Inhibited Multiple Myeloma Cell Growth Research Articles

Anticancer effect of minor phytocannabinoids in preclinical models of multiple myeloma.

Multiple myeloma (MM) is a blood cancer caused by uncontrolled growth of clonal plasmacells. Bone disease is responsible for the severe complications of MM and is caused by myeloma cells infiltrating the bone marrow and inducing osteoclast activation. To date, no treatment for MM is truly curative since patients relapse and become refractory to all drug classes. Cannabinoids are already used as palliative in cancer patients. Furthermore, their proper anticancer effect was demonstrated in many cancer models in vitro, in vivo, and in clinical trials. Anyway, few information was reported on the effect of cannabinoids on MM and no data has been provided on minor phytocannabinoids such as cannabigerol (CBG), cannabichromene (CBC), cannabinol (CBN), and cannabidivarin (CBDV). Scientific literature also reported cannabinoids beneficial effect against bone disease. Here, we examined the cytotoxic activity of CBG, CBC, CBN, and CBDV in vitro in MM cell lines, their effect in modulating MM cells invasion toward bone cells and the bone resorption. Subsequently, according to the in vitro results, we selected CBN for in vivo study in a MM xenograft mice model. Results showed that the phytocannabinoids inhibited MM cell growth and induced necrotic cell death. Moreover, the phytocannabinoids reduced the invasion of MM cells toward osteoblast cells and bone resorption in vitro. Lastly, CBN reduced in vivo tumor mass. Together, our results suggest that CBG, CBC, CBN, and CBDV can be promising anticancer agents for MM.

A Novel Indirubin-3-Monoxime Derivative Functions As Regulator of Proteostasis Via Targeting TRIM28 in Multiple Myeloma

Introduction: Multiple myeloma (MM) remains an incurable plasma cell malignancy. Agents disturbing the proteostasis play indispensable roles in MM treatment. Our previous studies have shown that the indirubin-3-monoxime (I3MO) works as a novel proteasome inhibitor that effectively inhibits MM cell growth. In this study, we aim to investigate the improvement of I3MO combined with histone deacetylase 6 (HDAC6) inhibitors, and clarified the molecular mechanisms. Methods: We constructed a compound 8b combined with I3MO and HADC6 inhibitor. The anti-MM effects of compound 8b were investigated in vivo and in vitro studies. Western blots, immunofluorescence, and proteasome activity assay were utilized to investigate the effects of 8b on MM cell proteasome activity, aggresome and autophagosome formation. RNA-seq and Dual-luciferase reporter gene assay were performed to identify the downstream targets of 8b treatment. We constructed MM cell lines with TRIM28 overexpression or shRNA knockdown. We further explored the function of TRIM28, one of the targets of 8b treatment, by ChIP-seq and IP-MS. Results: The novel I3MO derivative 8b more efficiently suppressed myeloma cell growth in vitro and in vivo. Compound 8b not decreased proteasome activity but inhibited the formation of aggresomes and autophagosomes. Interesting, we found that compound 8b efficiently inhibited TRIM28 promoter activity and repressed TRIM28 transcription level further. GEO data analysis demonstrated a positive correlation between TRIM28 and the expression of proteasome subunits as well as autophagy-related genes in MM. Further, we utilized ChIP-seq to confirm that TRIM28 could bind to the promoter region of multiple proteasome subunits-encoding genes, including PSMB1. Our data indicated that TRIM28 knockdown not only decreased the proteasome activity, but inhibited the formation of autophagosomes. Knockdown TRIM28 induced a higher sensitivity to BTZ treatment in MM cells. Furthermore, IP-MS with Co-IP analysis showed that TRIM28 interacts with 14-3-3ζ. TRIM28 acts as an E3 ligase in mediating the ubiquitination and degradation of 14-3-3ζ, a well-known negative regulator of autophagy. 14-3-3ζ degradation induced by TRIM28 activates autophagy in MM cells. These data suggested that TRIM28 suppressed by compound 8b would involve in proteostasis by regulation of proteasome activity and autophagy. Conclusions: Our study identified a novel compound 8b, I3MO combined with HDAC6i, efficiently suppressed MM cells survival. Additionally, we for the first time provided novel important insights for the roles of TRIM28 in MM pathobiology by regulation of proteasome activity and autophagy.

RING box protein-1(RBX1), a key component of SCF E3 ligase, induced multiple myeloma cell drug-resistance though suppressing p27

ABSTRACT Multiple myeloma (MM) is a clonal disease of plasma cells that remains, for the most part, incurable despite the advent of several novel therapeutics. The elevated expression of p27 and its association with cell-cycle arrest is speculated to be one of the major mechanisms by which MM cells escape the cytotoxic effects of therapeutic agents. In this study, we demonstrated that RBX1 silencing could inhibit MM cell growth and promote cell drug resistance. RBX1 directly interacted with and triggered the ubiquitination and degradation of p27, ultimately causing p27 reduction. Additionally, cell growth and apoptosis analysis indicated that the role of RBX1 in regulating myeloma cell proliferation and drug resistance resulted from p27 accumulation, which occurred in a Thr187 phosphorylation-dependent manner. Furthermore, the cell-cycle analysis demonstrated that RBX1 overexpression induced cells to enter the cell cycle (S-phase) and partially inhibited chemotherapeutic drugs-mediated cell cycle arrest. Notably, the forced expression of RBX1 also inhibited the cell adhesion-mediated elevation of p27 and induced the accumulation of adherent cells in apoptosis, especially the proteolytic cleavage of caspase-3. Additionally, RBX1 knockdown significantly inhibited myeloma development in SCID-Hu mice and in a human MM xenotransplant model. Overall, these in vitro and in vivo experiments indicated that the RBX1-p27 axis could be a central molecular mechanism by which RBX1 functions as a tumor promoter and stimulates cell growth in chemotherapeutic drugs treated MM cells.

Cytotoxic and Apoptotic Effects of Pinostilbene and Bortezomib Combination Treatment on Human Multiple Myeloma Cells.

Multiple myeloma (MM) is a cancer of plasma cells in the bone marrow characterized by bone lesions, hypercalcemia, anemia, and renal failure. Bortezomib (BTZ), a common treatment for MM, is a proteasome inhibitor that induces apoptosis in MM cells. However, high doses of BTZ can be very toxic, signifying a need for a synergistic drug combination to improve treatment efficacy. Resveratrol (RES), a phenolic compound found in grapes, has been shown to inhibit MM cell growth. We sought to identify a synergistic combination of BTZ with a RES derivative and analyze the effects on reducing viability and inducing apoptosis in human MM cells. BTZ as well as RES and its derivatives pinostilbene (PIN) and piceatannol (PIC) decreased MM cell viability in a dose- and time-dependent manner and increased expression of cleaved proapoptotic proteins poly(ADP-ribose) polymerase 1 (PARP1) and caspase-3 in a dose-dependent manner. The combination of 5 nM BTZ and 5 μM PIN was identified to have synergistic cytotoxic effects in MM RPMI 8226 cells. MM RPMI 8226 cells treated with this combination for 24 h showed increased cleaved PARP1 and caspase-3 expression and higher percentages of apoptotic cells versus cells treated with the individual compounds alone. The treatment also showed increased apoptosis induction in MM RPMI 8226 cells co-cultured with human bone marrow stromal HS-5 cells in a Transwell model used to mimic the bone marrow microenvironment. Expression of oxidative stress defense proteins (catalase, thioredoxin, and superoxide dismutase) in RPMI 8226 cells were reduced after 24 h treatment, and cytotoxic effects of the treatment were ameliorated by antioxidant N-acetylcysteine (NAC), suggesting the treatment impacts antioxidant levels in RPMI 8226 cells. Our results suggest that this combination of BTZ and PIN decreases MM cell viability synergistically by inducing apoptosis and oxidative stress in MM cells.

Protein arginine methyltransferase 1 is a therapeutic vulnerability in multiple myeloma.

Multiple myeloma (MM) is a devastating plasma cell malignancy characterized by the expansion of aberrant monoclonal plasma cells in the bone marrow, leading to severe clinical manifestations and poor prognosis, particularly in relapsed/refractory cases. Identifying novel therapeutic targets is crucial to improve treatment outcomes in these patients. In this study, we investigated the role of the protein arginine methyltransferase 1 (PRMT1) in MM pathogenesis and explored its potential as a therapeutic target. We observed that PRMT1, responsible for most asymmetric di-methylation in cells, exhibited the highest expression among PRMT family members in MM cell lines and primary MM cells. Importantly, PRMT1 expression was significantly elevated in relapsed/refractory patients compared to newly diagnosed patients. High expression of PRMT1 expression was strongly associated with poor prognosis. We found that genetic or enzymatic inhibition of PRMT1 impaired MM cell growth, induced cell cycle arrest, and triggered cell death. Treatment with MS023, a potent PRMT type I inhibitor, demonstrated a robust inhibitory effect on the viability of primary cells isolated from newly diagnosed and proteasome inhibitor-relapsed/refractory patients in a dose-dependent manner. Suppression of PRMT1 downregulated genes related to cell division and upregulated genes associated with apoptosis pathway. We also found that genes related to immune response and lymphocyte activation were significantly upregulated in PRMT1-suppressed cells. Notably, the activation status of T cells was strikingly enhanced upon co-culturing with PRMT1-KO MM cells. In vivo studies using a xenograft model revealed that targeting PRMT1 by either CRISPR/Cas9-mediated knockout or MS023 treatment significantly attenuated MM tumor growth and prolonged the survival of tumor-bearing mice. Histological analysis further confirmed increased apoptotic cell death in MS023-treated tumors. Collectively, our findings establish PRMT1 as an indispensable and novel therapeutic vulnerability in MM. The elevated expression of PRMT1 in relapsed/refractory patients underscores its potential as a target for overcoming treatment resistance. Moreover, our results highlight the efficacy of MS023 as a promising therapeutic agent against MM, offering new avenues for therapeutic approaches in relapsed/refractory MM.

Targeting ABCB6 with nitidine chloride inhibits PI3K/AKT signaling pathway to promote ferroptosis in multiple myeloma.

Since multiple myeloma (MM) remains a cureless malignancy of plasma cells to date, it becomes imperative to develop novel drugs and therapeutic targets for MM. We screened a small molecule library comprising 3633 natural product drugs, which demonstrated that Nitidine Chloride (NC), an extract from traditional Chinese medicine Zanthoxylum nitidum. We used Surface Plasmon Resonance-High Performance Liquid Chromatography-Protein Mass Spectrometry (SPR-HPLC-MS), Cellular Thermal Shift Assay (CETSA), molecular docking, and SPR assay to identify the potential targets of NC, in which ABCB6 was the unique target of NC. The effects of ABCB6 on cellular proliferation and drug resistance were determined by CCK8, western blot, flow cytometry, site-mutation cells, transmission electron microscopy, immunohistochemistry staining and xenograft model in vitro and in vivo. NC induced MM cell death by promoting ferroptosis. ABCB6 is the direct target of NC. ABCB6 expression was increased in MM samples compared to normal controls, which was significantly associated with MM relapse and poor outcomes. VGSK was the inferred binding epitope of NC on the ABCB6 protein. In the ABCB6-mutated MM cells, NC did not display cancer resistance, implying the vital role of ABCB6 in NC's bioactivity. Moreover, the silencing of ABCB6 significantly inhibited MM cell growth. Mechanistically, the direct binding of NC to ABCB6 suppressed PI3K/AKT signaling pathway to promote ferroptosis. In conclusion, ABCB6 can be a potential therapeutic target and prognostic biomarker in MM, while NC can be considered a novel drug for MM treatment.

MiR-203a-5p Inhibits Multiple Myeloma Cell Proliferation and Cell Cycle Progression via Targeting JAG1

To investigate the biological function of miR-203a-5p and the underlying mechanism in multiple myeloma (MM). Three miRNA expression profiles (GSE16558, GSE24371 and GSE17498) were downloaded from the GEO database. The three miRNA expression profiles contained 131 MM samples and 17 normal plasmacyte samples. The robust rank aggregation (RRA) method was used to identify the differentially expressed miRNAs between MM and normal plasmacytes. In order to carry out cytological experiments, MM cell line with stable over-expression of miR-203a-5p was constructed with lentivirus. Expression levels of miR-203a-5p in MM cells were quantified by qRT-PCR. The effects of miR-203a-5p on MM cells were investigated using assays of cell viability and cell cycle. Cell proliferation was measured using the Cell Counting kit (CCK)8 assay. The percentage of cells in each cell cycle was measured with a FACSCalibur system. Xenograft tumor models were established to evaluate the role of miR-203a-5p in tumorigenesis in vivo . To elucidate the underlying molecular mechanisms of miR-203a-5p in mediating cell proliferation inhibition and cell cycle arrest in MM, we used TargetScan and miRanda to predict the candidate targets of miR-203a-5p. The potential target of miR-203a-5p in MM cells was explored using the luciferase reporter assay, qRT-PCR, and Western blot. An integrated analysis of three MM miRNA expression datasets showed that the levels of miR-203a-5p in MM were notably downregulated compared with those in normal plasmacytes. Accordingly, the relative expression levels of miR-203a-5p were decreased in MM cell lines. In addition, overexpression of miR-203a-5p inhibited the proliferation and cell cycle progression of RPMI8226 and U266 cells. In vivo experiments demonstrated that upregulation of miR-203a-5p expression could significantly inhibit the tumorigenesis of subcutaneous myeloma xenografts in nude mice. Mechanistic investigation led to the identification of Jagged 1 (JAG1) as a novel and direct downstream target of miR-203a-5p. Interestingly, the reintroduction of JAG1 abrogated miR-203a-5p-induced MM cell growth inhibition and cell cycle arrest. Our data demonstrate that miR-203a-5p inhibits cell proliferation and cell cycle progression in MM cells by targeting JAG1, supporting the utility of miR-203a-5p as a novel and potential therapeutic agent for miRNA-based MM therapy.

MicroRNA-429 Regulates Invasion and Migration of Multiple Myeloma Cells via Bmi1/AKT Pathway

Background: miR-429-mediated progression of multiple myeloma (MM) was studied through mediating B cell-specific Moloney murine leukemia virus integration site 1 (Bmi1)/protein kinase B (AKT) pathway. Methods: miRNA or siRNA was delivered into MM cell lines to alter cellular proliferation, apoptosis, invasion and migration. Measurements of miR-429 and Bmi1 levels were performed. AKT and p-AKT expression change was measured after regulating miR-429. The interaction between miR-429 and Bmi1 was analyzed. Results: miR-429 elevation disrupted proliferation, anti-apoptosis, migration and invasion properties of MM cells, and inactivated AKT pathway. Bmi1 was a targeting partner of miR-429, which was highly expressed in MM. Bmi1 knockdown phenotyped the effects of overexpressed miR-429 on MM cells. AKT agonist SC70 reversed miR-429-regulated inhibition of MM cell growth. Conclusion: miR-429 suppresses the activation of Bmi1/AKT pathway to down-regulate the malignant functions of MM cells.

Targeting Iron Homeostasis As a Therapeutic Strategy in Multiple Myeloma

Multiple myeloma (MM) is a hematological cancer characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow (BM). Despite the survival improvement provided by current treatments, the majority of patients relapse and eventually become resistant to all treatments. Drug resistance and relapse are the main current challenges in the treatment of MM and development of new therapeutic interventions is a pressing need (Pinto et al., 2020 Cancers). Abnormal iron homeostasis is implicated in tumorigenesis and the progression of several cancers. Ironomycin is a small synthetic derivative of salinomycin that induces DNA damage and non-apoptotic cell death by sequestering iron in lysosomes (Mai et al., 2017 Nat Chem). Preclinical studies in Acute Myeloid Leukemia (Garciaz et al., 2022 Cancer Discovery) and Diffuse Large B-Cell Lymphoma (Devin et al., 2022 Can Res) have revealed the therapeutic interest of ironomycin in treating hematological malignancies. Here, we have evaluated the effect of ironomycin on MM cells, alone and in combination with standard anti-myeloma treatments. A list of 63 genes related to iron metabolism in cancer was defined. Using Maxstat R function, we showed that 6 genes out of the 63 investigated have a prognostic value in a cohort of newly diagnosed MM patients (n=345). Based on these prognostic genes, we created a gene expression profile (GEP)-based risk score as the sum of the beta coefficients weighted by ±1 according to the patient signal above or below the probe set Maxstat value as previously reported (Herviou et al., 2018 Clinical Epigenetics). This "Iron Score” (IS) was significantly associated with high-risk myeloma (OS and EFS) in 3 independent cohorts of newly diagnosed MM patients (TT2 cohort (N=345), HM cohort (N=206) and TT3 cohort (N=158)), as well as in a cohort of 188 patients at relapse treated with bortezomib monotherapy (Mulligan cohort N=188). GSEA analysis revealed that high-risk MM patients defined by iron score are significantly enriched in genes involved in cell cycle, TFRC targets and hypoxia (p < 0.001). We analyzed the therapeutic interest of ironomycin in 19 human myeloma cell lines (HMCLs). Ironomycin inhibits MM cell growth at nanomolar concentrations compared to other iron chelators. Ironomycin caused a significant cell growth inhibition mediated by cell cycle arrest and triggering of apoptosis, without significant ferroptosis induction. Moreover, cell proliferation arrest correlated with increased DNA damage and caspases activation, and a metabolic swift towards a glycolytic or quiescent metabolism. Iron could also play a role in transcriptional regulation of cancer cells mediated by epigenetic reprogramming. Iron dependent modifications of histone or DNA methylation have been shown to play a significant role in solid cancers. Ironomycin also induced a downregulation of major oncogenes of MM cells including MMSET, c-MYC and CCND1. Furthermore, ironomycin treatment resulted in an epigenetic dysregulation with H3K27ac, H3K4me3 and H3K9me3 downregulation. With major importance, we validated the therapeutic interest of ironomycin in primary MM cells from patients (n=5) without significant toxicity for non-tumor cells from the microenvironment. Furthermore, ironomycin presented a low toxicity on hematopoietic progenitors (CFU assays, n=5) compared to conventional treatment. We tested the therapeutic interest to combine ironomycin with conventional drugs used in MM treatment. The combination of ironomycin with IMiDs (lenalidomide and pomalidomide) had a synergistic effect on cell growth arrest and the activation of apoptotic mechanisms, associated with a marked reduction of several targets of IMiDs including Cereblon (CRBN), MYC, IRF4, IKZF1 and IKZF3. Our data demonstrate that targeting iron homeostasis using ironomycin could be of therapeutic interest in high-risk MM defined by the IS, especially in combination with IMiDs.

Abstract ND13: The discovery and characterization of CFT7455: A potent and selective degrader of IKZF1/3 for the treatment of relapsed/refractory multiple myeloma

Abstract Introduction: Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 inhibits the growth of multiple myeloma (MM) cells, confirming their dependency on IKZF1/3. IMiDs (lenalidomide, pomalidomide) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. However, most patients treated with lenalidomide or pomalidomide eventually develop progressive disease due to acquired resistance, underscoring the unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high binding affinity to cereblon (CRBN), rapid and deep IKZF1/3 degradation, and potent dose-dependent efficacy in vivo. Results: A series of novel benzoimidazolone-based CRBN ligands with potent binding affinity were discovered and their binding modes were informed by CRBN co-crystal structures. Although the benzoimidazolone-based CRBN binders did not exhibit IKZF1/3 degradation activity, structural insights into their unique binding modes and knowledge of the IKZF1/3 degradation pharmacophore were combined to enable identification of a novel benzoisoindolone-based ligand that exhibited a 10-fold potency increase in biochemical CRBN binding and a 30-fold potency increase in H929 MM cell growth inhibition when compared to lenalidomide. Additional rounds of structure-based drug design, degradation and phenotypic profiling led to the discovery of CFT7455, a highly potent, selective and orally bioavailable degrader of IKZF1/3. CFT7455 demonstrated an 800 and 1600-fold improvement in CRBN binding compared to pomalidomide in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced &amp;gt;75% degradation of IKZF1 within 1.5 hrs. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines, as well as H929 cells made resistant to IMiDs. In vivo, CFT7455 catalyzed deep and durable degradation of IKZF3, translating into potent antitumor activity in multiple myeloma xenograft models. CFT7455 also retained its activity in models resistant or insensitive to clinically approved IMiDs as single agent or in combination with standard of care agent dexamethasone. Conclusion: Overall, CFT7455 is a next generation IKZF1/3 degrader, with improved potency and anticancer efficacy in preclinical models compared to existing IMiDs. These features make CFT7455 an exciting drug candidate, as a single agent or for use in combination. CFT7455 is currently being studied in a Ph1 clinical trial. Citation Format: James A. Henderson, Scott J. Eron, Andrew Good, R Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Ashley A. Hart, Christina S. Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Adam S. Crystal, Roy M. Pollock, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. The discovery and characterization of CFT7455: A potent and selective degrader of IKZF1/3 for the treatment of relapsed/refractory 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 ND13.

Synergistic Effects of Resveratrol Derivatives with Bortezomib on Reducing Viability and Inducing Apoptosis in Multiple Myeloma Cells

Multiple myeloma (MM) is a cancer of plasma cells in the bone marrow characterized by bone lesions, hypercalcemia, anemia, and renal failure. Bortezomib (BTZ), a common treatment for MM, is a proteasome inhibitor that induces apoptosis in MM cells. However, high doses of BTZ can be toxic, signifying a need for a synergistic drug combination. Resveratrol (RES) is a phenolic compound found in grapes shown to inhibit MM cell growth and induce apoptosis in these cells. We hypothesize that combining BTZ with a RES derivative at certain concentrations is synergistic in reducing viability and inducing apoptosis in MM cells. To address this hypothesis, we performed viability assays to assess the effects of increasing concentrations of RES derivatives and BTZ on human MM cell viability over time (24, 48, 72 hours) and western blots to assess the compounds’ effects on inducing apoptosis in MM cells following 24 hour treatment. We found that BTZ as well as RES and its derivatives pinostilbene (PIN) and piceatannol (PIC) decreased MM cell viability in a dose‐ and time‐dependent manner and increased expression of cleaved proapoptotic proteins PARP1 and caspase‐3 in a dose‐dependent manner. We screened different combinations of RES derivatives with BTZ for synergistic effects on MM viability using CompuSyn and selected the combination of 5 nM BTZ and 5 μM PIN for further assessment as a synergistic treatment. In conclusion, a combination of low doses of BTZ and PIN synergistically decreases MM cell viability, possibly through inducing apoptosis.

Dysfunctional HDAC8 Impacts Genomic Integrity and Is a Novel Therapeutic Target in Multiple Myeloma

The histone modifications and associated changes in chromatin structure and function have emerged as important epigenetic mechanisms impacting gene expression and have significant translational relevance in cancers, including multiple myeloma (MM). Epigenetic intervention with histone deacetylases (HDACs) inhibitors is emerging as a promising therapeutic strategy in combination with current anti-myeloma agents. Although pan-HDAC inhibitors have been shown to be effective both in preclinical and clinical setting, they seem to be associated with toxicity. It is, therefore, extremely important to understand the biological and molecular roles of individual HDACs to then selectively target them to limit toxicities observed with pan-HDAC inhibitors. Based on our observation that elevated HDAC8 expression correlates with poor overall survival in MM patients in three different datasets including one publicly available dataset (GSE39754), we evaluated its functional role in MM.HDAC8, a member of class I HDAC isoenzymes, is responsible for the deacetylation of lysine residues on the N-terminal part of the core histones as well as non-histone proteins. We performed genetic modulation of HDAC8 by loss-of-function studies, using shRNA as well as siRNAs targeting HDAC8. Downregulation of HDAC8 in 3 different MM cell lines caused MM cell growth inhibition in a time-dependent manner which was associated with induction of cell apoptosis. Consistently, treatment with a selective and potent HDAC8 inhibitor (OJI-1) caused a significant inhibition of MM cell growth in a panel of 20 MM cell lines (IC50 = 80 nM) in a time- and dose-dependent manner, while having a minimal impact on six PBMC samples from healthy donors both in resting and activated state (IC50 = 150 nM).The mechanism of cell death was apoptosis as demonstrated by annexin-labeling. Importantly, both the HDAC8 knockdown and OJI-1 treatment inhibited DNA breaks as evidenced from γH2AX expression or a single cell gel electrophoresis method to visualize and quantitate DNA breaks. HDAC8 inhibition also caused inhibition of RAD51 foci and HR activity, as measured by strand-exchange assay. Interestingly, non-homologous end joining in MM cells was not impacted by these treatments. Consistent with these data, the overexpression of HDAC8 in MM as well as in normal cells increased DNA breaks and HR activity. Furthermore, the inhibition of HDAC8 (by knockdown and OJI-1) inhibited, whereas its overexpression increased genomic instability, as assessed by micronucleus assay, in surviving MM cells. We also demonstrate that HDAC8 interacts with RAD51 and impacts its acetylation. The treatment of MM cells with HDAC8 inhibitor (OJI-1) increased RAD51 acetylation. Next, we examined the in vivo efficacy of the HDAC8 conditional knockdown in a human xenograft mouse model, using H929 cells injected subcutaneously in SCID mice. HDAC8 knockdown not only caused a significant reduction in tumor growth but also increased survival (p=0.0016) compared to mice injected with control cells. Evaluation of tumors from these mice confirmed in vivo inhibition of DNA breaks and HR activity, and induction of apoptosis following HDAC8-knockdown. HDAC8 inhibitor OJI-1 also synergistically increased the cytotoxicity of existing MM drugs including dexamethasone, bortezomib and lenalidomide.In conclusion, our results demonstrate that elevated HDAC8 in MM cells is involved in inhibition of apoptosis but also contributes to increased DNA breaks and dysregulation of homologous recombination and genome stability. Therefore, HDAC8 is a novel target for therapeutic application in MM. Selective and potent HDAC8 inhibitor OJI-1 has shown a favorable therapeutic index with synergistic effect in combination with existing MM drugs. DisclosuresHajek: Pharma MAR: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Honoraria, Research Funding. Munshi: Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Karyopharm: Consultancy; Abbvie: Consultancy; Adaptive Biotechnology: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy; Legend: Consultancy.

OAB-032: Targeting GCK in RAS-mutant multiple myeloma offer a promising therapeutic approach

<h3>Background</h3> Next generation sequencing revealed frequent mutations of NRAS, KRAS or BRAF in up to 50% of newly diagnosed multiple myeloma (MM) patients. <h3>Methods</h3> Specific K- or N-RAS knockdowns led to strong decrease in MM cell viability if harbored the respective oncogenic isoform, but minimal effects were observed if only the wild-type isoform was present, presenting RAS signaling as the key driving oncogenes in the mutation bearing patients. Unfortunately, no inhibitor for RAS mutation is available for clinical use in MM. Therefore, key component in the RAS/MAPK pathway may represent an alternative therapeutic target for MM. Germinal center kinase (GCK) is an upstream activator in the MAPK pathway. <h3>Results</h3> Our data showed that GCK (MAP4K2) knockdown in MM cells induced MM cell growth inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins. Moreover, GCK silencing only led to significantly decreased GCK mRNA, however, did not affect IKZF1 expressions at mRNA level. We next tested the effects of GCK inhibitor TL4-12 on MM. Given the fact that RASMut MM cells have higher GCK expression level and are more sensitive to GCK knockdown, we compared mutated RAS MM cells with wild-type RAS MM cells. TL4-12 significantly inhibited the growth of mutated RAS MM cells, with IC50 5-10 fold lower compared to wild-type RAS MM cell lines. IKZF1/3 are the key targets of the immunomodulatory drugs (IMiDs), which are the backbone of MM therapy. IMiDs bind to cereblon (CRBN) and induce IKZF1/3 protein degradation, which subsequently lead to MM cell growth inhibition. Our data showed that GCK knockdown also downregulates IKZF1 protein level, indicating that IKZF1 is under the regulation of GCK. Concomitant treatment with proteasome inhibitor PS-341 blocked TL4-12 induced IKZF1 downregulation, suggesting that inhibition of GCK induces IKZF1 protein degradation. However, since IMiDs-resistant RPMI-8226 and JJN3 MM cells are sensitive to GCK inhibition, we hypothesized that the IKZF1 degradation mechanism induced by GCK inhibition is different from IMiDs and independent of CRBN. To confirm this hypothesis, we silenced CRBN in N-Rasmut H929 MM cells and examined the response to IMiDs and GCK inhibitor. CRBN silencing in H929 cells resulted in lenalidomide resistance. In contrast, CRBN silencing failed to rescue H929 MM from TL4-12 induced proliferation inhibition and IKZF1 downregulation, confirming that GCK regulated IKZF1 and cell growth is independent of CRBN. <h3>Conclusions</h3> Taken together, our data demonstrated that GCK inhibition induces cell growth inhibition and triggers apoptosis especially in RASmut MM cells. Importantly, GCK inhibition downregulates IKZF1 via a CRBN-independent mechanism. Our findings thus provide a rationale for the clinical evaluation of targeting GCK in RASmut MM patients and further mechanistic insight into the role of GCK in MM tumorigenesis as well as drug resistance.

P-046: B cell transcriptional coactivator POU2AF1 (BOB-1) modulates the protein synthesis and offers a potential vulnerability in multiple myeloma.

<h3>Background</h3> Multiple Myeloma (MM) is a disease driven by numerous genetic and epigenetic alterations, however true drivers of the disease have yet to be identified. <h3>Methods</h3> To identify new dependencies and actionable therapeutic targets in MM we integrated gene expression and genetic dependency (CRISPR KO). <h3>Results</h3> We found that many of the specific and potent dependencies in MM are transcription factors, especially those establishing plasma cell identity. Among others, the POU2AF1 gene, which encodes the OCA-B/BOB-1, a B cell transcriptional coactivator protein, represented the most striking dependency in MM. Although BOB-1 is expressed throughout B-cell development, we found it to be highly expressed in CD138+ plasma cells from patients with precursor conditions (MGUS and SMM) as well and established MM compared to normal plasma cells (NPC). Loss-of-function studies using shRNA, siRNAs as well as antisense GapMers specific for BOB-1 confirmed significant impact on MM cell viability. Transcriptomic analysis by RNA-sequencing revealed a small set of genes commonly modulated in MM cell lines upon BOB-1 depletion, including the XBP1- BHLHA15 axis involved in lipid synthesis and unfolded protein response (UPR). Interestingly, among the genes most significantly upregulated by BOB-1 depletion was heme oxygenase 1 (HMOX1), that was affected via the NRF2/Keap1 pathway. We observed that HMOX1 expression is significantly lower in MM cells from patients compared to normal plasma cells and correlates with poor clinical outcome, suggesting important role in MM. Moreover, we found that siRNA depletion of HMOX1 reverted the inhibition of MM cell growth caused by BOB-1 KD, confirming significant role for HMOX1 in the BOB-1 addiction observed in MM cells. Next, we performed gene set enrichment analysis (GSEA) and observed ribosome biogenesis pathways and mRNA translation and elongation processes, along with WNT and senescence pathways, to be significantly enriched among genes modulated by BOB-1 depletion in MM cells. Since high protein load is a feature of MM, we evaluated the role of BOB-1 in the translational efficiency of MM cells. In MM cell lines, BOB-1 knockdown decreased de novo protein synthesis, while its overexpression significantly enhances protein synthesis compared to control cells. As MM is characterized by excess production of monoclonal immunoglobulins, we evaluated impact of BOB-1 perturbation on intracellular kappa and lambda light chains production. We observed changes in the intracellular abundance of the light chains with BOB-1 modulation in all MM cell lines tested. As a result, BOB-1 depletion was associated with induction of resistance to proteasome inhibition. <h3>Conclusion</h3> In conclusion, we report BOB1 as a specific dependency in MM cells with potential role on modulating the protein load/capacity balance in MM cells and therefore the sensitivity to proteasome inhibition.

Apoptotic Extracellular Vesicles Ameliorate Multiple Myeloma by Restoring Fas-Mediated Apoptosis.

Apoptosis is critical for maintaining bodily homeostasis and produces a large number of apoptotic extracellular vesicles (apoEVs). Several types of cancer cells display reduced expression of Fas on the cell surface and are thus capable of escaping Fas ligand-induced apoptosis. However, it is unknown whether normal cell-derived apoEVs can regulate tumor growth. In this study, we show that apoEVs can induce multiple myeloma (MM) cell apoptosis and inhibit MM cell growth. Systemic infusion of mesenchymal stem cell (MSC)-derived apoEVs significantly prolongs the lifespan of MM mice. Mechanistically, apoEVs directly contact MM cells to facilitate Fas trafficking from the cytoplasm to the cell membrane by evoking Ca2+ influx and elevation of cytosolic Ca2+. Subsequently, apoEVs use their Fas ligand to activate the Fas pathway in MM cells, leading to the initiation of apoptosis. This study identifies the role of apoEVs in inducing MM apoptosis and suggests a potential for apoEVs to treat MM.

A First in Class APRIL Fully Blocking Antibody Targets Novel Immune Regulation of APRIL in Multiple Myeloma: Further Therapeutic Implication

A proliferation inducing ligand (APRIL), a critical paracine factor for human multiple myeloma (MM), is mainly secreted by myeloid cells, including macrophages and osteoclasts in the bone marrow (BM) microenvironment. Recently, an antagonistic APRIL monoclonal antibody (mAb) 01A significantly reduced myeloma burden in SCID-hu mice (Blood. 2016;127:3225-3236). This data suggests that the humanized 01A named BION-1301, which also potently (IC50 <1 nM) blocks APRIL binding to its receptors B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator (TACI) is likely to exert anti-MM activity in vivo. Here, we further characterize the novel inhibition by 01A and a hIgG4 chimeric version C4 on multiple immune cells affected by APRIL in the immunosuppressive MM BM milieu. First, using gene-modified RPMI8226 MM cells, 01A potently and selectively blocks growth and viability of APRIL-overexpressing but not BCMA-overexpressing RPMI8226 transfectants. Significantly, 01A inhibits MM cell growth and survival induced by osteoclasts, as a physiologically relevant endogenous source of APRIL. Next, we demonstrate that pretreatment of APRIL decreases MM cell lysis induced by Daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC). In addition, APRIL prevents anti-BCMA (J6M0)-induced ADCC against BCMA-expressing MM cell lines in a dose-dependent manner. Conversely, 01A or C4 (a surrogate of BION-1301) reverts APRIL-reduced MM cell lysis induced by J6M0 in the presence of PBMC or NK effector cells. These data indicate that targeting CD38 and BCMA may not be sufficient in MM. To further define the role of APRIL mediating anti-MM immune responses, we show that T cells do not express BCMA while natural T regulatory (Treg) cells significantly express higher TACI when compared with corresponding conventional T cells (Tcon) from the same individual (Abstract #102467). In parallel, detailed gene expression profiling in multiple solid tumors show that the tumor-resident Treg markers are significantly correlated with TACI, along with a strong association of APRIL expression levels with T cell inflamed signature. In addition to natural Treg, MM cell-induced Tregs (iTregs) also showed significantly increased TACI levels when compared with paired conventional T cells (Tcon) from the same individual in ex vivo cocultures. Importantly, APRIL further enhances MM cell-induced iTregs with IL-10-producing and CD15s+[JD2] FOXP3high fractions; conversely, 01A blocks APRIL-induced upregulation of iTreg. Osteoclasts also upregulated iTreg generation induced by MM cells. On the contrary, 01A or C4 partially blocks such induction. Importantly, APRIL directly promotes proliferation and viability of Treg via the induction of CCND1/2, BCL2, and BCL2L1/BCL-xL, which is blocked by 01A or C4. Finally, APRIL enhances the suppressive function of Treg on the proliferation of autologous conventional T cells, which could also be overcome by 01A or C4. Collectively, our data therefore demonstrate that APRIL, via TACI but not BCMA, critically modulates regulatory T cell function; and, importantly, 01A or C4 targets these immune regulatory subsets that have been linked to MM progression via APRIL-dependent manner. Moreover, since patients with active diseases have severe bone lesions induced by hyperactive osteoclasts, inhibiting the APRIL-TACI interaction may also overcome osteoclast-inhibited T cell killing on MM cells. Anti-APRIL Ab (BION-1301) will be entering clinical trial in late 2017 to target the immunosuppressive BM microenvironment and improve patient outcome in MM. DisclosuresDulos:Aduro Biotech Europe: Employment, Equity Ownership. Van Elsas:Aduro Biotech Europe: Employment, Equity Ownership. Richardson:Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees. Anderson:Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Multiple Myeloma Tumor Cells Are Selectively Killed By Pharmacologically-Dosed Ascorbic Acid

Background: High-dose chemotherapy to treat multiple myeloma (MM) can be life-threatening due to toxicities to normal cells and there is a need to target tumor cells more selectively and/or lower standard drug dosage without losing efficacy. We show that pharmacologically-dosed ascorbic acid (PAA) only in the presence of high intracellular iron leads to the formation of reactive oxygen species (ROS) resulting in cell death.Materials and Methods: CD138+ primary MM cells and CD138− non-MM cells were isolated from 9 MM, 2 smoldering MM, and 2 monoclonal gammopathy of undetermined significance (MGUS) patients. High doses of ascorbic acid alone and combination with standard therapeutic drugs were used to treat MM cells in vitro and in vivo . Electron microscope was used to detect cell necrosis and apoptosis as well as apoptosis-inducing factor 1 (AIF1) nuclear translocation. Overexpression (OE), empty vector (EV) and knockdown (KD) of ferroportin 1 (Fpn1) and AIF1 in MM cells were achieved by lentivirus infection. An In Vivo Imaging System (IVIS) was used to monitor tumor growth in vivo .Results: We show that PAA selectively kills CD138+ primary MM cells derived from MM and SMM, but not from MGUS patients. To confirm the capacity of PAA to induce MM cell death in vivo, xenografted MM mice were treated with PAA alone or combination with carfizomib or melphalan. We observed that PAA inhibited MM cell growth and extended MM mouse survival significantly. Importantly, PAA allowed a dramatic decrease in DNA-alkylating agent melphalan dosage (75%) without loss of efficacy. DFO, an iron chelator, abolished the ability of PAA to reduce cells viability in both EV and OE-Fpn1 OCI-MY5 cells pre-treated with iron. These results suggest that the mechanism of PAA killing of MM cells is indeed iron-dependent. Our data also show that PAA increases AIF1 cleavage and translocation from mitochondria to cytoplasm and nucleus. Overexpression of AIF1 in MM cells increases PAA sensitivity while KD of AIF1 prevents PAA induced MM cell death, indicating AIF1 plays an essential role in mediating PAA-induced MM cell death.Conclusion: Our findings show that PAA only kills tumor cells with high iron content, suggesting that iron is the initiator of PAA cytotoxicity. In addition, combination of PAA with standard therapeutic drugs, such as melphalan, might significantly reduce the dose of melphalan required. Because of the elevated intracellular iron in MM cells, PAA produces highly ROS leading to release of AIF1 from mitochondria of tumor cells with no toxicity to non-tumor cells with low iron content. These results provide a mechanistic rationale to pursue the therapeutic use of PAA in MM and other cancers with high iron content. This is first study testing directly PAA efficacy on primary cancer cells in vitro . DisclosuresNo relevant conflicts of interest to declare.

Preparation of caffeic acid phenethyl ester‐incorporated nanoparticles and their inhibitory effects on multiple myeloma cell growth

Multiple myeloma (MM) is a plasma cell cancer characterized by accumulation of malignant cells, preferentially in the bone marrow, that leads to severe bone disease in humans. This disease resulted in approximately 13,000 deaths last year in the United States. Caffeic acid phenethyl ester (CAPE), a phenolic compound found in bee glue, is reported to have an array of bioactive properties including anticancer effect. We previously reported that CAPE inhibits MM cell growth through induction of apoptosis and oxidative stress. It is established that iron oxide nanoparticles (IONPs) enhance drug delivery to tissues/cells and conjugation of a small peptide RGD with drug loaded IONPs improves cancer cell targeting. The purpose of this study is to improve the biocompatibility of CAPE through encapsulation onto IONPs (IONP/CAPE) and evaluate the cytotoxic and apoptogenic effects of IONP/CAPE with RGD conjugated as a targeting ligand for human MM cells. We hypothesize that RGD-IONP/CAPE may enhance the intracellular delivery of CAPE to MM cells thus demonstrating more potent inhibition on the growth of MM cells than CAPE alone. We prepared RGD-IONP/CAPE and characterized these NPs through determination of the hydrodynamic sizes and zeta potentials. We optimized the stability of these nanomaterial complexes with various stabilizers at different pH and evaluated the inhibitory effect of the most stable formulation on MM cell growth. Our results show that the developed RGD-IONP/CAPE were stable at neutral and basic conditions, and subject to release of CAPE at acidic pH (5.5) in vitro. The RGD-IONP/CAPE caused a decreased MM cell viability in a time (24, 48, 72 hours) and dose (1, 5, 10, 20 and 30 μM) -dependent manner. Interestingly, the growth-inhibitory effects of RGD-IONP/CAPE at 10 μM was equivalent to the parent drug at 30 μM, indicating a 3-fold enhancement in CAPE delivery efficiency under the experimental condition. Apoptogenic effect of RGD-IONP/CAPE was assessed using western blotting. Protein expression of apoptosis-executioner caspase 3 was examined, and the results indicated that caspase 3 was activated. In conclusion, we constructed a novel CAPE-loaded IONPs with RGD conjugation which showed more potent antimyeloma effect than parental CAPE.

Targeting the GCK pathway: a novel and selective therapeutic strategy against RAS-mutated multiple myeloma

In multiple myeloma (MM), frequent mutations of NRAS, KRAS, or BRAF are found in up to 50% of newly diagnosed patients. The majority of the NRAS, KRAS, and BRAF mutations occur in hotspots causing constitutive activation of the corresponding proteins. Thus, targeting RAS mutation in MM will increase therapeutic efficiency and potentially overcome drug resistance. We identified germinal center kinase (GCK) as a novel therapeutic target in MM with RAS mutation. GCK knockdown (KD) in MM cells demonstrated in vitro and in vivo that silencing of GCK induces MM cell growth inhibition, associated with blocked MKK4/7-JNK phosphorylation and impaired degradation of IKZF1/3, BCL-6, and c-MYC. These effects were rescued by overexpression of a short hairpin RNA (shRNA)-resistant GCK, thereby excluding the potential off-target effects of GCK KD. In contrast, overexpression of shRNA-resistant GCK kinase-dead mutant (K45A) inhibited MM cell proliferation and failed to rescue the effects of GCK KD on MM growth inhibition, indicating that GCK kinase activity is critical for regulating MM cell proliferation and survival. Importantly, the higher sensitivity to GCK KD in RASMut cells suggests that targeting GCK is effective in MM, which harbors RAS mutations. In accordance with the effects of GCK KD, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 and led to MM cell proliferation inhibition accompanied by induction of apoptosis. Here, our data identify GCK as a novel target in RASMut MM cells, providing a rationale to treat RAS mutations in MM. Furthermore, GCK inhibitors might represent an alternative therapy to overcome immunomodulatory drug resistance in MM.

MiR-17-3p promotes the proliferation of multiple myeloma cells by downregulating P21 expression through LMLN inhibition.

Multiple myeloma (MM), a hematological malignancy, has a poor prognosis and requires an invasive procedure. Reports have implicated miRNAs in the diagnosis, treatment and prognosis of hematological malignancies. In our study, we evaluated the expression profiles of miR‐17‐3p in plasma and bone marrow mononuclear cells of monoclonal gammopathy of undetermined significance (MGUS) and MM patients and healthy subjects. The results showed that the plasma and mononuclear cell expression levels of miR‐17‐3p in MM patients were higher than those in MGUS patients and normal controls. In addition, the expression of miR‐17‐3p was positively correlated with diagnostic indexes, such as marrow plasma cell abundance and serum M protein level, and positively correlated with the International Staging System stage of the disease. Receiver operating characteristic curve analysis suggested that miR‐17‐3p might be a diagnostic index of MM. Moreover, miR‐17‐3p regulated cell proliferation, apoptosis and the cell cycle through P21 in MM cell lines and promoted MM tumor growth in vivo. Furthermore, we predicted and verified LMLN as a functional downstream target gene of miR‐17‐3p. Negatively regulated by miR‐17‐3p, LMLN inhibits MM cell growth, exerting a tumor suppressive function through P21. Taken together, our data identify miR‐17‐3p as a promising diagnostic biomarker for MM in the clinic and unveil a new miR‐17‐3p‐LMLN‐P21 axis in MM progression.