Human MM Research Articles

Deregulation of Metalloproteinase Expression in Gray Horse Melanoma Ex Vivo and In Vitro.

The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial-mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq revealed the deregulation of several MPs in gray horse melanoma and, notably, a 125-fold upregulation of matrix metalloproteinase 1 (MMP1) that was further confirmed by RT-qPCR from additional tumor material. The IF staining of melanoma tissue versus intact skin for MMP1 and tumor marker S100 revealed MMP1 expression in all lesions. The co-expression of S100 was observed at different extents, with some tumors scoring S100-negative. The IF staining of primary tumor cells explanted from the tumors for MMP1 showed that the metalloproteinase is uniformly expressed in the cytoplasm of 100% of tumor cells. Overall, the presented data point to MP expression being deregulated in gray horse melanoma, and suggest that MMP1 has an active role in gray horse melanoma by driving EMT-mediated tumor cell dissemination via the degradation of the extracellular matrix. Whilst S100 is considered a reliable tumor marker in human MM, gray horse melanomas do not seem to regularly express this protein.

BCMA/CD47-directed universal CAR-T cells exhibit excellent antitumor activity in multiple myeloma

BackgroundBCMA-directed autologous chimeric antigen receptor T (CAR-T) cells have shown excellent clinical efficacy in relapsed or refractory multiple myeloma (RRMM), however, the current preparation process for autologous CAR-T cells is complicated and costly. Moreover, the upregulation of CD47 expression has been observed in multiple myeloma, and anti-CD47 antibodies have shown remarkable results in clinical trials. Therefore, we focus on the development of BCMA/CD47-directed universal CAR-T (UCAR-T) cells to improve these limitations.MethodsIn this study, we employed phage display technology to screen nanobodies against BCMA and CD47 protein, and determined the characterization of nanobodies. Furthermore, we simultaneously disrupted the endogenous TRAC and B2M genes of T cells using CRISPR/Cas9 system to generate TCR and HLA double knock-out T cells, and developed BCMA/CD47-directed UCAR-T cells and detected the antitumor activity in vitro and in vivo.ResultsWe obtained fourteen and one specific nanobodies against BCMA and CD47 protein from the immunized VHH library, respectively. BCMA/CD47-directed UCAR-T cells exhibited superior CAR expression (89.13-98.03%), and effectively killing primary human MM cells and MM cell lines. BCMA/CD47-directed UCAR-T cells demonstrated excellent antitumor activity against MM and prolonged the survival of tumor-engrafted NCG mice in vivo.ConclusionsThis work demonstrated that BCMA/CD47-directed UCAR-T cells exhibited potent antitumor activity against MM in vitro and in vivo, which provides a potential strategy for the development of a novel “off-the-shelf” cellular immunotherapies for the treatment of multiple myeloma.Graphic

Low Exposures to Amphibole or Serpentine Asbestos in Germline Bap1-mutant Mice Induce Mesothelioma Characterized by an Immunosuppressive Tumor Microenvironment.

We show that germline Bap1-mutant mice have enhanced susceptibility to MM upon minimal exposure to chrysotile asbestos, not only amphibole fibers. Chrysotile induced a more profound immune tumor response than crocidolite in Bap1-mutant mice by upregulating CD39/CD73-adenosine and Ccl2/Ccr2 pathways and recruiting more M2 macrophages, which together contributed to an immunosuppressive tumor microenvironment. Interrogation of human MM RNA-seq data revealed interconnected immunosuppressive pathways consistent with our mouse findings.

Establishment of an experimental model of canine malignant mesothelioma organoid culture using a three-dimensional culture method

Canine malignant mesothelioma (cMM) is a rare and drug-resistant malignant tumor. Due to few patients and experimental models, there have not been enough studies to demonstrate the pathogenesis of the disease and novel effective treatment for cMM. Since cMM resembles human MM (hMM) in histopathological characteristics, it is also considered a promising research model of hMM. Compared with conventional 2-dimensional (2D) culture methods, 3-dimensional (3D) organoid culture can recapitulate the properties of original tumor tissues. However, cMM organoids have never been developed. In the present study, we for the first time generated cMM organoids using the pleural effusion samples. Organoids from individual MM dogs were successfully generated. They exhibited the characteristics of MM and expressed mesothelial cell markers, such as WT-1 and mesothelin. The sensitivity to anti-cancer drugs was different in each strain of cMM organoids. RNA sequencing analysis showed cell adhesion molecule pathways were specifically upregulated in cMM organoids compared with their corresponding 2D cultured cells. Among these genes, the expression level of E-cadherin was drastically higher in the organoids than that in the 2D cells. In conclusion, our established cMM organoids might become a new experimental tool to provide new insights into canine and human MM therapy.

Abstract 4195: Evaluation of the anti-myeloma effects of high and metronomic selinexor alone and in combination with duvelisib in vitro and in vivo

Abstract Introduction: Selinexor (Karyopharm Therapeutics) is an FDA-approved oral selective inhibitor of nuclear export (SINE) compound. It specifically blocks XPO1 enforcing the nuclear retention of tumor suppressor proteins restoring their anti-cancer functions. It also attenuates the phosphoinositide-3 kinase (PI3K)-AKT pathway by activating PTEN. Although activation of PI3K signaling is often detected in hematological malignancies, there are no data regarding the combination of selinexor with the PI3K inhibitor duvelisib for MM treatment. Thus, we evaluated the anti-myeloma effects of selinexor in combination with duvelisib in vitro and also studied alternative schedules of selinexor with dexamethasone in vivo using our MM xenograft models LAGκ-2. Methods: The human MM cell line U266 was obtained from ATCC. Primary MM tumor cells were isolated from MM patients’ bone marrow (BM) aspirates. Cell viability was quantified using the MTS cell proliferation assay. For the in vivo studies, the human myeloma xenograft LAGκ-2 was surgically implanted into SCID mice. Selinexor was administered via oral gavage at 2.5 mg/kg 5x per week, 5 mg/kg 4x per week on consecutive days, or 10 mg/kg 2x per week with one day between treatments. Dexamethasone was administered via intraperitoneal injection at 4 mg/kg 2x per week except the vehicle control. Results and Discussion: MTS assays showed selinexor induced concentration dependent inhibition of cell viability/proliferation in the MM cell line MM1s at 0.1, 0.4 and 1.6 μM. Selinexor alone at 0.2 μM reduced cell proliferation of primary MM tumor cells obtained from BM mononuclear cells (MCs) from a patient with progressive disease. We next examined cell viability in the presence of selinexor and duvelisib. MM1s or fresh BM mononuclear cells were incubated with a fixed concentration of selinexor (0.1μM) with increasing concentrations of duvelisib (1.5 μM -25 μM) for 48 h. The results showed that duvelisib enhanced the cytotoxic effects of selinexor in a concentration dependent manner. We also evaluated dosing and scheduling of selinexor and dexamethasone in vivo using SCID mice bearing LAGκ-2 MM xenografts. When selinexor was combined with dexamethasone, lower more frequent dosing showed greater inhibition of tumor growth than mid-range and higher less frequent dosing of this SINE. This study illustrates that the combination of selinexor and duvelisib showed enhanced cytotoxic effects in vitro compared with single agent treatment, and we will further evaluate the anti-MM effects of selinexor in combination with duvelisib in vivo using our human MM xenograft models. These studies suggest that the combination of a SINE and PI3K inhibitor may be effective for MM treatment. Citation Format: Haiming Chen, Stacy Behare, Mingjie Li, Marissa Goldwater, Ava Hekmati, Jasmin Cao, Rocky Levin, Ning Xu, James Berenson. Evaluation of the anti-myeloma effects of high and metronomic selinexor alone and in combination with duvelisib in vitro and in vivo [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 4195.

Prevalence and Clinicopathologic Features of Canine Metastatic Melanoma Involving the Central Nervous System: A Retrospective Analysis and Comparative Review.

BackgroundCentral nervous system (CNS) involvement is the leading cause of death in malignant melanoma. Rodent models, while vital to mechanistic investigation, have had limited success identifying effective therapies for melanoma brain metastases. The companion dog with de novo melanoma is a promising complementary model for developmental therapeutic investigation, as these tumors occur in an immunologically outbred host that has shared environmental exposures with humans. However, relatively little is known regarding the prevalence and clinicopathological features of canine melanoma metastasis to the CNS. To further validate the dog as an appropriate model for human metastatic melanoma, the aims of this study were to determine the rate of CNS metastasis and associated clinicopathologic features in canine malignant melanoma.MethodsMedical records of dogs diagnosed with malignant melanoma from 1985-2019 at the University of California Davis Veterinary Medical Teaching Hospital were assessed retrospectively. Clinicopathologic features were compared between dogs with CNS metastasis (CNS+) and dogs without CNS metastasis (CNS-). Site of CNS involvement and associated neurological signs were analyzed via Wilcoxon-Mann-Whitney rank sum and Fisher’s exact tests. Survival data were analyzed via Kaplan-Meier estimates.ResultsCNS metastasis was identified in 38% of dogs in this study (20/53). The oral cavity was the most common site of primary melanoma in both groups [CNS+: n=12 (60%) vs. CNS-: n=22 (67%); p>0.99]. The total burden of metastatic disease was higher in the CNS+ group (CNS+: 4, 95% CI 3-5 vs. CNS-: 3, 95% CI 1-3; p<0.001). The cerebrum was the most common site of CNS metastasis (n=15, 75%) and seizures were the most observed neurological sign (n=9, 64%). There was no difference in overall survival between CNS+ and CNS- groups. However, the median survival time following onset of neurological signs was 9.5 days (95% CI 1-43), with 5 dogs euthanized within 24 hours of the onset of neurological signs.ConclusionsCanine and human MM patients share similar rates of CNS metastasis and clinical presentation. This study will guide clinical management of canines with malignant melanoma and inform future studies using dogs with spontaneously occurring melanoma as a preclinical model for human melanoma brain metastases.

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.

Ubiquitin Receptor PSMD4/Rpn10 Is a Novel Therapeutic Target in Myeloma

Background and Rationale 19S proteasome-associated Ubiquitin Receptor (UbR) PSMD4/Rpn10 chaperones ubiquitinated proteins to 20S proteasome for their degradation. Our earlier study showed that blockade of UbR Rpn13 triggers MM cell death and overcomes proteasome-inhibitor (PI)-resistance (Yan et al., Leukemia 2016; Yan et al., BCJ 2021). In this study, we utilized biochemical and genetic strategies as well as animal models to show that targeting UbR PSMD4 triggers anti-MM activity, including against PI-resistant MM cells.Materials and Methods Cell viability, drug sensitivity, and apoptosis assays were performed using WST/ CellTiter-Glo assay and Annexin V staining. MM cells were transiently transfected with control short interfering RNA (siRNA) PSMD4 ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. PSMD4 knockout HEK293 cell line was generated using CRISPR/Cas9. Doxcycycline (Dox)-inducible PSMD4-knockdown (KD) MM cell line was generated using short hairpin RNA (shRNA). Cell signaling and caspase activity were determined by western blotting. Compound X (Cpd X) was purchased from EMD Millipore, USA; and bortezomib was purchased from Selleck chemicals, USA. Alpha-assay kit was purchased from PerkinElmer, USA. In vivo studies were performed using a human MM xenograft model. Statistical significance was assessed with Student's t test.Results 1) GEP database on MM patients showed that PSMD4 expression inversely correlates with overall survival (n=175; p = 0.00064); 2) RT-PCR, immunoblotting, and immunohistochemistry of MM patient BM showed higher PSMD4 levels in patient MM cells and MM cell lines versus normal cells; 3) PSMD4-siRNA decreased viability of PI-resistant MM cells; 4) HEK293 cells carrying stable CRISPR/Cas9-PSMD4-knockout (KO), as well as MM cell lines (MM.1S, AMO-1) with Dox-inducible PSMD4-KD, showed increased polyubiquitinated proteins and reduced cell growth in both in vitro and animal models; 5)We designed an in vitro AlphaScreen® binding assay in HTS format to screen a library of 12K compounds, and identified novel PSMD4 prototype inhibitor Cpd X; 6) Binding and inhibition between compound Cpd X and PSMD4 was confirmed by pull-down, MS and FEB assays; 7) Treatment of MM and leukemic cell lines, as well as primary MM patient cells, with Cpd X significantly decreased their viability (IC50range 1.6µM to 9µM; p < 0.001 for all cell lines; n=3) without markedly affecting the viability of normal PBMCs, suggesting a favorable therapeutic index; 8) Cpd X inhibits proliferation of MM cells even in the presence of BM stromal or plasmacytoid dendritic cells (pDCs); 9) Cpd X inhibits PSMD4 without blocking 20S proteasomal catalytic activities or 19S proteasome-associated deubiquitinating enzymatic activities; 10) PSMD4 blockade via either Cpd X or siRNA/shRNA triggered MM cell death associated with induction of caspase-dependent- and unfolded protein response-related apoptosis; and finally, 11) Animal model studies show that Cpd X is well-tolerated, inhibits tumor growth, and prolongs survival.Conclusion Our preclinical data validates targeting UbR PSMD4/Rpn10 to enhance cytotoxicity and overcome PI resistance in MM. They provide the basis for further optimization studies of PSMD4 inhibitors for clinical application to improve MM patient outcome. DisclosuresChauhan: Oncopeptides: Consultancy; C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics: Consultancy. Anderson: Janssen: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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.

OAB-029: Bone marrow adipocytes induce metabolic reprogramming of multiple myeloma cells

<h3>Background</h3> Obesity-induced increases in bone marrow adipocyte (BMAd) numbers and volume are associated with an increased risk of MM. However, the associated molecular mechanisms have remained largely unknown. We hypothesize that BMAd support MM cells through metabolic reprogramming. <h3>Methods</h3> BM aspirates of MGUS, SMM, and NDMM patients were used to obtain mature BMAd or stromal cells (BMSCs). Murine BMSC cell-line OP9, MM cell-line 5TGM1, and human-origin MM cell-lines MM.1S, OPM2 were obtained from ATCC. Cell proliferation was assessed by CyQUANT Assay; lipolysis by Lipolysis Assay (Sigma); lipid uptake by flow cytometry (FCM) of fluorescent fatty acids (FA) BODIPY-C12 and -C16, or LipidTox. In-vivo efficacy of FA treatment was assessed in a MM CB17 SCID mouse plasmacytoma model. <h3>Results</h3> In-vitro co-culture revealed that BMSC-derived adipocytes (Ad) from MGUS/SMM and NDMM donors significantly increased the proliferation of MM.1S MM cells. Similarly, mature murine OP9 Ad cells also increased the proliferation of 5TGM1 murine MM cells. Interestingly, co-cultures showed dramatic decrease in LipidTox-stained lipid-droplet size distribution, suggesting increased lipolysis in Ad, further confirmed by increased glycerol content in conditioned media in the presence of MM cells. Moreover, BMAd from MGUS, SMM, and NDMM patients showed increased expression of genes responsible for lipolysis (NR1H3) and increased FA desaturation (SCD1, FASD2). Although MM cells lacked intracellular lipid storage, OPM2 and 5TGM1 MM cells rapidly took up BODIPY-C12 and -C16 FAs. The FA secreted from Ad were directly taken up by MM cells as shown by transfer of LipidTox-labeled lipids from OP9 Ad to unstained 5TGM1 or OPM2 MM cells as assessed by FCM. The addition of acipomox, a small-molecule inhibitor of lipolysis, decreased LipidTox signal in MM cells compared to untreated OP9 cells. FA are transported into cells through FATP (1-6) or CD36 receptors. Bioinformatics analysis of public Oncomine database showed that FATP1 and FATP4 were highly expressed in 21 human MM cell lines. MM cells from NDMM patients expressed high levels of FATP1 and FATP4. The uptake of BODIPY-C12 and -C16 by 5TGM1 or OPM2 MM cells was significantly reduced in the presence of Lipofermata, a pharmacological small-molecule inhibitor of FATP. Lipidomic analysis of BM aspirates from MM patients showed altered expression of various FA, including arachidonic acid (AA). Low doses of AA (0.125 - 2 µM) increased the proliferation and viability of MM cells whereas high doses (25- 100µM) dramatically decreased it, indicating a bimodal cellular effect of AA. Peritumoral AA treatment in a plasmacytoma model decreased tumor volume significantly. <h3>Conclusion</h3> We show that MM cells induce lipolysis in BMAd and that the released FFA are then taken up by MM cells through FATPs. Inhibition of either BMAd lipolysis or FFA transporter into MM cells could be a potential novel strategy to prevent MM progression.

P-093: Ubiquitin receptor PSMD4/Rpn10 as therapeutic target in Multiple Myeloma

<h3>Background</h3> Our prior studies showed that targeting Ubiquitin Receptor (UbR) Rpn13 upstream of the 20S proteasome signaling cascade overcomes proteasome-inhibitor (PI)-resistance (Yan et al. Leukemia 2016; Yan et al, 2021 BCJ–please complete reference details). Besides Rpn13, 19S-associated UbR PSMD4/Rpn10 also plays a key role in chaperoning ubiquitinated substrate proteins for downstream 20S proteasomal degradation. In the present study, we show that inhibition of PSMD4 triggers potent anti-MM activity using both in in vitro and in vivo models of MM, including against PI-resistant MM cells. <h3>Methods</h3> Cell viability, and apoptosis assays were performed using WST/ CellTiter-Glo assay, and Annexin V staining, respectively. PSMD4 knockout 293 cell line was generated using CRISPR/Cas9. Doxcycycline (Dox)-inducible PSMD4-knockdown (KD) MM cell line was generated using short hairpin RNA (shRNA). A xenograft human MM model was used to characterize the role of PSMD4 on tumor progression. Statistical significance was assessed with Student's t test. <h3>Results</h3> 1) MM patient gene expression profiling database showed that PSMD4 expression inversely correlates with overall survival (n=175; p=0.00064). 2) RT-PCR, immunoblotting, and immunohistochemistry of MM patient BM showed higher PSMD4 levels in patient MM cells and MM cell lines versus normal cells. 3) Transient transfection of MM cells including, bortezomib-resistant MM cells with PSMD4-siRNA decreased their viability; conversely, transfection with PSMD4-WT rescued cells from growth-inhibitory activity of PSMD4-siRNA. Western blotting confirmed knockdown of PSMD4 by PSMD4-siRNA versus scrambled (scr)-siRNA, and restoration of PSMD4 levels in cells transfected with PSMD4-WT versus PSMD4-siRNA. 4) CRISPR/Cas9-PSMD4-KO showed reduced cell growth. 5) Both PSMD4-KO and PSMD4-KD cells showed elevated levels of polyubiquitylated proteins, indicating blockade of proteasome-mediated protein degradation; 6) PSMD4 blockade triggered apoptosis, cell-cycle arrest, activation of caspases, and endoplasmic reticulum stress response signaling in MM cells; and finally, 7) Transplantation of Dox-inducible PSMD4-KD MM.1S cells in mice correlated with significantly reduced tumor growth. <h3>Conclusion</h3> Our preclinical data validates targeting UbR PSMD4/Rpn10 to enhance cytotoxicity and overcome PI resistance in MM, as well as provides the basis for the development and clinical evaluation of PSMD4 inhibitors to improve patient outcome.

Abstract LB110: Multiple myeloma cells depend on the NRF1-DDI2 proteasome stress response pathway for survival

Abstract Introduction: We previously showed that an imbalance between polyubiquitinated proteins awaiting proteasomal degradation and proteasome activity is a main determinant of proteasome inhibitor (PI) sensitivity in MM. While PIs have radically improved survival of MM patients, resistance is inevitable over time and understanding the molecular mechanisms underlying it is critically important to develop novel therapies to improve patient outcomes. The proteasome stress response (PSR) is an evolutionarily conserved homeostatic mechanism triggered by insufficient proteasome activity and aimed at restoring homeostasis via de novo proteasome biogenesis. The PSR master regulator NRF1 is constitutively degraded by the proteasome in homeostatic conditions, but in face of proteasomal insufficiency is cleaved by the aspartic protease DDI2, translocates to the nucleus, and induces proteasome subunit gene transcription. It is unknown whether blocking DDI2 or NRF1 could target an intrinsic vulnerability of MM. Materials and methods: We used human MM cell lines with distinct baseline sensitivity to PIs. NRF1 cleavage and nuclear localization were assessed via western blotting. We used CRISPR-Cas9 to knock out (KO) DDI2 and NRF1. Cell viability or tumor growth of DDI2/NRF1 KO cells versus cells edited with a non-targeting gRNA were compared in in vitro and in vivo growth competition studies, respectively. Addback studies were performed by stably expressing WT or aspartic protease dead DDI2 in DDI2 KO AMO1-VR monoclones. Chymotryptic-like proteasome activity was measured via cleavage of a fluorescent substrate upon incubation with protein lysates from DDI2 WT, KO, or addback cells. Results: We detected full-length and cleaved NRF1 in human MM cell lines, but not other hematologic cancer cell lines, suggesting constitutively active PSR in MM. Query of the dependency map (depmap.org) shows that MM is the cell line type most highly dependent on DDI2. Consistently, DDI2 or NRF1 KO is cytotoxic alone or in combination with PI across a panel of MM cells with distinct PI sensitivity, including de novo, PI-resistant KMS20. In vivo, DDI2 KO leads to reduced plasmacytoma formation in NOD SCID mice and results in prolongation of animal survival. While biallelic DDI2 or NRF1 KO monoclones could not be generated across 7 distinct MM cell lines, consistent with these genes being essential, we successfully established DDI2 KO monoclones in AMO1-VR, an isogenic AMO1 cell line variant adapted to grow in continuous bortezomib. Even in this cell line model of acquired PI resistance, DDI2 KO sensitized cells to PI carfilzomib. In this model, DDI2 KO blocks NRF1 cleavage and nuclear import, thereby impairing proteasome recovery after irreversible PI treatment and sensitizing to PI. Wild-type, but not catalytically-dead DDI2 addback rescues these phenotypes, confirming a causative link. Conclusions: Our data show that MM cells are intrinsically dependent on an intact DDI2/NRF1 axis to survive in baseline conditions and upon proteasomal inhibition. We propose that DDI2 is an unexplored, promising molecular target for MM treatment by disrupting the PSR and exacerbating proteotoxicity. This study provides the preclinical rationale for the development of novel therapeutics targeting the DDI2/NRF1 pathway to overcome acquired and de novo PI resistance and improve patient outcomes in MM. Citation Format: Tianzeng Chen, Matthew Ho, Jenna Briere, Maria Moscvin, Peter G. Czarnecki, Kenneth C. Anderson, T. Keith Blackwell, Giada Bianchi. Multiple myeloma cells depend on the NRF1-DDI2 proteasome stress response pathway for survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB110.

Germinal Center Kinase As a Therapeutic Target in Multiple Myeloma

Introduction: Despite many new drug approvals, Multiple Myeloma remains an incurable disease due to development of drug resistance. This process is driven by clonal evolution in which kinases play a major role. Germinal center kinase (GCK) is the founding member of the GCKs, a group of Ser/Thr kinases homologous to the saccharomyces cerevisiae Ste20p, and participates in B cell differentiation. Among human tissues, GCK is highly expressed in transformed lymphocytes, spleen and whole blood. However, the expression and function of GCK in human myeloma is totally unknown.Methods and Results: First we analyzed the expression level of GCK in human MM cells and found that GCK is abundantly expressed in all the MM cell lines we tested. To determine whether GCK is required for MM cell growth, we introduced inducible shRNA to knock down GCK expression in MM cells by lentiviral mediated transduction. MM cell lines MM.1S and RPMI-8266 were stably transduced with shRNA lentivirus generated from a robust inducible knockdown vector pLKO-tet-On. Doxycycline-induced GCK-tet-on-shRNA expression resulted in GCK protein knockdown and significant inhibition of cell growth by 75% in WST-1 proliferation assay. In accordance, cell cycle analysis revealed that the knockdown of GCK decreased cells in S-phase with concomitant increase in G0/G1 growth arrest (55% vs 67%). Importantly, GCK knockdown resulted in significant increase (3.21% vs 60.8%) of apoptotic cells suggesting that inhibition of GCK is a potential therapeutic target in MM cells. This was further confirmed by treating MM cells lines (MM.1S, RPMI-8266 and H929) with GCK inhibitors TL4-12, NG25 and Bay 61-3606. We found that these drugs dose dependently and significantly inhibit multiple myeloma cell growth, with IC50 7, 1.6 and 0.5 µM, respectively in H929 cells by WST-1 proliferation assay. Mechanistic studies showed that Bay 61-3606 dose-dependently decreases the protein levels of IKZF1/IKZF3 and c-Myc, increases p53 level and induces PARP cleavage resulting in cell cycle G1 arrest (60.8 vs. 69.8 with 1 µM) and apoptosis (9.16 vs. 29.4 at 1 µM) in MM.1S cells. To confirm the critical role of GCK in in vivo growth of MM cells, we generated subcutaneous MM xenografts in SCID Beige (CB17.Cg-PrkdcscidLystbg-J/Crl) mice using doxycycline-inducible GCK shRNA MM.1S cells. Doxycycline or vehicle treatment was started after the tumor was established on day 16. In contrast to vehicle-treated MM.1S-tet-on-shGCK or doxycycline-treated MM.1S-tet-on-sh-control tumors, doxycycline treated animals bearing MM.1S-tet-on-shGCK xenografts showed a significant inhibition (P<0.001) of tumor growth by 80% after 30 days. Immunohistochemistry staining of tumors confirmed the decrease of GCK and IKZF1 expression in doxycycline-treated mice bearing MM.1S-tet-on-shGCK tumors compared with tumors of vehicle- or non-doxycycline treated mice.Conclusion: Our findings show that GCK is a potential target for multiple myeloma therapy. GCK inhibition results in significant anti-MM effects by inducing cell cycle arrest and cell apoptosis accompanied by IKZF1/3 down regulation. Furthermore, the inhibition of in vivo tumor growth correlates with the doxycycline-induced GCK knockdown, further confirming the critical role of GCK in MM tumorigenesis. DisclosuresLentzsch:Amgen: Consultancy; BMS: Consultancy; Caelum Biosciences: Other: leadership position and stock.

RAR Gamma Activation Sensitizes Human Myeloma Cells to Carfilzomib Treatment through OAS-RNase L Innate Immune Pathway

Proteasome inhibitors (PIs) such as bortezomib (Btz) and carfilzomib (Cfz) kill MM cells by disrupting the degradation of misfolded proteins, presumably derived from high-level immunoglobulin production, which provides an appealing explanation for why MM cells are so uniquely sensitive to PIs. However, relapses are frequent and acquired resistance to PI treatment emerges in most patients. Therefore, identifying novel and safe drugs overcoming PI resistance in MM will aid in chemo-(re)sensitization, reducing PI-induced side effects, and maximizing the outcomes of PI therapy. Here we performed a high-throughput screen of 1855 FDA-approved drugs (Figure 1) and identified all-trans retinoic acid (ATRA), a classic pan-retinoic acid receptor (RAR) agonist used successfully to treat acute promyelocytic leukemia (APL), as a potent drug that enhanced MM sensitivity to Cfz-induced cytotoxicity and re-sensitized Cfz-resistant MM cells to Cfz in vitro. To determine which RARs are important for ATRA enhancement of Cfz-induced apoptosis in MM, esiRNAs of RARs for knocking down RARs and selective agonists of RARs were used in Cfz-treated MM cells. We identified that RARγ activation is important for ATRA sensitizing MM cells to Cfz treatment. To determine which signaling pathways are involved in ATRA-treated MM cells, gene-profiling data analysis of Cfz- versus ATRA+Cfz-treated MM cells was performed and real-time PCR was used to validate the microarray results. We found that ATRA treatment activated IFN-β response pathway (Figure 2), leading to upregulated expression of IRF1 and OAS1-3. Interestingly, similar to ATRA, IFN-β, which alone did not induce MM apoptosis, enhanced Cfz-induced MM cell apoptosis. Furthermore, using RNA integrity assay and dsRNA detection assay, we identified that ATRA treatment elevated the expression of OASs, which synthesized 2-5A upon binding to dsRNA induced by Cfz and resulted in cellular RNA degradation by RNase L and cell death (Figure 3). By knocking down each gene of OAS1-3, we demonstrated that OAS1 is essential for ATRA to sensitize MM cells to Cfz-induced apoptosis. Furthermore, we determined the impact and significance of RARγ and OAS1 in human MM pathogenesis and drug response by analyzing the gene-profiling data of 264 MM patients from Mulligan et al. datasets and 1,143 MM patients from MMRF coMMpass study IA13. In support of these findings, analyses of the large patient's gene-profiling datasets showed a strong and positive correlation between RARγ and OAS1 expression and patient's response to PI treatment (Figure 4). Finally, BMS961, a selective RARγ agonist, similar to ATRA, could also (re)sensitize MM cells to Cfz in vitro, and both ATRA and BMS961 significantly enhanced the therapeutic effects of Cfz in established MM in vivo (Figure 5). Thus, this study highlights the potential for RARγ agonists to sensitize MM and overcome MM resistance to Cfz treatment in patients. Disclosures No relevant conflicts of interest to declare.

Bortezomib Induces Anti-Multiple Myeloma Immune Response Mediated By Cgas/Sting Pathway Activation, Type I Interferon Secretion, and Immunogenic Cell Death: Clinical Application

Immune escape underlies progression of disease and resistance to therapy in MM, and dysfunction of both innate and adaptive immunity highlight the urgent need for scientifically-informed strategies to restore anti-MM immunity and improve patient outcome. Here we demonstrate that proteasome inhibitor bortezomib (BTZ) modulates the immunosuppressive bone marrow milieu by inducing immunogenic cell death (ICD) and a type-I interferon (IFN) response which contribute to its clinical efficacy. Moreover, we identified STING as a mediator of its anti-MM immune response, and provide the preclinical rationale for clinical trials of BTZ-STING agonist combination therapy. We first show that BTZ induces hallmarks of ICD in both human and murine MM cell lines, including exposure of endoplasmic reticulum protein calreticulin (CALR), that functions as an "eat me signal". Specifically, co-culture with BTZ-treated MM cells induced functional maturation of dendritic cells (DCs) and enhanced uptake of BTZ-treated MM cells, assessed by flow cytometry and confocal microscopy. Notably, these functional sequelae were abrogated when DCs were co-cultured with CALRKO MM cells; and add-back experiments by stable overexpression of CALR in KO clones confirmed the specific role of CALRin BTZ-induced immunogenicity. We next validated these findings in 2 in vivo syngeneic models. First, we observed that anti-MM activity of BTZ resulted in more potent murine 5TGM1 tumor cell shrinkage in immunocompetent than immunodeficient hosts, an effect mediated by ICD induction since it was abrogated in immunocompetent mice bearing CALRKO tumors. Second, in vitro BTZ-treated 5TGM1 cells were used as a vaccine to induce a protective anti-MM immune response: vaccination protected against tumor growth upon rechallenge with 5TGM1 cells; conversely, palpable tumors were detected in non-vaccinated mice by 1 week. Moreover, vaccination with BTZ-treated CALRKO 5TGM1 cells abrogated the protective effect of BTZ since only 50% mice were tumor free by 30 days. Consistently, ELISPOT assay on mice splenocytes confirmed that vaccination with BTZ-treated 5TGM1 cells triggered a MM-specific T cell response. Next, we performed RNAseq analysis of BTZ-treated vs untreated tumors from both CALRWT and CALRKO cells growing in immunocompetent mice; and then carried out an integrative analysis of RNAseq data from clinically-annotated MM patients (n=327) uniformly treated with BTZ-based regimens (IFM/DFCI 2009). We identified a specific ICD signature induced by BTZ only in CALRWT tumors in mice; and importantly, we found that increased expression of the human orthologs of this ICD signature was strongly and positively correlated with clinical outcome (overall survival (OS), p value=0.01). Moreover, the predictive value of this signature was confirmed in an independent dataset of BTZ-treated patients (GSE9782) (OS p value=0.024). Gene ontology analysis of the ICD signature genes revealed enrichment in inflammatory response pathways, including IFN stimulated genes (ISGs). Using RNAseq analysis and qRT-PCR validation, we showed that MM cells demonstrate a type-I IFN response after BTZ treatment which contributes to its in vivo efficacy, since neutralization of type-I IFNs signaling in both MM and host cells with a type-I IFNs receptor 1 (IFNAR) blocking antibody significantly reduced BTZ anti-MM activity. Finally, we showed that BTZ increases genomic instability/micronuclei formation in MM cells and activates innate cGAS/STING immune pathway, thereby stimulating a type-I IFN response. Conversely, STINGKO in MM cells abrogated this effect and decreased BTZ-triggered anti-MM T cell response. Moreover, expression of the ISGs included in ICD signature was positively correlated with STING expression in MM patients. Importantly, pharmacological activation of STING with STING agonist induced potent anti-MM activity in vivo; and combination of STING agonist with BTZ further potentiated this in vivo anti-MM response, with increased T cell infiltration into retrieved tumors, evidenced by IHC analysis. In conclusion, our studies delineate a novel mechanism whereby BTZ triggers anti-MM immune responses, and show that STING agonists can enhance this response. These findings provide the framework for clinical evaluation of STING agonists in combination with BTZ to induce potent anti-MM immune responses and thereby improve patient outcome. Disclosures Fulciniti: NIH: Research Funding. Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Chauhan:Oncopeptide AB: Consultancy; consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.. Munshi:Karyopharm: Consultancy; Takeda: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties; BMS: Consultancy. Anderson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees.

Proteomic analysis identifies mechanism(s) of overcoming bortezomib resistance via targeting ubiquitin receptor Rpn13.

Our prior study showed that inhibition of 19S proteasome-associated ubiquitin receptor Rpn13 can overcome bortezomib resistance in MM cells. Here, we performed proteomic analysis of Rpn13 inhibitor (RA190)-treated MM cells and identified an antioxidant enzyme superoxide dismutase (SOD1) as a mediator of Rpn13 signaling. SOD1 levels are higher in MM patient cells versus normal PBMCs; and importantly, SOD1 expression correlates with the progression of disease and shorter survival. Functional validation studies show that RA190-induced cytotoxicity in bortezomib-sensitive and -resistant MM cells is associated with decrease in SOD1 levels; conversely, forced expression of SOD1 inhibits RA190-induced cell death. Genetic knockdown and biochemical blockade of SOD1 with LCS-1 sensitizes bortezomib-resistant MM cells to bortezomib. SOD1 inhibitor LCS-1 decreases viability in MM cell lines and patient cells. LCS-1-induced cell death is associated with: (1) increase in superoxide and ROS levels; (2) activation of caspases, and p53/p21 signaling; (3) decrease in MCL-1, BCLxL, CDC2, cyclin-B1, and c-Myc; (4) ER stress response; and (5) inhibition of proteasome function. In animal model studies, LCS-1 inhibits xenografted bortezomib-resistant human MM cell growth and prolongs host survival. Our studies therefore show that targeting Rpn13 overcomes bortezomib resistance by decreasing cellular SOD1 levels, and provide the rationale for novel therapeutics targeting SOD1 to improve patient outcome in MM.

2024 - DEFICIENCY OF THE NOVEL TUMOR SUPPRESSOR C15ORF65 CAUSES MULTIPLE MYELOMA AND PLASMA CELL MALIGNANCY

The previously uncharacterized locus C15orf65 (murine Gm5918) is diminutive, yet highly conserved within and beyond the animal kingdom. We show that it encodes a heterochromatin-associated protein with a hydrophobic pocket and an aromatic cage in its domain of unknown function 4490 (DUF4490) that facilitate its action as a tumor suppressor for multiple myeloma and plasma cell malignancy. Hematopoietic loss of Gm5918 in mice results in the development of transplantable MM at high penetrance between 1-2 years of age. M-spikes developed in the animals as early as 2-3 months before death while Gm5918deficient kidneys showed light chain amyloidosis. 18F PET-CT of animals upon serum M-spike development revealed solitary medullary plasmacytomas or extensive skeletal remodeling, which we found to be accompanied by extramedullary disease on necropsy. Colony forming unit assays and serial competitive transplantation show the defect to be cell intrinsic and cytokine independent after malignant transformation, while IL-7 inducible before disease onset. CD138+ cells from sick animals showed derepression of PRC2 and H3K27me3 targets, and we observed reduced H3K27me3 in tumor-derived cell lines. C15orf65 homodimerizes and co-immunoprecipitates with histone H3 and EED, identifying it as a new class of histone-binding scaffold protein for PRC2-mediated methylation of H3K27. In human MM patients, low C15orf65 correlates with aggressive disease, short time to progression, and increased mortality. Re-expressing C15orf65 in human MM1.R cell lines inhibits growth and establishment post-passaging, while expression of DUF4490-mutant C15orf65 reverses the phenotype. We propose naming C15orf65 SCaffold for HIStone Modification 1 (SCHISM1) and the previously uncharacterized DUF4490 family as a Histone Tail Targeting PRC Scaffold (HTTPS) domain.

Abstract A144: The transcriptomic profile of peripheral T-cells that maintain dormant state of melanoma cells in patients treated with allogenic melanoma vaccine

Abstract Melanoma (MM) cells leave primary tumors early and evolve at different sites in parallel. However, timing in human MM is unknown. Lymphatic dissemination is fast shortly after the dermal invasion, but metastases may occur late. Such delay in metastases formation suggests possible host defense mechanism, or particular nature of non-actively proliferative (dormant) MM stem cell (MSC) population. T lymphocytes may also induce tumor dormancy. The proposed mechanism involves a cytostatic mode of tumor silencing resulting from the suppression of both neoplastic cell proliferation and cell cycle progression. Significant progress in the development of novel advanced MM therapies was made. However, not all patients benefit from the therapy, developing late metastases decades after treatment ends. Moreover, using the immunotherapy-based model of MM dormancy in mouse, vaccination against MM prevented tumor growth but failed to prevent tumor cell dissemination or elimination of all MM cells. We have developed therapeutic gene modified allogenic MM vaccine (AGI-101H) that has been tested since 1997 and resulted in long-term survival of a substantial fraction of patients. Genetic modification of vaccine cells to express designer cytokine cDNA encoding Hyper-IL-6 protein (comprising of IL-6 linked with a soluble IL-6 receptor) has altered AGI-101H cells phenotype towards MSC-like with high activity of aldehyde dehydrogenase isoenzyme (ALDH1A1). We have found significant induction of anti-MSCs response in immunized patients by a generation of functionally active cytotoxic CD8+T-cells and antibodies specific for ALDH1A1 in circulation. However, the AGI-101H mode of action is complex: besides inducing a direct anticancer response to MSC population, long-term vaccination may induce MM dormancy through T-cell mediated immunity. The above hypothesis is based on clinical observations of AHI-101H treated patient who was progression-free for many years, but injury induced metastases formation in the bruise. This patient developed skin MM in 1999. In 2002 in transit metastases appeared (resected). The patient was enrolled into AGI-101H program. In 2003 metastases in liver (resected), 2004 metastasis in ovary (resected). 11 years (2004-2015) no progression. In Nov. 2015 she had bicycle accident, trauma, and MM metastasis presenting with a bruise (resected). The treatment continues with no progression. The aim was to determine the molecular mechanisms of peripheral T-cell-mediated induction of MM dormancy induced by vaccination. Therefore, T-cell mRNA expression profiling of long-term surviving patients was carried out. PBMCs were isolated from treated patients (18), untreated MM patients (13) and healthy controls (8). Untouched T-cells were separated by magnetic beads and total RNA isolated. The transcriptome profiling was performed with Affymetrix HG U219 microarray (19,285 markers). Differential gene expression (DGE) analysis between all groups was conducted and validated with RT-qPCR and FACS. The transcriptomic results were further analyzed with Gene Set Enrichment Analysis (GSEA) tool. DGE analyses comparing AGI-101H vaccinated (AV) and nonimmunized (C) patients revealed 538 differentially expressed genes (DEGs), with 373 downregulated and 165 upregulated in AV (adj p &amp;lt; 0.05). Among these 538 markers, the expression of 14 (with |log2FC| &amp;gt; 1) is now being validated with RT-qPCR and FACS. Also, GSEA analysis revealed significant enrichment of “TNFa_signaling_via_NFkB”, “TGFb_signaling” and “G2/M_checkpoint” hallmark processes (MSigDB Hallmark gene sets) in AGI-101H vaccinated patients, that indicates considerable activation of antitumor response and prevention of conversion of MM dormanT-cells into proliferative metastases. Transcriptome profiling of peripheral T-cells in long-surviving MM patients immunized with AGI-101H suggests activation of the molecular mechanism that sustains MM cells in the non-actively proliferative dormant state. Citation Format: Andrzej A. Mackiewicz, Partycja Czerwinska, Marcin Rucinski, Katarzyna Gryska, Iga Grzadzielewska, Anna Jaworska, Jacek Mackiewicz. The transcriptomic profile of peripheral T-cells that maintain dormant state of melanoma cells in patients treated with allogenic melanoma vaccine [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A144.

New Cytotoxic Cyclic Peptide from the Marine Sponge-Associated Nocardiopsis sp. UR67.

A new cyclic hexapeptide, nocardiotide A (1), together with three known compounds—tryptophan (2), kynurenic acid (3), and 4-amino-3-methoxy benzoic acid (4)—were isolated and identified from the broth culture of Nocardiopsis sp. UR67 strain associated with the marine sponge Callyspongia sp. from the Red Sea. The structure elucidation of the isolated compounds were determined based on detailed spectroscopic data including 1D and 2D nuclear magnetic resonance (NMR) experimental analyses in combination with high resolution electrospray ionization mass spectrometry (HR-ESI-MS), while the absolute stereochemistry of all amino acids components of nocardiotide A (1) was deduced using Marfey’s method. Additionally, ten known metabolites were dereplicated using HR-ESI-MS analysis. Nocardiotide A (1) displayed significant cytotoxic effects towards the murine CT26 colon carcinoma, human HeLa cervix carcinoma, and human MM.1S multiple myeloma cell lines. The results obtained revealed sponge-associated Nocardiopsis as a substantial source of lead natural products with pronounced pharmacological activities.

Abstract 36: Preclinical models of multiple myeloma

Abstract Multiple myeloma is a clonal B-cell malignancy characterized by the accumulation of terminally differentiated, antibody-producing plasma cells in the bone marrow. Genetic mutations within the myeloma cells and their interaction with various cytokines and growth factors contribute to the invasiveness of the disease and enhanced drug resistance. Patients are generally asymptomatic until very late-state disease. However, once disease is present, localization in the bones, especially the spine, is common. There are few preclinical models that recapitulate human disease. In this work, we have evaluated the human MM.1S model and the murine 5TGM-1 model. MM.1S was derived from a 42-year-old African American woman and has been documented to express CD25, CD38, CD52, and CD59. It also expresses the glucocorticoid receptor and is dexamethasone sensitive. To more effectively monitor in vivo disease progression, we transfected the MM.1S line with luciferase (MM.1S-pMMP-LucNeo). With bioluminescence imaging (BLI) we track and monitor disseminated disease progression over time and find reproducible in vivo growth in SCID beige mice. The median tumor volume doubling time is ~2 days and median time to morbidity/mortality endpoint is 35-45 days. MM.1S-pMMP-LucNeo was also evaluated in NSG mice which show more aggressive disease onset (staging 7 days post implant vs. 15 days in SCID mice). In NSG mice the tumor volume doubling time is ~1.8 days and median time to endpoint is ~21 days, roughly half the overall survival time observed in the SCID mouse. In all mouse strains we have investigated, the animals are relatively asymptomatic throughout the lifetime of the study and only begin to present symptoms at late-stage disease, similar to how the disease manifests itself in humans. Common, late-stage clinical signs in the mice include lethargy and paralysis. BLI on these animals shows localization of the signal in the spine, long bones, and mandible. As in the clinic, bortezomib (Velcade®) has limited effect on disease progression in this model. Expression of CD138 is a hallmark of plasma cells and multiple myeloma cells. In the NSG study we determined, by flow cytometry, that &amp;gt;85% of the CD45- gate in the bone marrow was CD138+ 21 days post-implant, suggesting significant engraftment of the tumor cells within the bone marrow. 5TGM-1 is a syngeneic model generated from the C57BL/KaLwRij mouse which is predisposed to develop several monoclonal B-cell proliferative disorders, including a low percentage (0.5%) of the mice developing multiple myeloma. From this strain, several cell clones were isolated and called the 5TMM series, which included the 5T33MM clone. 5TGM-1 is a sub-clone of 5T33MM cells and we have used 5TGM-1-luc to monitor disease progression and localization in vivo. The 5TGM-1-luc model is aggressive and BLI has shown localization of disease in the long bones. We have compared this model in both the syngeneic C57BL/KaLwRij strain and the immune-deficient NIH III (triple deficient) strain. Disease progression behaves similarly in both strains of mice, with significant tumor cells localizing in the spine and the long bones. The model is sensitive to traditional chemotherapies like doxorubicin and cyclophosphamide, but bortezomib (Velcade) only produces modest activity and carfilzomib has shown little activity at the dose and schedule tested. A greater understanding of the preclinical behavior of these models, including response to standard of care and molecular profiling, will provide critical insight into designing more clinically relevant preclinical experiments. Citation Format: Erin Trachet, Maryland Rosenfeld Franklin. Preclinical models of multiple myeloma [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 36.