Primary Multiple Myeloma Patient Samples Research Articles

Abstract 4587: Development of highly selective, potent, orally available PIM1 inhibitor BLX0631 shows a therapeutics potential in multiple myeloma models

Abstract Background: The Proto-oncogenic PIMs family are nuclear and cytoplasmic Ser/Thr kinases (PIM1, PIM2, and PIM3) that are constitutively active and play a vital role in proliferation and survival in Multiple Myeloma (MM). Activated PIM1 kinase can induce progression of the cell cycle, inhibition of apoptosis, and modulation of other signal transduction pathways. Methods: BLX-0631 and a few analogs evaluated against a panel of MM cell lines (EJM, IM-9, L-363, LP-1, MM-1R, MM.1S, MOLP-2, NCI-H929, OPM-2, RPMI8226 and U-266) and primary MM patient samples by CellTiter-Glo® (CTG) assays. Cellular apoptosis, Cell cycle analysis, Flow Cytometry, and bone marrow microenvironment experiments on the most sensitive cells were performed. PIM1 and downstream targets by western blot, cell migration, and invasion on our lead PIM1 inhibitor BLX-0631 along with, kinome selectivity, ADME-Tox, PK, and in vivo MM.1S efficacy experiments were conducted. Results: The application of our empirical MolecuLernTM fragment library, PIM1 crystal structure, scaffold hopping, and the enumeration features of MolecuLernTM in combination with 3, 6, and 7-group analysis on pyridazine, lead to a synthesizable potential new lead series, representing Med Chem tractability and developable characteristics. Further, the consensus scoring, binding energies, and MolecuLern tractability filters were applied and led to the discovery of the BLX-0631 series that inhibits the PIM1 kinase. The active compounds, with their binding modes and lead optimization strategies, lead to the synthesis and testing of over 60 novel entities as PIM1 kinase inhibitors. Thus, based on our previous chemotype series and scaffold hopping method, the impact of changing the 3, 6, and 7 positions, while retaining the pyridazine core led to the discovery of BLX-0631 and its series of compounds exhibiting <5 nM potency. BLX-0631 in CTG assay demonstrated 100% cell growth inhibition across all MM cell lines tested. The lead compound BLX-0631 is a very potent inhibitor with an IC50 of 5.0/17/5.0 nM when tested against PIM1, PIM2, and PIM3 kinases respectively, and selective against a panel of 468 kinase panels. Dosing through IV and PO delivery routes, formulations, salt forms of BLX-0631 in mice, PK properties along with ADME-Tox, P450, and hERG results will be presented. In vivo MM.1S mouse xenograft models of BLX-0631 at 15, 30 and 45 mg/Kg, p.o, QD demonstrated TGI of 16.28%, 31.81% & 35.16% dose-dependently. Conclusions: The present study demonstrates that PIM1 plays a vital role in the proliferation and survival of Multiple Myeloma. BLX-0631 induces full inhibition of all MM cell lines, suppresses cancer cell proliferation in vitro, and exhibits promising efficacy in MM tumor models. BLX-0631, a nominated IND candidate can serve as a novel targeted agent for treating Multiple Myeloma patients. Citation Format: Zhaoang li, Chenyu Lin, David J. Bearss, Hariprasad Vankayalapati. Development of highly selective, potent, orally available PIM1 inhibitor BLX0631 shows a therapeutics potential in multiple myeloma models [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 4587.

Steroid-free combination of 5-azacytidine and venetoclax for the treatment of multiple myeloma.

Multiple Myeloma (MM) is an incurable plasma cell malignancy, that despite an unprecedented increase in overall survival, lacks truly risk-adapted or targeted treatments. A proportion of patients with MM depend on BCL-2 for survival and recently the BCL-2 antagonist venetoclax has shown clinical efficacy and safety in t(11;14) and BCL-2 overexpressing MM. However, only a small proportion of MM patients rely on BCL-2 (~20%), there is a need to broaden the patient population outside of t(11;14) that can be treated with venetoclax. Therefore, we took an unbiased screening approach and screened epigenetic modifiers to enhance venetoclax sensitivity in two non-BCL-2 dependent MM cell lines. The demethylase inhibitor 5-azacytidine was one of the lead hits from the screen, and the enhanced cell killing of the combination was confirmed in additional MM cell lines. Using dynamic BH3 profiling and immunoprecipitations we identified the potential mechanism of synergy is due to increased NOXA expression, through the integrated stress response. Knockdown of PMAIP1 or PKR partially rescues cell death of the venetoclax and 5-azacytidine combination treatment. The addition of a steroid to the combination treatment did not enhance the cell death and interestingly we found enhanced death of the immune cells with steroid addition, suggesting that a steroid-sparing regimen may be more beneficial in MM. Lastly, we show for the first time in primary MM patient samples, that 5-azacytidine enhances the response to venetoclax ex-vivo, across diverse anti-apoptotic dependencies (BCL-2 or MCL-1) and diverse cytogenetic backgrounds. Overall, our data identifies 5-azacytidine and venetoclax as an effective treatment combination and this could be a tolerable steroid-sparing regimen, particularly for elderly MM patients.

Abstract 2956: Superenhancer-drvien SMC4 promote cell growth and proliferation in multiple myeloma

Abstract Background Multiple myeloma (MM) is an incurable malignancy characterized by complex heterogeneous cytogenetic abnormalities. Defined as large clusters of cis-acting enhancers, super-enhancers (SEs) could promote oncogenic transcription to which cancer cells highly addicted. Structural maintenance of chromosome 4 (SMC4) is a core subunit of condensin complexes, which contributes to chromosome condensation and segregation. Here, we identified SMC4 as a novel SE-associated oncogene of myeloma cells, whose functional roles in MM remain largely elusive. Methods We performed H3K27ac ChIP-seq and RNA-seq on primary MM patient samples, MM cell lines, normal CD138+ plasma cell and lymphoma cell lines as the controls, then the ROSE analysis was used to annotate SEs and their associated genes. THZ1 as a transcriptional CDK7 inhibitor was used to further filter THZ1-responsive genes. Combined analysis of THZ1-sensitive and SE-associated gene uncovered a number of promising MM oncogenes as SMC4. The public data mining, RNA interference, CRISPR/Cas9 gene editing system, overexpression and a variety of cellular functional assays were performed to determine the oncogenic effects of SMC4 on MM malignant. Transcriptome analysis with SMC4 silencing and overexpression, and the underlying molecular mechanism of SMC4 oncogenic role in MM are ongoing. Results We have demonstrated SMC4 as one of the SE-associated genes specific to MM patient samples and cell lines. SMC4 was related to myelomagenesis with increased expression from MGUS, SMM to MM cases, and overexpression of SMC4 predicted poor overall survival of MM patients, suggesting its clinical relevance. SMC4 silencing in MM cells suppressed cell proliferation and lead to cell apoptosis. Repression of the SMC4-SE region also led to impairment of cell viability and cell growth, consistent with an oncogenic function. Conclusion In summary, our integrative approached by combing H3H27ac ChIP-seq, RNA-seq and THZ1-sensitive transcripts identified the landscape of SEs and novel oncogenic transcripts as SMC4 in MM. Mapping these acquired SEs and their associated genes may provide novel insight into understanding MM biology, and SMC4 may serve as novel therapeutic targets in MM. Citation Format: Yunlu Jia, Jianbiao Zhou, Tze-King Tan, Tae-Hoon Chung, Yongxia Chen, Enfan Zhang, Zhen Cai, Takaomi Sanda, Wee-Joo Chng. Superenhancer-drvien SMC4 promote cell growth and proliferation in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2956.

Transcriptional profiles define drug refractory disease in myeloma.

Identifying biomarkers associated with disease progression and drug resistance are important for personalized care. We investigated the expression of 121 curated genes, related to immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs) responsiveness. We analyzed 28 human multiple myeloma (MM) cell lines with known drug sensitivities and 130 primary MM patient samples collected at different disease stages, including newly diagnosed (ND), on therapy (OT), and relapsed and refractory (RR, collected within 12 months before the patients’ death) timepoints. Our findings led to the identification of a subset of genes linked to clinical drug resistance, poor survival, and disease progression following combination treatment containing IMIDs and/or PIs. Finally, we built a seven‐gene model (MM‐IMiD and PI sensitivity‐7 genes [IP‐7]) using digital gene expression profiling data that significantly separates ND patients from IMiD‐ and PI‐refractory RR patients. Using this model, we retrospectively analyzed RNA sequcencing (RNAseq) data from the Mulltiple Myeloma Research Foundation (MMRF) CoMMpass (n = 578) and Mayo Clinic MM patient registry (n = 487) to divide patients into probabilities of responder and nonresponder, which subsequently correlated with overall survival, disease stage, and number of prior treatments. Our findings suggest that this model may be useful in predicting acquired resistance to treatments containing IMiDs and/or PIs.

Super-Enhancer-Driven PPP1R15B As an Oncogenic and Potential Therapeutic Target in Multiple Myeloma

Background: Multiple myeloma (MM) is a hematological malignancy arising from immunoglobulin-secreting plasma cells. Despite recent progress in stem-cell transplantation, high-dose chemotherapy and novel therapies, MM remains incurable and most patients experience relapse. In addition of genetic alterations, growing evidence has suggested that alterations in epigenetic landscape contribute to pathogenesis of MM. Super enhancers (SE) are large clusters of putative enhancers with aberrantly strong binding of mediators and transcription-regulating proteins. MM cells are highly dependent on unfolded protein response (UPR) signaling pathways due to high level of endoplasmic reticulum (ER) stress. Phosphorylation of eIF2α can attenuate protein translation. The PPP1R15B (denoted as R15B hereafter) gene encodes a regulatory subunit of an eIF2α-specific phosphatase complex. In this study, we identified SE-driven oncogenes specific in MM with a particular focus on a candidate SE-associated gene R15B, whose functional roles in MM remain largely elusive.Methods: We performed H3K27Ac ChIP-seq on MM cell lines, primary MM patient samples and normal CD138+ plasma cells, memory B cells (controls). ROSE analysis was used to systematically annotate SEs and their associated genes between tumors and controls. A combination of public data mining, RNA interference (RNAi), overexpression and CRISPR/Cas9 technologies followed by functional assays were conducted to determine the oncogenic effects of R15B in MM. Transcriptome analysis of MM cell line NCI-H929 with R15B knockdown and scrambled control was performed. To further study the interactions between SE and its promoters, we are currently working on highly integrative chromatin immunoprecipitation (HiChIP) followed by sequencing.Results: We have identified R15B as one of the SE-associated genes specific to MM patient samples and MM cell lines. The expression of R15B was 6- to 50-fold higher in a panel of 8 MM cell lines than normal controls. SE activity was correlated with the expression level of R15B. Higher expression of R15B predicted poor overall survival of MM patients, suggesting its clinical relevance in MM pathogenesis. Knockdown and knockout of R15B significantly reduced cell viability, clonogenicity and induced G2/M phase cell cycle arrest. Gene Ontology (GO) enrichment and pathway analysis of downregulated genes in response to R15B knockdown indicates that these genes are significantly associated with G2/M checkpoint hallmark, oxidative phosphorylation, unfolded protein response and E2F1-related pathways. Loss of R15B may induce apoptosis via activation of UPR pro-apoptotic executors PUMA and NOXA. ChIP-qPCR assays indicates that transcription factor C/EBP-β is strongly enriched at R15B SE region. Furthermore, we examined the therapeutic effects of Salubrinal, a selective inhibitor of eIF2α phosphorylation, on MM cells. Salubrinal inhibited MM cell proliferation in a dose-dependent manner, and MM cell lines with higher R15B expression were more sensitive to Salubrinal than those with lower R15B level.Conclusions: Our integrative approaches identified R15B as a novel SE-driven oncogene, and may form a positive feedback loop with C/EBP-β. Salubrinal selectively inhibits proliferation of MM cells with high R15B expression. Hence, we propose that targeting R15B may serve as a new approach for effective anti-myeloma therapy, which warrants further clinical investigation. DisclosuresChng: Novartis: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Honoraria; Amgen: Honoraria, Research Funding.

Mapping the High-Risk Multiple Myeloma Cell Surface Proteome Identifies T-Cell Inhibitory Receptors for Immune Targeting

Multiple myeloma (MM) is an incurable malignancy of mature plasma cells. Despite major advances in the therapeutic armamentarium of MM, only 50% of patients survive more than 5 years after diagnosis, with significantly lower rates (21%) for high-risk patients.Chimeric Antigen Receptor (CAR) T-cell therapy targeting BCMA (B-cell maturation antigen) shows high response rates in relapsed/refractory patients. However, most patients have disease remission that lasts less than 18 months, prompting the search for additional and synergistic therapeutic approaches.We unbiasedly mapped the cell surface proteome of MM by integrating Mass-Spectrometry (MS) and RNA-seq analyses from 7 MM cell lines and 904 primary MM patient samples bearing high-risk cytogenetics. To identify cell surface proteins, we ran a pool of 4,761 proteins and 16,000+ transcripts through five repositories. An integrated scoring database was developed by scoring each ID based on the number of databases (0-5) it was identified in, with 0 if the molecule was not found in any and 5 if the protein was found in all five.We identified 402 proteins with a surface score of 3 or higher in MM cell lines and patient samples by transcriptomics and proteomics. We prioritized the 326 candidates that were more highly expressed in patients. Based on functional enrichment analyses, we found the proteins formed three main networks with immune mechanisms representing the largest cluster (227 out of 326 cell surface proteins) followed by transporters and adhesion proteins.Based on a pipeline we previously established (1), we further selected 97 candidates minimally expressed in normal tissues. This list included current therapeutic targets such as BCMA, SLAMF7, ITGB7 and LY9. Validation in primary patient samples by western blot and flow-cytometric analyses, enabled the identification of 10 top candidates (CCR1, CD320, FCRL3, IL12RB1, ITGA4, LAX1, LILRB4, LRRC8D, SEMA4A, SLAMF6) that resulted most frequently and highly expressed. We found that LAX1, LILRB4 and SEMA4A significantly impact myeloma patient overall survival based on Kaplan-Meier analysis in the MM Research Foundation (MMRF) cohort (2). CCR1, IL12RB1, LILRB4 and SEMA4A were upregulated by the treatment with Bortezomib or Venetoclax that conversely, decreased BCMA expression in MM U266 cells. By stratifying the patient population, we found that the SEMA4A and LAX1 were up-regulated in patients with t(4;14) compared to patients with no cytogenetic abnormality; LILRB4 in patients with t(14;16) and CCR1 patients with t(14;16) and t(14;20). By calculating co-expression levels CCR1-LILRB4 and CCR1-FCRL3 resulted co-expressed in 100% of patients. For safety purposes (3), we excluded candidates with high (>55%) protein abundance in highly-purified normal hematopoietic stem cells and activated T-cells, narrowing down the list to 6 top candidates (CCR1, FCRL3, IL12RB1, LILRB4, LRRC8D, SEMA4A).To define the function of this group of promising cell surface targets, we used a CRISPR/Cas9 inducible system in KMS11 MM cells. We found that knock-out of CCR1, LRRC8D and SEMA4A individually reduces the MM cell growth by ~60%, 50% and 50% respectively, and almost completely abrogates MM cell migration through porous chambers by >80%. By co-culturing irradiated KO and control MM cells with healthy donor T-cells we also found that lack of CCR1 increased T-cell proliferation by 50% compared to controls and enhanced killing of MM cells, suggesting that CCR1 may suppress T-cell mediated immune responses in addition to play a role in MM cell survival and migration.This study suggests the contribution of an altered MM surfaceome to disease development and may lead to potential novel immunotherapeutic approaches for high-risk MM.

P-098: Super-enhancer-driven PPP1R15B as an oncogenic and potential therapeutic target in Multiple Myeloma

<h3>Background</h3> Growing evidence suggests that alterations in epigenetic landscape contribute to pathogenesis of multiple myeloma (MM). MM cells are highly dependent on unfolded protein response signaling pathways due to high level of endoplasmic reticulum stress. Phosphorylation of eIF2a can attenuate protein translation. The PPP1R15B(denoted as R15B hereafter) gene encodes a regulatory subunit of eIF2a-specific phosphatase complex. In this study,we identified super enhancer (SE)-driven oncogenes specific in MM with a particular focus on a candidate gene R15B, whose functional roles in MM remain largely elusive. <h3>Methods</h3> We performed H3K27Ac ChIP-seq on MM cell lines, primary MM patient samples, normal CD138+ plasma cells and memory B cells. ROSE analysis was used to annotate SEs and their associated genes. A combination of public data mining, RNAi, overexpression and CRISPR/Cas9 technologies were conducted to determine the oncogenic effects of R15B in MM. Transcriptome analysis of MM cell line H929 with R15B KD and scrambled control was performed. To further study the interactions between SE and its promoters, we are currently working on HiChIP. <h3>Results</h3> We have identified R15B as one of the SE-associated genes specific to MM patient samples and cell lines. SE activity was correlated with the expression level of R15B. Higher expression of R15B predicted poor overall survival of MM patients, suggesting its clinical relevance in MM pathogenesis. R15B KD or KO significantly reduced cell viability, clonogenicity and induced G2/M arrest. ChIP-qPCR assays showed that C/EBP-β is strongly enriched at R15B SE region. We also found that salubrinal, a selective inhibitor of eIF2a phosphorylation, inhibited MM cell proliferation in a dose-dependent manner. <h3>Conclusions</h3> Our integrative approaches identified R15B as a novel SE-driven oncogene. We propose that targeting R15B may serve as a new approach for effective anti-myeloma therapy, which warrants further clinical investigation.

PIKfyve Inhibitors for the Treatment of Multiple Myeloma

IntroductionUsing a high throughput screening approach employing ~80 FDA approved, late stage clinical trial, or drugs of interest to multiple myeloma (MM), we examined single agent drug sensitivity of 35 MM and lymphoma cell lines and in 65 primary MM samples. We identified APY-0201, a PIKfyve inhibitor (P5I), as lethal at nanomolar concentrations in 48% of human myeloma cell lines (HMCLs) and 28.6% of primary MM samples after 24 hour of drug exposure. In 20% of patient samples, in vitro sensitivity was less than 100nM EC50. PIKfyve is a lipid and protein kinase that controls pleiotropic cellular functions, notably regulation of endomembrane homeostasis; mechanisms of sensitivity and resistance were explored.MethodsApilimod, a key P5I initially developed as an interleukin (IL)-12 and IL-23 inhibitor, has been used as an immunomodulatory agent in the treatment of inflammatory diseases and has been recently suggested for the treatment of neoplastic diseases, including B-cell Non-Hodgkin's Lymphoma (NHL). YM201636 has also been described as a potent P5I. The cellular efficacies of APY-0201, apilimod, and YM201636 in HMCLs were assessed using the CellTiter Glo assay (Promega), in 20-point 2-fold dilutions of drug concentrations at 72 hours; and HMCL P5I sensitivity was correlated with RNA-SEQ data publically available at www.keatslab.org. Cell lysates were extracted for western blot expression analysis of transcription factor EB (TFEB), cathepsin D, caspase 3, poly (ADP-ribose) polymerase (PARP), at different drug exposure time points (3, 6, 12, 24, 48, and 72 hours). Ex vivo sensitivity to APY-0201, the most potent studied P5I, was measured in CD138+ primary cells of 56 primary MM patients at 24 hours. In vivo activity of apilimod at 100-150 mg/kg and APY-0201 at 30-45 mg/kg was evaluated over 2 weeks in the Vk*MYC transgenic mouse model of MM. Tumor burden was measured by weekly evaluation of M-spike serum levels.ResultsAPY-0201 was the most potent P5I, followed by YM201636 and apilimod. However, dose-dependent inhibition of cell viability was observed in all HMCLs. APY-0201 EC50 ranged from 2.8 to 266.7nM (mean 123.4nM), 63.8 to 8425.8nM (mean 1329nM) for YM201636, and 62.5 to 3561.2nM (mean 2026nM) for apilimod. APY-0201 maximum inhibition ranged from 68.2 to 100% (mean 87.2%), 33.2 to 100% (mean 64.6%) for YM201636, and 56.7 to 100% (mean 83.6%) for apilimod. A significant correlation between low RNA-SEQ expression of TFEB, which regulates lysosomal biogenesis and autophagy, and lower sensitivity for APY-0201 was found (Wilcoxon signed-rank test p-value = 0.017). No correlation with baseline OSTM1, CLCN7 or SNX10 levels was seen.APY-0201 dose dependent sensitivities were observed in 37.5% of ex vivo patient samples. Of the 21 patients demonstrating sensitivity, 2 were inhibited at an EC50<10nM (median 8.58nM), 10 samples presented EC50 between 10 and 100nM (median 64.67nM), and 9 samples presented EC50>100nM (median 1000.16nM). Thus, 12 (21.4%) primary MM patient samples exhibited lethality with less than 100nM concentration of APY-0201 after 24 hours. P5I activity profile was not obviously correlated with clinical findings, such as stage of disease or genetic subtype, in either cell lines or primary samples.As demonstrated previously in B-cell NHL, treatment of the most sensitive cell line, KMS26, with apilimod and APY-0201 was associated with activation of TFEB, found in its dephosphorylated state. An accumulation of the inactive precursor procathepsin D was also observed in KMS26 following treatment, demonstrating a decrease in maturation of lysosomal proteases. Furthermore, the cytotoxic activity of both compounds was shown by induction of apoptosis makers, i.e. caspase 3 and PARP. In ongoing studies, in vivo activity was evident in 2 of 6 Vk*MYC transgenic MM mice treated with a P5I; dose finding and longer treatment duration studies are underway.ConclusionsInhibition of PIKfyve surprisingly demonstrated promising anti-myeloma activity in vitro, ex vivo and in vivo, thus providing a rationale for further evaluation in the treatment of MM. As reported by others in NHL, a novel mechanism of action involving disruption of endolysosomal function is suggested in PIKfyve mediated cytotoxicity in MM.AcknowledgmentThis work was supported in part by the Multiple Myeloma Research Foundation. DisclosuresSharik:Phosplatin Therapeutics: Research Funding. Sepetov:Nanosyn Inc.: Employment. Romanov:Nanosyn Inc.: Employment. Bergsagel:Phosplatin Therapeutics: Research Funding. Chesi:Phosplatin Therapeutics: Research Funding. Stewart:Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Roche: Consultancy.

Exploiting Protein Translation Dependence in Multiple Myeloma with Omacetaxine-Based Therapy.

The prognosis of patients with multiple myeloma who are resistant to proteasome inhibitors, immunomodulatory drugs (IMiD), and daratumumab is extremely poor. Even B-cell maturation antigen-specific chimeric antigen receptor T-cell therapies provide only a temporary benefit before patients succumb to their disease. In this article, we interrogate the unique sensitivity of multiple myeloma cells to the alternative strategy of blocking protein translation with omacetaxine. We determined protein translation levels (n = 17) and sensitivity to omacetaxine (n = 51) of primary multiple myeloma patient samples. Synergy was evaluated between omacetaxine and IMiDs in vitro, ex vivo, and in vivo. Underlying mechanism was investigated via proteomic analysis. Almost universally, primary patient multiple myeloma cells exhibit >2.5-fold increased rates of protein translation compared with normal marrow cells. Ex vivo treatment with omacetaxine resulted in >50% reduction in viable multiple myeloma cells. In this cohort, high levels of translation serve as a biomarker for patient multiple myeloma cell sensitivity to omacetaxine. Unexpectedly, omacetaxine demonstrated synergy with IMiDs in multiple myeloma cell lines in vitro. In addition, in an IMiD-resistant relapsed patient sample, omacetaxine/IMiD combination treatment resensitized the multiple myeloma cells to the IMiD. Proteomic analysis found that the omacetaxine/IMiD combination treatment produced a double-hit on the IRF4/c-MYC pathway, which is critical to multiple myeloma survival. Overall, protein translation inhibitors represent a potential new drug class for myeloma treatment and provide a rationale for conducting clinical trials with omacetaxine alone and in combination with IMiDs for patients with relapsed/refractory multiple myeloma.

Abstract B01: Nanoparticle multispecific T-cell engagers for the treatment of multiple myeloma

Abstract Introduction: Multiple myeloma (MM) is the second most prevalent hematologic malignancy. Chimeric antigen receptor (CAR)-T cells and bispecific T-cell engagers targeting BCMA, CS1, and CD38 are promising approaches for MM. However, using these targets individually limits the efficacy of these immunotherapy options due to the variable expression levels of these markers, leading to inefficient eradication of MM. Studies have attempted to develop CAR-T cells and T-cell engagers with multispecific targeting; however, these are still technically very challenging and are highly expensive. In this study, we developed nanoparticles with monovalent or multivalent specificity against MM targets and engage T cells, by chemical conjugation of monoclonal antibodies against each molecule to the surface of the liposome. Bi-specific nanoparticles with anti CD38/CD3, BCMA/CD3 or CS1/CD3 were termed nanoBiTEs, while the multispecific particles with anti BCMA/CS1/CD38/CD3 were termed nanoMuTEs. Methods: Liposomes were prepared using the thin-film hydration method followed by extrusion. nanoBiTEs and nanoMuTEs were developed by chemical conjugation of monoclonal antibodies against BCMA, CS1, and/or CD38; together with anti CD3 onto the liposomes. We have tested the expression of BCMA, CS1, and CD38 antigens in MM cell lines and primary MM patient samples, and we analyzed the binding of the CD38/CD3, BCMA/CD3, and CS1/CD3 nanoBiTEs, as well as the BCMA/CS1/CD38/CD3 nanoMuTEs to these cells by flow cytometry and confocal microscopy. Binding specificity was assessed by blocking the target with corresponding free antibodies. CD25 expression, a T-cell activation marker, was assessed for CD8 and CD4 T cells when incubated with or without nanoBiTEs and nanoMuTEs. Moreover, we tested the efficacy of the nanoBiTEs and nanoMuTEs against MM cells in vitro using the 3D tissue engineered bone marrow (3DTEBM) myeloma model. Finally, we tested the efficacy of the nanoBiTEs and nanoMuTE against MM tumors in vivo in xenograft MM models. Results: The expression of BCMA, CS1 and CD38 was variable in MM cell lines and patient samples, and so was the binding of each nanoBiTEs, while the binding of the nanoMuTE was higher and more universal than each nanoBiTE. Similar patterns were observed in activation of CD8 T cells (no significant activation was observed in CD4 T cells) and in eradication of MM cell lines and primary patient samples in vitro. In vivo, the nanoBiTEs extended the survival and reduced the tumor burden in the MM bearing mice compared to untreated and T cell alone groups, and nanoMuTE experiments are still ongoing. Conclusions: The nanoBiTEs and nanoMuTEs were bound specifically to MM cells and initiated CD8 T-cell activation that resulted in MM eradication in vitro and in vivo, while the effect of the nanoMuTEs was more universal and efficacious than each nanoBiTE. These nanoparticles are an inexpensive method of engaging T cells for the treatment of MM and represent a new class of immunotherapy that can be expanded to other tumors. Citation Format: Kinan Alhallak, Jennifer Sun, Barbara Muz, Christopher Egbulefu, Pilar de la Puente, Cinzia Federico, Justin King, Danny Kohnen, Ravi Vij, Samuel Achilefu, Abdel Kareem Azab. Nanoparticle multispecific T-cell engagers for the treatment of multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B01.

Super-Enhancer Profiling Identifies Novel Oncogenes and Therapeutic Targets in Multiple Myeloma

Background: Multiple myeloma (MM) is an aggressive neoplastic plasma cell cancer characterized by diversely cytogenetic abnormalities. MM can be divided into subtypes with immunoglobulin heavy chain (IGH) gene translocations involving CCND1-3, FGFR3/MMSET, MAFs and hyperdiploid myeloma containing trisomies of several odd numbered chromosomes 3, 5, 7, 9, 11, 15, 19, and 21. Although several new drugs have been introduced into clinic, treatment for MM patients remains challenge and refractory/resistant to therapy is often seen. Thus, a better understanding of the molecular pathogenesis of MM can lead to generate new prognostic classification and identify new therapeutic targets. Super-enhancers (SEs) are defined as large clusters of cis-acting enhancers, marked by high level bindings of acetylation of histone H3 lysine 27 (H3K27ac) and mediator complex. SEs have been shown to control genes for maintaining cellular identity and also key tumor drivers in various malignancies. Methods: H3K27Ac ChIP-seq and RNA-seq were performed on primary MM patient samples, MM cell lines. Normal plasma cells and lymphoma cell lines were served as controls. We systematically compared SEs and their associated genes of normal and cancerous tissue. THZ1, a CDK7 inhibitor, was used to efficiently down-regulate SE-associated genes. Combinatory analysis of THZ1-sensitive and SE-associated gene revealed a number of promising MM oncogenes. CRISPR/Cas9 technology and ectopic expression experiments in conjunction with cellular functional assays were performed to determine the effects of candidate SE-genes on MM cells. Circularized chromatin conformation capture followed by sequencing (4C-seq) was applied to explore the direct contact of SE and promoter. Results: SE analysis uncovered some cell lineage-specific transcription factors (TFs) and known oncogenes in MM. Several key TFs, including IRF4, PRDM1, MYC and XBP1, were identified in most MM samples, confirming the origin of MM cells. These data reinforce the concept that SE establishment is a key component of MM biology. The acquisition of SEs around oncogene drivers is widely observed during tumorigenesis. ST3GAL6 and ADM were two known oncogenic drivers in myeloma cells, which were associated with super-enhancers in all MM samples but not in normal plasma cell and lymphoma cells. We also found SE constituents for multiple subtype-specific key oncogenes such as CCND1 in t(11;14) cells, C-MAF in t(14;16) cells, and NSD2 and FGFR3 in t(4;14) cells. Furthermore, THZ1 showed prominent anti-neoplastic effect against MM cells. SE-associated genes were more sensitive to THZ1 compared with those genes associated with typical enhancers (TEs). By overlapping THZ1-sensitve gene with SE-associated genes, we identified a number of novel MM oncogenes, including MAGI2, EDEM3, HJURP, LAMP5, MBD1 and UCK2 as a potential druggable kinase. The expression level of MAGI2 and HJURP confers poor prognosis in several MM datasets. MAGI2 silencing in MM cells decreased cell proliferation and induced apoptosis. qRT-PCR and Western blot analysis confirmed the overexpression of HJURP in t(4;14) cells relative to non-t(4;14) MM cells. Furthermore, 4C-seq analysis revealed the physical interaction between HJURP-SE and promoter and THZ1 treatment diminished this interaction. Motif search at SE constituents revealed a highly significant enrichment of NSD2 recognition. Significant reduction of NSD2 binding at HJURP-SE region was observed in KMS11 infected with NSD2-specific shRNAs. Interestingly, blocking SE sites by CRISPR/Cas9i or silencing HJURP by shRNA led to decreased HJURP expression and cell apoptosis, whereas overexpression of this gene promoted cell growth. Taken together, our data demonstrated that HJURP is a novel SE-associated oncogene in t(4;14) MM. Conclusions: Our integrative approaches by combing H3K27Ac ChIP-seq, RNA-seq and THZ1-sensitive transcript defined the landscape of SE and identified SE-associated novel oncogenes, as well as lineage-specific TFs in MM. Furthermore, we also revealed subtype-specific SE-driving oncogenic program in MM. Taken together, these results not provide novel insight into the MM pathology, but also offer novel, potential therapeutic targets, such as MAGI2, and HJURP for the treatment of MM patients. Disclosures No relevant conflicts of interest to declare.

Targeting the Leukemia Antigen PR1 with Immunotherapy for the Treatment of Multiple Myeloma.

Purpose: PR1 is a human leukocyte antigen (HLA)-A2 nonameric peptide derived from neutrophil elastase (NE) and proteinase 3 (P3). We have previously shown that PR1 is cross-presented by solid tumors, leukemia, and antigen-presenting cells, including B cells. We have also shown that cross-presentation of PR1 by solid tumors renders them susceptible to killing by PR1-targeting immunotherapies. As multiple myeloma is derived from B cells, we investigated whether multiple myeloma is also capable of PR1 cross-presentation and subsequently capable of being targeted by using PR1 immunotherapies.Experimental Design: We tested whether multiple myeloma is capable of cross-presenting PR1 and subsequently becomes susceptible to PR1-targeting immunotherapies, using multiple myeloma cell lines, a xenograft mouse model, and primary multiple myeloma patient samples.Results: Here we show that multiple myeloma cells lack endogenous NE and P3, are able to take up exogenous NE and P3, and cross-present PR1 on HLA-A2. Cross-presentation by multiple myeloma utilizes the conventional antigen processing machinery, including the proteasome and Golgi, and is not affected by immunomodulating drugs (IMiD). Following PR1 cross-presentation, we are able to target multiple myeloma with PR1-CTL and anti-PR1/HLA-A2 antibody both in vitro and in vivoConclusions: Collectively, our data demonstrate that PR1 is a novel tumor-associated antigen target in multiple myeloma and that multiple myeloma is susceptible to immunotherapies that target cross-presented antigens. Clin Cancer Res; 24(14); 3386-96. ©2018 AACR.

Abstract 5818: Globally optimizing therapeutic combinations against bortezomib-resistant multiple myeloma using a quantitative parabolic optimization platform

Abstract Multiple myeloma is an incurable hematological malignancy that relies on drug combinations as first and secondary lines of treatment. The inclusion of proteasome inhibitors, such as bortezomib, into these drug combination regimens has improved median survival. Resistance to bortezomib, however, is a common occurrence that ultimately contributes to treatment failure. Thus, there remains a need to identify improved drug combinations that may serve as later lines of treatment for improved treatment against bortezomib-resistant multiple myeloma. We have developed the quantitative parabolic optimization platform (QPOP) to optimize drug combinations against bortezomib-resistant multiple myeloma. By mapping phenotypic output data to parabolic response surfaces, QPOP is able to deterministically optimize drug combinations as well as drug dosages. By continuously optimizing in multiple systems of interest, from in vitro to in vivo, drug combinations can be globally optimized for greater efficacy in increasingly complex biological systems. While QPOP does not rely on molecular mechanism modeling or prediction, identified optimal drug combinations can reverse DNA hypermethylation and silencing of tumor suppressors that occurs following acquired bortezomib-resistance in multiple myeloma. Furthermore, this drug combination is broadly effective across a range of primary multiple myeloma patient samples. Beyond bortezomib-resistant multiple myeloma, global optimization of drug combinations by QPOP can serve to improve drug combination design across a range of other cancers and diseases through a continuous optimization process across the entire drug development pipeline. Citation Format: Masturah Rashid, Tan Boon Toh, Lissa Hooi, Aleidy Silva, Yanzhou Zhang, Neerja Karnani, Sudhakar Jha, Chih-Ming Ho, Wee Joo Chng, Dean Ho, Edward Kai-Hua Chow. Globally optimizing therapeutic combinations against bortezomib-resistant multiple myeloma using a quantitative parabolic optimization platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5818.

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.

The Dual PIM/PI3-K Inhibitor Ibl-202 Overcomes Microenvironmental Mediated Resistance in Multiple Myeloma and Prevents PIM1 Induced CXCR4 Upregulation

Background:The interaction of multiple myeloma (MM) cells with bone marrow (BM) cells along with factors in the BM milieu such as chemokines and cytokines play a crucial role in both progression of MM and drug resistance. Activation of the PI3-K/Akt survival pathway is a characteristic of both human MM cell lines and patient samples. This activation can be linked to BM microenvironmental signalling and use of proteasome inhibitors in treatment, suggesting this as a crucial point of therapeutic intervention to abrogate growth and survival signals in MM. However, the efficacy of such therapeutics has been modest and is likely to be compromised by the stimulation of compensatory signalling pathways, such as the PIM kinases, which like the PI3-K/Akt pathway are also induced by BM microenvironmental influences and share similar downstream targets. These proto-oncogenic kinases are constitutively active and play an important role in proliferation and survival in MM. The influence of these kinases on homing and migration has been observed in other malignancies, this has yet to be reported in MM. Here we report the effects of a dual inhibitor of PIM/PI3-K, IBL-202, and provide novel insights into effects on cell survival, signaling and migration.Methods:We investigated the effect of IBL-202 against a panel of MM cell lines (MM.IS, NCI-H929s, KMS11 and RPMI-8226) and primary MM patient samples. The in vitro efficacy of IBL-202 was compared to that of single pan-PIM inhibitors pPIMi and AZD1208 and also the pan-PI3-K inhibitor GDC-0941. Apoptosis was measured with AnnexinV staining and cell cycle analysed with Edu/DAPI staining.To mimic BM microenvironmental conditions MM cells were cultured under hypoxic conditions (1% O2) and in co-culture with the human stromal cell line HS5.Surface expression of CXCR4 was assessed in MM cell lines by flow cytometry. PIM kinases, pCXCR4 and downstream targets of PIM/PI3-K were examined by western blot. Transwell migration assays were carried out in the presence of 50ng SDF-1α for 4h @ 37o C.Results:Simultaneous inhibition of PIM and PI3-K using IBL-202 in vitro was significantly more potent at inducing apoptosis than GDC-0941, pPIMi or AZD1208 in all MM cell lines tested. IC50 values were under 1μM for IBL-202 at 48h whilst in comparison the pan PIM inhibitors pPIMi and AZD1208 scored IC50 values between 5 and 10μM. The IC50 for GDC-0941 was on average 5μM (Figure 1).At the molecular level there was a notable decrease in phosphorylation of known PIM/PI3-K targets Akt (Ser473), Bad (Ser112) and the translational targets S6 (Ser235/236) and 4EBP1 (Thr37/46). The levels of total proteins were unchanged.Treatment with increasing doses of IBL-202 led to a marked reduction in cells in S phase of the cell cycle. These changes were paralled by down regulation of the cell cycle promoting proteins cyclin D1 and c-myc.IBL-202 was also effective in inducing apoptosis in primary MM patient samples (n=4) after just 24h as assessed by Annexin-V staining (Figure 2).To explore the role of the BM microenvironment we co-cultured MM cell lines with HS5s. This led to strong induction of PIM2 in MM cells. While MM cells in this setting were protected from Bortezomib-induced cell death, the apoptotic effect of IBL-202 was enhanced. In a further effort to mimic the tumour microenvironment we cultured MM cell lines in hypoxia. This may be of particular relevance as Pim-1 has been reported to be a pivotal regulator involved in hypoxia-induced chemoresistance. MM cells were further sensitised to IBL-202 in hypoxia. In addition, hypoxia increased the surface expression of CXCR4, a chemokine receptor critical for homing of MM cells to the bone marrow, with a concomitant increase in PIM1. Treatment of MM cell lines with IBL-202 reduced the level of PIM1 and CXCR4 Ser339 phosphorylation, along with down regulation of CXCR4 surface expression resulting in reduced migration of MM cells along an SDF-1 gradient.Conclusion:Together these data provide direct evidence of the potency of IBL-202 in MM in conditions that mimic the BM microenvironment. Moreover, they indicate a potential role for PIM kinases in facilitating dissemination and invasiveness of MM by CXCR4 and provide an added rationale for targeting PIM kinases in MM. [Display omitted] [Display omitted] DisclosuresO'Neill:Inflection Biosciences: Employment.

TORC1 and class I HDAC inhibitors synergize to suppress mature B cell neoplasms

Enhanced proliferative signaling and loss of cell cycle regulation are essential for cancer progression. Increased mitogenic signaling through activation of the mTOR pathway, coupled with deregulation of the Cyclin D/retinoblastoma (Rb) pathway is a common feature of lymphoid malignancies, including plasmacytoma (PCT), multiple myeloma (MM), Burkitt's lymphoma (BL), and mantle cell lymphoma (MCL). Here we evaluate the synergy of pharmacologically affecting both of these critical pathways using the mTOR inhibitor sirolimus and the histone deacetylase inhibitor entinostat. A dose-matrix screening approach found this combination to be highly active and synergistic in a panel of genetically diverse human MM cell lines. Synergy and activity was observed in mouse PCT and human BL and MCL cell lines tested in vitro, as well as in freshly isolated primary MM patient samples tested ex vivo. This combination had minimal effects on healthy donor cells and retained activity when tested in a co-culture system simulating the protective interaction of cancer cells with the tumor microenvironment. Combining sirolimus with entinostat enhanced cell cycle arrest and apoptosis. At the molecular level, entinostat increased the expression of cell cycle negative regulators including CDKN1A (p21) and CDKN2A (p16), while the combination decreased critical growth and survival effectors including Cyclin D, BCL-XL, BIRC5, and activated MAPK.

Novel Targeting Of Phospho-Marcks Overcomes Drug Resistance and Induces Anti-Tumor Activity In Preclinical Models Of Multiple Myeloma

Multiple myeloma (MM) remains incurable due to the development of a drug-resistant phenotype after prolonged therapy. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a protein kinase C (PKC) substrate that plays an important role in cell adhesion, spreading and invasion. Our previous studies found that overexpression of phospho-MARCKS (pMARCKS) was detected in developed drug resistant MM cell lines (RPMI-8226 R5, MM.1R) relative to their parental drug sensitive cell lines (RPMI-8226S, MM.1S). We hypothesized that pMARCKS is involved in chemo- and novel drug resistance in MM. To further evaluate the drug resistance, we exposed both RPMI-8226 R5 and MM.1R cell lines to varying dosages of bortezomib, dexamethasone, doxorubicin, and lenalidomide. By MTT assay, both resistant cell lines were found to have significantly higher viability to all 4 drugs compared to their respective non-resistant lines. In addition, Western blot analysis showed increased pMARCKS expressions in all 3 bortezomib resistant cell lines 8226.BR, OPM2.BR, and ANBL-6.BR as compared to their respective bortezomib sensitive cell lines. We next acquired MM patient samples collected at diagnosis and at relapse after bortezomib treatment, and investigated their pMARCKS expression with immunoblotting analyses. The patient samples collected from relapse after bortezomib treatment had higher pMARCKS expression than those collected at diagnosis. Furthermore, we studied additional 3 primary MM patient samples with high pMARCKS expressions and 3 with low expressions for their vaibility after a 36 hour bortezomib treatment, and found that the samples with high pMARCKS expressions were more resistant to bortezomib than those with low pMARCKS expressions (mean IC50 of 7.1 nM and mean IC50 of 4.8 nM, respectively; p = 0.042). Importantly, combination of a low dosage of bortezomib (5.0 nM) with either 2.5 uM or 5.0 uM of enzastaurin (an inhibitor of phospho-PKC), displayed a synergistic cytotoxicity on myeloma cells with high pMARCKS expressions. To further elucidate the role of pMARCKS in drug resistance, we knocked down pMARCKS expression by transfecting siMARCKS into 8226 R5 and MM.1R cells. Following the knockdown, both cell lines had significantly lower viability after treatment with either bortezomib, dexamethasone, doxorubicin, or lenolidomide, in comparison to empty vector controls. FACS analysis and annexin V assay of the knockdown cells and the control cells from both cell lines showed significantly induction of G1/S cell cycle arrest and apoptosis in the knockdown cells. The immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) DNA-qPCR analysis further demonstrated that pMARCKS regulates SKP2 expression through binding with E2F1, mediating SKP2/p27Kip1 cell cycle pathway. Finally, we investigated the effect of inhibition of pMARCKS in a 8226 R5 xenograft model of SCID mice. Mice injected with shMARCKS-transfected 8226 R5 cells and received bortezomib showed significant retardation of tumor growth and prolonged survival compared to the control groups. Taken together, our data indicate that pMARCKS is constitutively activated in resistant and relapsed MM cells and contributes to drug resistance by regulating E2F1 mediated SKP2/p27Kip1 cell cycle pathway, thus providing a preclinical rationale for targeting pMARCKS as a promising approach in patients with refractory/relapsed MM. Disclosures:Reece:BMS: Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Millennium: Research Funding; Novartis: Honoraria; Onyx: Honoraria.

Genome-wide studies in multiple myeloma identify XPO1/CRM1 as a critical target validated using the selective nuclear export inhibitor KPT-276

RNA interference screening identified XPO1 (exportin 1) among the 55 most vulnerable targets in multiple myeloma (MM). XPO1 encodes CRM1, a nuclear export protein. XPO1 expression increases with MM disease progression. Patients with MM have a higher expression of XPO1 compared with normal plasma cells (P<0.04) and to patients with monoclonal gammopathy of undetermined significance/smoldering MM (P<0.0001). The highest XPO1 level was found in human MM cell lines (HMCLs). A selective inhibitor of nuclear export compound KPT-276 specifically and irreversibly inhibits the nuclear export function of XPO1. The viability of 12 HMCLs treated with KTP-276 was significantly reduced. KPT-276 also actively induced apoptosis in primary MM patient samples. In gene expression analyses, two genes of probable relevance were dysregulated by KPT-276: cell division cycle 25 homolog A (CDC25A) and bromodomain-containing protein 4 (BRD4), both of which are associated with c-MYC pathway. Western blotting and reverse transcription-PCR confirm that c-MYC, CDC25A and BRD4 are all downregulated after treatment with KPT-276. KPT-276 reduced monoclonal spikes in the Vk*MYC transgenic MM mouse model, and inhibited tumor growth in a xenograft MM mouse model. A phase I clinical trial of an analog of KPT-276 is ongoing in hematological malignancies including MM.

The PI3K inhibitor GDC-0941 combines with existing clinical regimens for superior activity in multiple myeloma

The phosphatidylinositol 3'-kinase (PI3K) pathway is dysregulated in multiple myeloma (MM); we therefore tested a highly selective class I PI3K inhibitor, GDC-0941, for anti-myeloma activity. Functional and mechanistic studies were first performed in MM cell lines, then extended to primary MM patient samples cultured in vitro. GDC-0941 was then assessed as a single agent and in various combinations in myeloma tumor xenograft models. We show p110 α and β are the predominant PI3K catalytic subunits in MM and that a highly selective class I PI3K inhibitor, GDC-0941, has robust activity as a single agent to induce cell cycle arrest and apoptosis of both MM cell lines and patient myeloma cells. Mechanistic studies revealed an induction of cell cycle arrest at G0/G1, with decreased phospho-FoxO1/3a levels, decreased cyclin D1 and c-myc expression, and an increase in the cell cycle inhibitor, p27kip. Induction of apoptosis correlated with increased expression of the pro-apoptotic BH3-only protein BIM, cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP). In vitro, GDC-0941 synergized with dexamethasone (Dex) and lenalidomide (combination index values of 0.3-0.4 and 0.4-0.8, respectively); in vivo GDC-0941 has anti-myeloma activity and significantly increases the activity of the standard of care agents in several murine xenograft tumor models (additional tumor growth inhibition of 37-53% (Dex) and 22-72% (lenalidomide)). These data provide a clear therapeutic hypothesis for the inhibition of PI3K and provide a rationale for clinical development of GDC-0941 in myeloma.

Phagocytosis Is A Mechanism of Action for Daratumumab

AbstractAbstract 4054Daratumumab (DARA) is a human IgG1 CD38 antibody with broad-spectrum killing activity. DARA induces killing of CD38-expressing tumor cells, including fresh cells from multiple myeloma (MM) patient samples, via diverse mechanisms. These prominently include the Fc-dependent effector mechanisms complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers et al. J. Immunol. 2011). In this study we show that DARA is also able to induce tumor cell killing via antibody-dependent cellular phagocytosis (ADCP) as an additional Fc-dependent effector mechanism.In a first set of experiments, we studied ADCP with human macrophages as effector cells. Calcein-AM-labeled Daudi tumor cells, a Burkitt's lymphoma cell line, were mixed with human macrophages in the absence or presence of DARA. Specific DARA-induced phagocytosis was analyzed in flow cytometry by measuring the percentage of calcein-AM+/CD11b+ double-positive (DP) macrophages. Both classical GM-CSF activated and alternative M-CSF activated macrophages mediated DARA-specific ADCP of the Burkitt's lymphoma cells. To further explore the in vivo contribution of ADCP in the mechanism of action (MoA) of DARA, we studied DARA mediated phagocytosis with murine macrophages. In vitro ADCP with M-CSF-stimulated bone marrow-derived murine macrophages showed a dose-dependent DARA-specific effect on the Burkitt's lymphoma cell lines Ramos and Daudi, resulting in up to 24% and 43% DP macrophages and a 25% and 50% tumor cell reduction, respectively. Furthermore, dose-dependent DARA-specific phagocytosis was observed with patient-derived MM cell lines L363 and UM9, which were transduced with CD38 to obtain levels CD38 expression as they are generally observed in primary MM patient samples. With life-cell imaging we found that ADCP of Daudi and Ramos cells occurred very rapid and efficiently. Interestingly, our recordings document that single macrophages could engage multiple target cells and that they were able to engulf up to six tumor cells sequentially in a 30 min period. This suggests that ADCP might be a very potent MoA of DARA in vivo, which we are currently studying in a mouse xenograft model.In conclusion, in addition to CDC and ADCC, we now show that DARA can also induce killing of CD38 expressing tumor cells via phagocytosis. This very fast and potent MoA might contribute to the treatment efficacy of DARA in hematological tumors, especially at sites where high numbers of macrophages reside, such as the bone marrow. Disclosures:Overdijk:Genmab BV: Employment. Verploegen:Genmab BV: Employment. Groen:Genmab BV: Research Funding. Martens:Genmab BV: Research Funding. Lammerts van Bueren:Genmab BV: Employment. Bleeker:Genmab BV: Employment. Parren:Genmab BV: Employment.