Preclinical Models Of Multiple Myeloma Research Articles

Abstract 1237: IGM-2644, a CD38xCD3 bispecific IgM T cell engager, shows enhanced anti-tumor activity compared to daratumumab in preclinical models of multiple myeloma

Abstract Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow. CD38 is an attractive therapeutic target for MM due to its high expression on plasma cells, but it is also expressed on multiple subsets of normal immune cells. Approved therapies including anti-CD38 monoclonal antibodies such as daratumumab are available, however resistance inevitably develops, and toxicities such as reduced immune cell counts put patients at increased risk for infections. To address this and other issues associated with current therapies, we are developing IGM-2644, an engineered high affinity, high avidity bispecific anti-CD38xCD3 IgM T cell engager (TCE) antibody for the treatment of plasma cell disorders. IGM-2644 kills CD38-expressing tumor cells through complement-dependent cytotoxicity (CDC) and T cell-dependent cellular cytotoxicity (TDCC). Live cell imaging of tumor cells in the presence of normal human serum as a source of complement has shown that IGM-2644, compared to daratumumab, has exhibited superior CDC with faster kinetics and higher maximal killing, as well as increased activity on tumor cell lines that express low levels of CD38. IGM-2644 also has displayed enhanced cellular dependent cytotoxicity over daratumumab across tumor cell lines with a range of CD38 expression levels in vitro. In ex vivo assays with MM patient bone marrow mononuclear cells (BMMC), IGM-2644 has shown superior tumor cell depletion compared to daratumumab even with low T cell to tumor cell ratios. Furthermore, IGM-2644 can deplete tumor cells in BMMC from daratumumab-refractory MM patients. Similarly, using in vivo humanized xenograft tumor mouse models with various levels of CD38 expression, greater anti-tumor activity with IGM-2644 over daratumumab was observed. When compared to daratumumab, IGM-2644 demonstrated lower immune cell fratricide in an in vivo xenograft tumor mouse model, suggesting a potentially differentiated safety profile. In summary, IGM-2644 is a bispecific anti-CD38xCD3 IgM TCE antibody that demonstrates superior tumor cell killing when compared to daratumumab in vitro and especially in xenograft tumor models in vivo with low CD38 expression while having low immune cell depletion, potentially reducing the risk of infections. The safety and efficacy of IGM-2644 has been initially studied in a phase 1 clinical trial for relapsed/refractory (r/r) MM. Applicability of IGM-2644 for the treatment of other plasma cell disorders is currently being explored, including autoimmune diseases. Citation Format: Kevin C. Hart, Daniel Santos, Keyu Li, Rui Yun, Min Chai, Andras Kabai, Gene Li, Poonam Yakkundi, Roel P. Funke, Yinghui Guan, Thomas Manley, Liqin Liu, Angus M. Sinclair, Albert F. Candia, Bruce Keyt, Maya F. Kotturi. IGM-2644, a CD38xCD3 bispecific IgM T cell engager, shows enhanced anti-tumor activity compared to daratumumab in preclinical models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1237.

Designing combination therapies for cancer treatment: application of a mathematical framework combining CAR T-cell immunotherapy and targeted radionuclide therapy.

Cancer combination treatments involving immunotherapies with targeted radiation therapy are at the forefront of treating cancers. However, dosing and scheduling of these therapies pose a challenge. Mathematical models provide a unique way of optimizing these therapies. Using a preclinical model of multiple myeloma as an example, we demonstrate the capability of a mathematical model to combine these therapies to achieve maximum response, defined as delay in tumor growth. Data from mice studies with targeted radionuclide therapy (TRT) and chimeric antigen receptor (CAR)-T cell monotherapies and combinations with different intervals between them was used to calibrate mathematical model parameters. The dependence of progression-free survival (PFS), overall survival (OS), and the time to minimum tumor burden on dosing and scheduling was evaluated. Different dosing and scheduling schemes were evaluated to maximize the PFS and optimize timings of TRT and CAR-T cell therapies. Therapy intervals that were too close or too far apart are shown to be detrimental to the therapeutic efficacy, as TRT too close to CAR-T cell therapy results in radiation related CAR-T cell killing while the therapies being too far apart result in tumor regrowth, negatively impacting tumor control and survival. We show that splitting a dose of TRT or CAR-T cells when administered in combination is advantageous only if the first therapy delivered can produce a significant benefit as a monotherapy. Mathematical models are crucial tools for optimizing the delivery of cancer combination therapy regimens with application along the lines of achieving cure, maximizing survival or minimizing toxicity.

Early Intervention with Celmods, but Not Imids, Prevents Relapse to Forimtamig Driven By GPRC5D-Negative Myeloma Cells

Despite novel therapeutic options leading to a substantial increase in survival rates, multiple myeloma (MM) still represents an incurable disease. T cell bispecific antibodies (TCBs) have become a novel therapeutic option for relapsed refractory myeloma (RRMM) patients based on their promising objective response rates (ORR), favorable safety profile and off-the-shelf availability as compared to CAR-T cell therapies. Although BCMA- and GPRC5D-targeted TCBs have been reported to induce deep clinical responses, antigen drift represents a tumor intrinsic resistance mechanism limiting durability of responses 1. Here, we report that the combination of forimtamig, a 2:1 GPRC5D-targeted TCB, with CELMoDs, but not IMIDs, prevents tumor relapse driven by target-negative myeloma cells in a preclinical model of multiple myeloma. In order to mimic acquired TCB resistance in vivo, stem-cell humanized mice were engrafted with NCI-H929 tumors and treated once weekly (QW) with fixed duration forimtamig at 0.1 mg/kg for 6 cycles with a treatment-free follow up of more than 2 weeks. Single administration of forimtamig reduced tumor load by 80% at the end of cycle 1 (C1). At the end of cycle 4 (C4), all mice achieved a complete response (CR) with no detectable tumors. However, between C4 and cycle 6 (C6), tumor escape was observed in 47% of animals and progression free survival rate (PFS) at study termination was 53%. Relapsed tumors were subjected to quantitative immunohistochemistry and while prevalence for BCMA was comparable to baseline, GPRC5D expression was lost. To evaluate if cereblon modulation represents a strategy to improve PFS, we combined fixed-duration forimtamig with either pomalidomide (pom) or iberdomide (iber) at C1 day 1 (C1D1). Combination with pom and iber increased anti-tumor response compared to monotherapy during C1 deleting 88% and 92% of tumors, respectively. PFS for the combination with pom was not improved (~50%) as tumor escape was observed after treatment was stopped at C6. In contrast, no tumor relapse was observed for the combination with iber increasing PFS to 100%. Notably, as compared to forimtamig monotherapy, combination with pom and iber further increased serum levels of IFNg, IP-10, IL-2 and TNFa 48h after TCB dosing. We next explored if a sequential start of the combination would impact PFS and cytokine release and started co-administration at C3 instead of C1. Forimtamig induced strong tumor regressions in 90% of animals during C1-C4, but tumor escape was observed in 100% of animals starting after C4. When combined with pom or iber, PFS rates increased to 30% and 60%, respectively. Overall cytokine release was significantly lower compared to C1 and neither addition of pom nor iber led to an increase in cytokine production as compared to forimtamig monotherapy. To explore the potential of CELMoD combination at significantly lower TCB starting and target doses, we introduced step up dosing for forimtamig reflecting clinically relevant exposures and combined with thrice weekly (Q3W) or five time a week (Q5W) dosing of mezigdomide (mezi) starting at C1D1. Although forimtamig induced transient tumor regressions at C1D1 as well C1D15, mice showed progressive disease at the end of C1 and only 20% of mice achieved a CR at the end of the study. In contrast, combination with mezi led to rapid onset of tumor regression during C1 correlating with significantly improved PFS rates of 80% (Q3W) and 100% (Q5W). Cytokine release was measured after each step up and target dose and a maximal 2-fold increase in combination with mezi was detected. Interestingly, in contrast to IL-2 or IP-10, serum levels of IL-8 as well as MIP-1a decreased for the Q3W mezi schedule. Taken together, our data suggest that combination with CELMoDs but not IMIDs can prevent relapse to forimtamig driven by GPRC5D negative tumor cells. We confirm timing of intervention with CELMoDs to have an significant impact on PFS and cytokine release and suggest a broad therapeutic window using low dose forimtamig and intermittent dosing of iberdomide or mezigdomide.

Bivalent BCMA Binding and Low Affinity CD3 T-Cell Engagement By Abbv-383 Drives Sustained Activation with Reduced T-Cell Exhaustion in Preclinical Models of Multiple Myeloma

Introduction: ABBV-383 is an IgG4-based CD3-bispecific T-cell engager (TCE) that has shown promising efficacy and a favorable safety profile in heavily pre-treated patients with relapsed/refractory multiple myeloma (MM). ABBV-383 possesses a unique combination of a low-affinity CD3δε antibody coupled to a bivalent, high-affinity BCMA engager arm in a 2+1 format. Prior pre-clinical studies demonstrated that ABBV-383 induces potent redirected T-cell killing of MM tumors with reduced cytokine release. Here, we provide additional evidence that bivalent BCMA binding by ABBV-383 reduces the negative impact of soluble BCMA and low affinity CD3 T-cell engagement drives sustained T-cell activation with reduced immune exhaustion. Methods: Structural variants of ABBV-383 were created, including 1) the bivalent BCMA targeting antibody of ABBV-383 replaced by proximal and distal monovalent VH targeting molecules, and 2) BCMA antibody structures coupled to high-and low affinity CD3 variants ( Figure). For functional testing, ABBV-383 and the structural variants were added to PBMCs or T-cells from healthy donors to serve as effector cells in co-culture with BCMA-positive MM tumor cell lines. Tumor cell killing and T-cell activation were determined by flow cytometry, and cytokine production was assessed using Luminex instrumentation. Recombinant soluble BCMA (sBCMA) was utilized in the experimental models to evaluate the extent of BCMA TCE suppression. To define the relationship between CD3-affinity and T-cell exhaustion, multi-dimensional flow cytometry was performed in long-term, 28-day co-cultures with irradiated tumor cells replated every 7 days. Results: T-cell binding was shifted by >100x with substitution of the high affinity CD3 antibody compared to ABBV-383. While all antibody variants tested induced T-cell specific MM cell killing in a BCMA-dependent manner, the negative impact of soluble BCMA on tumor killing was reduced with ABBV-383 due to its bivalent interaction with tumor cells compared to the monovalent TCE formats. In addition, the low CD3 binding affinity of ABBV-383 lowered T-cell activation antigen expression and reduced inflammatory cytokine production but resulted in similar maximal cytotoxicity of tumor cells in co-cultures of healthy and MM patient samples compared to high-affinity CD3 targeting. While maximal tumor cytotoxicity was similar, ABBV-383 was found to significantly delay the induction of a critical exhaustion-inducing transcription factor, TOX, in long-term assays. T-cell population changes were observed as early as 3 days with decreased effector memory cells (CD45RA-CD62L-) and increased naïve T-cell populations (CD62L+CD45RA+) compared to high-affinity CD3 targeting. On day 21, the high-affinity CD3 x bivalent BCMA molecule resulted in 85% of the CD8+ T-cell population expressing the transcription factor TOX compared to only 15% for ABBV-383 co-cultures. TOX induces several gene families with chromatin-modulating capacity that drive the distinct transcriptional trajectory leading to T-cell dysfunction during chronic stimulation. Simultaneous expression of multiple checkpoint inhibitory proteins is indicative of T-cell exhaustion. Triple positive CD8+ T-cells expressing LAG3, PD-1 and CTLA4 were significantly reduced by ABBV-383 versus the high-affinity CD3 variant ( Figure). Conclusions: High avidity BCMA binding coupled to low affinity T-cell engagement distinguishes the resulting mechanism of action for ABBV-383 in MM. Here, we show that the presence of bivalent-BCMA reduced the negative impact of soluble BCMA, reduced inflammatory cytokine production, and dampened the emergence of TOX+ CD8+ T-cells indicating that ABBV-383 structure maximizes its clinical potential as a safe and effective MM therapy.

Differential Chromatin Organization between t(4;14) and Non-t(4;14) Multiple Myeloma Driven By the Histone Methyltransferase NSD2

Background: The incidence of multiple myeloma (MM) with t(4;14) translocation is ~15% of newly diagnosed MM (NDMM), and is associated with poor prognosis and high-risk disease. Recent analysis of t(4;14) genomics indicate that the location of the chromosome 4 (chr4) breakpoint can significantly segregate patient outcome regardless of therapeutic intervention. Translocation of IGH regulatory elements from chr14, drive the expression of NSD2 (chr4), a histone (H3) methyltransferase responsible for di-methylation of H3 lysine 36 (H3K36me2), a mark of active chromatin. Depending upon the location of the chr4 breakpoint, different isoforms of NSD2 are expressed, including full-length (NSD2-FL) and various N-terminus truncations (NSD2-TR). The mechanisms of how the location of the chr4 breakpoint and the functionalities of NSD2 isoforms may contribute to a higher risk biology in t(4;14) MM patients is not known. Here we compare and contrast the 3D chromosomal organization of t(4;14)-NSD2-FL, t(4;14)-NSD2-TR and non-t(4;14) in pre-clinical MM models. Results: Comparative analysis of chromosome conformation capture (Hi-C) indicates that there is a decrease in medium-to-long range chromosomal looping interactions accompanied by an increase in short-range interactions from t(4;14) vs. non-t(4;14) models (Figure A). Accordingly, relative quantification of chromosomal contact probabilities of intra-chromosomal (cis) contacts increase in frequency from non-t(4;14) (77%) vs. t(4;14)-NSD2-FL (87%) vs. t(4;14)-NSD2-TR (89%). Evaluation of active/inactive (A/B) 3D genomic compartments between non-t(4;14) and t(4;14) models suggest significant compartment switches (~30% genome; combined A-to-B and B-to-A). Analysis of gene neighborhoods through topologically associated domains (TADs) suggests that there are decreases in contacts at the TAD boundary regions in t(4;14)-NSD2-TR vs. t(4;14)-NSD2-FL vs. non-t(4;14) models. TAD sizes are also generally smaller in size, from t(4;14)-NSD2-TR vs. t(4;14)-NSD2-FL vs. non-t(4;14). Complimentary analysis of activity by contact (ABC) to assess the potential influence of proximal and distal enhancers on gene activity confirms there is a decrease in ABC scores when comparing t(4;14) vs. non-t(4;14). Assessment of global chromatin accessibility (ATAC-Seq) indicate significant consensus open peaks (~135k), differentially accessible (DA) peaks (~2.6k), and differential closed peaks (~8.8k) in t(4;14) vs. non-t(4;14). Principal component (PC) analysis demonstrated a unique grouping of t(4;14) models compared to a very heterogenous distribution of non-t(4;14) models. DA regions in t(4;14) were found to be primarily more enriched in both intronic and intergenic regions, and within proximal and distal promoters. Analysis of histone marks was used to assess differences in global active enhancers (H3K4me1, H3K27ac), repressive chromatin (H3K9me3, H3K27me3), and active transcription (H3K36me2/3, H3K4me3) between non-t(4;14) and t(4;14). Active marks were shown to be increased, and repressive marks decreased in t(4;14) vs. non-t(4;14) models that also correlated with DA peaks. Enhancer ranking suggests the frequency of potentially poised superenhancers increase in t(4;14) vs. non-t(4;14). Differential expression (DE) analysis between t(4;14) and non-t(4;14) was highly correlative with DA peaks (Figure B). In addition, distinctive transcriptional signatures between non-t(4;14), t(4;14)-NSD2-FL, and t(4;14)-NSD2-TR were identified. Conservative (DA+DE) pathway enrichment suggests multiple and distinct biological processes including cell adhesion through adherens junctions, cell-cycle regulation through mitotic checkpoints, chromosomal reorganization through depolymerization of the nuclear lamina, and epithelial to mesenchymal transition. Conclusions: Systematic chromosomal re-arrangement and accessibility driven by differential epigenetic deposition of histone regulatory marks, differentiates t(4;14) from non-t(4;14) MM biology. These analyses suggest that given the nature of the translocation, and additionally the location of the chr4 breakpoint, result in major 3D chromosomal structural rearrangement, mediating biological programs that may explain the differences between successful and poor interventional therapies for this subset of MM disease.

OA-09 Combination of forimtamig with standard of care improves depth and duration of response in preclinical models of multiple myeloma

OA-09 Combination of forimtamig with standard of care improves depth and duration of response in preclinical models of multiple myeloma

CFT7455, a novel IKZF1/3 degrader, demonstrates potent activity in peripheral and CNS models of NHL as a single agent or in combination with clinically approved agents

Immunomodulatory drugs, such as pomalidomide and lenalidomide are approved for the treatment of multiple subtypes of NHL. These drugs induce an interaction between Ikaros family zinc finger proteins 1 and 3 (IKZF1/3) and cereblon (CRBN), an E3 ligase, resulting in IKZF1/3 degradation. Clinically, immunomodulatory drugs are active as single agents or when used in combination with several classes of targeted therapies, in both first line and relapsed/refractory NHL subtypes. We previously described the preclinical characterization of CFT7455 as a novel IKZF1/3 degrader with 800-fold increased potency over pomalidomide in CRBN binding. We demonstrated that this increased activity translates into dramatically greater efficacy compared to pomalidomide in cellular and in vivo preclinical models of multiple myeloma and NHL. Here we show that the increased catalytic activity and improved potency of CFT7455 also translates into potent anti-tumor activity as a single agent or in combination with clinically approved agents in multiple in vivo models of NHL. In addition, the enhanced activity of CFT7455 demonstrates CNS activity in both the Raji and OCI-Ly10 intracranial xenograft models. CFT7455 (100 µg/kg/day) led to a significant increase in survival probability in comparison to pomalidomide (3000 µg/kg/day) and two investigational IKZF1/3 degraders in both models. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Aggressive T-cell non-Hodgkin lymphoma, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. M. J. Thomenius Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics S. Perino Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics J. Kirby Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics R. Agafonov Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics P. Chaturvedi Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics S. Eron Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics A. Good Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics J. A. Hart Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics M. He Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics A. Phillips Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics D. A. Proia Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics J. A. Henderson Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics C. Nasveschuk Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics S. L. Fisher Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics R. M. Pollock Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics

Abstract ND09: M3913 induces the maladaptive unfolded protein response through a novel mechanism resulting in strong anti-tumor activity

Abstract Therapeutic strategies based on exacerbated stress signaling may represent novel and effective treatment paradigms in oncology. Here, we disclose for the first time the endoplasmic reticulum (ER) stress modulator (ERSM) M3913 that was discovered and developed following a phenotypic screening campaign. Mechanistically, M3913 engages an ER transmembrane protein not yet implicated in cancer biology to induce a Ca2+ shift from the ER towards the cytoplasm, resulting in an unfolded protein response and subsequent antitumor activity in sensitive preclinical models. We detected concurrent elevation of bona fide ER stress markers by several methods including qRT-PCR, Western Blotting analysis, and unbiased transcriptome analysis. Genetic removal of M3913’s putative ER transmembrane target abrogated the M3913-mediated, but not the Thapsigargin-mediated, ER stress response. PK/PD studies confirmed a dose- and time-dependent upregulation of ER stress markers in preclinical cancer models in vivo. As a monotherapy, M3913 induced full and partial tumor regression in preclinical models of multiple myeloma, non-small-cell lung cancer, triple-negative breast cancer, and other cancers. An in vitro combination screen indicated combination potential of M3913 with standard-of-care agents and novel candidate drugs. IND-enabling toxicology and additional mechanism-of-action studies in rat and minipig uncovered specific target organs for M3913 in line with expression of the target in human tissues. This presentation details mechanistic profiling data from M3913, reveals its putative ER-resident target and discusses forward translation studies that support M3913 as a novel therapeutic option for hard-to-treat subtypes of a variety of cancers. Citation Format: Frank Czauderna, Richard Schneider, Shivapriya Ramaswamy, Susanne Brandstetter, Elise Drouin, Olga Bogatyrova, Johanna Mazur, Qing Sun, Catherine Jorand-Lebrun, Eva Sherbetjian, Jonny Nachtigall, Carolyn Wing, Christian Hildebrand, Peter Ellinghaus, Isabelle Lemoine, Mac Johnson, Russell Hoover, Michael Clark, Ralph Lindemann. M3913 induces the maladaptive unfolded protein response through a novel mechanism resulting in strong anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND09.

Abstract 6253: SP-3164, a novel molecular glue degrader with activity in preclinical models of multiple myeloma

Abstract Multiple myeloma (MM) makes up approximately 10% of all hematologic malignancies, with increasing incidence worldwide. Though treatment options for newly diagnosed patients have evolved in recent years, they are not curative, and relapse is common with 2,500-3,000 relapsed or refractory (R/R) MM US patients seeking novel treatments, annually. Cereblon (CRBN)-binding Ikaros and Aiolos (I/A) protein degraders (i.e., lenalidomide (LEN) and pomalidomide (POM)) have had great success in treating MM. However, due to the unstable chiral nature of the CRBN-binding moiety, common to these molecules, many exist as racemic mixtures (1:1 mixture of interconverting (R)- and (S)-enantiomers). A growing body of evidence suggests that the (S)- enantiomer is the active anticancer species, while the (R)-enantiomer is inactive and potentially results in undesired activity. The ability to stabilize the active (S)- enantiomer represents a potential strategy to improve therapeutic outcomes. We are developing SP-3164, the deuterium-stabilized (S)-enantiomer of avadomide (AVA), an I/A-degrading molecular glue which has been studied in over 400 patients, demonstrating safety and activity in hematologic diseases. Our in vitro data demonstrate that SP-3164 more potently and rapidly degrades the cancer-promoting transcription factors, I/A, compared to LEN, POM, and AVA, while the stabilized (R)-enantiomer, SP-3165, neither binds CRBN nor degrades I/A. In preliminary in vivo studies, SP-3164 elicited significant tumor growth inhibition (TGI). Here, we investigate the activity of SP-3164 in MM, compared to established treatment regimens. SP-3164’s potential to degrade Aiolos in MM.1S cells was compared to SP-3165, AVA, and POM. SP-3164 rapidly and robustly degraded Aiolos in a time- and dose-dependent manner, more potently than AVA and POM, while SP-3165 demonstrated minimal Aiolos degradation. To investigate the effect of SP-3164 on cell survival, we treated NCI-H929 cells with SP-3164 (0.1 µM, 1 µM, and 10 µM) and SP-3165 (1 µM) and assessed apoptosis by flow cytometry. SP-3164 induced dose-dependent early and late apoptosis after 72 hours, while SP-3165 showed no induction of apoptosis. SP-3164’s in vivo activity was assessed and compared to SP-3165, LEN, and POM in an NCI-H929 mouse xenograft model. SP-3164 showed significant TGI as a single agent while SP-3165 seemed to support tumor growth. LEN and POM demonstrated minimal TGI. When combined with dexamethasone (DEX), SP-3164 had improved TGI compared to the combination of POM+DEX. These preclinical data demonstrate that SP-3164 is a potent CRBN-binding molecular glue with the ability to rapidly degrade cancer-promoting transcription factors and induce apoptosis in vitro. In vivo, SP-3164 has superior single agent and combination activity compared to approved molecular glues. These data support the continued development as a novel therapy in MM. Citation Format: Aundrietta D. Duncan, Daniela Y. Santiesteban, Nadeem Q. Mirza, Steve Horrigan, Sheila DeWitt, Vincent Jacques, P. Leif Bergsagel. SP-3164, a novel molecular glue degrader with activity in preclinical models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6253.

Abstract 6268: TT125-802 is a potent and highly selective CBP/p300 bromodomain inhibitor for the treatment of castration resistant prostate cancer and haematological malignancies

Abstract The paralogous lysine acetyltransferases CREB-binding protein (CBP) and p300 are key epigenetic regulators involved in diverse signaling pathways in cancer. The bromodomain (BRD) of CBP/p300 serves as an acetyl-lysine “reader” that allows CBP/p300 to bind chromatin at acetylated histone and non-histone proteins leading to the regulation of gene transcription. Indeed, CBP/p300 are critical co-activators of nuclear receptors, including the androgen receptor (AR) in castration resistant prostate cancer (CRPC). Thus, inhibition of CBP and p300 is an emerging therapeutic strategy to block the transactivation activity of the AR in CRPC. In addition, inhibition of the CBP/p300 BRD has been described as a potential therapeutic strategy to treat multiple myeloma (MM) through transcriptional suppression of interferon regulatory factor 4 (IRF4) and concomitant repression of its target genes MYC and MYB. TT125-802 is a highly selective and potent, oral small molecule inhibitor of the BRD of CBP/p300. In a BROMOscan assay against a panel of 40 BRD-containing proteins, TT125-802 revealed the unique selectivity to the BRD of CBP/p300 with a minimal off-target binding to all other BRDs, including BET proteins.TT125-802 shows selective anti-proliferative activity in AR-dependent prostate cancer cell lines (22Rv-1, C4-2, and LNCaP) and inhibits AR target gene expression (KLK2, KLK3, TMPRSS2, and MYC) in a dose-dependent manner (IC50 of 2 to 33 nM). AR-negative prostate cancer cell lines (DU-145 and PC-3) are insensitive to TT125-802 in vitro, pointing to an AR-selective mode of action. In an in vivo model of CRPC, TT125-802 inhibited tumor growth when administered orally to human C4-2 xenograft-bearing mice. Daily dosing of TT125-802 was well tolerated with stable bodyweights and platelet counts. In addition, preclinical studies in CRPC patient-derived xenograft (PDX)-bearing mice showed that the combination treatment of TT125-802 and enzalutamide had a synergistic effect on tumor growth inhibition compared to single agent treatments. TT125-802 reduced mRNA expression of the AR-target genes in tumor samples and decreased plasma PSA levels compared to enzalutamide alone, and the combination reduced levels even further. In a preclinical model of MM (OPM2), TT125-802 had a dose-dependent effect on tumor growth, inducing tumor regressions at the highest dose. Target genes such as MYC, MYB, and IRF4 were potently downregulated in tumors. We conclude that TT125-802 is a novel, highly selective inhibitor of the BRD of CBP/P300. It has therapeutic potential as monotherapy in prostate cancer and multiple myeloma and in combination with next-generation AR inhibitors for patients with lethal prostate cancer. A FIH study of TT125-802 in cancer patients is on track to start in 2023. Citation Format: Sara Laudato, Dorothea Gruber, Thomas Bohnacker, Martin Schwill, Charles-Henry Fabritius, Raquel Herrador, Katrin Westritschnig, Thushara Pattupara, Vikram Ayinampudi, Stefanie Flückiger-Mangual. TT125-802 is a potent and highly selective CBP/p300 bromodomain inhibitor for the treatment of castration resistant prostate cancer and haematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6268.

PARP1 and POLD2 as prognostic biomarkers for multiple myeloma in autologous stem cell transplant.

Multiple Myeloma (MM) is an incurable plasma cell malignancy often treated by autologous stem cell transplant (ASCT). Clinical response to ASCT has been associated with DNA repair efficiency. Here we interrogated the role of the base excision DNA repair (BER) pathway in MM response to ASCT. Across 450 clinical samples and six disease stages, expression levels of genes in the BER pathway were found to be highly upregulated during the development of MM. In a separate cohort of 559 patients with MM treated with ASCT, expression of BER pathway members MPG and PARP3 was positively associated with overall survival (OS) while expression of PARP1, POLD1, and POLD2 was negatively associated with OS. In a validation cohort of 356 patients with MM treated with ASCT, PARP1 and POLD2 findings were replicated. In patients with MM who never received ASCT (n=319), PARP1 and POLD2 were not associated with OS, suggesting that the prognostic effect of these genes may be treatment-dependent. In preclinical models of MM, synergy was observed in anti-tumor activity when poly (ADPribose) polymerase (PARP) inhibitors (olaparib, talazoparib) were used in combination with melphalan. The negative prognosis associated with PARP1 and POLD2 expression along with the apparent melphalan-sensitizing effect of PARP inhibition may suggest this pathway as a potential biomarker in patients with MM in the setting of ASCT. Further understanding of the role of the BER pathway in MM is vital to improve therapeutic strategies related to ASCT.

BRD9 Degradation Disrupts Ribosome Biogenesis in Multiple Myeloma.

BRD9 is a defining component of the noncanonical SWI/SNF complex, which regulates gene expression by controlling chromatin dynamics. Although recent studies have found an oncogenic role for BRD9 in multiple cancer types including multiple myeloma, its clinical significance and oncogenic mechanism have not yet been elucidated. Here, we sought to identify the clinical and biological impact of BRD9 in multiple myeloma, which may contribute to the development of novel therapeutic strategies. We performed integrated analyses of BRD9 in vitro and in vivo using multiple myeloma cell lines and primary multiple myeloma cells in established preclinical models, which identified the molecular functions of BRD9 contributing to multiple myeloma cell survival. We found that high BRD9 expression was a poor prognostic factor in multiple myeloma. Depleting BRD9 by genetic (shRNA) and pharmacologic (dBRD9-A; proteolysis-targeting chimera; BRD9 degrader) approaches downregulated ribosome biogenesis genes, decreased the expression of the master regulator MYC, and disrupted the protein-synthesis maintenance machinery, thereby inhibiting multiple myeloma cell growth in vitro and in vivo in preclinical models. Importantly, we identified that the expression of ribosome biogenesis genes was associated with the disease progression and prognosis of patients with multiple myeloma. Our results suggest that BRD9 promotes gene expression by predominantly occupying the promoter regions of ribosome biogenesis genes and cooperating with BRD4 to enhance the transcriptional function of MYC. Our study identifies and validates BRD9 as a novel therapeutic target in preclinical models of multiple myeloma, which provides the framework for the clinical evaluation of BRD9 degraders to improve patient outcome.

A Review of The Synergistic Effects of Curcumin with Proteasome Inhibitors in Multiple Myeloma Preclinical Models.

Proteasome inhibitors are the cornerstone of multiple myeloma treatment, but challenges still remain despite the increased survival rates. We conducted a review on the role of curcumin, a natural product, as an adjunct to bortezomib and carfilzomib in preclinical multiple myeloma models. Four studies reviewed showed enhanced anticancer effects when curcumin was combined with bortezomib compared to either treatment alone. Two additional studies showed similar results with carfilzomib. Synergistic mechanisms include inhibition of NF-kB, IL-6-induced signaling pathways, JNK pathway modulation, and increased cell cycle arrest.

Regulation of bone homeostasis by MERTK and TYRO3

The fine equilibrium of bone homeostasis is maintained by bone-forming osteoblasts and bone-resorbing osteoclasts. Here, we show that TAM receptors MERTK and TYRO3 exert reciprocal effects in osteoblast biology: Osteoblast-targeted deletion of MERTK promotes increased bone mass in healthy mice and mice with cancer-induced bone loss, whereas knockout of TYRO3 in osteoblasts shows the opposite phenotype. Functionally, the interaction of MERTK with its ligand PROS1 negatively regulates osteoblast differentiation via inducing the VAV2-RHOA-ROCK axis leading to increased cell contractility and motility while TYRO3 antagonizes this effect. Consequently, pharmacologic MERTK blockade by the small molecule inhibitor R992 increases osteoblast numbers and bone formation in mice. Furthermore, R992 counteracts cancer-induced bone loss, reduces bone metastasis and prolongs survival in preclinical models of multiple myeloma, breast- and lung cancer. In summary, MERTK and TYRO3 represent potent regulators of bone homeostasis with cell-type specific functions and MERTK blockade represents an osteoanabolic therapy with implications in cancer and beyond.

CFT7455, a Novel IKZF1/3 Degrader, Demonstrates Potent Anti-Tumor Activity in Models of Non-Hodgkin's Lymphoma As a Single Agent or in Combination with Clinically Approved Agents

Immunomodulatory drugs, such as pomalidomide and lenalidomide are approved for the treatment of multiple subtypes of non-Hodgkin's lymphoma (NHL). These drugs induce an interaction between Ikaros family zinc finger proteins 1 and 3 (IKZF1/3) and cereblon (CRBN), an E3 ligase, resulting in IKZF1/3 degradation. IKZF1/3 are master transcription factors that play a key role in B and T-cell differentiation and maintain levels of interferon regulatory factor 4 (IRF4), a critical regulator in NHL. Clinically, immunomodulatory drugs have shown activity as single agents and when used in combination with several classes of targeted therapies, such as rituximab, in both first line and relapsed/refractory NHL subtypes. We previously described the preclinical characterization of CFT7455 as a novel IKZF1/3 degrader with 800-1600-fold increased potency over pomalidomide in CRBN binding and cellular IKZF1 degradation assays. We demonstrated that this increased activity translated into dramatically greater efficacy compared to pomalidomide in cellular and in vivo preclinical models of multiple myeloma and NHL. Here we show that the increased catalytic activity and improved potency of CFT7455 also translates into potent anti-tumor activity as single agent or in combination with clinically approved agents in central nervous system (CNS) and subcutaneous models of NHL. In both the Raji and OCI-Ly10 CNS xenograft models, CFT7455 (100 µg/kg/day) led to a significant increase in survival in comparison to pomalidomide (P=0.002, P=0.0002). The combination of immunomodulatory drugs with CD20, BTK, or HDAC targeting agents shows promising clinical activity in the treatment of NHL. Combination studies were conducted, dosing CFT7455 in combination with rituximab, ibrutinib, or the HDAC inhibitor romidepsin in several models of NHL. In the mantle cell lymphoma (MCL) model, Mino, the combination of CFT7455 with rituximab demonstrated synergistic activity and complete tumor regression. In the diffuse large B-cell lymphoma (DLBCL) model, TMD8, the combination of ibrutinib and CFT7455 was synergistic and resulted in a significant increase in survival probability. In the CNS NHL model, OCI-Ly10, the combination of CFT7455 with rituximab or ibrutinib also led to a significant increase in survival probability highlighting the therapeutic potential of this combination. Synergistic activity was also observed with the combination of CFT7455 and romidepsin in two models of anaplastic large cell lymphoma (ALCL). In conclusion, CFT7455 is a potent, selective catalytic degrader of IKZF1/3, with single agent and combination antitumor activity in DLBCL, ALCL, and MCL models, supporting clinical investigation of CFT7455 for NHL.

Phase I Study of a Novel Oral Proteasome Inhibitor TQB3602 in Relapsed/Refractory Multiple Myeloma

Background: TQB3602 is a novel oral proteasome inhibitor with an acridine ring in addition to the main structure of ixazomib, which maintains better metabolic stability and higher activity and selectivity in preclinical models of multiple myeloma. TQB3602 consists of the L-malate ester with active boronic acid compound WXFL10410333 selectively targeting 20S proteasome enzyme. This is the first-in-human study to evaluate the safety, tolerability and pharmacokinetics of TQB3602 and assess the efficacy of TQB3602 combined with immunomodulators/dexamethasone regimen in relapsed/refractory multiple myeloma (RRMM). Study Design and Methods: This study is a multi-center open-label Phase I study in RRMM, consisting of the dose escalation phase and dose expansion phase. The inclusion criteria mainly include: 1) 18 to 75 years old male or female, 2) ECOG score of 0 to 2, 3) Life expectancy ≥12 weeks, 4) RRMM had received at least two treatment regimens (including lenalidomide or thalidomide, bortezomib, and glucocorticoid). The exclusion criteria mainly include: 1) grade ≥ 2 peripheral nerve diseases, 2) grade > 1 diarrhea during the screening period, 3) ixazomib has been administered less than five half-lives before the first dose of TQB3602, 4) previous allogeneic hematopoietic stem cell transplantation. TQB3602 was administered alone in 3+3 dose escalation (at a starting dose of 0.5mg on days 1, 8 and 15 in a 28-day cycle) until toxicity or disease progression in the dose escalation phase. According to prior lines of therapy, patients with RRMM were divided into two dose expansion cohorts, including TQB3602/lenalidomide/dexamethasone and TQB306/pomalidomide/dexamethasone (TQB3602 6mg on days 1, 8, 15, lenalidomide 25mg daily or pomalidomide 4mg daily on days 1 to 21, dexamethasone 20mg on days 1-2, 8-9, 15-16, 22-23 in a 28-day cycle). Results: The study enrolled 56 patients with RRMM including 25 patients in the dose escalation and 31 patients in the dose expansion between May 28, 2020 and July 20, 2022. The demographics and baseline characteristics were shown in table 1. Patients received single-agent TQB 3602 0.5mg to 7 mg on days 1, 8 and 15 of a 28-day cycle and two dose-limiting toxicities occurred at 7 mg, including diarrhea and grade 2 peripheral neuropathy. The maximum tolerated dose of TQB3602 was determined as 6.0mg. The terminal half-life was 23 to 152 hours after multiple dosing and the mean half-life was approximately 82 hours for 6mg TQB3602. The dose expansion cohorts recruited 29 RRMM patients receiving TQB3602/pomalidomide/dexamethasone regimen and two RRMM patients receiving TQB3602/lenalidomide/dexamethasone regimen. Among 14 response-evaluable patients in the dose expansion cohorts, eight RRMM (57%) achieved partial response or better (figure 1). The most common adverse events included thrombocytopenia, neutropenia, anemia, diarrhea, hypokalemia, fatigue and peripheral neuropathy. Adverse events of grade ≥3 included thrombocytopenia, neutropenia, anemia, elevated transaminase, diarrhea and pulmonary infection. Conclusions: TQB3602 is generally well tolerated. The combination therapy has shown preliminary efficacy in RRMM, and further clinical trials are in progress. (NCT04275583) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Multi-Kinases Inhibitor ARQ531 Enhanches Anti-MM Activity of Imids By Dual Deregulation of MAP- and Cyclin-Dependent Kinases Activities

Introduction: Kinases inhibitors are emerging as promising therapeutic tools for various blood tumors. We previously reported that a novel small tyrosine kinase inhibitor, ARQ531, constrains leukemia cells proliferation and survival by disrupting multiple tumor-addicted programs based on its kinase inhibition profile. As result we pursued its application in pre-clinical models of Multiple Myeloma (MM). Methods: Inhibitory effects of ARQ531 on cell viability were investigated in a panel of MM cell lines and primary tumor cells (collected from NDMM and RRMM patients) by CellTiterGlo luminescent assay over a range of drug concentrations and time. Specific transcriptomic profiling of ARQ531-treated MM cells was performed by RNA-Seq analysis. Molecular mechanism of this small molecule on MM cells was further investigated by qPCR and western blot analyses. The therapeutic efficacy of ARQ531 combination with different anti-MM drugs was also explored and CalcuSyn software was employed to confirm synergistic effects. Mice engrafted with MM cells were used to determine in-vivo anti-MM activity of ARQ531 in combination with lenalidomide. Results: ARQ531 inhibited cell viability of MM cell lines (n=20) with IC50 value of 3±1 µM. Furthermore, inhibitory effects of ARQ531 on cell viability were evaluated in a panel of primary MM cells with different mutational status (including those with RAS mutations), with an IC50s value ranging from 0.9 to 4 µM. Consistent with the effect on cell viability, ARQ531 increased the apoptotic rate of MM cells tested. Moreover, anti-MM effects of ARQ531were not reduced in presence of bone marrow stromal cells (BMSCs), and, more importantly such treatment showed a good therapeutic window. A transcriptome profiling analysis of ARQ 531-treated cells demonstrates wide oncogenic transcriptional programs dysregulation, as well as specific interference with kinases involved in cell cycle progression including MAP Kinases and CDK. Given that these kinases are targetable resistance mechanisms for immunomodulatory drugs (IMiDs) in MM, we combined ARQ531 with lenalidomide, pomalidomide or iberdomide observing synergistic effects. Different gene-editing approaches further supported ARQ531-identified profiling as crucial for observed anti-MM activity of tested drugs. We also confirmed that co-treatment in vivo leads to impaired engraftment and prolonged survival in a MM xenograft mouse model, by disrupting key cell proliferation pathways including c-MYC. Conclusions: Our data show that the small multi-kinases inhibitor ARQ531 has a promising therapeutic activity against MM in preclinical models thus supporting the feasibility of targeting oncogenic driven programs in MM cells with ARQ531 alone or in combinations with IMiDs.

Genome-Wide CRISPR-Cas9 Screen Identifies KDM6A As a Modulator of Daratumumab Sensitivity in Multiple Myeloma

Daratumumab (Dara) is the first-in-class monoclonal antibody targeting CD38, which triggers both direct and immune-mediated cytotoxicity of multiple myeloma (MM) cells in the bone marrow (BM) milieu. Importantly, Dara combination therapies have a high frequency and extent of response in both relapsed/refractory and newly diagnosed MM. However, relapse is commonly observed due to the development of Dara resistance. As an immune-targeting drug, the anti-tumor activity of Dara is closely associated with the expression of CD38, with nonresponders having a lower expression of CD38. To identify novel regulators mediating CD38 expression and Dara sensitivity in MM cells, we performed a genome-wide CRISPR-Cas9 knockout (KO) screen in vitro. We identified 46 different sgRNAs that were positively correlated with MM cytotoxicity triggered by Dara and primary NK cells. Using the cells with the lowest expression of CD38 (bottom 5%), we performed a second round of screening and found that KDM6A was the top-ranked gene out of the set of overlapping genes from these two screenings. We confirmed that CD38 expression was significantly decreased (>2 times, p<0.01) by KDM6A KO and that re-introducing KDM6A into KDM6A KO cells restored the expression level of CD38 and Dara-induced cytotoxicity. KDM6A encodes lysine-specific demethylase 6A, which demethylates lysine 27 of histone H3 (H3K27) to promote gene expression. To confirm that CD38 expression was altered by the methylation status of H3K27, we compared the CD38 promoter area between KDM6A KO and control cells using ChIP-seq and ChIP qPCR and found that the H3K27me3 level was higher in KDM6A KO cells. H3K27 is methylated by EZH2, and we found that EZH2 inhibitor treatment significantly decreased the H3K27me3 level of the CD38 promoter area. Moreover, EZH2 inhibitor treatment upregulated both CD38 mRNA and protein expression in KDM6A KO cells. Importantly, EZH2 inhibitor also restored the sensitivity of KDM6A KO cells to Dara-mediated NK cell cytotoxicity in vitro and in xenograft mouse model. We next re-introduced CD38 into KDM6A KO cells and found that CD38 overexpression only partially restored the sensitivity of KDM6A KO cells to Dara treatment. This suggested that besides CD38 downregulation, there are other mechanisms underlying Dara resistance induced by KDM6A KO. We performed RNA-seq in KDM6A KO cells and found that CD48, an NK-activating ligand, was downregulated. Overexpression of CD48 in KDM6A KO cells significantly increased secretion of Granzyme B and Perforin, restoring the activity of NK cells and re-sensitizing KDM6A KO cells to Dara treatment. These data suggest that KDM6A not only regulates CD38 expression, but also NK cell activity by CD48 regulation, thereby modulating the efficacy of Dara. Taken together, our studies demonstrate that KDM6A regulates CD38 and CD48 expression in MM. Moreover, we validate that combination treatment with FDA-approved EZH2 inhibitor and Dara can overcome Dara resistance in preclinical MM models, providing the rationale for combination clinical trials to overcome Dara resistance and improve patient outcome.

Abstract 5588: PD-1 blockade synergizes with IMiDs to enhance bispecific T cell engager immune responses to Vk*MYChCRBN multiple myeloma by preventing T cell exhaustion

Abstract Bispecific T cell engagers (TCE) are novel immunotherapies for cancer that redirect a synthetic immune response against tumors. Early clinical trial data show that BCMA-targeting TCEs are highly effective immunotherapies for treating relapsed and refractory multiple myeloma (MM). Thus, it is important to define combinatorial approaches that increase the efficacy and durability of TCE. We recently investigated a murine anti-BCMA TCE in the immunocompetent IMiD-sensitive Vk*MYChCRBN model of MM. Our data show that anti-BCMA TCE was safe and efficacious in most mice, but failed in those with high-tumor burden, consistent with clinical reports of TCE in leukemia. We tested the combination of anti-BCMA TCE and IMiDs to provide an additive anti-tumor effect and stimulatory cytokines, such as IL-2 in the tumor environment. This combination expanded cytolytic T cells and improved activity even in IMiD-resistant high-tumor burden cases. Yet, survival was only marginally extended due to T cell exhaustion. We hypothesized that blockade of the inhibitory immune checkpoint PD-1 would synergize with TCE and IMiDs to potentiate the response directed by the TCE by reinvigorating exhausted T cells. We evaluated the efficacy of the triple therapy combination in Vk*MYChCRBN: the anti-BCMA TCE, pomalidomide, and anti-PD1. Checkpoint blockade alone, or in combination with TCE did not improve tumor control or survival compared to control treatments. The triple combination proved most effective at controlling tumor growth, inducing a complete response after 2 weeks of treatment, and was curative in 40% of the mice. Longitudinal analysis of the immune response showed that the addition of anti-PD1 to TCE-IMiDs rescued T cell expansion at tumor sites and sustained expression of activation markers after repeated doses. Longitudinal analysis of blood and bone marrow (BM) cytokines showed the typical profile associated with cytokine release syndrome (CRS) during T cell immunotherapy. Of note, we observed IL-2, IFN-γ, and GM-CSF production was sustained in the BM tumor environment in the triple combination. We suspect that these cytokines, especially IL-2, are key to sustaining non-exhausted effector T cell proliferation in response to TCE therapy. Expectedly, we observed toxicity with the elevated cytokine release in the triple combination. that we anticipate could be controlled with step-up dosing or CRS mitigation treatment. We conclude that combination of PD1 blockade, IMiDs and TCE provides superior efficacy in the immunocompetent preclinical model of MM, Vk*MYChCRBN, while increasing inflammatory pathways that can lead to CRS. Finally, as we found this combination to be curative in some mice, we will investigate whether this combination immunotherapy also enhanced an endogenous anti-tumor immune response leading to protective immunosurveillance. Citation Format: Erin W. Meermeier, Seth J. Welsh, Meaghen Sharik, Megan T. Du, Chang-Xin Shi, Bryant Chau, Feng Wang, Matthew Wheeler, Natalie Bezman, Pavel Strop, P. Leif Bergsagel, Marta Chesi. PD-1 blockade synergizes with IMiDs to enhance bispecific T cell engager immune responses to Vk*MYChCRBN multiple myeloma by preventing T cell exhaustion [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 5588.

Abstract 5352: Genome-wide CRISPR-cas9 screening identifies KDM6A as a modulator of CD38 expression in multiple myeloma: Therapeutic implications

Abstract Daratumumab (Dara) is the first-in-class monoclonal antibody targeting CD38 which triggers both direct and immune mediated cytotoxicity in preclinical in vitro and in vivo models of multiple myeloma (MM) in the bone marrow (BM) milieu. Importantly, Dara combination therapies can achieve high frequency and extent of response in both relapsed/refractory and newly diagnosed MM. However, relapse of MM is commonly observed due, at least in part, to downregulation of CD38 on MM cells. Here we utilize genome-wide CRISPR-cas9 knockout (KO) screening to identify novel regulators mediating CD38 expression and Dara sensitivity in MM cells. We identified 46 different sgRNAs to be positively correlated with MM cytotoxicity triggered by Dara and NK cells treatment. We further performed second round screening using the cell population with lowest 5% CD38 expression after CRISPR lentiviral library transduction. Importantly, KDM6A was the top ranked of the overlapping genes from these two screenings. Next, we individually KO of KDM6A in MM cells and found that both mRNA and protein levels of CD38 were significantly decreased, as well as the cell surface CD38 expression detected by flow cytometry. Re-introduction of KDM6A into KDM6A KO cells restored the expression level of CD38 and Dara-induced cytotoxicity, indicating that KDM6A positively regulates CD38 expression in MM cells. Since KDM6A regulates demethylation of lysine 27 at histone H3 (H3K27) to activate gene expression, we next examined whether CD38 expression was altered by methylation status of H3K27. From our ChIP-seq and ChIP Q-PCR data, we identified that H3K27me3 level was highly enriched on the promotor area of CD38 in KDM6A KO cells compared to control cells. These data indicate that KDM6A modulates CD38 expression by regulating H3K27me3 level on the CD38 promotor, thereby mediating sensitivity of MM cells to Dara-induced cytotoxicity. H3K27me3 is regulated by the balance of demethylase (KDM6A) and methyltransferase (EZH2). However, downregulating H3K27me3 on the CD38 promoter by enhancing KDM6A activity is challenging. Therefore, we next explored whether EZH2 inhibitor can restore CD38 expression and overcome Dara resistance in KDM6A KO cells. Indeed, H3K27me3 level on the CD38 promotor area was significantly decreased by EZH2 inhibitor. Moreover, EZH2 inhibitor treatment upregulated both CD38 mRNA and protein levels in KDM6A KO cells. Importantly, EZH2 inhibitor also restored sensitivity of KDM6A KO cells to Dara-mediated NK cell cytotoxicity. Taken together, our studies demonstrate that KDM6A regulates H3K27me3 level on CD38 promotor area and CD38 expression in MM. Moreover, we validate that combination treatment with EZH2 inhibitor and Dara can overcome Dara resistance in preclinical MM models, providing the rationale for combination clinical trials to overcome Dara resistance and improve patient outcome. Citation Format: Jiye Liu, Lijie Xing, Teru Hideshima, Kenneth Carl Anderson. Genome-wide CRISPR-cas9 screening identifies KDM6A as a modulator of CD38 expression in multiple myeloma: Therapeutic implications [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 5352.