anti-MM Efficacy Research Articles

EZH2 inhibition induces senescence via ERK1/2 signaling pathway in multiple myeloma.

Epigenetic modifications play an important role in cellular senescence, and enhancer of zeste homolog 2 (EZH2) is a key methyltransferase involved in epigenetic remodeling in multiple myeloma (MM) cells. We have previously demonstrated that GSK126, a specific EZH2 inhibitor, exhibits anti-MM therapeutic efficacy and safety in vivo and in vitro; however, its specific mechanism remains unclear. This study shows that GSK126 induces cellular senescence in MM, which is characterized by the accumulation of senescence-associated heterochromatin foci (SAHF) and p21, and increased senescence-associated β galactosidase activity. Furthermore, EZH2 is inhibited in ribonucleotide reductase regulatory subunit M2 (RRM2)-overexpressing OCI-MY5 and RPMI-8226 cells. RRM2 overexpression inhibits the methyltransferase function of EZH2 and promotes its degradation through the ubiquitin-proteasome pathway, thereby inducing cellular senescence. In this senescence model, Lamin B1, a key component of the nuclear envelope and a marker of senescence, does not decrease but instead undergoes aberrant accumulation. Meanwhile, phosphorylation of extracellular signal-regulated protein kinase (ERK1/2) is significantly increased. The inhibition of ERK1/2 phosphorylation in turn partially restores Lamin B1 level and alleviates senescence. These findings suggest that EZH2 inhibition increases Lamin B1 level and induces senescence by promoting ERK1/2 phosphorylation. These data indicate that EZH2 plays an important role in MM cellular senescence and provide insights into the relationships among Lamin B1, p-ERK1/2, and cellular senescence.

Tinostamustine (EDO-S101), an Alkylating Deacetylase Inhibitor, Enhances the Efficacy of Daratumumab in Multiple Myeloma by Upregulation of CD38 and NKG2D Ligands.

Multiple myeloma is a malignancy characterized by the accumulation of malignant plasma cells in bone marrow and the production of monoclonal immunoglobulin. A hallmark of cancer is the evasion of immune surveillance. Histone deacetylase inhibitors have been shown to promote the expression of silenced molecules and hold potential to increase the anti-MM efficacy of immunotherapy. The aim of the present work was to assess the potential effect of tinostamustine (EDO-S101), a first-in-class alkylating deacetylase inhibitor, in combination with daratumumab, an anti-CD38 monoclonal antibody (mAb), through different preclinical studies. Tinostamustine increases CD38 expression in myeloma cell lines, an effect that occurs in parallel with an increment in CD38 histone H3 acetylation levels. Also, the expression of MICA and MICB, ligands for the NK cell activating receptor NKG2D, augments after tinostamustine treatment in myeloma cell lines and primary myeloma cells. Pretreatment of myeloma cell lines with tinostamustine increased the sensitivity of these cells to daratumumab through its different cytotoxic mechanisms, and the combination of these two drugs showed a higher anti-myeloma effect than individual treatments in ex vivo cultures of myeloma patients' samples. In vivo data confirmed that tinostamustine pretreatment followed by daratumumab administration significantly delayed tumor growth and improved the survival of mice compared to individual treatments. In summary, our results suggest that tinostamustine could be a potential candidate to improve the efficacy of anti-CD38 mAbs.

A NOTCH3-CXCL12-driven myeloma-tumor niche signaling axis promotes chemoresistance in multiple myeloma.

Multiple myeloma (MM) remains incurable due to disease relapse and drug resistance. Notch signals from the tumor microenvironment (TME) confer chemoresistance, but the cellular and molecular mechanisms are not entirely understood. Using clinical and transcriptomic datasets, we found that NOTCH3 is upregulated in CD138+ cells from newly diagnosed MM (NDMM) patients compared to healthy individuals and increased in progression/relapsed MM (PRMM) patients. Further, NDMM patients with high NOTCH3 expression exhibited worse responses to bortezomib (BOR)-based therapies. Cells of the TME, including osteocytes, upregulated NOTCH3 in MM cells and protected them from apoptosis induced by BOR. NOTCH3 activation (NOTCH3OE) in MM cells decreased BOR anti-MM efficacy and its ability to improve survival in in vivo myeloma models. Molecular analyses revealed that NDMM and PRMM patients with high NOTCH3 exhibit CXCL12 upregulation. TME cells upregulated CXCL12 and activated the CXCR4 pathway in MM cells in a NOTCH3-dependent manner. Moreover, genetic or pharmacologic inhibition of CXCL12 in NOTCH3OE MM cells restored sensitivity to BOR regimes in vitro and in human bones bearing NOTCH3OE MM tumors cultured ex vivo. Our clinical and preclinical data unravel a novel NOTCH3-CXCL12 pro-survival signaling axis in the TME and suggest that osteocytes transmit chemoresistance signals to MM cells.

Pharmacologic targeting of the p62 ZZ domain enhances both anti-tumor and bone-anabolic effects of bortezomib in multiple myeloma.

Multiple myeloma (MM) is a malignancy of plasma cells whose antibody secretion creates proteotoxic stress relieved by the N-end rule pathway, a proteolytic system that degrades N-arginylated proteins in the proteasome. When the proteasome is inhibited, protein cargo is alternatively targeted for autophagic degradation by binding to the ZZ-domain of p62/ sequestosome-1. Here, we demonstrate that XRK3F2, a selective ligand for the ZZ-domain, dramatically improved two major responses to the proteasome inhibitor bortezomib (Btz) by increasing: i) killing of human MM cells by stimulating both Btz-mediated apoptosis and necroptosis, a process regulated by p62; and ii) preservation of bone mass by stimulating osteoblast differentiation and inhibiting osteoclastic bone destruction. Co-administration of Btz and XRK3F2 inhibited both branches of the bimodal N-end rule pathway exhibited synergistic anti-MM effects on MM cell lines and CD138+ cells from MM patients, and prevented stromal-mediated MM cell survival. In mice with established human MM, co-administration of Btz and XRK3F2 decreased tumor burden and prevented the progression of MM-induced osteolytic disease by inducing new bone formation more effectively than either single agent alone. The results suggest that p62-ZZ ligands enhance the anti- MM efficacy of proteasome inhibitors and can reduce MM morbidity and mortality by improving bone health.

Antibody-Mediated Nanodrug of Proteasome Inhibitor Carfilzomib Boosts the Treatment of Multiple Myeloma.

Multiple myeloma (MM) is the second most common hematological malignancy. For relapsed and refractory MM, a proteasome inhibitor, carfilzomib (CFZ), has become one of the few clinical options. CFZ suffers, nevertheless, metabolic instability and poor bioavailability and may induce severe cardiovascular and renal adverse events. Here, we report that daratumumab (Dar)-decorated polypeptide micelles (Dar-PMs) mediate the targeted delivery of CFZ to CD38-positive MM, effectively boosting its anti-MM efficacy. CFZ-loaded Dar-PMs (Dar-PMs-CFZ) exhibited an average diameter of ca. 80 nm and Dar density-dependent cell endocytosis and anti-MM activity, in which over 6-fold greater inhibitory effect to LP-1 and MM.1S MM cells than nontargeted PMs-CFZ control was achieved at a Dar density of 3.2 (Dar3.2-PMs-CFZ). Interestingly, Dar3.2-PMs-CFZ markedly enhanced the growth inhibition of orthotopic LP-1 MM in mice and significantly extended the median survival time compared with PMs-CFZ and free CFZ (95 days vs 60 and 54 days, respectively). In line with its high MM targetability and anti-MM efficacy, Dar3.2-PMs-CFZ revealed little toxic effects and effectively prevented osteolytic lesions. The antibody-targeted nanodelivery of a proteasome inhibitor appears to be an appealing strategy to treat multiple myeloma.

Deep dive into the immune response against murine mesothelioma permits design of novel anti-mesothelioma therapeutics.

Given the need to improve the efficacy of standard-of-care immunotherapy (anti-CTLA-4 + anti-PD-1) in human malignant pleural mesothelioma (hMPM), we thoroughly characterized the immunobiology of the AB12 murine mesothelioma (MM) model, aiming to increase its accuracy in predicting the response of hMPM to immunotherapy and in designing novel anti-hMPM treatments. Specifically, we used immunologic, transcriptomic and survival analyses, to synchronize the MM tumor growth phases and immune evolution with the histo-molecular and immunological characteristics of hMPM while also determining the anti-MM efficacy of standard-of-care anti-hMPM immunotherapy as a benchmark that novel therapeutics should meet. We report that early-, intermediate- and advanced- AB12 tumors are characterized by a bell-shaped anti-tumor response that peaks in intermediate tumors and decays in advanced tumors. We further show that intermediate- and advanced- tumors match with immune active ("hot") and immune inactive ("cold") hMPM respectively, and that they respond to immunotherapy in a manner that corresponds well with its performance in real-life settings. Finally, we show that in advanced tumors, addition of cisplatin to anti CTLA-4 + anti PD-1 can extend mice survival and invigorate the decaying anti-tumor response. Therefore, we highlight this triple combination as a worthy candidate to improve clinical outcomes in hMPM.

γ-secretase inhibitors augment efficacy of BCMA-targeting bispecific antibodies against multiple myeloma cells without impairing T-cell activation and differentiation

We here defined the impacts of γ-secretase inhibitors (GSIs) on T-cell-dependent BCMA-specific multiple myeloma (MM) cell lysis and immunomodulatory effects induced by bispecific antibodies (BisAbs). GSIs-induced membrane BCMA (mBCMA) accumulation reached near maximum within 4 h and sustained over 42h-study period on MM cell lines and patient MM cells. GSIs, i.e., 2 nM LY-411575 or 1 μM DAPT, robustly increased mBCMA densities on CD138+ but not CD3+ patient cells, concomitantly with minimum soluble/shed BCMA (sBCMA) in 1 day-culture supernatants. In ex vivo MM-T-cell co-cultures, GSIs overcame sBCMA-inhibited MM cell lysis and further enhanced autologous patient MM cell lysis induced by BCMAxCD3 BisAbs, accompanied by significantly enhanced cytolytic markers (CD107a, IFNγ, IL2, and TNFα) in patient T cells. In longer 7 day-co-cultures, LY-411575 minimally affected BCMAxCD3 BisAb (PL33)-induced transient expression of checkpoint (PD1, TIGIT, TIM3, LAG3) and co-stimulatory (41BB, CD28) proteins, as well as time-dependent increases in % effector memory/central memory subsets and CD8/CD4 ratios in patient T cells. Importantly, LY41157 rapidly cleared sBCMA from circulation of MM-bearing NSG mice reconstituted with human T cells and significantly enhanced anti-MM efficacy of PL33 with prolonged host survival. Taken together, these results further support ongoing combination BCMA-targeting immunotherapies with GSI clinical studies to improve patient outcome.

NK cells enhance CAR-T cell antitumor efficacy by enhancing immune/tumor cells cluster formation and improving CAR-T cell fitness

BackgroundChimeric antigen receptor (CAR)-T cell immunotherapy has modified the concept of treatment in hematological malignancies. In comparison with pediatric patients, where responses are maintained over many years, older patients, such...

CD38-specific Chimeric Antigen Receptor Expressing Natural Killer KHYG-1 Cells: A Proof of Concept for an "Off the Shelf" Therapy for Multiple Myeloma.

Chimeric antigen receptor (CAR) T cells are highly successful in the treatment of hematologic malignancies. We recently generated affinity-optimized CD38CAR T cells, which effectively eliminate multiple myeloma (MM) cells with little or no toxicities against nonmalignant hematopoietic cells. The lack of universal donors and long manufacturing times however limit the broad application of CAR T cell therapies. Natural killer (NK) cells generated from third party individuals may represent a viable source of “off the shelf” CAR-based products, as they are not associated with graft-versus-host disease unlike allogeneic T cells. We therefore explored the preclinical anti-MM efficacy and potential toxicity of the CD38CAR NK concept by expressing affinity-optimized CD38CARs in KHYG-1 cells, an immortal NK cell line with excellent expansion properties. KHYG-1 cells retrovirally transduced with the affinity-optimized CD38CARs expanded vigorously and mediated effective CD38-dependent cytotoxicity towards CD38high MM cell lines as well as primary MM cells ex vivo. Importantly, the intermediate affinity CD38CAR transduced KHYG-1 cells spared CD38neg or CD38int nonmalignant hematopoietic cells, indicating an optimal tumor nontumor discrimination. Irradiated, short living CD38CAR KHYG-1 cells also showed significant anti-MM effects in a xenograft model with a humanized bone marrow-like niche. Finally, CD38CAR KHYG-1 cells effectively eliminated primary MM cells derived from patients who are refractory to CD38 antibody daratumumab. Taken together, the results of this proof-of-principle study demonstrate the potential value of engineering affinity-optimized CD38CARs in NK cells to establish effective anti-MM effects, with an excellent safety profile, even in patients who failed to response to most advanced registered myeloma therapies, such as daratumumab.

Oncolytic Reovirus Sensitizes Multiple Myeloma Cells to Anti-PD-L1 Therapy

Adaptive resistance mediated by inhibitory ligands such as programmed death 1 ligand (PD-L1) has emerged as an important mechanism of malignant cell survival. This has spurred the development of new agents that disrupt the PD-L1/PD-1 immune checkpoint. Analysis of patient specimens from clinical trials of novel immune checkpoint inhibitors indicates that high basal expression of PD-L1 on tumor cells may predict sensitivity to and be necessary to elicit significant clinical benefit from this drug class. Notably, many multiple myeloma (MM) cell lines and primary CD138+ cells from MM patients do not overexpress PD-L1 compared to normal plasma cells and this may preclude patients with MM from optimally benefitting from immune checkpoint inhibitor therapy. These data suggest that strategies that transiently increase PD-L1 levels could potentially sensitize malignant cells with low PD-L1 expression to anti-PD-1/PD-L1 blockade. The oncolytic reovirus-based anticancer agent Reolysin is known to have significant immunomodulatory effects and has demonstrated promising preclinical efficacy in MM models and favorable safety and tolerability in early MM clinical trials. We demonstrated that Reolysin selectively replicates in MM cells and possesses significant activity in preclinical in vitro and in vivo MM models. These findings established the framework for an ongoing investigator-initiated phase 1b clinical study of Reolysin in combination with bortezomib and dexamethasone in patients with relapsed/refractory MM. Recent gene ontology analyses of RPMI-8226 and U266 MM cells treated with Reolysin revealed that reovirus exposure triggers a highly significant transient increase in CD274 (PD-L1) in MM cell lines. Reolysin-mediated PD-L1 upregulation was confirmed by immunoblotting, qRT-PCR, and flow cytometric analyses in MM cell lines and primary patient specimens treated with Reolysin. Increased PD-L1 expression was also detected by immunohistochemistry in MM tumor samples collected from mice treated with Reolysin. A comparison of the anti-MM effects achieved by live reovirus versus UV-inactivated reovirus demonstrated that live reovirus is required to decrease MM cell viability and upregulate PD-L1 expression. Our data demonstrate proof of concept that reovirus infection and replication in MM cells can efficiently and selectively upregulate PD-L1 levels in malignant cells with low target expression.We hypothesized that Reolysin treatment could be used as a precision priming strategy to potentiate the anti-MM efficacy of PD-1/PD-L1 targeted therapy by promoting myeloma immune recognition and PD-L1 upregulation. To investigate this therapeutic approach, 5TGM1-luc murine MM cells were injected IV into immunocompetent mice to generate MM bone disease. After disease was established, mice were randomized into groups and treated with vehicle, Reolysin (5 x 108 TCID50, Q7D), murine anti-PD-L1 antibody (200 mg/mouse, Q2D) or the combination for 5 weeks. Mice treated with the combination demonstrated decreased disease burden as measured by bioluminescent imaging and also showed reduced IgG2bk levels (specific IgG secreted by 5TGM1 cells) by ELISA. Importantly, the combination also led to increased overall animal survival compared to vehicle control and either single agent treatment (P<0.01). Analysis of bone marrow specimens from mice in all experimental groups showed that Reolysin stimulated elevated PD-L1 levels in vivo in a manner that was directly linked to the enhanced efficacy of the combination. Our findings demonstrate that Reolysin has dramatic PD-L1-related immune priming effects in clinically relevant models of MM and support its use as a precision agent to sensitize MM cells to immune checkpoint inhibitor therapy. Based on these promising data, we are currently planning a clinical study of Reolysin in combination with bortezomib and a PD-1 inhibitor in patients with relapsed/refractory MM. DisclosuresKelly:Amgen: Honoraria; Abbvie: Honoraria; Pharmacyclics: Honoraria; Jannsen: Honoraria.

Preclinical Evaluation of the First in Class BMI1 Inhibitor PTC-028 Confirms Potent Efficacy to Target BMI1-Addiction in Multiple Myeloma

Background:Treatment modalities for multiple myeloma (MM) patients suffering from refractory or high-risk disease are still limited. Although translational research efforts previously identified key players and promising drug targets in MM biology (e.g. IRF4, MYC, FOXM1), direct modulation of these factors is impeded by the lack of clinically effective inhibitors. Initially linked to the pathogenesis of MM more than a decade ago, the polycomb group protein BMI1 represents a prominent intrinsic driver of MM without a suitable clinical grade inhibitor. We recently confirmed BMI1 overexpression as a hallmark of MM and proved its essential role in MM cell survival using a small molecule inhibitor that affects BMI1 transcript levels (PTC209). Based on these observations, here, we explored the anti-myeloma efficacy of the first in class BMI1 protein inhibitor PTC028 and confirmed its applicability to target a so far not drug-able key molecule in MM.Methods:The anti-MM efficacy of PTC028 (kindly provided by PTC Therapeutics Inc.) was studied in vitro using human MM cell lines (HMCLs) and primary MM cells. Candidate gene expression levels were analyzed by quantitative PCR (qPCR), Western Blot and/or flow cytometry (FC). Mechanistic studies were conducted via FC and immunoblot experiments. Confirmatory studies are employed using lentiviral transduction of BMI1 overexpression or knockout plasmids, respectively.Results:PTC028 induced a time-dependent reduction of BMI1 protein levels in HMCLs. Evaluation of the in vitro efficacy demonstrated potent anti-MM activity in all primary MM cells and HMCLs tested (median IC50: 39 nM, range: 11-102 nM). This activity persisted in the presence of bone marrow stromal cells and in proteasome inhibitor (PI) resistant HMCLs. Importantly, median IC50 values were significantly reduced compared to PTC209 (median IC50 680 nM, P <0.05) confirming superior activity of PTC028. We also observed a significant correlation between PTC028 IC50 values and the reduction of BMI1 protein levels in HMCLs (R=0.86, P=0.01), suggesting that a rapid drop of BMI1 levels could serve as predictive marker for treatment efficacy.Mechanistically, treatment with PTC028 led to an accumulation of cells in the G2/M phase of the cell cycle and induction of apoptosis (verified by the presence of cleaved PARP and depolarization of the mitochondrial membrane potential). To better segregate the sequence of these events we performed time course experiments with U266 and KMS-12-BM cells. This demonstrated rapid induction of G2/M cell cycle arrest (evident 6h post treatment) and subsequent induction of apoptosis. Western Blot analysis demonstrated accumulation of CDK1 and Cyclin B1 in line with the G2/M cell cycle stop. In addition, we evaluated MCL1 protein levels due to its known degradation during mitotic arrest and indeed observed a time dependent downregulation of this key MM survival factor. In contrast, neither BIM nor BAX were found to be deregulated. Further validation of these results was obtained by qPCR analysis. PTC028 induced rapid upregulation of mitosis associated genes (CCNB1, AURKA, BIRC5) 6h and 24h post treatment indicating mitotic arrest which was accompanied by MCL1 downregulation. No impact was found on the gene expression levels of BMI1 and other cell cycle (CCND1, CDKN2A, CDKN1A, CDKN1B) or apoptosis (BAX, BIM, NOXA) related genes.Due to the close interaction between BMI1, MYC and FOXM1 we were finally interested whether PTC028 also affects these key factors. Intriguingly, PTC028 treatment led to rapid downregulation of both proteins suggesting that targeting of BMI1 via PTC028 offers a novel strategy to attack this MM high-risk network.Conclusion:This study demonstrates potent preclinical activity of the first in class BMI1 inhibitor PTC028 in a panel of HMCLs, primary MM cells, PI-resistant HMCLs and in the presence of BMSCs. This suggests efficacy in the context of the MM microenvironment and drug resistance. Moreover, our data point to significant impact on a currently not targetable high-risk network consisting of BMI1, MYC and FOXM1. These results strongly support further evaluation of PTC028 in the clinical setting. Detailed results about the impact of PTC028 on BMI1 associated signaling networks as well as preliminary in vivo efficacy results will be provided at the meeting. DisclosuresWeetall:PTC Therapeutics: Employment. Branstrom:PTC Therapeutics: Employment. Zojer:Celgene, Janssen, Amgen, Takeda, BMS: Consultancy, Honoraria, Research Funding.

Humanized Anti-CD26 Monoclonal Antibody Has Anti-Myeloma Efficacy for the Treatment of High-Risk Multiple Myeloma

Background: Despite remarkable progress in treatment options of multiple myeloma (MM) over the last decade with proteasome inhibitors and immunomodulatory drugs (IMiDs), most of patients with high-risk multiple myeloma (MM) relapse and ultimately become refractory. Recently, novel monoclonal antibody directly targeting CS1(elotuzumab) or CD38 (daratuzumab) induced durable responses in patients with multidrug-refractory MM. CD26, a 110-kDa transmembrane glycoprotein which is expressed on several tumor cells including malignant lymphoma, has been implicated in tumorigenesis, whereas its role in plasma cell malignancies has not been characterized yet. Our recent work has revealed that CD26 is intensely expressed on activated osteoclasts (OCs) in MM. In addition, in vitro studies showed that although CD26 expression is only slightly detected on MM cell lines cultured alone, it is intensely and uniformly expressed on MM cell lines co-cultured with OCs. In this study, we clarify the direct on-tumor and immune mechanism of action with huCD26mAb, a humanized monoclonal IgG1 antibody specifically targeting CD26, alone or in combination with approved agents in high-risk MM.Methods and Results: Immunostaining on bone marrow biopsy specimens showed that CD26 is expressed on plasma cells in several MM patients, but not in those of healthy individuals. So, we first analyzed the effect of huCD26mAb on survival of MM cell lines (KMS18, KMS26, KMS27, KMS28, KMS34, U266). HuCD26mAb had no direct effect on proliferation in any of tested CD26- MM cell lines cultured alone, but it inhibited growth of CD26+ MM cell lines co-cultured with OCs at higher concentrations (>10μg/ml), as measured by the MTT assay. Next, we examined the ability of huCD26mAb to lyse CD26 positive MM cell lines by antibody-dependent cellular cytotoxicity (ADCC) using the calcein-AM release assay. HuCD26mAb, but not isotype control IgG1, triggered ADCC against CD26+ KMS18, KMS26, KMS28, KMS34 and U266 with t(4;14) by natural killer (NK) effector cells in a dose-dependent manner with lytic activity starting at 0.0001μg/ml and maximum lysis at 10μg/ml and in an effector to target (E/T) ratio-dependent manner. Moreover, huCD26mAb dose-dependently induced lysis of lenalidomide-resistant MM.1R via ADCC. We further asked whether pretreatment with conventional or novel agents is able to induce superior ADCC activity by huCD26mAb. CD26+ KMS18, KMS26 and KMS28 were pretreated with bortezomib (3nM), lenamlidomide (0.5μM) or dexamethasone (25nM) overnight. Indeed, subsequent MM cell lysis triggered by huCD26mAb (10μg/ml) was significantly augumented. Treatment with lenalidomide (0.5μM) followed by huCD26mAb (10μg/ml) also markedly reduced the viable MM.1R in the presence of NK effector cells. The synergistic ADCC efficacy of huCD26mAb in combination with lenalidomide resulted in 2.0-fold increase in MM.1R lysis. In addition, pretreatment of NK effector cells with lenalidomide also potentiated ADCC against MM.1R mediated by huCD26mAb in a dose-dependent fashion. Complement dependent cytotoxicity (CDC) of CD26+ MM cells by huCD26mAb was also tested using human serum as a source of complement. However, in the presence of human serum, the incubation with huCD26mAb or isotype IgG1 exerted no effect on MM cell lysis. These results indicate that huCD26mAb (≦10μg/ml) kills CD26+ MM cells by inducing ADCC but not CDC, regardless of sensitivity or resistance to conventional or novel agents. Finally, to further determine whether huCD26mAb alone or combination with lenalidomide affects side population (SP) fractions of drug-resistant MM cells, we first examined CD26 expression in SP and main population (MP) fractions by flow cytometry. Both CD26+ RPMI8226 and KMS11 exhibited SP fractions which equally expressed CD26 at high levels as MP fractions. So, we examined ADCC activity by huCD26mAb (10μg/ml) against these SP fractions, mixed with NK effector cells. Interestingly, although lenalidomide alone did not decrease the ratio of SP fractions, huCD26mAb substantially reduced their ratio and its further reduction was observed with both in combination against CD26+ RPMI8226 and KMS11.Conclusions: HuCD26mAb induced significant anti-MM efficacy chiefly through ADCC. CD26 may be a novel valuable target for antibody therapy and huCD26mAb, alone or in combination, may be a promising immunotherapeutic strategy for the treatment of high-risk MM. DisclosuresMorimoto:Y's Therapeutics: Consultancy, Research Funding. Yamada:Y's therapeutics: Consultancy, Research Funding.

Finding the optimal partner to pair with bispecific antibody therapy for multiple myeloma.

BCMA/CD3ε-targeted bispecific antibody (BsAb) therapy represents a promising T-cell redirecting immunotherapy to treat relapsed and refractory multiple myeloma (MM). However, rational combination strategies will most likely be key to achieve a long-lasting immune response. In this issue, Meermeier and colleagues investigate BsAb therapy in a syngeneic MM model and elucidate that partnering with cyclophosphamide is associated with tempered activation, mitigated exhaustion of T-cells, and is superior to pomalidomide or bortezomib in enhancing durable anti-MM efficacy.

Novel Small Molecular Compound AE-848 Potently Induces Human Multiple Myeloma Cell Apoptosis by Modulating the NF-κB and PI3K/Akt/mTOR Signaling Pathways.

BackgroundWe aimed to investigate the anti-multiple myeloma (MM) activity of the new small molecular compound AE-848 (5-bromo-2-hydroxyisophthalaldehyde bis[(1-methyl-1H-benzimidazol-2-yl)hydrazone]) and its underlying anti-MM mechanism.MethodsCell viability and apoptosis were detected and quantified by using MTT and flow cytometry, respectively. JC-1 dye-related techniques were used to assess mitochondrial membrane potential (MMP). Western blotting was applied to detect the expression of NF-κB and PI3K/Akt/mTOR pathway-associated proteins. The in vivo activity of AE-848 against MM was evaluated in a MM mouse model.ResultsApplication of AE-848 into the in vitro cell culture system significantly reduced the viability and induced apoptosis of the MM cell lines, RPMI-8226 and U266, in a dose- and time-dependent manner, respectively. JC-1 dye and Western blotting analysis revealed that AE-848 induced the cleavage of caspase-8, caspase-3, and poly ADP-ribose polymerase (PARP), resulting in loss of mitochondrial membrane potential (MMP). Both the NF-κB and PI3K/AKT/mTOR signaling pathways were involved in AE-848-induced apoptosis of U266 and RPMI8226 cells. Moreover, AE-848 leads to cell cycle arrest of MM cells. Its anti-MM efficacy was further confirmed in a xenograft model of MM. AE-848 administration significantly inhibited MM tumor progression and prolonged the survival of MM-bearing mice. More importantly, our results demonstrated that AE-848 markedly induced primary MM cell apoptosis.ConclusionOur results for the first time showed that the small compound AE-848 had potent in vitro and in vivo anti-myeloma activity, indicating that AE-848 may have great potential to be developed as a drug for MM treatment.

Efficacy and Safety of Nivolumab Combined with Daratumumab with or without Low-Dose Cyclophosphamide in Relapsed/Refractory Multiple Myeloma; Interim Analysis of the Phase 2 Nivo-Dara Study

Efficacy and Safety of Nivolumab Combined with Daratumumab with or without Low-Dose Cyclophosphamide in Relapsed/Refractory Multiple Myeloma; Interim Analysis of the Phase 2 Nivo-Dara Study

Optimal Dual-Targeted CAR Construct Simultaneously Targeting Bcma and GPRC5D Prevents Bcma-Escape Driven Relapse in Multiple Myeloma

Multiple myeloma (MM) remains generally incurable, calling for the development of novel treatment strategies such as chimeric antigen receptor (CAR) T cell therapy. Most clinically tested CAR T cell therapies for MM target B cell maturation antigen (BCMA), but despite high response rates, many patients relapse (Raje N. NEJM 2019). BCMA negative-low MM cells are implicated as a reservoir preceding relapse (Brudno J. JCO 2018; Cohen A. JCI 2019). Our aims are to (1) evaluate whether upfront simultaneous targeting of an additional antigen such as G protein-coupled receptor class C group 5 member D (GPRC5D; Smith EL. Sci Trans Med 2019) can mitigate BCMA escape-mediated relapse in MM, and (2) compare dual targeting strategies to identify an optimal approach. Dual targeting for CD19/CD22 malignancies has been investigated, and multiple approaches are feasible; however, approaches have yet to be comprehensively compared head to head. Here, we compare 2 parallel production and 3 single-vector dual targeting strategies (Fig. 1A). To enhance clinical translatability, all strategies are built on the BCMA(125)/4-1BBζ CAR (BCMA scFv 125; Smith EL. Mol Ther 2018), which is currently under multi-center clinical investigation (NCT03430011; Mailankody S. ASH 2018). We confirmed that all dual targeted approaches lyse, proliferate, and secrete polyfunctional cytokines specifically in response to BCMA and GPRC5D mono- and dual-positive cell lines and/or primary patient MM aspirate samples. Activity in vivo was confirmed using the bone marrow-tropic OPM2 MM model (endogenously BCMA+GPRC5D+). In all experiments MM cells (2 x 106) were injected IV into NSG mice and engrafted/expanded for 14 days before treatment. A high dose of all dual targeted CAR T cell approaches (3 x 106 CAR+) induced long-term disease control (median overall survival (mOS) BCMA(125) non-signaling del control 32d vs other groups mOS not reached; p &amp;lt; 0.05). Prevention of latent BCMA escape-mediated relapse was evaluated by re-challenge of previously treated long-surviving mice with 2 x 106 OPM2 BCMA CRISPR KO (OPM2BCMA KO) cells at day 100 without re-treatment. While mice previously treated with BCMA(125)/41BBζ CAR T cells succumbed to OPM2BCMA KO disease, dual targeted approaches prevented OPM2BCMA KO growth (mOS BCMA mono-targeted arm 37d post re-challenge vs other groups mOS not reached; p &amp;lt; 0.05). To better recapitulate human MM and distinguish among dual targeting approaches, we modeled established BCMA heterogeneous disease by spiking 5-10% OPM2BCMA KO into bulk OPM2WT cells for injection. Each OPM2 population was modified to express distinct luciferases for simultaneous in vivo monitoring by bioluminescent imaging (BLI). Treatment with a moderate (5 x 105) dose of CAR T cells eradicated OPM2WT cells in all groups, but anti-GPRC5D CARs with CD28 co-stimulation, whether included within a mixed T cell population or in a bicistronic construct (Fig. 1A ii, iv), failed to control OPM2BCMA KO cells (Fig. 1B). Correspondingly, 4-1BB-only containing CAR T cells had increased in vivo expansion (2.1-4.1-fold increase CAR T cell BLI at day 7 over CD28 containing groups; p &amp;lt; 0.05). As this result is likely from greater activation-induced cell death in the CD28-containing approaches that was not rescued by 4-1BB, we later compared 4-1BB-only containing approaches (Fig. 1A i, iii, v). These 3 dual targeting approaches effectively controlled OPM2WT disease at moderate (1 x 106 CAR+) and low (2.5 x 105 CAR+) doses. However, when using a sub-therapeutic dose (2.5 x 105 CAR+) in the OPM2BCMA KO-spiked model, the tandem scFv-single stalk design was least effective in controlling OPM2BCMA KO disease (Fig 1C). At a dose that is sub-therapeutic to control OPM2WT disease (1 x 105 CAR+), the bicistronic dual 4-1BB design (Fig. 1A iii) was more effective in eradicating tumor compared with the parallel production approach (6-fold difference tumor BLI at day 28; p &amp;lt; 0.05). These results indicate that upfront dual targeting of BCMA/GPRC5D with CAR T cells can mitigate BCMA escape-mediated relapse in a model of MM. While parallel infusion of separate BCMA- and GPRC5D-targeted CAR T cells is effective, a single bicistronic vector encoding two 4-1BB-containing CARs avoids the practical challenges of parallel manufacturing, and uniquely may provide superior anti-MM efficacy. Figure Disclosures Fernandez de Larrea: Takeda: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Brentjens:JUNO Therapeutics: Consultancy, Patents &amp; Royalties, Research Funding; Celgene: Consultancy. Smith:Celgene: Consultancy, Patents &amp; Royalties, Research Funding; Fate Therapeutics and Precision Biosciences: Consultancy.

Geranylgeranyl Diphosphate Synthase Inhibitor and Proteasome Inhibitor Combination Therapy in Multiple Myeloma

Introduction: Multiple myeloma (MM) is characterized by proliferation of malignant plasma cells that produce monoclonal protein (MP). MM cells, compared with non-secretory cells, have a lower threshold for induction of the pro-apoptotic components of the unfolded protein response pathway (UPR) as a consequence of near-maximal expression of the protective UPR elements. Proteasome inhibitors (PIs) are widely used in the treatment of MM and have been shown to disrupt the UPR. As an alternative strategy by which to target protein homeostasis in MM, we have focused on the development of geranylgeranyl diphosphate synthase (GGDPS) inhibitors. Inhibition of GGDPS results in disruption of Rab protein geranylgeranylation, which in turn results in disruption of MP trafficking, leading to accumulation of intracellular MP, induction of the UPR and apoptosis. We have previously reported preclinical studies with our lead GGDPS inhibitor (GGDPSi), RAM2061, which demonstrated the agent's metabolic stability, prolonged half-life (plasma elimination half-life of 29.2 (+6.0) hours (hrs)), systemic distribution, in vivo disruption of geranylgeranylation and anti-tumor efficacy in a mouse MM xenograft model (Haney et al., ASH 2018, abstract 215). We hypothesized that the combination of GGDPSi and PI therapy would result in enhanced disruption of the UPR and increase anti-MM efficacy. Methods: MTT assays were conducted to evaluate the cytotoxic effects of combining RAM2061 with the PI bortezomib (Bor) in human MM cell lines (RPMI-8226, MM.1S) and isobologram analysis was performed. The effects of RAM2061 and/or Bor on markers of the UPR were evaluated via qRT-PCR (ATF4, ATF6, CHOP, PERK, IRE1) and immunoblot analysis (ATF4, IRE1, phosphorylated eIF2α). Apoptotic markers (cleaved caspases 3, 8 and 9 and cleaved PARP) were evaluated by immunoblot analysis. Cell assays were performed using both concurrent (both drugs added at the start of the 48 hr incubation period) and RAM2061 pre-treatment (RAM2061 added at the start, Bor added after 24 hrs) approaches. To evaluate the in vivo activity of the novel combination therapy, a flank xenograft model was used. NOD/SCID mice were subcutaneously inoculated in the flank with MM.1S cells. Once tumors became palpable, mice were randomly divided into four treatment groups (n=8 per group): control (PBS), RAM2061 (0.08 mg/kg IV two times/week (wk)), Bor (0.3 mg/kg SQ two times/wk) and RAM2061/Bor combination. Tumor volume (TV) and animal body weight were recorded three times/wk. Mice were euthanized when tumors reached 2000 mm3. Blood samples from day 9 post-initiation of treatment were analyzed via ELISA for human λ light chain. Blood samples collected at time of euthanasia were analyzed for hematologic, renal and hepatic parameters. Results: Isobologram analysis revealed a primarily additive effect with concurrent GGDPSi/PI incubations and primarily synergistic effect with the RAM2061 pre-treatment approach. The effect of Bor on augmenting RAM2061-induced upregulation of UPR and apoptotic markers was both concentration- and time-dependent. While there was in general an enhanced induction of apoptosis with the combination, Bor partially abrogated the RAM2061-induced upregulation of UPR markers at the tested time points. Studies are ongoing to determine whether this effect is associated with an accelerated time course of UPR activation in the presence of Bor. Consistent with our previous in vivo studies, the primary toxicity associated with RAM2061 treatment was hepatic transaminase elevation. No additional toxicity was observed when Bor was combined with RAM2061 based on animal weight, hematological, renal or hepatic parameters. Tumor growth over time was significantly decreased in the RAM2061 (p=0.0009) and RAM2061/Bor (p=0.0002) treatment groups compared to control animals and this corresponded to prolonged time to sacrifice (p=0.09 for RAM2061 group and p=0.003 for RAM2061/Bor group). Consistent with TV, mean plasma human λ light chain levels were decreased in the RAM2061 and RAM2061/Bor treatment groups compared to the control mice (p&lt;0.05). Conclusion: These studies support further investigation into the therapeutic potential of GGDPSi/PI-based combination therapy for MM. Further studies are needed to dissect the mechanisms underlying the interactions between the two classes of drugs on the UPR and apoptotic pathways. Disclosures Holstein: Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Celgene: Consultancy; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Sorrento: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees.

Novel Allosteric Inhibitors of Heat Shock Protein 70 As Agents to Probe Protein Homeostasis and Overcome Proteasome Inhibitor Resistance in Multiple Myeloma

Introduction: Clinical efficacy of proteasome inhibitors (PIs) has established multiple myeloma (MM) as the paradigm cancer type in which to therapeutically target protein homeostasis. However, PI resistance is one of the key clinical challenges in this disease. While mechanisms of PI resistance appear complex, proteomic analysis showed multiple heat shock proteins to be highly upregulated in PI-resistant cell line models (Soriano et al, Leukemia (2016) 30:2198). Furthermore, HSPA1B, which encodes a cytosolic Hsp70, is one of the genes which best-stratifies PI-sensitive versus resistant MM cell lines (Mitra et al, Blood Cancer J (2017) 7:e581). These findings support the hypothesis that Hsp70 family chaperones may be critical in maintaining plasma cell protein homeostasis by reducing unfolded protein burden in the PI-refractory setting, indicating potential as a therapeutic target. Targeting Hsp70 may also therefore be effective in combination with PIs or other agents targeting MM protein homeostasis. Here, we profiled a series of novel, allosteric Hsp70 inhibitors of the “JG” class (Shao et al, J Med Chem (2018) 61:6163). We aimed to evaluate their role as anti-myeloma therapeutics as well as their utility as tool compounds to better define targetable protein homeostasis networks in MM.Methods: “JG” compounds were screened in quadruplicate against a panel of 13 cell lines: 9 MM, one AML, 3 immortalized normal bone marrow stromal, in 384-well plates with viability measured by CellTiter-Glo. The parental compound JG98 was tested in combination with the p97 inhibitor CB-5083, the PI bortezomib, and Hsp90 inhibitor 17-DMAG in RPMI-8226, MM1.S, KMS34, HS5, and CMK cell lines for 72 hours, with synergy and antagonism quantified using the ZIP score method. Murine studies were performed using luciferase-labeled RPMI-8226 implanted in NOD scid gamma (NSG) mice (n = 5 mice per arm). For cell surface mass spectrometry experiments, RPMI-8226 and AMO-1 cells were treated with JG342 at the LD20 for 48 hours prior to biotinylation of cell surface glycoproteins (n=3), followed by enrichment for biotinylated proteins with Neutravidin beads and LC-MS sample processing and analysis. For pulsed-SILAC experiments (n = 2), MM1.S cells were grown in light medium for six doublings, followed by exchange to heavy medium (Arg10 and Lys8) with JG342 treatment at the LD10 or DMSO, with timepoint collections performed at 16, 21, and 26 hours, followed by sample processing and LC-MS analysis. All mass spectrometry data was analyzed in MaxQuant and statistical analysis was performed in Perseus and R.Results: Supporting our hypothesis, drug screening of 16 JG compounds revealed consistently greater potency versus a PI-resistant AMO-1 cell line versus the parental line (Fig. A). We focused on the lead compound JG342, which exhibits sub-µM LC50s (140-500 nM) against a panel of myeloma cell lines and a favorable therapeutic index against bone marrow stromal cells. However, all tested JG molecules, including JG342, showed unfavorable pharmacokinetics in NSG mouse models and could not demonstrate in vivo anti-MM efficacy at achievable doses. Therefore, we focused on JG molecules as tool compounds to probe protein homeostasis in MM and further validate Hsp70 as a therapeutic target in this disease. Combination assays using JG98, bortezomib, CB-5083, and 17-DMAG revealed surprisingly diverse, cell line-specific protein homeostasis wiring networks leading to differential synergism and antagonism in dual therapies targeting the proteostasis machinery. To evaluate potential combination effects of Hsp70 inhibition with immunotherapies, we employed cell surface proteomics and found that JG342 leads to broad changes in the MM surface proteome including decrease in expression of BCMA (Fig. B). Lastly, we employed pSILAC mass spectrometry to profile JG342 mediated changes in protein synthesis and degradation. Calculated protein half-lives revealed a distinct subset of differentially regulated proteins after Hsp70 inhibition including aminotransferases and DNA chaperones.Conclusions: Inhibition of Hsp70 is a promising therapeutic strategy to overcome PI resistance in myeloma, but new molecules with more favorable in vivo properties are needed to benefit patients. The “JG” molecules also provide a novel set of tool compounds for exploring protein homeostasis biology not only in MM but across biological systems. [Display omitted] DisclosuresWiita:Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

The JAK Inhibitor Blocks PD-L1, PD-L2 and CD44 Expression in Multiple Myeloma (MM) and Sensitizes MM Cells to Lenalidomide and Steroids

Introduction: Ruxolitinib (RUX) is an inhibitor of the Janus kinase family of protein tyrosine kinases (JAKs) that is effective for the treatment of myeloproliferative diseases. Immunomodulatory drugs including lenalidomide (LEN) and corticosteroids (dexamethasone [DEX]) have shown efficacy in the treatment of multiple myeloma (MM). We have demonstrated that the combination of the RUX, LEN and steroids shows both preclinical and clinical efficacy for the treatment of patients with relapsed and refractory MM. The immune inhibitory proteins PD-L1 and PD-L2 and CD44 are highly expressed in MM; moreover, increased expression of these proteins is associated with resistance to treatment in MM. In this study, we investigated the effects of RUX on expression of PD-L1, PD-L2 and CD44 in MM, and the effect of RUX in combination with LEN and DEX on growth of human MM in vivo.Methods: Bone marrow mononuclear cells (BMMCs) were purified from aspirates from MM patients with progressive disease (PD) or in complete remission (CR) before and after treatment with RUX in combination with LEN and methylprednisolone (MP). The cells were stained with antibodies directed against human PD-L1, PD-L2 and CD44 and flow cytometric analysis (FCA) was performed. MM biopsies or BMMCs were analyzed using immunofluorescent analysis (IFA). Total RNA was extracted from monocytes. Quantitative PCR (qPCR) was measured for gene expression of PD-L1, PD-L2 and CD44 and the housekeeping gene HPRT1 with TaqMan technology following standard qPCR protocol. The human MM xenograft LAGκ-2 was surgically implanted into the left superficial gluteal muscle of anaesthetized naive SCID mice. LEN was administered via oral gavage daily (15 mg/kg) and DEX was administered intraperitoneally daily (1 mg/kg). RUX was given via intraperitoneal (IP) injection twice daily (1.5 mg/kg). Seven days following tumor implantation, mice were treated with vehicle alone, single agent RUX, LEN or DEX or using combination treatments.Results: We examined PD-L1 gene expression in MM patients with PD or in CR and those with monoclonal gammopathy of undetermined significance (MGUS). The results showed that PD-L1 gene expression was markedly increased in BMMCs from MM patients with PD compared with those patients in CR or with MGUS. We determined the effects of RUX on expression of PD-L1 and CD44. The FCA data showed the percentage of PD-L1+ BMMCs was markedly reduced in MM patients treated with RUX, LEN and MP compared to those before starting treatment whereas there were decreased CD44+ cells in the MM patients after this treatment compared to those before treatment with this combination. IFA further confirmed that PD-L1+ and CD44+ BMMCs staining was markedly deceased in MM patients treated with this three drug combination compared to those without treatment. We further investigated the in vitro effects of RUX on gene expression of PD-L1, PD-L2 and CD44 in MM cell lines and primary MM cells co-cultured with THP1 monocytes. MM cells showed marked upregulation of these genes following co-culture with THP1 cells. RUX treatment markedly reduced PD-L1, PD-L2 and CD44 gene expression in the MM tumor cells co-cultured with monocytes compared with cells not treated with the JAK1/2 inhibitor. To evaluate these drugs in vivo, the human MM xenograft LAGκ-2 model was used. The mice were then treated with RUX, LEN or DEX alone, doublets or the combination of all three drugs. RUX alone produced minimal anti-MM effects whereas the doublets showed more anti-MM effects than any single agent, and the combination of all three drugs showed the most marked anti-MM effects.Conclusion: The PD-L1/PD-1 pathway delivers inhibitory signals that regulate both peripheral and central tolerance and inhibit anti-tumor immune-mediated responses. This study demonstrated that the JAK inhibitor RUX downregulated expression of PD-L1, PD-L2 and CD44 in MM tumor cells which should help overcome the immune resistance generated by these proteins for patients with this B-cell malignancy. We also demonstrated the combination of RUX with LEN and DEX showed enhanced anti-MM efficacy in vivo. These results also suggest that JAK inhibitors may be effective for treating MM patients through their ability to reduce expression of checkpoint proteins involved in the development of immune resistance that frequently occurs in MM patients. DisclosuresChen:OncoTracker: Equity Ownership. Li:OncoTracker: Equity Ownership. Sanchez:OncoTracker: Equity Ownership. Wang:OncoTracker: Equity Ownership. Berenson:Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria; Daiichi: Speakers Bureau; Takeda: Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau.

CD26 is a potential therapeutic target by humanized monoclonal antibody for the treatment of multiple myeloma

CD26, a 110-kDa transmembrane glycoprotein that is expressed on several tumor cells including malignant lymphoma, has been implicated in tumorigenesis: however, little is known regarding its role in multiple myeloma (MM). Recently, we identified CD26 expression on human osteoclasts (OCs) and demonstrated that humanized IgG1 monoclonal antibody targeting CD26, huCD26mAb, inhibits human OC differentiation. Herein, we show that CD26 expression was present on plasma cells in the bone marrow tissues of MM patients. In vitro immunostaining studies revealed that although CD26 expression was low or absent on MM cell lines cultured alone, it was intensely and uniformly expressed on MM cell lines co-cultured with OCs. The augmented CD26 expression in MM cells was exploited to enhance anti-MM efficacy of huCD26mAb via a substantial increase in antibody-dependent cytotoxicity (ADCC) but not complement-dependent cytotoxicity (CDC). Moreover, huCD26mAb in combination with novel agents synergistically enhanced huCD26mAb induced ADCC activity against CD26+ MM cells compared with each agent alone. huCD26mAb additionally reduced the ratio of the side population (SP) fraction in CD26+ MM cells by ADCC. Finally, huCD26mAb significantly reduced the MM tumor burden and OC formation in vivo. These results suggest that CD26 is a potential target molecule in MM and that huCD26mAb could act as a therapeutic agent.