JJN3 Cells Research Articles

HSV1716 Prevents Myeloma Cell Regrowth When Combined with Bortezomib In Vitro and Significantly Reduces Systemic Tumor Growth in Mouse Models

Multiple myeloma remains largely incurable due to refractory disease; therefore, novel treatment strategies that are safe and well-tolerated are required. Here, we studied the modified herpes simplex virus HSV1716 (SEPREHVIR®), which only replicates in transformed cells. Myeloma cell lines and primary patient cells were infected with HSV1716 and assessed for cell death using propidium iodide (PI) and Annexin-V staining and markers of apoptosis and autophagy by qPCR. Myeloma cell death was associated with dual PI and Annexin-V positivity and increased expression of apoptotic genes, including CASP1, CASP8, CASP9, BAX, BID, and FASL. The combination of HSV1716 and bortezomib treatments prevented myeloma cell regrowth for up to 25 days compared to only transient cell growth suppression with bortezomib treatment. The viral efficacy was tested in a xenograft (JJN-3 cells in NSG mice) and syngeneic (murine 5TGM1 cells in C57BL/KaLwRijHsd mice) systemic models of myeloma. After 6 or 7 days, the post-tumor implantation mice were treated intravenously with the vehicle or HSV1716 (1 × 107 plaque forming units/1 or 2 times per week). Both murine models treated with HSV1716 had significantly lower tumor burden rates compared to the controls. In conclusion, HSV1716 has potent anti-myeloma effects and may represent a novel therapy for multiple myeloma.

Inhibition of the Protein Arginine Methyltransferase PRMT5 in High-Risk Multiple Myeloma as a Novel Treatment Approach.

Multiple myeloma (MM) is an incurable clonal plasma cell malignancy. Subsets of patients have high-risk features linked with dismal outcome. Therefore, the need for effective therapeutic options remains high. Here, we used bio-informatic tools to identify novel targets involved in DNA repair and epigenetics and which are associated with high-risk myeloma. The prognostic significance of the target genes was analyzed using publicly available gene expression data of MM patients (TT2/3 and HM cohorts). Hence, protein arginine methyltransferase 5 (PRMT5) was identified as a promising target. Druggability was assessed in OPM2, JJN3, AMO1 and XG7 human myeloma cell lines using the PRMT5-inhibitor EPZ015938. EPZ015938 strongly reduced the total symmetric-dimethyl arginine levels in all cell lines and lead to decreased cellular growth, supported by cell line dependent changes in cell cycle distribution. At later time points, apoptosis occurred, as evidenced by increased AnnexinV-positivity and cleavage of PARP and caspases. Transcriptome analysis revealed a role for PRMT5 in regulating alternative splicing, nonsense-mediated decay, DNA repair and PI3K/mTOR-signaling, irrespective of the cell line type. PRMT5 inhibition reduced the expression of upstream DNA repair kinases ATM and ATR, which may in part explain our observation that EPZ015938 and the DNA-alkylating agent, melphalan, have combinatory effects. Of interest, using a low-dose of mTOR-inhibitor, we observed that cell viability was partially rescued from the effects of EPZ015938, indicating a role for mTOR-related pathways in the anti-myeloma activity of EPZ015938. Moreover, PRMT5 was shown to be involved in splicing regulation of MMSET and SLAMF7, known genes of importance in MM disease. As such, we broaden the understanding of the exact role of PRMT5 in MM disease and further underline its use as a possible therapeutic target.

Targeting DNA2 Overcomes Myeloma Cells' Metabolic Reprogramming in Response to DNA Damage

DNA damage resistance is a major barrier to effective DNA-damaging anticancer therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense therapy targeting ILF2, an important DNA damage regulator in MM (Marchesini et. al., Cancer Cell 2017). We continuously treated JJN3 and KMS11 cells with an ILF2-targeting antisense oligonucleotide (ILF2 ASOs) or control non-targeting antisense oligonucleotide (NT ASOs). Whereas KMS11 cells maintained a high level of DNA damage activation and a significantly increased rate of apoptosis after 3 weeks of ILF2 ASOs treatment, JJN3 cells overcame ILF2 ASO-induced DNA damage activation and became resistant to ILF2 ASOs treatment.To evaluate whether continuous ILF2 ASOs exposure could lead to the selection of MM clones intrinsically resistant to ILF2 ASO-induced DNA damage, we performed single-cell RNA seq (scRNA-seq) analysis of JJN3 cells treated with NT or ILF2 ASOs for 3 weeks. Our analysis divided JJN3 cells into 2 main clusters that were independent of treatment (Fig. 1A), suggesting that persistent exposure to ILF2 ASOs did not induce clonal selection. Differential gene expression analysis of NT ASO- and ILF2 ASO-treated cells in each of these clusters revealed that DNA damage resistant ILF2 ASO-treated cells had significantly upregulated oxidative phosphorylation (OXPHOS), DNA repair signaling, and reactive oxidative species (ROS). Consistent with these results, metabolomic analysis of JJN3 cells after long-term exposure to ILF2-ASOs showed a significant enrichment of tricarboxylic acid cycle (TCA) intermediates (Fig. 1B). ILF2-ASO-resistant MM cells were significantly more sensitive to the OXPHOS inhibitor IACS-010759 than ILF2-ASO-sensitive cells were. These data suggest that MM cells can undergo an adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage.We then hypothesized that ILF2-ASO-resistant cells' metabolic reprogramming relies on the repair of DNA damage induced by ILF2 depletion or by the generation of ROS from activated mitochondrial metabolism and that targeting DNA repair proteins involved in these processes overcomes DNA damage resistance. We used a CRISPR/Cas9 library screening strategy to identify DNA repair genes whose loss of function suppresses MM cells' ability to overcome ILF2-ASO-induced DNA damage. Compared with those in NT-ASO-treated cells, DNA2-targeting sgRNAs were significantly depleted after 3 weeks of treatment in ILF2-ASO-treated JJN3 cells but not in ILF2-ASO-treated KMS11 cells. These data suggest that DNA2 is needed to promote resistance to ILF2 depletion. Accordingly, the DNA2 inhibitor NSC105808 (NSC) significantly enhanced ILF2-ASO-induced apoptosis in JJN3 cells. These data gain added significance in light of previous findings that DNA2 is a nuclear and mitochondrial DNA nuclease/helicase that enables cancer cells to counteract the DNA replication stress and mitochondrial oxidative DNA damage induced by DNA-damaging agents. Accordingly, we observed that DNA2 was mainly localized into the mitochondria of MM cells.To dissect the mechanisms of DNA2 inhibition-induced synthetic lethality, we evaluated whether DNA2 activity is essential to maintain activated OXPHOS, which ILF2-ASO-resistant cells require to survive. The quantification of mitochondrial respiratory activity in NT-ASO-and ILF2-ASO-treated MM cells exposed to NSC for 72 hours showed that DNA2 activity inhibition significantly decreased the oxygen consumption rate while increasing ROS production in only ILF2-depleted cells. Transmission electron microscopy analysis showed that NSC-treated ILF2-depleted cells had fragmented mitochondrial cristae structures, whose perturbations affect the OXPHOS system structure and impair cell metabolism. These data suggest that DNA2 is essential to counteract oxidative DNA damage and maintain mitochondrial respiration after MM cells' metabolic reprogramming.In conclusion, our study has revealed a novel mechanism through which MM cells can overcome DNA damage activation. Further studies will clarify whether targeting DNA2 is synthetically lethal in tumors with increased demand of mitochondrial metabolism. [Display omitted] DisclosuresKonopleva: Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Sanofi: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Agios: Other: grant support, Research Funding; Cellectis: Other: grant support; Rafael Pharmaceuticals: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Stemline Therapeutics: Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; KisoJi: Research Funding.

TRIM33 Loss in Multiple Myeloma Impairs the DNA Damage Response Resulting in Sensitivity to PARP and ATR Inhibitors

IntroductionChromosomal instability is a hallmark of Multiple Myeloma (MM), with most patients displaying cytogenetic abnormalities which can arise due to DNA damage response (DDR) defects. TRIM33 is an E3 ligase and transcriptional co-repressor located on chromosome 1p13.2, a region frequently deleted in MM. Previous studies have shown that TRIM33 plays a role in the DDR and can regulate chromosomal stability, but its precise function remains unknown. In this study we investigated the impact of TRIM33 loss in MM on genomic stability and DDR pathways and whether this could be exploited therapeutically.MethodsThe CoMMpass dataset (IA15 release) was screened to identify patients with copy number (CN) loss of TRIM33 and this was correlated with overall survival (OS) and structural variants. TRIM33 shRNA knockdown models were established in JJN3 and U266 cells. The effect on DDR signalling was determined by western blotting and immunofluorescence. The Selleckchem DNA Damage/Repair Compound Library was screened on the JJN3 model in a high-throughput manner using the CellTox™ Green cytotoxicity assay. Validation of selected compounds was performed using CellTiter® Glo viability assay or clonogenic assays. Combination indices (CI) were calculated using CompuSyn software.ResultsData on CN, OS and structural variants were available for 730 newly diagnosed MM patients and of these, 69 (9.5%) were identified to have a CN loss of TRIM33. These patients have poorer OS compared to those without TRIM33 loss (52.3 months vs 72.6 months; p<0.0001). Moreover, they exhibit a significantly higher median number of structural variants (deletions, duplications, inversions, and translocations; 38 vs 26; p<0.0001), indicative of increased chromosomal instability.Our data in MM cell lines has shown that TRIM33 is rapidly recruited to chromatin within 5 minutes of induced DNA damage. TRIM33 knockdown led to an increase in 53BP1 foci formation and endogenous γH2AX (P<0.001) indicating unrepaired DNA double-strand breaks (DSBs) typical of a DDR defect. In response to these DSBs both ATM and ATR kinases were activated as demonstrated by increased pKAP1 Ser824 and pCHK1 Ser345 respectively (p<0.001). Additionally, we observed a reduction in RAD51 (p<0.05) indicative of a potential defect in the DSB repair pathway homologous recombination (HR).To identify therapeutic vulnerabilities relating to TRIM33 loss, we performed a high-throughput screen to assess sensitivity to 160 unique DNA damaging compounds. TRIM33 knockdown cells exhibited increased sensitivity to 27 compounds across a range of drug classes. Additional studies confirmed that compared to control cells, TRIM33 knockdown sensitized cells to the PARP inhibitor Olaparib and ATR inhibitors BAY-1895344 and VE-821. Further investigation with VE-821 demonstrated that whilst treatment induced PARP cleavage and DSBs in both control and knockdown cells within 48 hours, knockdown cells exhibited significantly more pCHK1 Ser345 inhibition (p<0.01). Furthermore, combining VE-821 with bortezomib yielded synergistic effects in TRIM33 knockdown cells across a range of doses (CI range 0.57-0.9) while no synergy was observed in control cells (CI>1 for all combinations).ConclusionWe have identified a subset of MM patients with TRIM33 loss who display high-risk disease characterized by chromosomal abnormalities and defective DDR. Alongside this we have identified PARP and ATR inhibitors as therapeutic vulnerabilities in cell line models of TRIM33 loss. Moreover, we demonstrate that ATR inhibition increases the efficacy of bortezomib in TRIM33 knockdown cells. Further investigation into these compounds could lead to novel therapies for patients with TRIM33 loss. DisclosuresNo relevant conflicts of interest to declare.

Development and Validation of [18f](2 S,4 R)-4-Fluoroglutamine in Multiple Myeloma Mouse Models

Glutamine (Gln) addiction has been recently described as a typical metabolic feature of MM by our group. In order to sustain high Gln demand, MM cells upregulate the expression of the Gln transporters ASCT2, SNAT1 and LAT1 and are characterized by fast Gln uptake. Currently, 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) is the standard tracer for the positron emission tomography/computed tomography (PET/CT) scan to detect both medullary and extramedullary disease in MM patients. However, FDG-PET has some limitations, since there is a portion of MM patients who are false-negative. For these reasons, the aim of our study is to characterize Gln metabolism in vivo in different MM mouse models and investigate the possible use of Gln as a PET tracer in MM.To this purpose, we have firstly synthesized enantiopure (2 S,4 R)-4-fluoroglutamine (4-FGln) and validated it in human MM cell lines (RPMI8226 and JJN3) comparing its uptake with that of 3H-labelled Gln. Both Gln and 4-FGln were actively accumulated by MM cells. Inhibition analysis revealed that ASCT2 was the major entry route of both compounds.We then carried out the radiosynthesis of [18F]4-FGln and tested its uptake for MM detection by PET in comparison with [18F]FDG. Firstly, we characterized [18F]4-FGln kinetics using immunodeficient NOD/SCID mice subcutaneously injected with JJN3. In this xenograft model, [18F]FDG- and [18F]4-FGln-PET were performed after plasmacytomas became palpable. In tumor, [18F]4-FGln uptake peaked at 25 min slowly declining thereafter. The Logan plot confirmed linearity starting at 15 min from injection, consistent with largely reversible tracer exchange.Thereafter, [18F]FDG and [18F]4-FGln distribution were assessed in both syngeneic (murine Vk12598 cells) and xenograft ( human JJN3 cells) MM models.C57BL/6 mice were injected intravenously with Vk12598 cells obtained from transgenic Vk*MYC mice. Mice were monitored weekly for M-spike and MM progression by retro-orbital bleeding. At weeks 3, 4 and 5, mice underwent PET with [18F]FDG and [18F]4-FGln on consecutive days. The expression of ASCT2, checked in the femur of Vk12598 MM bearing mice, increased along with MM progression. Uptake of both tracers was measurable in the spleen of MM mice by week 3, reached a peak at week 4 and declined thereafter. In JJN3 model, all the tumors were positive for [18F]FDG and displayed [18F]4-FGln uptake (18F]4-FGln: tumor to muscle ratios (T/M): 1.6 ± 0.1; [18F]FDG: T/M: 3.5 ± 1.1). Tumor volume (896.8 ±349.00 mm3), [18F]4-FGln uptake and [18F]FDG uptake (T/M: 2.3 ± 0.3 and 7.1 ± 2.6, respectively) increased after 1 week ([18F]4-FGln p = 0.0156; [18F]FDG p = 0.0167).Thereafter, the effect of bortezomib (BOR) was investigated to evaluate the potential use of [18F]4-FGln to monitor anti-MM treatment. JJN3-bearing mice were treated with BOR (1mg/kg) by intravenous injections twice weekly. Before and after treatment, animals performed the PET acquisitions with [18F]FDG and [18F]4-FGln on consecutive days and were sacrificed for post-mortem analysis on day 7. As expected, bortezomib treatment reduced tumor size compared with vehicles (172.0 ± 86.6 vs 687.9 ± 286.5 mm3; p = 0.0006).PET analysis performed after BOR treatment showed that BOR significantly reduced the uptake of both radiopharmaceuticals in comparison with vehicles (p< 0.05). BOR-treated mice were classified as responders and non responders according to the adapted RECIST score. Interestingly, responder mice showed a reduction of [18F]4-FGln T/M ratio and [18F]4-FGln-related tumor volume. On the contrary, all mice displayed increased [18F]FDG parameters independently from the response. Lastly, tracer overlap analysis showed that the area of relative exclusive uptake of [18F]4-FGln decreased in responders compared to non responders (9% versus 32%, respectively, p=0.04), while that of [18F]FDG increased in responders compared to non responders (82% versus 23%, respectively, p=0.04).Our data indicate that [18F](2 S,4 R)-4-FGln is a new PET tracer in pre-clinical MM models, providing the rationale to design studies in MM patients. DisclosuresGiuliani: Celgene: Membership on an entity's Board of Directors or advisory committees, Other: congress, Research Funding; Millenium Pharmaceutical: Other: clincial studies; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical studies, congress, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Other: clinical studies; Bristol Mayers Squibb: Other: congress.

N-Cadherin Stabilizes β-Catenin and Promotes β-Catenin/TCF Transcriptional Activation and Cell Adhesion-Mediated Drug Resistance in Multiple Myeloma

Introduction:Inappropriate activation of Wnt/β-catenin signaling plays a role in some cancers. β-catenin (β-cat) levels in the cell can be regulated by a cadherin-mediated sequestration into membrane-bound and free cytosolic pools, with the later translocating to the nucleus and driving TCF-mediated transcriptional activity following Wnt signal transduction. While sequencing has shown that MM lacks the mutations that typically lead to constitutive β-cat activation seen in other cancers, we and others have demonstrated that Wnt/β-catenin signaling is nonetheless activated in MM and can regulate MM growth. The mechanism driving β-cat stabilization and activation in MM is unclear. E- and N-cadherin (N-cad) expression is elevated in MM compared to plasma cells from healthy donors. We hypothesized that that cadherins can regulate Wnt/β-catenin signaling in MM.Materials and Methods:We detected different forms of β-cat expression in a panel of human MM cell lines (HMCLs) and CD138 PC from MM patients by several approaches. Cadherin gain- or loss-of-function MM models were produced by expressing wild-type N-cad in MMS1 and ARP1 (lack endogenous N-cadherin expression) using a lentiviral system to create stable cell lines (N-Cad/MMS1 and N-cad/ARP1) and empty vector control (EV/MMS1, and EV-ARP1). We knocked down N-cadherin in the JJN3 cell line expressing high level of endogenous N-cadherin using shRNA specific for N-cad (shNcad/JJN3) or scrambled control shRNA (shCont/JJN3) by lentiviral-mediated transfection. We used a TCF reporter system to evaluate β-cat transcriptional activity as previously described.Results:We surveyed 25 HMCLs and CD138-selected plasma cells from 72 newly diagnosed MM for active β-cat with an antibody that specifically recognizes the unphosphorylated active form of β-cat. Higher levels of cytosolic and/or nuclear β-cat protein were seen in 13 of 25 (52%) HMCLs and 36 of 72 (50%) primary MM PC. Correlation of β-cat protein levels with global mRNA expression levels in primary PC revealed significant correlation with only one gene, CDH2 (N-cad). Remarkably, those primary MM with high β-cat levels but low CDH2 levels expressed high levels of E-cadherin/CHD1 mRNA. This posed the question of whether CDH2 is a direct target of TCF/β-cat transcriptional activity or whether high levels of CDH2 lead to increased levels of β-cat protein via sequestration. Both CDH2 mRNA and protein were correlated with β-cat protein but not β-cat mRNA in 23/25 HMCLs. Co-immunoprecipitation revealed that N-cad and β-cat complexes could be identified in HMCLs and primary MM. Consistent with N-cad-mediated stabilization of β-cat both total and unphosphorylated β-cat levels and TCF activity were significantly elevated in N-cad/MMS1 and N-Cad/ARP1 cells relative to controls. In contrast, shRNA mediated knockdown of N-cad led to a loss of both N-cad and β-cat protein levels and TCF-dependent transcription activity relative to controls. These findings provide evidence that β-cat/TCF signaling can be regulated by N-cad in MM. CDH2 mRNA is significantly elevated in the MS and HY subgroups of MM. To search for a potential mechanism of CDH2 transcriptional regulation in MS MM, we compared TCF activity and β-cat protein levels in MS versus non-MS HMCLs. TCF activity and active β-cat were elevated in MS versus non-MS forms of MM and B-cell lymphoma lacking N-cadherin. To determine if MMSET is required to up-regulate N-cad expression, we depleted the full-length MMSET protein in KMS11 cells. The results revealed a dramatic loss of total and unphosphorylated β-cat protein, but not mRNA, and loss of both CDH2 mRNA and protein relative to controls. These data suggest that MMSET can regulate the transcription of the CDH2 gene. MMS1 and ARP1 cells stably expressing N-cad exhibited enhanced adhesion to bone marrow stromal cells and decreased sensitivity to bortezomib (Bzb). In contrast, blocking N-cadherin-mediated adhesion by CDH2 shRNA increased sensitivity to Bzb. These results suggests that N-cad/β-cat complexes can contribute to adhesion-mediated drug resistance in MM.Conclusion: Taken together, these findings establish that β-cat is stabilized by N-cadherin-, and likely E-cadherin-, adhesins junction formation in MM. This in turn leads to an increased pool of β-cat that can drive TCF transcriptional activation as well participate in cadherin-mediated cell adhesion and drug resistance. DisclosuresDavies: Amgen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Walker: Bristol Myers Squibb: Research Funding; Sanofi: Speakers Bureau.

18F](2S,4R)-4-Fluoroglutamine as a New Positron Emission Tomography Tracer in Myeloma.

The high glycolytic activity of multiple myeloma (MM) cells is the rationale for use of Positron Emission Tomography (PET) with 18F-fluorodeoxyglucose ([18F]FDG) to detect both bone marrow (BM) and extramedullary disease. However, new tracers are actively searched because [18F]FDG-PET has some limitations and there is a portion of MM patients who are negative. Glutamine (Gln) addiction has been recently described as a typical metabolic feature of MM cells. Yet, the possible exploitation of Gln as a PET tracer in MM has never been assessed so far and is investigated in this study in preclinical models. Firstly, we have synthesized enantiopure (2S,4R)-4-fluoroglutamine (4-FGln) and validated it as a Gln transport analogue in human MM cell lines, comparing its uptake with that of 3H-labelled Gln. We then radiosynthesized [18F]4-FGln, tested its uptake in two different in vivo murine MM models, and checked the effect of Bortezomib, a proteasome inhibitor currently used in the treatment of MM. Both [18F]4-FGln and [18F]FDG clearly identified the spleen as site of MM cell colonization in C57BL/6 mice, challenged with syngeneic Vk12598 cells and assessed by PET. NOD.SCID mice, subcutaneously injected with human MM JJN3 cells, showed high values of both [18F]4-FGln and [18F]FDG uptake. Bortezomib significantly reduced the uptake of both radiopharmaceuticals in comparison with vehicle at post treatment PET. However, a reduction of glutaminolytic, but not of glycolytic, tumor volume was evident in mice showing the highest response to Bortezomib. Our data indicate that [18F](2S,4R)-4-FGln is a new PET tracer in preclinical MM models, yielding a rationale to design studies in MM patients.

P-016: The role [18F]-(2S,4R)-4-Fluoroglutamine as a new positron emission tomography tracer in Myeloma in vivo models.

<h3>Background</h3> The high glycolytic activity of multiple myeloma (MM) cells is the rationale for use of Positron Emission Tomography (PET) with 18F-fluorodeoxyglucose ([18F]FDG) to detect both bone marrow (BM) and extramedullary disease. However, FDG-PET has some limitations, since there is a portion of MM patients who are negative, and new tracers are actively searched. Glutamine (Gln) addiction has been recently described as a peculiar metabolic feature of MM cells. Yet, the possible exploitation of Gln as a PET tracer in MM has never been assessed thus far and is investigated in this study. <h3>Methods</h3> We have firstly synthesized enantiopure (2S,4R)-4-fluoroglutamine (4-FGln) and validated it as a Gln transport analogue in human MM cell lines, comparing its uptake with that of 3H-labelled Gln. We then radiosynthesized of [18F]4-FGln and tested its uptake for MM detection by PET in two different murine models and we checked the effect of Bortezomib treatment. <h3>Results</h3> Both [18F]4-FGln and [18F]FDG clearly identified the spleen as site of MM cell colonization in C57BL/6 mice challenged with syngeneic Vk12598 cells and assessed by PET. NOD.SCID mice subcutaneously injected with human MM JJN3 cells, showed high values of both [18F]4-FGln and [18F]FDG uptake to T/M: 2.3 ± 0.3 and 7.1 ± 2.6, respectively. With both [18F]4-FGln and [18F]FDG radiotracers, BOR treated animals displayed Standard Uptake Values (SUV) mean values significantly lower than controls at post treatment PET. However, [18F]4-FGln better correlated with the tumour volume in NOD.SCID mice, and a reduction of glutaminolytic, but not of glycolytic, tumour volume was evident in mice showing the highest response to Bortezomib. <h3>Conclusion</h3> Our data indicate that [18F](2S,4R)-4-FGln is a new PET tracer in pre-clinical MM models, yielding a rationale to design studies in MM patients.

P-106: Targeting DNA2 overcomes myeloma cells’ metabolic reprogramming in response to DNA damage

<h3>Background</h3> DNA damage resistance is a major barrier to effective DNA-damaging anticancer therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense therapy targeting ILF2, an important DNA damage regulator in 1q21 MM. <h3>Method</h3> We continuously treated MM cells with an ILF2-targeting antisense (ILF2-ASO) or control non-targeting antisense (NT-ASO) for 3 weeks to see if ILF2-ASO exposure leads to the selection of MM clones intrinsically resistant to DNA damage or activates compensatory mechanisms to overcome ILF2 depletion-induced DNA damage. <h3>Results</h3> Single-cell RNA sequencing (scRNA-seq) analysis revealed that DNA damage-resistant ILF2-ASO-treated cells had significantly upregulated oxidative phosphorylation (OXPHOS), DNA repair signaling, and reactive oxidative species (ROS). Metabolomic analysis of MM cells after long-term exposure to ILF2-ASO showed a significant enrichment of tricarboxylic acid cycle (TCA) intermediates. Consistent with these results, ILF2-ASO-resistant MM cells were significantly more sensitive to the OXPHOS inhibitor IACS-010759 than ILF2-ASO-sensitive cells were. These data suggest that MM cells can undergo an adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage. We then hypothesized that ILF2-ASO-resistant cells' metabolic reprogramming relies on the repair of DNA damage induced by ILF2 depletion or by the generation of ROS from activated mitochondrial metabolism and that targeting DNA repair proteins involved in these processes overcomes DNA damage resistance. We used a CRISPR/Cas9 screening strategy to identify DNA repair genes whose loss of function suppresses MM cells' ability to overcome ILF2-ASO-induced DNA damage. Compared with those in NT-ASO-treated cells, DNA2-targeting sgRNAs in ILF2-ASO-treated JJN3 cells were significantly depleted after 3 weeks of treatment, suggesting that DNA2 is needed to promote resistance to ILF2 depletion. Accordingly, the DNA2 inhibitor NSC105808 (NSC) significantly enhanced ILF2-ASO-induced apoptosis. To dissect the mechanisms of DNA2 inhibition-induced synthetic lethality, we evaluated whether DNA2 activity is essential to maintain activated OXPHOS, which ILF2-ASO-resistant cells require to survive. The quantification of mitochondrial respiratory activity in NT-ASO- and ILF2-ASO-treated MM cells exposed to NSC for 3 days showed that DNA2 activity inhibition significantly decreased the oxygen consumption rate while increasing ROS production in only ILF2-depleted cells. Transmission electron microscopy analysis showed that NSC-treated ILF2-depleted cells had fragmented mitochondrial cristae structures, whose perturbations affect the OXPHOS system structure and impair cell metabolism. <h3>Conclusion</h3> In conclusion, our study has revealed a novel mechanism through which MM cells counteract oxidative DNA damage and maintain mitochondrial respiration after metabolic reprogramming.

P-054: TRIM33 loss in Multiple Myeloma is associated with defective DNA repair and sensitivity to PARP inhibition

<h3>Background</h3> Chromosomal instability is a hallmark of Multiple Myeloma (MM) with most patients displaying cytogenetic abnormalities which can often act as prognostic indicators. Such abnormalities can arise due to defects in the DNA Damage Response (DDR). TRIM33 is an E3 ligase and transcription co-repressor located on chromosome 1p13.2, a region frequently deleted in MM. Previous studies have shown that TRIM33 is involved in PARP-dependent DDR and regulation of chromosomal stability. Here, we investigated the influence of TRIM33 loss in MM, focusing on its role in the DDR and whether this could be exploited therapeutically. <h3>Methods</h3> The CoMMpass dataset (IA15 release) was screened to identify patients with copy number (CN) loss of TRIM33 and this was correlated with survival, structural variants and common cytogenetic abnormalities. TRIM33 shRNA knockdown models were established in JJN3 and U266 cells for in vitro studies. Protein expression and interactions were assessed by co-immunoprecipitation, western blotting and/or immunofluorescence. Clonogenic survival assays were used to assess response to Olaparib. <h3>Results</h3> Previously we identified a subset of MM patients with TRIM33 loss and identified that these patients exhibit significantly more chromosomal structural variants (deletions, inversions, duplications and translocations) (p<0.0001) and a significantly poorer overall survival (p<0.0001). Additionally, we have determined the frequency of common recurrent primary and secondary cytogenetic abnormalities for these patients. No recurrent primary cytogenetic abnormalities were associated with TRIM33 loss. However, high-risk secondary chromosome 1 aberrations were associated with loss of TRIM33, both del(1p) and gain(1q) (p<0.0001 and p=0.0474 respectively). In vitro, TRIM33 knockdown resulted in increased formation of 53BP1 foci and increased γH2AX expression (p<0.001) indicating unrepaired DNA damage typical of a DDR defect. Following induced DNA damage using 2Gy irradiation (IR), TRIM33 is recruited to chromatin within 5 minutes, with levels returning to basal by 30 minutes. The chromatin remodelling enzyme ALC1 is known to regulate sensitivity to PARP inhibition and TRIM33 is required for its timely removal from sites of damage. TRIM33 transiently interacts with ALC1 within 15 minutes of 2Gy IR. TRIM33 knockdown did not affect ALC1 expression. However, knockdown did sensitize MM cells to the PARP inhibitor Olaparib reducing the IC50 from 1.7µM to 780nM. <h3>Conclusion</h3> Here, we show that a subgroup of MM patients have TRIM33 loss and these patients have high-risk disease and poor outcome. We show that TRIM33 is recruited to damaged chromatin where it regulates ALC1 activity. Therefore, TRIM33 loss results in a DDR defect leading to chromosomal abnormalities. However, TRIM33 loss-associated DDR defects can be exploited therapeutically using Olaparib which is currently approved for the treatment of BRCA1/2 mutated breast and ovarian cancers.

5,6-Epoxycholesterol Isomers Induce Oxiapoptophagy in Myeloma Cells.

Simple SummaryAs the second most frequent hematological malignancy, multiple myeloma remains incurable with recurrent patient relapse due to drug resistance. Therefore, the development of novel and potent therapies is urgently required. Herein, we demonstrated the anti-tumor activity of 5,6 α- and 5,6 β-epoxycholesterol isomers against human myeloma cells. Our results highlighted a striking anti-myeloma efficiency of these bioactive molecules and their added value in future potential treatments including combination therapy of multiple myeloma.Multiple myeloma (MM) is an incurable plasma cell malignancy with frequent patient relapse due to innate or acquired drug resistance. Cholesterol metabolism is reported to be altered in MM; therefore, we investigated the potential anti-myeloma activity of two cholesterol derivatives: the 5,6 α- and 5,6 β-epoxycholesterol (EC) isomers. To this end, viability assays were used, and isomers were shown to exhibit important anti-tumor activity in vitro in JJN3 and U266 human myeloma cell lines (HMCLs) and ex vivo in myeloma patients’ sorted CD138+ malignant cells. Moreover, we confirmed that 5,6 α-EC and 5,6 β-EC induced oxiapoptophagy through concomitant oxidative stress and caspase-3-mediated apoptosis and autophagy. Interestingly, in combination treatment a synergistic interaction was observed between 5,6 α-EC and 5,6 β-EC on myeloma cells. These data highlight a striking anti-tumor activity of 5,6 α-EC and 5,6 β-EC bioactive molecules against human myeloma cells, paving the way for their potential role in future therapeutic strategies in MM.

MAF and NF-κB Signaling Pathways Regulate Expression of APOBEC3B in Multiple Myeloma

IntroductionAPOBEC mutagenesis is a source of mutations in ~50% of human cancers, resulting from the over-expression of APOBEC proteins, including APOBEC3B (A3B). High expression of A3B is associated with poor prognosis and drug resistance in many cancers. In multiple myeloma, the chromosomal translocation t(14;16) leads to the over-expression of the transcription factor MAF through juxtaposition with the IGH super-enhancer. We have shown that in this poor prognosis sub-group A3B is also over-expressed and there is an enrichment for APOBEC signature mutations and a higher mutational load. It is not clear whether over-expression of A3B results from direct activation by MAF or indirectly through downstream pathways. In other cancers, mitosis, cell-cycle related gene expression, and the PKC/NF-κB signaling pathway may also affect expression of A3B.MethodsGene expression profile (GEP, U133 Plus 2.0) of 50 human myeloma cell lines (HMCLs) and 1310 myeloma patients was performed. mRNA levels of MAF and the AID /APOBEC family was investigated by qPCR in HMCLs. Western blotting and a DNA cytosine deaminase activity assay were used to detect A3B protein and analyze A3B enzymatic activity which results in mutagenesis, respectively. The effect of MAF on expression of A3B was investigated in inducible TRIPZ lentiviral shRNA c-MAF knockdown in HMCLs with high expression of MAF. Bortezomib, a proteasome inhibitor, and phorbol 12-myristate 13-acetate (PMA), a PKC dependent activator of the NF-κB signaling pathway, was used to investigate A3B expression in TRIPZ lentiviral shRNA MAF (MF KO) and A3B (A3B KO) knockdown HMCLs. Localization of A3B in regular HMCLs and cell lines with over-expression of A3B (generated using ViraPower T-Rex lentiviral expression system) was investigated by immunofluorescent analysis (IFA) using laser confocal and high resolution microscopy.ResultsBy microarray, high levels (log2>10) of A3B mRNA were found in approximately half of multiple myeloma patients. The majority of t(14;16) MAF patients had high levels of A3B. Increased expression of A3B was also detected in 72% of HMCLs. In t(14;16), MAF, and t(14;20), MAFB, HMCLs there was a strong positive correlation between MAF and A3B mRNA expression (r2= 0.51 and r2=0.43, respectively). On the basis of levels of MAF expression two groups of HMCLs were selected. Group 1 with high expression of MAF (MF) with the presence of a t(14;16) ARP1, CAG, JJN3, OCMY5, MM1S, and RPMI8226. Group 2 had low expression of MAF (non-MF) and was represented by cell lines with t(11;14) - U266, XG1 and KMS12BM; t(4;14) - KMS28PE and H929; or t(14;20) SACHI. We found a strong positive correlation (r2=0.68) between MAF and A3B mRNA expression in MF cell lines and no such correlation in the non-MF group (r2=0.09). Upon down-regulation of MAF by shRNA, JJN3 and RPMI8226 showed reduced expression of A3B protein (-33% and -29%, respectively) by 72 hours with accompanying reduction in cytosine deaminase activity (-23% and -26%, respectively). There was a positive correlation between expression of MAF protein and A3B protein in JJN3 MAF KO r2= 0.21, in RPMI8226 MAF KO r2=0.50; and a positive correlation between MAF protein and DNA cytosine deaminase activity in JJN3 MAF KO r2=0.21 and in RPMI8226 MAF KO r2=0.34. Bortezomib reduced expression of MAF and A3B and DNA cytosine deaminase activity in JJN3 and RPMI8226 cell lines. PMA increased the expression of A3B mRNA in HMCLs within 6 hours of treatment and lasted for 7 days after treatment. The highest sensitivity of A3B to PMA was detected in JJN3 and U266 cell lines, with increases of 4.4 and 8.2-fold, respectively, after 6 hours of treatment. Protein levels of A3B were increased 2.4-fold in both cell lines but DNA cytosine deaminase activity was increased (27%) only in JJN3 cells after PMA treatment.ConclusionsKnock down of MAF results in decreased expression and activity of A3B, indicating that MAF is a regulator of A3B expression in multiple myeloma. Expression of A3B in t(14;16) cases is dependent upon MAF expression but can also be increased through activation of the PKC/NF-κB signaling pathway, resulting in increased DNA damage. Understanding the mechanisms responsible for mutagenesis in the t(14;16) subgroup through A3B, which results in new clone formation and drug resistance in multiple myeloma will be highly beneficial in designing new therapeutic strategies. DisclosuresDavies:Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Morgan:Bristol Myers: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria.

Glutamine Catabolism Maintains MCL-1 Expression in Glucose-Deprived Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy accounting for 11,000 deaths annually in the US with the majority succumbing to disease due to the development of resistance. A key component to the development of resistance lies in ineffective engagement of the BCL-2 family of apoptosis regulators. We and others have demonstrated that glucose maintains levels of a key resistance-promoting BCL-2 family member, myeloid cell leukemia factor 1 (MCL-1). Expression of MCL-1 promotes resistance of MM to a wide range of therapeutics. MM, like other cancers, exhibits elevated glucose consumption associated with manifestation of the Warburg effect. We previously reported that myeloma cells maintain the glycolytic phenotype by achieving constitutive plasma membrane localization of GLUT4, and GLUT4 inhibition or glucose deprivation suppressed MCL-1 expression. In this study, we further investigated the mechanism underlying the suppression of MCL-1 expression upon glucose deprivation.L363 and KMS18 cell lines are sensitive to glucose deprivation, exhibiting significant cytotoxicity upon glucose deprivation and demonstrating rapid reduction of MCL-1 protein in contrast to JJN3 and KMS11 cells that are resistant to glucose deprivation. Knockdown of MCL-1 in JJN3 dramatically induced apoptosis while overexpression of MCL-1 in L363 significantly reversed cell death upon glucose deprivation, supporting a role for MCL-1 suppression in mediating glucose-deprivation elicited cell death. We next proceeded to investigate the metabolic pathways contributing to the persistence of MCL-1 in the glucose-insensitive lines. Since glutamine is an important carbon source that can compensate for glucose deprivation, we examined glutamine uptake in L363 and JJN3 cells. Evaluation of 3H-glutamine uptake revealed that JJN3 cells exhibit higher glutamine uptake than L363 cells. In addition, we performed 13C-based metabolic flux analysis in order to identify metabolites that may be contributing to the maintenance of MCL-1 in glucose-deprived cells. Interestingly the level of glutathione (GSH) was much higher in JJN3 than in L363 that was maintained even upon glucose deprivation. Since GSH is a major cellular reactive oxygen species (ROS) scavenger, we evaluated levels of ROS using a cyto-roGFP construct and found that it was significantly elevated upon glucose withdrawal in L363 but not in JJN3. In addition, treating glucose deprived L363 with cell-permeant GSH monoethyl ester (GSH-MEE) rescued MCL-1 expression, further supporting a role for ROS in suppressing MCL-1 expression.Glutamine uptake evaluation revealed that glutamine consumption was maintained upon glucose withdrawal in both JJN3 and L363, so we were then interested in the TCA cycle-related differences in glutamine metabolism between JJN3 and L363. The level of pyruvate dehydrogenase alpha 1 (PDHA1) phosphorylation decreased upon glucose deprivation only in JJN3 cells, which activates PDHA1 and allows pyruvate to flow into the TCA cycle. In accordance with this, treatment of JJN3 with dichloroacetate (DCA), which inhibits pyruvate dehydrogenase kinase and therefore activates PDHA1, restored MCL-1 expression and prevented cell death of L363 during glucose deprivation. To study how TCA cycle helps maintain the GSH level, glutamate dehydrogenase (GDH) knockdown cells were generated because GDH catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and glutamate is the precursor for GSH. Interestingly, in GDH knockdown L363 cells, treatment of DCA was not able to reverse MCL-1 protein level upon glucose deprivation. Taken together, our observations suggest that glucose-deprived resistant cells sustain higher levels of GSH via greater glutamine uptake, thereby scavenging ROS to sustain cell viability; while sensitive cells cannot make GSH effectively, leading to increased ROS level, decreased MCL-1 expression, and ultimately, apoptosis.In sum, we have discovered that targeting glucose entry elicits apoptosis via an induction of ROS leading to suppression of MCL-1. These observations provide further rationale for the development of GLUT4-specific biologics to target aberrant glucose metabolism in myeloma and the need to further understand how glutamine metabolism and the TCA cycle contribute to maintaining MCL-1 expression. DisclosuresNo relevant conflicts of interest to declare.

The IL15 Superagonist Complex ALT-803 Potently Enhances the Proliferative Capacity and Activity of in Vitro Expanded Natural Killer Cells

IntroductionWe previously reported on the treatment of 8 relapsing high-risk multiple myeloma (HRMM) patients with ex vivo activated/expanded NK cells (ENKs). We demonstrated that interleukin-2 (IL2) provided suboptimal cytokine support post adoptive transfer of ENKs. Aiming to improve upon prior data we hypothesize that the novel IL15 superagonist complex ALT-803 will provide superior support compared to IL2 and enhance ENK activity, proliferation and persistence.MethodsTo compare the activity of IL2 and ALT-803 on ENKs, we performed long-term in vitro culture studies. Analogous to the 8-day clinical production method, healthy donor (HD) peripheral blood mononuclear cells (PBMCs) were first co-cultured with the leukemic cell line K562 modified to express both membrane-bound IL15 and the costimulatory molecule 4-1BBL, and high-dose IL2. We deliberately chose to maintain IL2 in the expansion method rather than substituting ALT-803 because our FDA-approved GMP-grade expansion was validated using IL2. On expansion Day 8 (the day clinical ENK products are infused), the ENKs were either maintained at high-dose (300U/mL) IL2, or otherwise cultured at low-dose (20U/mL) IL2 or 35ng/mL ALT-803, in all conditions without K562 stimulator cells. Proliferation (fold-change from baseline) was measured on Days 14, 21, and 28, and expression of key activating and inhibitory markers was assessed via flow cytometry. Relative cytolytic ability was determined via 4-hour chromium (Cr)-release assays against K562 and the MM cell lines OPM2, JJN3, and U266.ResultsProliferation of ALT-803-ENKs was superior to both IL2-ENK conditions at all assessed time points; on Day 28, fold-expansion of ALT-803-ENKs was 753, versus 156- and 557-fold for ENKs maintained in 20U or 300U/mL IL2, respectively, Figure 1A. Immunophenotyping studies demonstrated significant differences between ENKs cultured in 20U/mL IL2 versus ALT-803-treated ENKs, including activation (CD26, CD69, DNAM1, NKp30) and functional (CD16, perforin, granzyme, TRAIL) molecules. ALT-803-treated ENKs maintained an activated phenotype, whereas IL2 ENKs (20U/mL) exhibited reduced expression of several markers by Day 14, with further decreases noted on Days 21 and 28. Representative examples are depicted in Figure 1B. Morphologically, IL2-ENKs acquired apoptotic features after extended culture, whereas ALT-803-ENKs did not. Though cytolytic ability was variable on Days 14 and 21, the activity of ALT-803-ENKs exceeded that of IL2-ENKs (20U/mL) against K562, OPM2, JJN3, and U266 cell lines on Day 28. High-dose IL2-ENKs exhibited a similar phenotype and cytolytic ability compared to ALT-803-ENKs.ConclusionsWe compared clinically-relevant doses of IL2 and ALT-803 to determine whether ALT-803 is superior at maintaining ENK activity and proliferative capacity. ALT-803 ENKs exhibited robust proliferation through Day 28 and sustained expression of key activation and functional markers, highlighting the superiority of ALT-803 in this setting. After four weeks of continuous culture, ALT-803-ENKs maintained greater cytolytic ability against MM cell lines. Though high-dose IL2 is capable of sustaining ENK activation state in vitro, this dose is not achievable in humans. Unlike IL2, ALT-803 does not induce expansion of T regulatory cells, and has a more favorable toxicity profile. Importantly, ALT-803 stimulated and maintained expression of CD16, indicating that it would be well-suited to pair with monoclonal antibodies for the support of antibody-dependent cellular cytotoxicity. Further studies are in progress to study the effects of ALT-803 on ENKs in the presence of suppressive cytokines/cells to determine if ALT-803 is able to overcome the effects of the tumor microenvironment. [Display omitted] DisclosuresWong:Altor BioScience: Employment, Equity Ownership. Jeng:Altor Bioscience: Employment, Equity Ownership. Shrestha:Altor Bioscience: Employment, Equity Ownership. Davies:Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Morgan:Bristol Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Aplidin (Plitidepsin) Regulates Viability and Function of Myeloma Cells and Bone Cells in Combination with Other Anti-MM Drugs

In multiple myeloma (MM) the clonal expansion of malignant plasma cells within the bone marrow results in the development of lytic bone lesions due to exacerbated bone resorption. Despite recent progress in its treatment, MM remains incurable and the associated bone disease persists even after complete remission. Thus, identification of new therapeutic approaches that suppress MM cell growth and protect the bone is an unmet need. Aplidin, a novel anti-cancer compound isolated from the marine tunicate Aplidium albicans, exhibits anti-MM activity even in MM cells resistant to conventional chemotherapy and is currently in phase III clinical trials in Europe. However, the effects of Aplidin on bone cells and MM-induced bone disease are unknown. The goal of the current study was to examine the effect of Aplidin, alone or in combination with other anti-MM drugs, on myeloma cells and bone cells (osteocytes, osteoblasts, and osteoclasts) using in vitro approaches.Aplidin decreased the number of human JJN3 and murine 5TGM1 MM cells with an EC50~10nM, determined by MTT assay, due to decreased cell viability, assessed by Trypan blue exclusion, reaching a 20% increase in MM cell death after 48h. Treatment with glucocorticoids (GC) (10-5M) or Bortezomib (3nM) also decreased MM viability and enhanced the anti-MM activity of Aplidin by 1.6-fold and 2.5-fold, respectively. In addition, Aplidin at >10nM induced MLO-A5 osteocytic dead up to 15% and in combination with GC up to 50%; whereas, in contrast, Bortezomib did not affect osteocyte viability and partially prevented the increase in MLO-A5 cell death induced by Aplidin. Further, 1nM Aplidin increased OB-6 osteoblastic cell death up to 20% and decreased their matrix mineralization capacity, quantified by Alizarin red. These effects were enhanced when Aplidin was combined with GC, but partially prevented by combination of Aplidin with Bortezomib. Cells of the osteoclastic lineage exhibited higher sensitivity to Aplidin in comparison to MM cells or osteoblastic cells, and thus it was tested at lower concentrations. Specifically, 50 pM Aplidin decreased the number of hematopoietic precursors by 15-20%, as quantified by enumerating GM-CSF colonies in cultures established from murine bone marrow. Moreover, Aplidin, at concentrations as low as 0.01 pM, reduced osteoclast precursor commitment and differentiation by 10%, as quantified by enumerating the number of osteoclast precursors maintained in the presence of M-CSF for 3 days with or without Aplidin. In addition, Aplidin decreased by 40% the number of TRAP positive osteoclasts generated after 4 days in the presence of M-CSF and sRANKL; and blunted the rapid (5-15min) increased in ERK phosphorylation induced by Rankl, but did not affect RANKL-induced p38 phosphorylation, suggesting that Aplidin inhibited RANKL-induced differentiation of osteoclast precursors by preventing ERK activation. Lastly, to test the effects of Aplidin on osteoclasts function, mature osteoclasts generated in the presence of M-CSF/sRANKL were detached by trypsinization, replated on bovine dentin slides and exposed to Aplidin for 3 and 7 days. 10pM Aplidin reduced the number of mature osteoclasts by 60% and markedly decreased the number of resorption pits.In summary, Aplidin (1nM and up) decreases MM cell viability and GC or Borteozmib enhances its anti-MM activity. In addition, Aplidin (10nM and up) decreases osteocytic cell viability and this effect is exacerbated by GC but partially prevented by Bortezomib. Moreover, Aplidin (1nM and up) induces osteoblastic cell death and decreases their mineral production, and these effects are increased by GC, but partially prevented by Bortezomib. Furthermore, Aplidin (0.01pM and up) reduces osteoclast precursor commitment and differentiation, and inhibits mature osteoclast resorption activity. Taken together these findings suggest that combining Aplidin with proteosomal inhibitors may be a novel therapy for MM to enhance anti-tumor responses, decrease bone destruction by inhibiting bone resorption, and prevent potential side effects of Aplidin on cells of the osteoblastic lineage. DisclosuresDelgado-Calle:PharmaMar: Research Funding. Galmarini:PharmaMar: Employment. Roodman:PharmaMar: Research Funding. Bellido:PharmaMar: Research Funding.

Myeloma-Secreted Galectin-1 Potently Interacts with CD304 on Monocytic Myeloid-Derived Suppressor Cells.

Progression of multiple myeloma is regulated by factors intrinsic to the clonal plasma cells (PC) and by the immune effector cells in the tumor microenvironment. In this study, we investigated the interaction between CD304 expression on myeloid-derived suppressor cells (MDSC) and galectin-1 from malignant PCs in the context of autologous stem cell transplantation (ASCT) for multiple myeloma. Using high-throughput screening, CD304 expression on circulating monocytic MDSCs (M-MDSC; CD14+HLA-DRlow/-) was compared before and after ASCT. There was a significantly higher M-MDSC expression of CD304 before ASCT and a clear correlation between circulating pre-ASCT M-MDSC frequency and serum galectin-1 concentration. Treatment of pre-ASCT M-MDSCs, but not post-ASCT M-MDSCs, with galectin-1 in vitro expanded the M-MDSC population and increased expression of CD304. High galectin-1 expression by malignant PCs was associated with poor clinical outcomes. M-MDSC development and expression of CD304 were differentially induced when healthy donor peripheral blood mononuclear cells were cultured with the human multiple myeloma cell lines RPMI-8226 and JJN3, which express high and low galectin-1, respectively. Inhibition of galectin-1 reduced M-MDSC proliferation induced by RPMI-8226 cells but not by JJN3 cells, and blockade of CD304 reduced M-MDSC migration induced by RPMI-8226 cells but not by JJN3 cells. In addition, blockade of CD304 reversed suppression of the in vitro cytotoxic effect of melphalan by pre-ASCT M-MDSCs. Our data demonstrate that multiple myeloma-derived galectin-1 could mediate the tumor-promoting effect of M-MDSCs through its interaction with CD304 on M-MDSCs and contribute to multiple myeloma progression after ASCT.See related Spotlight on p. 488.

Atpase Family AAA Domain-Containing Protein 2 (ATAD2) As a Novel Target in Multiple Myeloma

Multiple myeloma (MM) is a genomically heterogenous malignancy characterized by a number of copy number alterations (CNA). Moreover, there is a clear evolution of genomic changes that may affect prognosis. To identify the drivers of this inherent genomic instability, we applied an integrated genomics approach utilizing genomic data for copy number changes and transcriptomic profile. We first identified genes whose expression correlated with total copy number events in a patient dataset (gse26863, n=246). We then applied those genes to identify the genes whose elevated expression correlated with both overall and event free survival in two different datasets (IFM70, n=170; gse2408, n=559). Elevated expression of this 30 gene signature also correlated with poor overall as well as event free survival in a third myeloma dataset (MMRF; P&amp;lt;0.0005 for both EFS and OS). We have begun to validate these genes for impact on genomic instability and on MM cell growth and survival. Here, we present the functional validation of AAA domain containing protein 2 (ATAD2), which is one of the top-most genes in our 30 gene signature in MM and has also been part of a chromosomal instability signature representing six different cancer types (Nature Genetics, 38: 9, 2006) and a mitotic chromosomal instability signature identified in breast cancer [Sci Transl Med, 2013]. Using The Cancer Genome Atlas data we have also correlated elevated ATAD2 expression with poor OS in pulmonary and pancreatic cancers (P&amp;lt;0.02). ATAD2 is a member of the AAA ATPase family of proteins containing two conserved ATPase domains and a bromodomain. Bromodomain containing proteins are involved in the regulation of gene expression and are frequently dysregulated in MM as well as other cancers. ATAD2 bromodomain selectively recognizes acetylated histone 4 (acetylated at K5 and K12) and has been shown to regulate many cellular processes including cell proliferation. ATAD2 has been shown to interact with and stimulate transcriptional activity of MYC and has also been shown to contribute to invasion and migration in several cancers. We have confirmed elevated ATAD2 expression relative to normal PBMC by Western blotting in all twelve myeloma cell lines tested. To further delineate its role in MM, we evaluated the impact of its knockdown on various parameters of growth and genome maintenance using shRNAs. Relative to control shRNA, knockdown with multiple different shRNAs resulted in near complete cell death in 3 MM cell lines (JJN3, H929 and RPMI) in five days. Reduced cell viability was accompanied by increased apoptosis as seen by annexin V/PI staining. Relative to control, ATAD2 knockdown in RPMI cells led to increase in the apoptotic cell fraction by 56% and in H929 cells by 42%. To investigate genomic impact of elevated ATAD2 expression, the live cell fraction from control and ATAD2-knockdown cells was evaluated, right after selection, for impact on the expression of γ-H2AX (a DNA break marker), pRPA32 (a marker of DNA end resection, a distinct step in the initiation of homologous recombination; HR) and recombinase RAD51 (a key player in HR). ATAD2-knockdown in H929 cells inhibited spontaneous DNA breaks, DNA end resection as well as RAD51 expression, suggesting that elevated ATAD2 contributes to increased spontaneous DNA damage and HR activity observed in MM cells. To further confirm its impact on genome stability, the live cell fraction from control and knockdown cells was evaluated for micronuclei (a marker of genomic instability). Relative to control shRNA-transduced cells, knockdown of ATAD2 cells reduced micronuclei by 58% in H929 and 53% in JJN3 cells. In summary, we demonstrate that elevated ATAD2 contributes to dysregulation of DNA repair and genome stability and is required for MM cell survival, indicating that it is a promising target to inhibit growth and reduce genomic evolution in myeloma. Disclosures Munshi: BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties; C4: Current equity holder in private company; Janssen: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.

ILF2 Antisense Oligonucleotide Therapy and a CRISPR/Cas9-Based Screening for DNA Repair Effectors Identify Synthetic Lethal Approaches Enhancing Myeloma Cells Sensitivity to DNA Damage

In previous studies of recurrently amplified 1q21 genes in multiple myeloma (MM), we identified ILF2 (Interleukin Enhancer Binding Factor 2) as a key modulator of the DNA repair pathway, which promotes adaptive responses to genotoxic stress in a dose-dependent manner, explaining why 1q21 patients benefit less from high-dose chemotherapy than non-1q21 patients do (Marchesini, Cancer Cell 2017). These findings prompted us to develop strategies for blocking ILF2 signaling to enhance the effectiveness of available DNA-damaging agent-based treatments. Given that ILF2 is selectively overexpressed in 1q21 MM cells and is not easily amenable to small-molecule or antibody depletion, we collaborated with IONIS Pharmaceuticals to develop antisense nucleotides targeting ILF2 (ILF2 ASOs). To exclude on-target toxicities that could arise from ILF2 inhibition, we injected 14 different ASOs targeting mouse ILF2 into male Balb/c mice. Of the 14 ILF2 ASOs we tested, 6 did not induce either notable histopathological findings or hematological and biochemical alterations, which suggests that ILF2 inhibition is well-tolerated in normal tissues. Thus, ILF2 ASOs were used for functional validation studies in myeloma. Consistent with our previous work using ILF2-targeting shRNAs, we observed that ILF2 ASO-induced ILF2 depletion resulted in significantly inhibited cell proliferation, increased ATM/Chk2 pathway activation, γH2AX accumulation, and caspase 3-mediated apoptosis in KMS11 and JJN3 cells and sensitized these cells to melphalan, bortezomib and olaparib treatment (Fig 1). However, whereas KMS11 cells had a high level of DNA damage activation and a significantly higher apoptosis rate after more than 2 weeks of ILF2 ASO treatment, JJN3 cells overcame ILF2 ASO-induced DNA damage activation and apoptosis and became resistant to ILF2 ASO treatment. To gain insights into the molecular mechanisms by which MM cells can overcome ILF2 ASO-induced DNA damage activation, we subjected ILF2 ASO-treated KMS11 and JJN3 cells to RNA sequencing analysis at early and late treatment times. We found that the genes that were significantly downregulated in JJN3 but not KMS11 cells treated with ILF2 ASOs for more than 2 weeks as compared with those treated for 1 week were mostly involved in the regulation of the DNA damage response (Fig 2). These findings suggest that MM cells can activate compensatory mechanisms to overcome the deleterious effects of DNA damage and survive. To identify DNA repair effectors whose loss of function suppresses 1q21 MM cells' capability to overcome ILF2 ASO-induced DNA damage, we performed a CRISPR/Cas9 screening using a pool of single-guide RNAs (sgRNAs) targeting 196 genes involved in the DNA damage response. Using the drugZ algorithm to assess differences in the representation of all sgRNAs between cells treated with NT or ILF2 ASOs for 3 weeks (Fig 3), we found that sgRNAs targeting the DNA replication helicase/nuclease 2 (DNA2) were among the most significantly depleted sgRNAs in ILF2 ASO-treated JJN3 cells, whereas sgRNAs targeting the Fanconi anemia core complex-associated protein 24 (FAAP24) were significantly depleted in ILF2 ASO-treated KSM11 cells. Using the DNA2 inhibitor C5, we further validated that targeting DNA2 significantly enhances ILF2 ASO-induced apoptosis in JJN3 cells (Fig 4). Functional validation experiments using inducible sgRNAs are ongoing to evaluate whether the inhibition of DNA2 or FAAP24 is a synthetic lethal approach to targeting 1q21 MM cells in the setting of therapies with DNA-damaging agents. Collectively, our study demonstrates that ILF2 ASO therapy may be exploited to optimize the use of DNA-damaging agents in patients with 1q21 MM. Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Colla:Abbvie: Research Funding; Amgen: Research Funding; IONIS: Other: Intellectual property and research material IONIS).

18f]-(2S,4R)-4-Fluoroglutamine As a New Positron Emission Tomography Tracer in Multiple Myeloma

High glycolitic activity of multiple myeloma (MM) cells is the rationale for the use of Positron Emission Tomography (PET) with 18F-fluorodeoxyglucose ([18F]FDG) to detect both medullary and extramedullary disease. However, FDG-PET has some limitations, since there is a good portion of MM patients who are false-negative. Besides enhanced glycolysis, glutamine (Gln) addiction has been recently described as a metabolic feature of MM by our group. To sustain high Gln demand, MM cells increase the expression of several Gln transporters (ASCT2, SNAT1, LAT1) and are endowed with fast Gln uptake. Yet, at variance with other Gln-addicted cancers, the possible exploitation of Gln as a PET tracer in MM has never been assessed and was investigated in this study. To this purpose, we have firstly synthesized enantiopure (2S,4R)-4-Fluoroglutamine (4-FGln) and validated it as a Gln analogue in human MM cell lines (RPMI8226 and JJN3) comparing its uptake with that of 3H-labelled Gln. The intracellular levels of 4-FGln were determined by HPLC-MS/MS employing a HILIC gradient separation and multiple reaction monitoring (MRM) detection. Both Gln and 4-FGln were actively accumulated by MM cells and exhibited a strong reciprocal competition, pointing to shared transporters. Inhibition analysis revealed that ASCT2 was the major entry route of both compounds, with minor contributions from the other transporters. However, compared with Gln, 4-FGln exhibited higher affinity for both ASCT2 and LAT1 transporters. On the basis of these results, we then tested [18F]4-FGln uptake for MM detection by Positron Emission Tomography (PET) in two different in vivo murine models. Firstly, to investigate sensitivity of human MM to [18F]4-FGln in vivo, JJN3 cells were subcutaneously injected in immunodeficient NSG mice In this xenograft model, [18F]4-FGln- and[18F]FDG-PET scans were performed after plasmacytomas became palpable and repeated after one week. All the tumours were positive for [18F]FDG and displayed [18F]4-FGln uptake with Standard Uptake Values (SUV) of 1.21±1.9 and 0.99±0.07 after 2 weeks, respectively. Thereafter, the effect of bortezomib (BOR) was investigated to evaluate the potential use of [18F]4-FGln to monitor anti-MM treatment. Ten NGS mice were injected with JJN3 cells and, after 14 days, treated twice weekly with BOR, 1mg/kg, or vehicle for two weeks. PET scans were performed before and after 5 and 12 days of BOR treatment. As expected, BOR reduced tumour size as compared to vehicle. At the first post-BOR PET scan, [18F]4-FGln (SUV mean: pre 0.85±0.31; post 0.45±0.10, P&lt;0.05), but not [18F]FDG (SUV mean: pre 0.97±0.38, post 0.75±0.14) was already significantly reduced: [18F]FDG and [18F]4-FGln uptake was reduced of 22 and 45% respectively. With both radiotracers, BOR treated animals displayed SUV mean values significantly lower than those of vehicle treated animals at post treatment PET (SUV means [18F ]FDG: BOR 0.75±0.14; vehicle 1.27±0.34, P&lt;0.05; SUV mean [18F]4-FGln: BOR 0.45±0.10 ; vehicle: 0.73±0.18 ; P &lt;0.05). Thereafter, to mimic BOR-resistant MM in a syngeneic mouse model, C57BL/6 mice were injected intravenously with Vk12598 cells obtained from transgenic Vk*MYC mice repeatedly treated with sub-optimal doses of BOR. Upon injection into C57BL/6 mice, Vk12598 cells colonize the BM without lytic lesions and extensively colonize the spleen generating an aggressive MM that brings animals to death within five weeks. PET scans were performed with [18F]4-FGln and [18F]FDG before Vk*MYC MM cells injection and after three, four and five weeks. Blood samples for M-spike evaluation were obtained in parallel. Four weeks after MM cells injection a significant increase of both [18F]4-FGln and [18F]FDG uptake was detected in spleens (SUV mean: 1.14±0.23, P=0.018; 0.94±0.24, P= 0.005). In both MM models, the volume of distribution of [18F]4-F-Gln did not overlap that of [18F]FDG. In conclusion, our data indicate that [18F]-(2S,4R)-4-Fluoroglutamine is a new potential PET tracer in pre-clinical MM models especially of extramedullary disease, either in a BOR-sensitive or in a BOR-resistant context, supporting the exploitation of Gln addiction for diagnostic purposes in MM patients. Disclosures Giuliani: Janssen: Research Funding.

Syndecan-1 Is Critical in ARF6-Dependent Macropinocytosis Driven By KRAS Mutation in the Pathophysiology of Multiple Myeloma

Syndecan-1 (SDC1), also known as CD138, is a member of integral membrane heparin sulfate proteoglycans constantly expressed in plasma cells (PCs) and a primary diagnostic marker for human multiple myeloma (MM). We here further define new functions of SDC1 in the MM pathobiology. Firstly, flow cytometry and qRT-PCR analysis showed that SDC1 is expressed at relatively higher levels in AMO-1, U266, OPM2, H929, MM1S, and MM1R MM cells when compared with JJN3, RPMI 8226, and ANBL6 MM cells. SDC1 levels are comparable in paired MM cell lines sensitive or resistant to current anti-MM therapies including lenalidomide, pomalidomide, and bortezomib. Significantly increased SDC1 mRNA levels in advanced MM stages (p&lt;0.05) were further correlated with elevated soluble SDC1 protein levels in patient serum by ELISA. As expected, higher soluble SDC1 was also detected in culture media (CM) from MM cell lines with higher mRNA levels. Next, the effects of SDC1 were studied by SDC1 knockout (KO) in OPM2, JJN3 and H929 cells via CRISPR/Ca9 gene modification, followed by RNA-Seq analysis. Neglectable shed SDC1 in CM of all SDC1 KO MM cells confirm null SDC1 expression. Expression of anti-apoptosis gene BCL2L1, cell cycle genes (CCND1, CCND2), and transcription factor RELA gene were decreased in SDC1 KO vs control MM cells. Permanent SDC1 KO cells were eventually derived, indicating additional SDC1 function besides its role in MM cell growth and survival. KEGG pathway analysis associated with genes downregulated following SDC1 KO showed biological processes (BPs) enrichment in ECM-receptor interaction (hsa04512; p&lt; 0.001), cell adhesion molecules (hsa04514; p&lt;0.001), focal adhesion (hsa04510; p&lt;0.001), cytokine-cytokine receptor interaction (hsa04060; p=0.005), chemokine signaling pathway ( hsa04062; p=0.006), gap junction (hsa04540; p=0.002), axon guidance (hsa04360; p=0.016), JAK-STAT signaling pathway (hsa04630; p=0.026), lysosome (hsa04142; p=0.047).Specifically, IL-21R, related to JAK-STAT signaling pathway and cytokine-cytokine receptor interaction, was significantly decreased in SDC1 KO MM cells, as validated by qRT-PCR and human receptor array analysis. IL-21R contains the common cytokine-receptor gamma-chain shared by the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, indicating potential cross-talks between MM cells and surrounding immune cells via SDC1. Since its natural ligand IL-21 is mainly secreted by non-myeloma bone marrow (BM) accessory cells, SDC1 could also modulate interactions between myeloid lineages and MM cells via IL-21/IL-21R circuit in the BM microenvironment. Of note, other key MM antigens, i.e., CD38, BCMA, SLAMF7 were affected at mRNA levels in SDC1 KO vs control MM cells. Moreover, human receptor array data showed decreased expression in Flt-3L, DR6, Endoglin, GITR, HVEM, IL-2RG, IL-17RA, IL-21R, PECAM-1, PDGFRB, RAGE, Trappin-2 and µPAR in SDC1 KO MM cells. BPs through GO analysis in these downregulated receptors were cell activation (GO:0001775), cell surface receptor signaling pathway (GO:0007166), and immune system process (GO:0002370). KEGG analysis showed that those receptors molecular were enriched in cytokine-cytokine receptor interaction pathway (KEGG:04060).Consistent with RNA seq data, µPAR, an important factor of ARF6-dependent trafficking, was also found significantly downregulated in SDC1 KO MM cells. Since ARF6 activation regulates macropinocytosis, an essential metabolic pathway fueling Ras-driven cancer cells, these data suggest that SDC1 may involve in ARF6-dependent macropinocytosis in MM cells. ARF6 is induced by KRAS mutation, we thus checked macropinocytic index in KRAS-mutated MM cell lines. Increased macropinocytosis occur in KRAS-mutated MM cells (KMS28-BM, MM1S, MM1R) compared with KRAS WT OPM2 and KMS12-BM. Importantly, macropinocytosis was inhibited following SDC1 depletion in KRAS-mutated MM cells, indicating that SDC1 critically mediates KRAS-driven macropinocytosis in MM cells. These data highlight the requirements for SDC1 to mediate nutrient-scavenging macropinocytosis in MM cells, most prominently harboring KRAS-mutation. Taken together, our results identify new functions of SDC1 which are crucial to enhance myeloma cell fitness and adaptation to various conditions in the BM milieu, thereby further supporting SDC1 targeted immunotherapy in MM. Disclosures Munshi: Celgene: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Abbvie: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Adaptive: Consultancy. Anderson:Celgene: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau.