Panel Of Multiple Myeloma Cell Lines Research Articles

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

Abstract Background: The Proto-oncogenic PIMs family are nuclear and cytoplasmic Ser/Thr kinases (PIM1, PIM2, and PIM3) that are constitutively active and play a vital role in proliferation and survival in Multiple Myeloma (MM). Activated PIM1 kinase can induce progression of the cell cycle, inhibition of apoptosis, and modulation of other signal transduction pathways. Methods: BLX-0631 and a few analogs evaluated against a panel of MM cell lines (EJM, IM-9, L-363, LP-1, MM-1R, MM.1S, MOLP-2, NCI-H929, OPM-2, RPMI8226 and U-266) and primary MM patient samples by CellTiter-Glo® (CTG) assays. Cellular apoptosis, Cell cycle analysis, Flow Cytometry, and bone marrow microenvironment experiments on the most sensitive cells were performed. PIM1 and downstream targets by western blot, cell migration, and invasion on our lead PIM1 inhibitor BLX-0631 along with, kinome selectivity, ADME-Tox, PK, and in vivo MM.1S efficacy experiments were conducted. Results: The application of our empirical MolecuLernTM fragment library, PIM1 crystal structure, scaffold hopping, and the enumeration features of MolecuLernTM in combination with 3, 6, and 7-group analysis on pyridazine, lead to a synthesizable potential new lead series, representing Med Chem tractability and developable characteristics. Further, the consensus scoring, binding energies, and MolecuLern tractability filters were applied and led to the discovery of the BLX-0631 series that inhibits the PIM1 kinase. The active compounds, with their binding modes and lead optimization strategies, lead to the synthesis and testing of over 60 novel entities as PIM1 kinase inhibitors. Thus, based on our previous chemotype series and scaffold hopping method, the impact of changing the 3, 6, and 7 positions, while retaining the pyridazine core led to the discovery of BLX-0631 and its series of compounds exhibiting <5 nM potency. BLX-0631 in CTG assay demonstrated 100% cell growth inhibition across all MM cell lines tested. The lead compound BLX-0631 is a very potent inhibitor with an IC50 of 5.0/17/5.0 nM when tested against PIM1, PIM2, and PIM3 kinases respectively, and selective against a panel of 468 kinase panels. Dosing through IV and PO delivery routes, formulations, salt forms of BLX-0631 in mice, PK properties along with ADME-Tox, P450, and hERG results will be presented. In vivo MM.1S mouse xenograft models of BLX-0631 at 15, 30 and 45 mg/Kg, p.o, QD demonstrated TGI of 16.28%, 31.81% & 35.16% dose-dependently. Conclusions: The present study demonstrates that PIM1 plays a vital role in the proliferation and survival of Multiple Myeloma. BLX-0631 induces full inhibition of all MM cell lines, suppresses cancer cell proliferation in vitro, and exhibits promising efficacy in MM tumor models. BLX-0631, a nominated IND candidate can serve as a novel targeted agent for treating Multiple Myeloma patients. Citation Format: Zhaoang li, Chenyu Lin, David J. Bearss, Hariprasad Vankayalapati. Development of highly selective, potent, orally available PIM1 inhibitor BLX0631 shows a therapeutics potential in multiple myeloma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4587.

Multiple Myeloma Derived Extracellular Vesicle Uptake by Monocyte Cells Stimulates IL-6 and MMP-9 Secretion and Promotes Cancer Cell Migration and Proliferation.

Multiple Myeloma (MM) is an incurable haematological malignancy caused by uncontrolled growth of plasma cells. MM pathogenesis is attributed to crosstalk between plasma cells and the bone marrow microenvironment, where extracellular vesicles (EVs) play a role. In this study, EVs secreted from a panel of MM cell lines were isolated from conditioned media by ultracentrifugation and fluorescently stained EVs were co-cultured with THP-1 monocyte cells. MM EVs from three cell lines displayed a differential yet dose-dependent uptake by THP-1 cells, with H929 EVs displaying the greatest EV uptake compared to MM.1s and U266 EVs suggesting that uptake efficiency is dependent on the cell line of origin. Furthermore, MM EVs increased the secretion of MMP-9 and IL-6 from monocytes, with H929 EVs inducing the greatest effect, consistent with the greatest uptake efficiency. Moreover, monocyte-conditioned media collected following H929 EV uptake significantly increased the migration and proliferation of MM cells. Finally, EV proteome analysis revealed differential cargo enrichment that correlates with disease progression including a significant enrichment of spliceosome-related proteins in H929 EVs compared to the U266 and MM.1s EVs. Overall, this study demonstrates that MM-derived EVs modulate monocyte function to promote tumour growth and metastasis and reveals possible molecular mechanisms involved.

NK92 Expressing Anti-BCMA CAR and Secreted TRAIL for the Treatment of Multiple Myeloma: Preliminary In Vitro Assessment.

Multiple myeloma (MM) has witnessed improved patient outcomes through advancements in therapeutic approaches. Notably, allogeneic stem cell transplantation, proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies have contributed to enhanced quality of life. Recently, a promising avenue has emerged with chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA), expressed widely on MM cells. To mitigate risks associated with allogenic T cells, we investigated the potential of BCMA CAR expression in natural killer cells (NKs), known for potent cytotoxicity and minimal side effects. Using the NK-92 cell line, we co-expressed BCMA CAR and soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) employing the piggyBac transposon system. Engineered NK cells (CAR-NK-92-TRAIL) demonstrated robust cytotoxicity against a panel of MM cell lines and primary patient samples, outperforming unmodified NK-92 cells with a mean difference in viability of 45.1% (±26.1%, depending on the target cell line). Combination therapy was explored with the proteasome inhibitor bortezomib (BZ) and γ-secretase inhibitors (GSIs), leading to a significant synergistic effect in combination with CAR-NK-92-TRAIL cells. This synergy was evident in cytotoxicity assays where a notable decrease in MM cell viability was observed in combinatorial therapy compared to single treatment. In summary, our study demonstrates the therapeutic potential of the CAR-NK-92-TRAIL cells for the treatment of MM. The synergistic impact of combining these engineered NK cells with BZ and GSI supports further development of allogeneic CAR-based products for effective MM therapy.

Higher CD56 Expression on Multiple Myeloma Cells Increases CD38 Expression, Reduces Intracellular NAD+ Levels, and Enhances the Efficacy of Daratumumab-Based Treatment Strategies

NCAM1 (Neural Cell Adhesion Molecule 1), also known as CD56, is a member of the immunoglobulin superfamily and is deregulated in many tumors. In multiple myeloma (MM), high levels of CD56 are observed on MM cells in 70% of patients. This molecule has several functions including tumor growth, adhesion and response to therapy, as suggested by several studies. However, an extensive analysis of the specific landscape underlying its presence and its role as a prognostic biomarker is lacking, especially in MM. Here, we evaluated CD56 surface levels on tumor cells collected from 136 newly diagnosed MM patients (Policlinic San Martino Hospital, Genoa, Italy) and found that low CD56 expression significantly correlated with extramedullary disease (EMD) (p = .016) while high CD56 levels were associated with a lower risk of disease progression or death (p = .003). When evaluating the surface expression of MM cells, we surprisingly found a positive correlation between CD56 and CD38 levels (p < .0001), which was replicated in a panel of MM cell lines (p = .02). To gain insight into the biological significance of this, we challenged a panel of MM cell lines with Daratumumab (DARA), a standard-of-care CD38 antibody that kills MM cells through multiple mechanisms including antibody-dependent cellular cytotoxicity (ADCC). Given the correlation between the expression of CD56 and CD38, we hypothesized that higher CD56 expression would lead to enhanced DARA-mediated ADCC. We performed standard 3-hour ADCC assays using NK cells from healthy donors as effector cells and CD56-positive or -negative cell lines. Myeloma cells were stained with calcein-AM and co-cultured with NK cells at indicated effector:target (E:T) ratios. The assay was assessed in the presence or absence of 1μg/mL Daratumumab. We found that MM cell lines with high expression of CD56 were more sensitive to DARA-mediated NK cell killing (p = .004 at ratio 1:1 E:T). Consistently, MM patients with higher levels of CD56 (N = 8) had better overall response rates to DARA-based therapies compared to patients with lower levels (N = 9). CD56 and CD38 are not known to interact, so this correlation may be mediated by the NAD+ biosynthesis pathway. CD38 is a critical enzyme for breaking down NAD + in cells. We found that ectopic expression of CD56 led to lower intracellular NAD + levels (MM1S overexpressing CD56 p = .0875; RPMI 8226 overexpressing CD56 p = .0003) and increased CD38 enzymatic activity (MM1S overexpressing CD56 p = .0035; RPMI 8226 overexpressing CD56 p = .0478), as measured by a cyclase activity assay. As a result, CD56 surface levels affected MM cells' sensitivity to NAD +-lowering agents, including next-generation NAMPT inhibitors. Indeed, MTS assays confirm that CD56-overexpressing MM cells were more vulnerable to these small molecules, whereas CD56 silencing made cells more resistant. In conclusion, our data suggest CD56 levels as an important determinant of sensitivity to DARA-based therapies. Moreover, our results suggest the next-generation NAMPT inhibitors as an additional therapeutic strategy for CD56-expressing MM patients.

Selinexor Enhances Natural Killer Cell Function Against Multiple Myeloma Cells

Introduction: Natural killer (NK) cells are innate immune effector cells which can mediate antibody dependent cellular cytotoxicity (ADCC) and lyse cancer cells. Harnessing NK cell function against multiple myeloma (MM) is of high interest and multiple strategies are currently under development. Exportin-1 (XPO1) is a nuclear export protein which inhibits the function of tumour suppressor proteins and facilitates oncogene mRNA translation. Selinexor is a first-in-class XPO1 inhibitor approved for the treatment of relapsed or refractory MM that induces cancer cell apoptosis. In addition, selinexor has recently been shown to promote NK cell cytotoxicity against B cell lymphoma cells. This study aimed to determine whether selinexor augments NK cell activation against MM cells both alone, and in combination with the anti-CD38 antibody daratumumab. Methods: A panel of MM cell lines (MM.1S, U266 and RPMI-8226) were incubated with clinically relevant concentrations of selinexor for 24 hours, then either assessed for expression of ligands for NK cell receptors (KIR, NKG2A, NKG2D and NKp46), or co-cultured with primary human NK cells ± daratumumab. Following co-culture, NK-mediated lysis of MM cells was assessed by propidium iodide staining and NK cell degranulation assessed by CD107a expression. Results: Selinexor pre-treatment of MM cells significantly increased NK cell degranulation (p<0.05) and enhanced NK specific lysis of MM targets (p<0.05). Furthermore, selinexor pre-treatment significantly (p<0.05) enhanced NK cell activation in combination with daratumumab compared to daratumumab alone. Importantly, selinexor did not alter surface expression of CD38 on MM cell lines. To investigate the mechanism behind this enhanced NK cell function, we assessed the effect of selinexor on the expression of NK cell ligands by MM cells. Selinexor increased the expression of ULBP1/2/5/6 (2-fold, p<0.001) which bind to the NK cell activating receptor NKG2D. In addition, selinexor decreased MM cell expression of HLA-E (30% decrease, p<0.05) which binds to the inhibitory NK receptor NKG2A. Conclusions: These data suggest that selinexor may promote the efficacy of NK targeted therapeutics in multiple myeloma.

Novel Combinatorial Therapeutic Options in NEAT1-Depleted Multiple Myeloma Cells By the Integration of in Vitro and in silico Drug Screening Approaches

Multiple myeloma (MM) is an incurable plasma cells malignancy characterized by high genomic instability and clinical heterogeneity. Transcriptional landscape in MM is characterized by a marked de-regulation of long noncoding RNAs (lncRNAs) that influence disease progression and therapy resistance. We have shown that the lncRNA NEAT1 is overexpressed in MM patients and demonstrated that NEAT1 silencing negatively impact MM cells viability, both in vitro and in vivo suggesting that it may result in higher sensitivity to standard MM treatments. Herein, we adopted in vitro high throughput (HTS) and in silico drug screening approaches to explore the NEAT1 role in chemo-resistance scenario and identify novel putative therapeutic combination strategies. For the HTS approach, a library of 320 selective inhibitors covering 123 targets involved in proliferative and survival pathways, was screened in NEAT1-KD AMO-1 MM cells and their relative scramble condition. Silencing of total NEAT1 was achieved through the gymnotic delivery of LNA-gapmeRs. Synergy score, calculated with Bliss analysis, identified 19 compounds having a synergistic effect, in combination with NEAT1 KD, leading to a decreased MM cells proliferation, compared to single treatments. These inhibitors span different cellular targets, grouped into five categories: cell cycle checkpoint, transcriptional regulators, protein tyrosine kinases, receptors channels, epigenetics regulators. Among the top scoring candidates, Aurora kinase A inhibitors were the most promising compounds able to individually exert a synergistic activity with total NEAT1-KD in vitro. Additionally, transcriptomic analysis was performed by bulk RNA sequencing in NEAT1-KD AMO-1 cell line. Libraries with optimal quality and quantity were run on NextSeq 500. Differential gene expression analysis identified 752 down-regulated genes and 957 up-regulated genes. This signature was used to query the Connectivity Map (CMap) database. Compounds mimicking NEAT1 KD transcriptional signature were selected with the highest connectivity score (> 90). Among the top scoring molecules, Aurora kinase A (AURKA) inhibitors were identified as able to mimic NEAT1 KD transcriptional signature. Both approaches identified AURKA inhibitors as promising compounds able to affect NEAT1 functions. Based on this evidence, we started the validation of two different AURKA inhibitors on a panel of MM cell lines. Cell proliferation was assessed after 96 hours of NEAT1 silencing and 72 hours of drug treatment with Trypan-exclusion method and with live-cell imaging and analysis by taking advantage of Incucyte S3 TM technology. The synergy score was calculated with CompuSyn software. WB was used to assess the drugs on-target activity and qRT-PCR was used to validate the efficiency of NEAT1 KD. These data, suggest that NEAT1 KD and treatment with AURKA inhibitors can have similar biological outcome and can affect similar molecular pathways on MM cells. Overall, our results indicate that NEAT1 silencing combined with Aurora kinase A inhibition could represent a promising therapeutic option for MM patients, supporting the need to further characterize the molecular mechanisms underlying the synergistic effect observed in MM cells.

The Potent Dihydroorotate Dehydrogenase Inhibitor, Hosu-53, Exhibits Compelling Monotherapy Efficacy in Multiple Myeloma and Augments CD47 Targeted Therapy

Worldwide, multiple myeloma (MM) is the second most common hematological malignancy characterized by the expansion of aberrant mature plasma cells in the bone marrow. Despite the FDA approval of several drugs with different mechanisms of action such as immunomodulatory drugs (IMiDs) and proteosome inhibitors (PIs), MM remains a challenging incurable disease where most patients ultimately relapse and become refractory to available . The resistant nature of MM underscores the need for novel therapeutic strategies that can directly affect MM cells while enhancing the outcome with MM current standard of care (SOC). Among the most promising MM SOC agents is monoclonal antibodies (mAb) such as the CD38 antibody daratumumab, which significantly improved the management of newly diagnosed and relapsed/refractory MM. Therefore, we aimed to develop novel therapies that can enhance mAb therapy in MM. Dihydroorotate dehydrogenase (DHODH) is an enzyme which mediates the fourth and rate-limiting step in the de novo pyrimidine synthesis pathway converting dihydroorotate to orotate, the precursor of uridine. Pyrimidine starvation using DHODH inhibitors has been shown to have potent antiproliferative activity in several malignancies including acute myeloid leukemia (AML) and MM. Thus, we developed a novel potent DHODH inhibitor, HOSU-53 for the treatment of hematological malignancies. We previously discovered the ability of HOSU-53 to modulate surface CD38 expression and found that HOSU-53 had potent monotherapy activity in MM and provided impressive synergy in combination with daratumumab using the NCI-H929 MM cell line derived xenograft (CDX) model (2022 AACR annual meeting abstracts). Herein, we further expanded our studies to pre-clinically develop HOSU-53 as a new potential MM therapy and explore additional synergistic immunotherapy combinations that could maintain efficacy in the case of anti-CD38 therapy resistance. We evaluated the in vitro antiproliferative efficacy of HOSU-53 against a panel of MM cell lines and found nanomolar potency validating that HOSU-53 would have monotherapy efficacy. Indeed, we conducted in vivo studies using two additional MM CDX subcutaneous (s.c) models, OPM-2 and RPMI-8226, and found significant tumor delay and survival advantage. In the OPM-2 model, HOSU-53 has a median survival of 54-days compared to vehicle at 28-days, while the median survival for HOSU-53 was 60-days compared to vehicle at 26-days in the RPMI-8226 model. We further verified HOSU-53 efficacy using the disseminated MM1.S luciferase CDX model and found a significant prolonged survival in the HOSU-53 cohort (median survival53-days) compared to vehicle (median survival28-days) that was further enhanced with isatuximab combination resulting in superior survival benefit (median survival 69-days). Given the continuous challenge in maintaining a durable response with anti-CD38 therapies due to resistance, we sought to explore additional combination regimens to maximize HOSU-53 efficacy. Currently there is significant clinical interest in CD47 antibody therapy such as magrolimab for both solid tumors and hematological malignancies. Our previous work suggested a strong synergy between CD47 antibodies and HOSU-53 in AML with curative potential (2022 ASH annual meeting abstracts), and hypothesized that we would observe similar synergy with HOSU-53 in combination with anti-CD47 in MM. Indeed, we found complete tumor regression in all mice treated with HOSU-53 + B6.H12 anti-CD47 therapy in the NCI-H929 s.c CDX model and significant prolonged survival and reduced bioluminescence in the NCI-H929 luciferase disseminated CDX model. Together, these two studies validate a potential clinical benefit to explore HOSU-53 + anti-CD47 regimen in MM patients. Furthermore, we observed that calreticulin (pro-phagocytosis signal) was modulated post HOSU-53 in vitro treatment suggesting its role in the observed synergy between HOSU-53 and CD47 blockade therapy. In summary, we show compelling survival benefit for HOSU-53 as a monotherapy which is further enhanced when combined with anti-CD38 or anti-CD47 therapies. HOSU-53 is expected to enter phase 1 clinical trials in 2024 and our data is supportive for its expansion into MM.

The NAD + Metabolic Dependency Restriction Overcomes Drug-Resistance Phenotype of Multiple Myeloma CELLS

Introduction: We previously reported that Nicotinamide Adenine Dinucleotide (NAD +), a master regulator of catabolic metabolism and several enzymatic activities, plays a crucial role for Multiple Myeloma (MM) cells growth; indeed, small molecules targeting the rate-limiting NAD + producer enzyme NAMPT, have potent anti-MM activity. However, targeting dysregulated metabolic landscape of tumors has proven to be a major challenge as suggested by existence of alternative NAD + production routes which confer resistance to NAMPT inhibitors. As result here we focused on whole NAD + metabolic dependency of MM cells by investigating relevance of a comprehensive NAD + metabolic restriction as strategy to enhance activity of currently used anti-MM strategies. Methods: whole transcriptomic analysis of the NADome signature was performed on MM patients by probing MMRF CoMMpass study; its correlation with clinical data (OS and PFS) was also investigated. Next, GSEA algorithm on biological modules was run on MM patients carrying low- vs high-NADome signature. Chemical and genome editing-approaches using CRISPR-Cas9-based strategy were both used to investigate relevance of our findings. Then, an anti-MM drugs screen was used to challenge fully-NAD depleted MM cells using different viability tools. Mechanistic studies were also performed using qPCR, western blot, immunofluorescence analyses, targeted enzymatic assays and in-vivo xenograft MM models. Finally, clinical benefits of our findings were accomplished by challenging publicly available dataset on ASCT-treated MM patients. Results: RNAseq analysis on CoMMPass dataset sharply divided MM patients according to their comprehensive NADome signaling landscape: high-NAD producers showed poorer outcome than those expressing low mRNA levels, both in terms of PFS and OS as well. In line with these data, a panel of MM cell lines showed that pervasive NAPRT levels reduced NAMPT inhibitors activity thus supporting a complete NAD +-depletion for more effective strategies also in in-vivo xenograft models. PROGENy analysis revealed an increased NF-KB activity in NAPRT-depleted cells which in turn resulted in higher oxygen reactive species generation from these cells. Indeed, NAPRT KO cells showed oxidative damage accumulation, with increased malondialdehyde (MDA) activity and lower endogenous antioxidant defenses as suggested by glutathione reductase, glutathione peroxidase and ratio of reduced GSH to oxidized glutathione (GSSG) ratio measurements. Next, mitochondrial-energetic status was also tested by measuring electron transport chain (ETC) Complex I and Complex II activities: a shift towards oxidative metabolism was observed. Overall these data phenocopy the energetic status observed in drug-resistant cells; as result NAPRT-KO makes MM cells resistant to Melphalan. In such a context, the use of NAMPT-inhibitors was able to overcome drug-resistance by re-sensitizing cells to anti-MM activity of DNA damaging agents. Finally, a focused analysis on ASCT-treated MM patients, revealed poor outcome for “high-NAD + producers” thus supporting clinical relevance for NAD + metabolic restriction in these patients. Conclusions. Our data provide evidence that NAD + biosynthesis shapes metabolic phenotypes of MM cells, with its impairment resulting in metabolic reprogramming and cells viability injury. As result, a comprehensive NAD + depletion with dual NAPRT and NAMPT inhibition primes MM cells to alkylating agents which still represent the backbone strategy for ASCT-eligible patients.

Long Noncoding RNA LINC01410 Interacts with the Minichromosome Maintenance (MCM) Complex to Promote Tumor Cell Growth in Multiple Myeloma

The human genome contains thousands of loci that produce long non-protein coding RNAs (lncRNAs) that play critical roles in healthy cell function as well as tumorigenesis. We have recently described the lncRNA landscape in newly diagnosed multiple myeloma (MM), identified their role as independent risk predictors for clinical outcome, and validated the functional contribution of specific lncRNAs to MM cell growth and viability. Although these examples suggest the significance of lncRNAs in MM pathobiology, the vast majority of lncRNA genes have not been functionally tested. To identify lncRNAs that promote MM cell growth, we performed a large-scale cell viability screen of lncRNAs using CRISPR interference (CRISPRi) to inhibit gene expression. To find lncRNA targets, we first interrogated our large RNA-seq dataset from 360 newly diagnosed MM patients and identified 913 lncRNA transcripts expressed in primary MM cells. We designed the CRISPRi library to target the transcription start site of these 913 lncRNA genes, each with 7 sgRNAs. We screened 3 MM cell lines (H929, KMS12BM, KMS11) and identified 32 lncRNA loci that modified cell growth upon CRISPRi targeting, with 7 lncRNAs impacting all three cell lines tested. We then evaluated the differential expression of these lncRNAs in primary MM cells compared to normal plasma cells (PC). We observed that LINC01410 was significantly overexpressed in MM patients compared to normal PC, especially in MM patients with t(4;14) translocations, and was an independent risk predictor that highly correlated with overall survival in newly diagnosed MM patients. We next knocked down LINC01410 using gapmeR antisense oligonucleotides (ASO) in a panel of MM cell lines and observed time-dependent inhibition of MM cell viability, including in the t(4;14) cell lines. As LINC01410 appears to localize to both the cytosol and nucleus, we also utilized siRNAs to specifically target the cytosolic isoform and observed a robust reduction of cell growth, suggesting that both cellular localizations are important for the activity of LINC01410. Transcriptomic analysis indicated a set of genes modulated in response to LINC01410 depletion, with an enrichment in genes involved in the cell cycle and DNA replication. An RNA-protein pull down (RPPD) assay identified three out of six MCM complex subunits (MCM4, MCM5, MCM7) as relevant protein interactors of LINC01410 and was confirmed using RNA-immunoprecipitation (RIP) assay. The MCM complex is a DNA helicase essential for genomic DNA replication, and we hypothesize that LINC01410 affects cell proliferation by interacting with this complex. We are now investigating this functional interaction by looking at the impact of LINC01410 modulation on the assembly of the complex, by using integrated mass spectrometry analysis, and on its ability to load onto chromatin by using chromatin immunoprecipitation sequencing (ChIP-Seq). In conclusion, we report the identification of LINC01410 as a novel lncRNA dependancy that supports MM cell growth via interacting with the MCM complex and controlling the cell cycle/DNA replication axis. Promising developments in the field of lncRNA inhibitors may allow forthcoming clinical applications.

The Novel Trifunctional Anti-BCMA NK Cell Engager SAR'514 Has Potent in-Vitro and in-Vivo Anti-Myeloma Effect through Dual NK Cell Engagement

Multiple Myeloma (MM) is the second most prevalent hematopoietic malignancy, representing 10% of total blood cancers. Despite the emergence of new therapies in the recent years, it is still an incurable disease, with a 5-year overall survival of 56% in patients (https://cancerstatisticscenter.cancer.org/). Thus, innovative approaches that would be safe, potent and with long lasting beneficial effects are required. BCMA (B Cell Maturation Antigen) is a cell surface receptor highly and selectively expressed on normal and malignant plasma cells and which function is to promote cell proliferation and survival upon binding of its ligands APRIL (A PRoliferation Inducing Ligand) and BAFF (B cell Activating Factor). BCMA has a very high prevalence among MM patients and its expression on tumor cells is maintained after treatments with standard of care such as anti-CD38 therapies, or even BCMA-targeting agents (Cohen AD et al. J Clin Invest 2019). The ability of NK cells to intrinsically kill tumor cells, leaving healthy cells unharmed, with minimal pro-inflammatory cytokine release induction as compared to T cell-based therapies makes NK cells ideal immune cells for a safe and efficacious therapeutic approach. We developed a NK Cell Engager (NKCE) SAR'514 that activates NK cells through a dual engagement of NKp46 and CD16a, two major NK cell activating receptors highly expressed on NK cells in MM patients, and which redirects the activated NK cells to engage and kill BCMA+ tumor cells. The trifunctional NK cell engager molecule SAR'514 was designed to specifically and efficiently activate NK cells to induce potent cytotoxic activity against BCMA+ MM cells. SAR'514 exhibits high affinity for the 3 targets BCMA, CD16 and NKp46. The ability of SAR'514 to induce NK cell-mediated cytotoxicity in vitro was tested against a panel of MM cell lines with low, medium and high BCMA expression levels and compared to an afucosylated reference compound. SAR'514 exhibited a strong and similar potency against all tested cell lines and a better potency compared to this comparator (EC50 0.4 pM vs 7.8 pM) demonstrating the superiority of a dual targeting NK cell engager molecule as compared to an antibody approach. An optimized NK cell activation was obtained with a dual engagement of both NKp46 and CD16a compared to single CD16a or NKp46 engagement or the combination of two molecules engaging CD16a or NKp46. Binding of SAR'514 on NK cells promoted a strong NK cell activation and induced cytotoxic activity only in the presence of MM target cells with no fratricide effect on NK cells. This antitumor activity is associated with very low IL-1b, IL-6, TNFa and IFNg cytokine release (in the low pg/mL range) in human PBMC and whole blood settings in the presence of BCMA+ tumor target cells, and a good safety profile. In addition, the in vivo anti-tumor activity of an anti-murine NKp46 surrogate NK cell engager molecule at 0.05, 0.5 and 5 mg/kg was investigated versus a control isotype in huFcgR transgenic mice engrafted with the EL4-huBCMA murine thymoma model. As compared to the control group in which only 20% of mice survived with a median survival day of 44.5 days, SAR'514 induced a significant mouse survival from 0.5 mg/kg to 5 mg/kg with an overall survival of 90% and a median survival day over 90 days. In summary, these results demonstrate the efficacy of SAR'514 for controlling MM tumors in vivo and provide consistent support for its clinical development.

BRD9 Is Essential for Ribosome Biogenesis and the Survival of Multiple Myeloma Cells

BRD9 is a component of the noncanonical SWI/SNF (ncSWI/SNF) complex, which is a chromatin-remodeling complex that modulates genomic architecture and DNA accessibility. Since BRD9 has been shown to play a critical tumor-promoting role in various cancers, we investigated its biologic impact in multiple myeloma (MM) cells. We found that BRD9 was highly expressed in patient MM cells compared to normal plasma cells (GSE39754, P = 0.045). Moreover, higher BRD9 expression was associated with poor overall survival (GSE39754, P = 0.049) in MM patients. We next confirmed that genetic (shRNA) and pharmacological (dBRD9-A) depletion of BRD9 significantly reduced growth in a panel of MM cell lines and patient MM cells. Furthermore, dBRD9-A induced G1 cell-cycle arrest and apoptosis in OPM2 and H929 cells, even in the presence of anti-apoptotic cytokines (IL-6, IGF-1), or bone marrow stromal cells. In a murine xenograft model, dBRD9-A treatment significantly inhibited OPM2 tumor growth compared with vehicle control (P = 0.001 and P < 0.001, respectively) without significant body weight loss. Kaplan-Meier curves and log-rank analysis showed a significantly prolonged overall survival in the dBRD9-A treatment cohort compared with the vehicle control cohort (P < 0.001). Having nominated BRD9 as a potential therapeutic target, we next investigated the cellular mechanisms impacted by BRD9 depletion by RNA-seq analyses. After BRD9 knockdown (shRNA) or dBRD9-Aapplication, a total of 162 and 197genes were commonly up- or downregulated, respectively, compared with DMSO-treated control cells (Padj < 0.05). Importantly, we identified that ribosome biogenesis and rRNA processing were the most significantly downregulated pathways. Notably, target genes of MYC, the master regulator of ribosome biogenesis, were also markedly downregulated. Furthermore, we observed that the expression of representative proteins (i.e., MYC, RRS1, and BOP1) was also consistently decreased by dBRD9-A in vitro and in vivo. Indeed, we observed a significant decrease in total pre-rRNA levels and nucleolar number by dBRD9-A treatment in OPM2 and H929 cells, indicating ribosome biogenesis was positively regulated, at least in part, by BRD9. OPM2 cells treated with dBRD9-A had fewer ribosomal subunits and fewer actively translating polysomes compared with DMSO-treated controls, as determined by polysome profiling of cytosolic extracts. Furthermore, AgNOR staining of tumors harvested from our murine xenograft model demonstrated that dBRD9-A treatment reduced the number of nucleolar organizer regions in vivo. To understand how BRD9 regulates the expression of ribosome biogenesis genes, we next performed ChIP-seq. We found that many of the genes downregulated by dBRD9-A had BRD9 occupancy at their promoters compared to other intact genes. Notably, genes with BRD9 signals in their promoter regions were enriched for ribosome biogenesis and rRNA processing and were downregulated by dBRD9-A treatment. Although GLTSCR1, another ncBAF-specific component, was still able to form a complex even after BRD9 depletion, ChIP-seq revealed a significant loss of GLTSCR1 binding throughout the genome after treatment with dBRD9-A. Thus, we hypothesized that BRD9 is required for the localization of the ncSWI/SNF complex to target gene regions. Indeed, BRD9 co-immunoprecipitated with the positive transcription elongation factors, RNAPII, BRD4, and MYC in MM cells. Interestingly, the interaction between BRD9 and BRD4 was enhanced by an HDAC inhibitor, while BRD9 depletion attenuated the interaction between GLSTCR1 and BRD4, suggesting that BRD9 interacts with the acetylated form of BRD4, stabilizing its binding to the ncBAF complex and thereby supporting the initiation of transcription of target genes. Indeed, ChIP-seq analysis demonstrated that BRD9-bound sites preferentially co-localized with ChIP peaks of BRD4, MYC, and RNAPII to the promoter regions of all ribosome biosynthesis genes. In summary, our data identify a novel function for BRD9 in promoting ribosome biogenesis in MM. BRD9 depletion impairs ncBAF localization to its genomic targets, thereby disrupting ribosome biogenesis machinery, resulting in significant cell growth inhibition. Our study therefore provides the rationale for further evaluation of BRD9 degraders as a novel treatment strategy in MM.

Identification of the Proteasome Subunits PSMB4 and PSMD4 As Novel Targets in Multiple Myeloma Patients Carrying 1q21 Amplification

Gain and/or amplification of the 1q21 (1q21+) region is one of the most frequent secondary cytogenetic abnormalities present in multiple myeloma (MM) patients. Interestingly, it has been reported that the incidence of the amplification of chromosome 1q (Amp1q) in patients with pre-malignant smoldering myeloma (SMM) is about 40%. Several data indicate that the presence of Amp1q is associated with a worse clinical outcome and a poor response to therapies including proteasome inhibitors (PIs). Recently, a study showed that newly diagnosed MM (NDMM) carrying Amp1q receiving carfilzomib-based treatment have an early disease progression and shorter overall survival, compared to those with 1q21 gain. The genes driving amplification of the 1q21 region have not been fully elucidated. This study aims to identify novel targets in the 1q21 region possibly correlated to PIs treatment. We purified CD138+ plasma cells (PCs) by immunomagnetic beads from bone marrow (BM) aspirates. We used a total cohort of 29 MM patients: 11 SMM and 18 NDMM. Fluorescent in situ hybridization analysis was performed on all the patients. 48% (14/29) patients carried 1q21+. A score indicating the number of 1q21 copies was calculated based on the FISH hybridization pattern of each patient. The expression profile of all 29 samples was generated using GeneChip ClariomD Arrays (Affymetrix Inc., Santa Clara, CA, USA). The smar package was used to identify differentially expressed genes between 1q21+ and control samples. The expression profile of our cohort showed that the expression of both proteasome subunits, PSMB4 and PSMD4 was significantly upregulated in MM patients Amp1q compared to control patients (PSMB4 p=0.0006; PSMD4p=<0.0001 respectively). Additionally, we found a strong positive correlation between gene expression levels and 1q21 copy number for the proteasome subunits PSMB4 (p=<0.0001, r=0.5631) and PSMD4 (p=<0.0001, r=0.6391). Interestingly, the PSMB4 and PSMD4 expression levels were independent of the disease stage (SMM vs MM) and the expression was driven exclusively by the 1q21 copy number. We evaluated PSMB4 and PSMD4 mRNA and protein expression levels in a 1q21 wild-type MM cell line (OCI-MY5) and a panel of MM cell lines carrying 1q21+. Our results showed that PSMB4 and PSMD4 expression levels were higher in cell lines with Amp1q, following a 1q21 copy number fashion. Subsequently, we assessed the role of PSMB4 and PSMD4 to the sensibility of PI bortezomib in two myeloma cell lines carrying 1q21+. Our results showed that bortezomib treatment downregulates the protein expression of PSMD4 while PSMB4 was unaffected. Moreover, the MTT viability test correlated with the anti-myeloma effect of bortezomib. Furthermore, we generated a JJN3 PSMB4 and PSMD4-knockdown MM cell line using short hairpin RNA (shRNA) lentivectors. An empty vector was used as a control plasmid. Cell viability and apoptosis assays were performed using MTT assay and Apo 2.7 staining. Our functional studies showed that shPSMB4 and shPSMD4 decreased MM cell viability when treated with PI. Moreover, both shPSMB4 and shPSMD4 knockdown cells showed elevated levels of polyubiquitylated proteins, indicating blockade of proteasome-mediated protein degradation. In conclusion, our study identified proteasome subunits PSMB4 and PSMD4 to be significantly upregulated in MM patients carrying 1q21+, correlated with 1q21 copy number but not with disease stage. Therefore, targeting PSMB4 and PSMD4 could be a strategy to treat MM patients with 1q21+.

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.

Abstract CT186: Pharmacokinetic (PK) profile of a novel IKZF1/3 degrader, CFT7455, enables significant potency advantage over other IKZF1/3 degraders in models of multiple myeloma (MM) and the results of the initial treatment cohort from a first-in-human (FIH) phase 1/2 study of CFT7455 in MM

Abstract Introduction: CFT7455 is a highly potent and novel Ikaros Family Zinc Finger Protein 1/3 (IKZF1/3) degrader. In xenograft models, CFT7455 has more potent IKZF1/3 degradation compared to other degraders. Early observations from the FIH clinical trial (NCT04756726) along with supporting translational studies are presented here. Methods: Pre-clinical studies comparing CFT7455 and CC-92480 in both in vitro and xenograft models were performed. The pre-clinical studies’ results coincided with observations from the on-going clinical trial. The clinical trial is an open-label, Phase 1/2, multi-center, FIH study in heavily pretreated relapsed/refractory (R/R) MM and non-Hodgkin’s lymphoma (NHL) patients evaluating safety, tolerability, and PK of CFT7455. Eligible MM patients are R/R to therapy and are not candidates for regimens known to provide clinical benefit. A starting dose of 50 μg QD 21 days on/7 days off (21/7) in 28-day cycles was administered. Results: CFT7455 and CC-92480 showed similar cereblon binding profiles and in vitro IKZF1/3 degradation kinetics, translating into sub-nanomolar GI50 values in proliferation assays across a panel of MM cell lines. In the NCI-H929 xenograft model, 100 μg/kg/day of CFT7455 resulted in durable tumor regressions, while 1000 μg/kg/day of CC-92480 gave tumor stasis. Similar results were seen in a systemic model of MM, MM1.S. Both compounds achieved &amp;gt;95% IKZF3 degradation in tumors 4h post dose. At 48h post dose, CFT7455 was more effective than CC-92480 in maintaining IKZF3 degradation (65% vs. 6% respectively). When levels of CFT7455 and CC-92480 in plasma and tumor were compared, CFT7455 concentrations were &amp;gt; DC80 in tumor 48h post dose, while CC-92480 levels were undetectable in tumor and plasma, demonstrating CFT7455 has longer exposure resulting in sustained IKZF1/3 degradation in pre-clinical models. In cohort A, 5 heavily pre-treated MM patients (pts) received single agent CFT7455. 4 pts have received at least 3 cycles, with 2 pts receiving 5 cycles. Neutropenia (grade 4) was observed in 3/5 pts without coincident fever or infection. Additionally, a 2-4 fold accumulation in plasma CFT7455 exposure at steady state was observed. Early pharmacodynamic (PD) data demonstrates deep persistent degradation of IKZF3 (~100%) and serum free light chain reduction (up to 72%) in response to treatment. Stable disease has been observed in 34 pts, suggesting clinical benefit. Conclusions: While CFT7455 showed clinical benefit at 50 ug with deep target degradation, neutropenia was dose limiting. PK/PD modeling suggests alternative dosing regimens may result in increased tolerability with preserved efficacy, and evaluation of them is underway. Updated results will be presented at the meeting. Citation Format: Sagar Lonial, Shambavi Richard, Jeffrey V Matous, Andrew J. Yee, Urvi Shah, Neha Mehta-Shah, Thomas Martin, Eli Muchtar, Sikander Ailawadhi, Paul G. Richardson, Manisha Bhutani, Samantha Perino, Jason Kirby, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, Matthew Schnaderbeck, Michael R. Palmer, Cathleen Gorman, Oliver Schoenborn-Kellenberger, Amanda Hoerres, Stewart L. Fisher, Roy M. Pollock, Adam Crystal, Michelle Mahler, Jesus Bardeja. Pharmacokinetic (PK) profile of a novel IKZF1/3 degrader, CFT7455, enables significant potency advantage over other IKZF1/3 degraders in models of multiple myeloma (MM) and the results of the initial treatment cohort from a first-in-human (FIH) phase 1/2 study of CFT7455 in MM [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 CT186.

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

Abstract Introduction: Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 inhibits the growth of multiple myeloma (MM) cells, confirming their dependency on IKZF1/3. IMiDs (lenalidomide, pomalidomide) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. However, most patients treated with lenalidomide or pomalidomide eventually develop progressive disease due to acquired resistance, underscoring the unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high binding affinity to cereblon (CRBN), rapid and deep IKZF1/3 degradation, and potent dose-dependent efficacy in vivo. Results: A series of novel benzoimidazolone-based CRBN ligands with potent binding affinity were discovered and their binding modes were informed by CRBN co-crystal structures. Although the benzoimidazolone-based CRBN binders did not exhibit IKZF1/3 degradation activity, structural insights into their unique binding modes and knowledge of the IKZF1/3 degradation pharmacophore were combined to enable identification of a novel benzoisoindolone-based ligand that exhibited a 10-fold potency increase in biochemical CRBN binding and a 30-fold potency increase in H929 MM cell growth inhibition when compared to lenalidomide. Additional rounds of structure-based drug design, degradation and phenotypic profiling led to the discovery of CFT7455, a highly potent, selective and orally bioavailable degrader of IKZF1/3. CFT7455 demonstrated an 800 and 1600-fold improvement in CRBN binding compared to pomalidomide in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced &amp;gt;75% degradation of IKZF1 within 1.5 hrs. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines, as well as H929 cells made resistant to IMiDs. In vivo, CFT7455 catalyzed deep and durable degradation of IKZF3, translating into potent antitumor activity in multiple myeloma xenograft models. CFT7455 also retained its activity in models resistant or insensitive to clinically approved IMiDs as single agent or in combination with standard of care agent dexamethasone. Conclusion: Overall, CFT7455 is a next generation IKZF1/3 degrader, with improved potency and anticancer efficacy in preclinical models compared to existing IMiDs. These features make CFT7455 an exciting drug candidate, as a single agent or for use in combination. CFT7455 is currently being studied in a Ph1 clinical trial. Citation Format: James A. Henderson, Scott J. Eron, Andrew Good, R Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Ashley A. Hart, Christina S. Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Adam S. Crystal, Roy M. Pollock, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. The discovery and characterization of CFT7455: A potent and selective degrader of IKZF1/3 for the treatment of relapsed/refractory multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND13.

Aberrant CDK7 Activity Drives the Cell Cycle and Transcriptional Dysregulation to Support Multiple Myeloma Growth: An Attractive Molecular Vulnerability

Multiple myeloma (MM) cells are characterized by cell cycle dysregulation, epigenetic heterogeneity, and perturbation of the transcriptional landscape. We have previously shown that chemical and genetic perturbation of transcriptional regulator CDK7 significantly and selectively impacted MM cell growth and viability, supporting it as a pharmacologically relevant target for MM. Indeed, selective CDK7 inhibitor YKL-5-124 was active against a large panel of MM cell lines and primary MM cells, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs). The efficacy of YKL-5-124 was confirmed in vivo in several murine models of MM, including disseminated models.Gene expression analysis after CDK7 inhibition in several MM cell lines revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells, supporting CDK7 as a central hub in the oncogenic CDK-pRb-E2F pathway in MM cells, with its expression and activity positively correlated with E2F transcriptional output in patient MM cells. Importantly, dual inhibition with low doses of YKL-5-124 and BRD4 inhibitor JQ1, displayed superior activity against a panel of MM cell lines and primary MM cells compared to single perturbation alone by both converging on a subset of key SE-associated dependencies as well as impacting distinct oncogenic expression programs.To identify synthetically lethal targets and mechanisms of resistance to CDK7 inhibition, we performed a genome-wide CRISPR-Cas9 knockout screen in the MM1S cells treated with YKL-5-124 or DMSO. We found that BCL6, NFKBIA and B, TRAF2, TSC1 and CSNK2A1, a subunit of CK2, were top synthetically lethal hits; whereas deletion of RB1, SF3B3 and the DNA-binding transcriptional activator TADA2A that regulates RNA-pol II transcription, led to resistance to YKL-5-124. Molecular mechanisms of intrinsic and acquired resistance to CDK7 inhibition are now under investigation and will be presented.In conclusion, our study demonstrates CDK7 as an attractive molecular vulnerability in MM that can be exploited therapeutically alone or in combination. DisclosuresShirasaki: FIMECS: Consultancy. Chesi: Novartis: Consultancy, Patents & Royalties: human CRBN transgenic mouse; Pfizer: Consultancy; Pi Therapeutics: Patents & Royalties: Genetically engineered mouse model of myeloma; Abcuro: Patents & Royalties: Genetically engineered mouse model of myeloma; Palleon Pharmaceuticals: Patents & Royalties: Genetically engineered mouse model of myeloma. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: 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; 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. Mitsiades: Arch Oncology: Research Funding; Karyopharm: Research Funding; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Honoraria; Abbvie: Research Funding; FIMECS: Consultancy, Honoraria; Nurix: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; H3 Biomedicine: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; BMS: Research Funding; TEVA: Research Funding. Munshi: Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy.

Identifying Long Noncoding RNA Dependencies Using CRISPR Interference (CRISPRi)-Based Platform in Multiple Myeloma

Identifying Long Noncoding RNA Dependencies Using CRISPR Interference (CRISPRi)-Based Platform in Multiple Myeloma

PSMB4 and PSMD4 Are Correlated with 1q21 Amplification in CD138 + Plasma Cells: New Potential Druggable Targets in Myeloma Patients

The amplification of the 1q21 (amp1q21) region is one of the most acquired cytogenetic abnormalities (CA) in multiple myeloma (MM) associated with a worse patient outcome and disease progression. Moreover, different studies have demonstrated that the number of copies (CN) 1q21 (gain1q21: three copies or amp1q21: ≥ four copies) have a different impact in the response to anti-MM therapies. Particularly, it has been proposed that in MM patients, additional copies of 1q21 may be associated with the resistance to proteasome inhibitor (PI) treatment as bortezomib. A recent study showed that newly diagnosed MM (MMD) patients carrying amp1q21 but not gain1q21 receiving carfilzomib-based treatment have an early disease progression with shorter overall survival.Previous studies underlined that the amplification of 1q21 can lead to the overexpression and/or dysregulation of several candidate genes associated with cell proliferation, apoptosis, and drug resistance. Here we aim to identify 1q21 target genes possibly correlated to the response to PI therapy.We evaluated a total cohort of 29 primary plasma cells (PCs) purified from bone marrow (BM) blood aspirates from 11 smoldering MM (SMM) and 18 MMD. The median age of our cohort was 70 years (range: 38-86). Fluorescence in situ hybridization (FISH) analysis was performed to access the presence or absence of copy number alteration (CNA) in the 1q21 region in all patients. 14 out of 29 patients carried 1q21 CNA (5 with gain1q21 and 9 with amp1q21). A score reflecting the number of 1q21 copies was calculated based on the hybridization pattern.The transcriptional profiles of the 29 BM PCs samples were generated on GeneChip ClariomD Arrays (Affymetrix Inc., Santa Clara, CA, USA). The samr package was used in R for call genes as differentially expressed between 1q21 CN-altered and wild-type samples. The correlation between the 1q21 copy number score and the gene expression levels was performed. Moreover, we have evaluated by FISH the 1q21 CNA in a panel of human myeloma cell lines (HMCLs): OCY-MY5, JJN3, RPMI-8226, NCI-H929, and OPM2. JJN3 were transfected with a control vector and PSMB4 and PSMD4 short hairpin RNA (shRNA) lentivectors. The gene and protein expression levels of PSMB4 and PSMD4 in MM cell lines were analyzed by qRT-PCR and Western Blot, respectively. Cell viability and proliferation were assessed using MTT assay and flow cytometry.Our bioinformatic analyses highlighted the overexpression of different genes (IL6R, ILF2, BCL9, MCL1, CSk1B, ADAR1, ARNT, ANP32E) in the 1q21 CNA samples with respect to the controls, as already reported in the literature. Our analysis showed a significantly higher expression of two proteasome subunits (PSMB4 and PSMD4) in patients with 1q21 CNA when compared with patients without (PSMB4 p=0.0006; PSMD4 p=<0.0001). Patients with amp1q21 showed a higher expression of PSMB4 when compared to the patients with gain1q21 (p=0,007). In our cohort, gene expression profile analysis also showed a strong positive correlation between gene expression levels and 1q21 CN for the proteasome subunits PSMB4 (p=<0.0001, r=0.5631) and PSMD4 (p=<0.0001, r=0.6391). Interestingly, we found that the PSMB4 and PSMD4 expression level was independent of the disease stage (SMM vs MM) and was only driven by 1q21 CN.We have evaluated PSMB4 and PSMD4 mRNA and protein expression levels in a 1q21 wild-type cell line (OCY-MY5) and in a panel of MM cell lines carrying different degrees of 1q21 CN (in order: JJN3, U266, RPMI-8226, OPM2, and NCI-H929). The mRNA expression level of PSMB4 and PSMD4 was higher in cell lines carrying 1q21 amp, following a 1q21 copy number fashion. Similar results were obtained when protein levels of MM cell lines were analyzed by Western Blot. To further determine the potential role of both proteasome subunits in the pathogenesis of amp1q21, we generated a PSMB4-shRNA and PSMD4-shRNA knockdown stable MM cell lines. Functional studies showed that blockade of PSMB4 and PSMD4 decreased MM cell viability.In conclusion, our study identified proteasome subunits PSMB4 and PSMD4 to be significantly upregulated in MM patients carrying amp1q21, correlated with 1q21 copy number but not with disease stage. In addition, knockdown of both, PSMB4 and PSMD4 decreased MM cell proliferation. Therefore, targeting PSMB4 and PSMD4 could be a strategy to treat MM patients with ampq21 DisclosuresGiuliani: Celgene: Membership on an entity's Board of Directors or advisory committees, Other: congress, Research Funding; Bristol Mayers Squibb: Other: congress; GSK: Other: clinical studies; Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical studies, congress, Research Funding; Millenium Pharmaceutical: Other: clincial studies.

OAB-009: Genome-wide CRISPR interference screen identifies RNA Regulator of Lipogenesis (RROL) as a leading LncRNA dependency in Multiple Myeloma

OAB-009: Genome-wide CRISPR interference screen identifies RNA Regulator of Lipogenesis (RROL) as a leading LncRNA dependency in Multiple Myeloma

Abstract LB007: CFT7455: A novel, IKZF1/3 degrader that demonstrates potent tumor regression in IMiD-resistant multiple myeloma (MM) xenograft models

Abstract Introduction Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 in MM cells inhibits growth, confirming their dependency on IKZF1/3. IMiDs (lenalidomide[len], pomalidomide[pom]) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. Most patients treated with len or pom eventually develop progressive disease due to acquired resistance, underscoring an unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high affinity cereblon (CRBN) binding, rapid IKZF1/3 degradation, and potent in vivo efficacy. Results CFT7455 demonstrated an 800 to 1600-fold improvement in CRBN binding compared to pom in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced &amp;gt;75% degradation of IKZF1 within 1.5 hr. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines as well as H929 cells made resistant to IMiDs. In RPMI-8226 MM mice xenografts, CFT7455 (0.1 mg/kg/day) resulted in deep, durable degradation of IKZF3 (21% and 9.5% of vehicle levels at 4 and 24 hr, respectively). Protein levels for IRF4, an essential TF in MM, declined over 7 days with daily CFT7455 treatment to 8% of vehicle levels. Dose dependent efficacy ranged from 0.003-0.1 mg/kg/day, with tumor regression evident at doses ≥0.01 mg/kg/day. Pom was inactive in this model at a human equivalent dose (3 mg/kg/day), with no observed tumor shrinkage in these mice following 17 days of dosing. Switching pom treatment to CFT7455 (0.1 mg/kg/day) on Day 18 led to tumor regression in 67% of animals on Day 28 and 100% tumor regression on Day 35, demonstrating that CFT7455 penetrates large tissues and is efficacious in rapidly growing, IMiD resistant tumors. In the H929 tumor xenograft model, administration of CFT7455 (0.1 mg/kg/day) promoted tumor regression (95% tumor growth inhibition by 7 days); dosing was stopped after 21 days. On Day 63, half the tumors remained below their starting tumor volume. Additionally, CFT7455 demonstrated durable tumor regression in the aggressive MM1.S systemic MM tumor model. In mice bearing RPMI-8226 xenograft tumors, the combination of CFT7455 (QD) and dexamethasone (QW) was more active, and demonstrated a significant survival improvement, compared to either agent alone. Conclusions CFT7455 is a highly potent, catalytic degrader of IKZF1/3, with marked antitumor activity as a single agent and in combination with dexamethasone. Importantly, CFT7455 retains activity in models resistant or insensitive to IMiDs. These results warrant investigation of CFT7455 as a therapeutic approach for MM and a clinical study is planned. Citation Format: James A. Henderson, R. Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Scott J. Eron, Andrew Good, Ashley A. Hart, Christina Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Roy M. Pollock, Adam S. Crystal, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. CFT7455: A novel, IKZF1/3 degrader that demonstrates potent tumor regression in IMiD-resistant multiple myeloma (MM) xenograft models [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 LB007.