenta Therapeutics: Employment, Equity Ownership.

Combined Inhibition of Notch Signaling and Bcl-2/Bcl-xL Results in Synergistic Antimyeloma Effect

Signaling through the receptor/transcriptional regulator Notch plays an important role in tumor cell survival. Recent studies have demonstrated that pharmacological inhibition of the Notch pathway with γ-secretase inhibitor (GSI) induces apoptosis of multiple myeloma (MM) cells via upregulation of the proapoptotic protein Noxa. ABT-737, a novel BH3 mimetic, was shown to block Bcl-2 and Bcl-xL and induce MM cell apoptosis. Here, we investigated whether the inhibition of Notch signaling could enhance the proapoptotic effect of ABT-737. The antimyeloma effect of ABT-737 on MM cell lines or primary cells was substantially increased by the addition of Notch inhibitor. The synergistic effect of the GSI+ABT-737 combination was mediated by activation of Bak and Bax and release of cytochrome c. While toxic for MM cells, the combination of GSI and ABT-737 did not affect survival of peripheral blood mononuclear cells isolated from healthy donors. In vivo experiments using xenograft and SCID-hu models of MM demonstrated a significant antitumor effect of the GSI/ABT-737 combination as compared to the effect of Notch or Bcl-2/Bcl-xL inhibitors alone. Thus, this drug combination may be therapeutically beneficial for patients with MM.