Myeloma Cell Lines Research Articles

Abstract 5277: The ROCK-2 inhibitor belumosudil exerts a direct antimyeloma effect and improves isatuximab-mediated cytotoxicity against multiple myeloma cells

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterized by clonal accumulation of malignant plasma cells in the bone marrow. Isatuximab (Sarclisa®) is a monoclonal antibody used for the treatment of MM. Isatuximab binds to CD38 (highly expressed on MM cells) and induces tumor cell killing via several mechanisms including antibody dependent cellular cytotoxicity (ADCC) and direct apoptosis. Belumosudil (RezurockTM) is a ROCK2 inhibitor that is approved for the treatment of chronic graft versus host disease (cGVHD). Recent reports suggest that inhibition of ROCK2 induces apoptosis and prevents growth of MM cells. Here, we investigated the potential antimyeloma effect of belumosudil alone or in combination with isatuximab with different readouts of viability, apoptosis and cytotoxicity. The potential cytotoxic effect of belumosudil (as single agent or in combination with isatuximab) against MM cells was assessed in vitro by flow cytometry apoptosis assay, CellTiter-Glo® luminescent cell viability assay or long-term cytotoxic assay (Incucyte). The effect of belumosudil on healthy donor-derived NK cells viability was also assessed by flow cytometry. Belumosudil reduced the number and viability of several MM cell lines without affecting healthy donor NK cell number and viability. Moreover, belumosudil induced apoptosis of MOLP-8 MM cells in a dose-dependent manner and further increased MOLP-8 cell apoptosis when combined with isatuximab after 2, 3 and 4 days of in vitro treatment. Belumosudil induced killing of MM cells in a dose-dependent manner over time (up to 4 days), independently of the presence of NK cells, as measured by Incucyte-based cytotoxicity assay. In addition, the combination of belumosudil (3.3 µM) with isatuximab (10 µg/ml) in presence of NK cells enhanced the killing of different MM cell lines over time compared to single agents.Finally, the expression of CD38 on MM cells has been documented to be decreased after treatment with anti-CD38 antibodies. Interestingly, analysis of CD38 expression on MM cells indicated that belumosudil induces an increase of CD38 on the surface of MOLP-8 MM cells and prevents isatuximab-induced decrease of CD38 expression. These data suggest that belumosudil has a direct antimyeloma effect and increases isatuximab-mediated cytotoxicity against MM cells, without affecting the viability of NK cells. Taken together, this study provides evidence of the therapeutic potential of belumosudil in combination with isatuximab in the setting of MM. Citation Format: Marco Meloni, Pauline Perrin, Claire Nouguier, Stephane Poirier, Monsif Bouaboula, Kamlesh Bisht, Helgi Van de Velde, Angela Virone-Oddos, Marielle Chiron. The ROCK-2 inhibitor belumosudil exerts a direct antimyeloma effect and improves isatuximab-mediated cytotoxicity against multiple myeloma cells [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 5277.

Abstract 2374: Bispecific and trispecific GPRC5D antibodies with potent cell-killing activity against multiple myeloma

Abstract Background: GPRC5D is a G protein-coupled receptor that is expressed on multiple myeloma cells but absent from most healthy tissues except for hair follicles. It is an attractive target for anti-tumor modalities including T cell-engaging bispecific antibodies. Recent clinical data demonstrate that combination therapy of T cell-engaging molecules individually targeting GPRC5D and BCMA on tumor cells offers unprecedented therapeutic benefits in relapsed/refractory multiple myeloma patients. A trispecific antibody that simultaneously engages these molecules while activating T cells is expected to provide comparable and additional benefits as a monotherapy. Methods: Multipass membrane proteins are valuable therapeutic targets but are largely inaccessible for antibody discovery due to structural complexity and high conservation. We developed an antibody discovery platform (MPS) tailored for membrane proteins that utilizes advanced immunization techniques including DNA, mRNA, and Lipoparticles (virus-like particles) and evolutionarily divergent host species. From the parental antibodies isolated, we engineered panels of GPRC5DxCD3 bispecific and GPRC5DxBCMAxCD3 trispecific antibodies using multiple formats and CD3 arms that encompass different geometries and binding stoichiometries. GPRC5D multispecific antibodies were tested for target specificity using Integral Molecular’s Membrane Proteome Array (MPA), and epitope residues were defined using alanine scanning mutagenesis. Both bispecific and trispecific antibodies were tested for in vitro potency and cytokine release using MM.1R and RPMI 8226 multiple myeloma cell lines. In vivo efficacy for a subset of antibodies was determined using xenograft models in PBMC-engrafted mice. Results: We immunized chickens with GPRC5D to obtain high-titer immune responses and configured resulting antibodies as T cell-engaging bispecific molecules with a CD3 binding arm. A subset of GPRC5DxCD3 bispecific antibodies displayed potent T cell-mediated cytotoxicity with picomolar potency on multiple myeloma cell lines. Antibody specificity profiling showed high specificity binding, and epitope mapping helped to explain why only some GPRC5D antibodies were active as multispecific antibodies. Bispecific molecules were then re-engineered as trispecific antibodies with additional binding to BCMA, a well-validated multiple myeloma target. GPRC5DxBCMAxCD3 trispecifics showed improved cytotoxic activity against multiple myeloma cells. Both bispecific and trispecific antibodies demonstrated a large window between cell killing and cytokine release. We will present in vivo efficacy data for a panel of bi- and trispecific molecules against MM.1R xenografts in PBMC-engrafted mice. Citation Format: Hayley Roth, Daniel Rogers, Ileine Sanchez, Breanna Tyrell, Trevor Barnes, Ami Snyder, Nenna Nowgu, Kyle Doolan, Benjamin J. Doranz, Ross Chambers, Joseph B. Rucker. Bispecific and trispecific GPRC5D antibodies with potent cell-killing activity against 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 2374.

Abstract 2739: XON27, a novel and potent immunotherapy against hematologic malignancies

Abstract Background: Over the past decades, intensive research has led to the development of innovative therapies (monoclonal and bispecific antibodies, CAR T cells) that have increased the survival rate of patients with multiple myeloma (MM) and B cell lymphoma. However, despite this progress, the majority of patients relapse and become refractory due to the development of resistance. Polyclonal antibodies, with their ability to target multiple antigens, can counteract this resistance. Here we evaluate the safety and efficacy of XON27, a first-in-class glyco-humanized polyclonal antibody (GH-pAb), in MM and other blood cancers. Material and Methods: The ability of XON27 to induce antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and apoptosis was tested in a panel of hematological malignancy cells and peripheral blood mononuclear cells (PBMC). Specific binding to human blood cancers was assessed using tissue microarrays. To study resistance mechanisms, MM cell lines were cocultured with macrophages in the presence or absence of XON27 and macrophage. To study relapse mechanisms and tumorigenicity, repeated in vitro administration of XON27 was evaluated in KMS-12-BM. The in vivo efficacy of XON27 was evaluated in KMS-12-BM NMRI nude mice model. Pharmacokinetics and safety of this drug were assessed in non-human primate after repeated IV dosing up to 60mg/kg.Results: XON27 induces potent and specific cytotoxicity in several blood malignancy cell lines including human MM cells via multiple mechanisms: CDC (EC50=35.84 ug/ml), apoptosis (IC50=100 ug/ml) and ADCP (up to 40% of cell death at 50 ug/ml). The cytotoxicity induced by XON27 in the KMS-12-BM was significantly more potent compared to daratumumab (anti-CD38) in both CDC and apoptosis assays, XON27 was able to kill up to 100% of cancer cells without affecting PBMC. XON27 showed cross-reactivity with blood cancer patient biopsies without staining healthy tissue. In addition, repeated administrations of XON27 demonstrate its ability to effectively kill tumor cells after each administration. No resistance is induced after repeated treatments. Interestingly, XON27 blocked the proliferation of residual tumor cells for up to 45 days after the second treatment at 50 ug/ml compared to the control condition, demonstrating sustainable effect of XON27 on tumorigenicity. The in vitro efficacy of XON27 was translated into in vivo efficacy in a murine MM xenograft model. It induced a significant reduction in tumor growth of up to 75%. XON27 was also characterized by a high tolerability profile and satisfactory exposure in primates. Conclusion: Although many therapeutic advances have been made in the field of blood cancers, myeloma remains an incurable disease with a real need for new innovative therapies. XON27 represents a promising and selective immunotherapy which may provide an effective and safe treatment against refractory myeloma. Citation Format: Carine Ciron, Pierre Morice, Françoise Shneiker, Odile Duvaux, Firas Bassissi. XON27, a novel and potent immunotherapy against hematologic malignancies [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 2739.

Abstract 4180: Evaluation of CD38 pharmacodynamics in multiple myeloma using PET with a Ga-68 labelled peptide

Abstract Introduction: Multiple myeloma (MM) is a disease of plasma cells that primarily arises in the bone marrow. CD38 is a receptor expressed uniformly and with high density in MM. Several CD38 targeted therapeutics are in clinic for MM. However, how those therapeutics engage or modulate CD38 is poorly understood particularly in lesions that cannot be assessed with routine blood testing. CD38 specific radiotracers for positron emission tomography (PET) could be helpful for detection of MM and for assessing therapy response early by measuring CD38 pharmacodynamics non-invasively. Here, we report the development of a first-in-class peptide-based PET radiotracer, [68Ga]AJ206, evaluation of its pharmacokinetics (PK), specificity for non-invasive CD38 detection, and its potential to evaluate therapy induced CD38 pharmacodynamics in mouse models of MM. Methods: A bicyclic peptide, AJ206, was synthesized and its binding affinity for CD38 was determined by surface plasmon resonance. AJ206 was labelled with Gallium-68 in high radiochemical yields and purity. In vitro uptake of [68Ga]AJ206 was carried out in four MM cell lines with variable CD38 expression (MOLP8, MM1S, RPMI8226, and U266). PK and biodistribution of [68Ga]AJ206 were evaluated by PET in cell line derived xenografts, in patient derived xenografts (PDXs) and in a disseminated disease model. PET data was corroborated by ex vivo biodistribution studies. [68Ga]AJ206 in vivo specificity for CD38 was confirmed by co-injection of non-radioactive AJ206 (1 mg/kg) and cross-correlative immunohistochemistry (IHC) of matched xenografts. Furthermore, ability of [68Ga]AJ206 to quantify CD38 pharmacodynamics was established by treating PDX models with all-trans retinoic acid (ATRA) and cross validating PET imaging observations with IHC. Results: AJ206 exhibits high affinity (KD,19 nM) for human CD38. In vitro binding assays showed variable [68Ga]AJ206 uptake in MM cells that correlated with receptor density measured by flow cytometry. Dynamic PET-MR imaging revealed high and specific uptake of [68Ga]AJ206 in MOLP8 tumor within 5 min that retained for at least 90 min. In contrast, [68Ga]AJ206 exhibited rapid clearance from normal tissues providing high contrast images at 60 minutes and a high tumor-to-muscle ratio of 27.1±2.7. Tumor uptake was reduced by > 80 % in mice receiving blocking dose, confirming the specificity of the radiotracer. Also, [68Ga]AJ206 could detect CD38 expression in different MM xenografts, PDXs and disseminated disease models which was corroborated by flow cytometry and IHC findings. Moreover, [68Ga]AJ206 successfully quantified the increased CD38 expression in PDXs following ATRA treatment. Conclusion: [68Ga]AJ206 is a CD38 specific high affinity peptide-based PET imaging agent that can be used to non-invasively quantify CD38 pharmacodynamics in MM, with potential applications in other diseases with CD38 involvement. Citation Format: Ajay Kumar Sharma, Kuldeep Gupta, Akhilesh Mishra, Gabriela Lofland, Ian Marsh, Dhiraj Kumar, Gabriel Ghiaur, Philip Imus, Steven P. Rowe, Robert Hobbs, Christian B. Gocke, Sridhar Nimmagadda. Evaluation of CD38 pharmacodynamics in multiple myeloma using PET with a Ga-68 labelled peptide [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 4180.

Abstract 2305: Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry

Abstract Characterizing the heterogeneity in human leukemias is critical for understanding mechanisms of disease onset, uncovering biomarkers for disease diagnosis, and guiding research into novel treatment approaches. While extensive research focus has been placed on characterizing the molecular heterogeneity in leukemia by modern omics technologies, comparatively less emphasis has been placed on studying the diversity in holistic morphological changes that occur in single cells in the context of disease. Here, we report on a novel approach to understanding and characterizing different hematological malignancies using ghost cytometry, a recently developed high-content flow cytometric method that leverages high-speed, artificial intelligence (AI) driven morphological characterization and analysis of single cells. We performed morphometric profiling on human bone marrow samples from patients with T and B-cell acute lymphoblastic leukemia (ALL, n=5), acute myeloid leukemia (AML, n=5), multiple myeloma (MM, n=5) and healthy bone marrow mononuclear cell (BM-MNC) controls (n=6). Morphological profiles from individual cells were analyzed by unsupervised machine learning (universal manifold approximation and projection, UMAP) and compared within disease subsets and against controls. We identified disease-specific cell populations from a mixture of healthy control and disease samples without any labeling, based on completely label-free information. For validation of the specific cell subsets, we overlaid known cell surface markers (e.g. CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM). The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population. We also identified a unique cell subpopulation in the disease-specific cell population that could not be distinguished by existing markers. We also analyzed three human myeloma cell lines (AMO1, KMS, and L363) for comparison to the primary MM samples. In the UMAP analysis using LF-GC data, in all three types of cell lines, Bortezomib-resistant cell lines exhibited different distributions from the control parental cell lines. In addition, we observed distinct morphological profiles between primary myeloma samples and myeloma cell lines. Here we present a novel cell characterization approach for human hematological diseases that leverages AI-based, label-free, high-speed morphological characterization of single cells. We demonstrate that the approach can be used to identify subtle morphological differences in blast cells from a range of blood cancer subtypes. Application of morphological profiling and AI can be used to identify measurable, disease-related changes in these diseases that have diagnostic, drug screening, and therapeutic monitoring potential. Citation Format: Asako Tsubouchi, Juho J. Miettinen, Keisuke Wagatsuma, Satoru Akai, Yuri An, Mika Uematsu, Philipp Sergeev, Dimitrios Tsallos, Markus J. Vähä-Koskela, Sadao Ota, Caroline A. Heckman. Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry [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 2305.

Abstract 660: OPN-6602, a potent dual EP300/CBP bromodomain inhibitor, targets multiple myeloma through concomitant suppression of IRF4 and c-MYC

Abstract Multiple myeloma (MM) is an aggressive hematological cancer derived from malignant plasma cells in the bone marrow. E1A-binding protein (EP300) and CREB-binding protein (CBP) are transcription coactivators that contain both epigenetic writer histone acetyltransferase (HAT) and reader bromo- (BRD) domains that regulate transcription of genes via chromatin remodeling. EP300/CBP inhibition causes cell cycle arrest and apoptosis in MM through suppression of interferon regulatory factor (IRF4) and concomitant repression of c-MYC, rendering EP300/CBP as a novel target for MM. OPN-6602 is a potent, selective, and orally active small molecule dual EP300/CBP inhibitor discovered through structure guided drug design. Its binding interaction exhibits low nM activity on EP300 (Kd= 1.5 nM) and CREBBP (Kd= 1 nM), with > 200-fold less activity against BRD4. OPN-6602 binding to EP300 is particularly potent by Surface Plasmon Resonance analysis, displaying a Kd of 0.87 nM, with a fast association rate (1.5 E+06/Ms) and a slow dissociation rate (1.3 E-03/s) with a residence time of 13 min. It potently inhibits cell growth in MM cell lines OPM-2 (IC50 ~ 4 nM) and MM.1S (IC50 ~ 47 nM) as well as histone acetylation (IC50 ~ 2 nM) via BRD intramolecular modulation of HAT activity. OPN-6602 demonstrated potent inhibition of H3K27Ac in both LK2 (CBP-deficient lung cancer cells, IC50 = 11 nM) and in OPM-2 (IC50 = 1.6 nM). OPN-6602 also potently inhibited H3K27ac expression in PBMCs with IC50s of 0.75 and 1.4 nM, respectively. The effects of OPN-6602 with dexamethasone (dex) or lenalidomide (len) on the growth of OPM-2 and MM1.S cells were evaluated in a matrix combination study. As single agents, maximal growth inhibition reached 60-75% following dex treatment and only 30-40% following len treatment. In vitro combination studies utilizing a systematic assessment of a broad range of OPN-6602 concentrations in combination with dex or len demonstrated strong synergy. Synergy was observed between OPN-6602 at 3.7 nM and len through a concentration range from 0.027 to 20 µM. Synergy was observed with 1.2 or 3.7 nM of OPN-6602 in combo with dex concentrations from 0.055 to 40 µM. In the OPM-2 xenograft model, OPN-6602 inhibits tumor growth by 30-111% at doses from 2.5-60 mg/kg QD. This growth inhibition correlated with downregulation of IRF4 and cMYC in tumor samples analyzed by QPCR analysis. The pharmacokinetic profile is favorable for oral QD dose administration due to a high Cmax (19 µg/mL at 24 mg/kg) and short half-life (T1/2 = 1.5 hr). A first-in-human study of OPN-6602 in patients with MM is planned for 2024. Citation Format: Bernice Matusow, Wayne Spevak, Chao Zhang, Yan Ma, Rafe Shellooe, James Tsai, Pei Pei Li, Pan Yu Chen, Gaston Habets, Christine Nichols, Parmveer Singh, Kerry Inokuchi, Jackie Walling, Jason Halladay, Gideon Bollag. OPN-6602, a potent dual EP300/CBP bromodomain inhibitor, targets multiple myeloma through concomitant suppression of IRF4 and c-MYC [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 660.

Abstract 2727: Pre-clinical development of a novel anti-GPRC5D inducing potent anti-tumor effect through enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) for multiple myeloma

Abstract Multiple myeloma (MM), characterized by clonal accumulation of malignant plasma cells in the bone marrow, is the second most prevalent hematopoietic malignancy. Despite emerging therapies, MM remains an incurable disease with a 5-year overall survival of 56% for relapsed and/or refractory disease (RRMM) patients. Thus, innovative approaches that would be safe and potent are required. GPRC5D (G Protein-Coupled Receptor Class C Group 5 Member D) is an orphan GPCR protein involved in keratin synthesis with limited expression in healthy tissues. GPRC5D is expressed at very high prevalence among MM patients and its expression on tumor cells is maintained after treatments with standard of care agents such as anti-CD38, or BCMA-targeting therapies. The ability of NK cells to kill tumor cells leaving other cells unharmed and to induce minimal pro-inflammatory cytokine release as compared to T cell-based therapies, makes NK cells ideal immune cells for a safe and efficacious therapeutic approach. Like Sarclisa®, other therapies engaging NK cells and mediating tumor cell killing through antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism have demonstrated their efficacy in clinic. Here we report the design, preclinical characterization, and development of a novel anti-GPRC5D antibody inducing potent cytotoxic activity against MM cells. SAR446523 is an IgG1-based antibody with an engineered Fc domain for enhanced ADCC function and targeting human GPRC5D on tumoral plasma cells. SAR446523 demonstrated high affinity for GPRC5D target and increased affinity for both high and low affinity FCGR3A-158V and 158F variants of CD16a receptor. The ability of SAR446523 to induce ADCC in vitro was assessed in a panel of MM cell lines exhibiting low, medium, and high GPRC5D expression. SAR446523 induced strong potency against all tested cell lines (picomolar range), regardless of the levels of GPRC5D. In addition, SAR446523 induced higher cytotoxic activity compared to a clinical benchmark compound. This anti-tumor activity was associated with very low IL-6, TNFa and IFNg pro-inflammatory cytokine release, much lower compared to a CD3xGPRC5D T-cell engager in in vitro assay using human peripheral blood mononuclear cells (PBMC) in presence of GPRC5D positive cells. In addition, the in vivo anti-tumor activity of SAR446523 was investigated in a NK humanized NOG huIL15 transgenic mouse model bearing disseminated MM cells. SAR446523 led to a robust antitumoral efficacy with a significant improved mouse survival. These data demonstrating potent anti-tumor activity and favorable safety profile of this novel anti-GPRC5D antibody suggest that SAR446523 may be a promising therapy for patients with RRMM and provide consistent support for clinical development. Citation Format: Helene Bonnevaux, Elisabeth Remy, Theo Pizette, Sophie Bouvier, Klervi Desrumeaux, Laurent Bassinet, Celine Nicolazzi, Pauline Rettman, Zandra Klippel, Marco Meloni, Marielle Chiron, Angela Virone-Oddos. Pre-clinical development of a novel anti-GPRC5D inducing potent anti-tumor effect through enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) for 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 2727.

Abstract 1691: p300 catalytic inhibition selectively targets IRF4 oncogenic activity in multiple myeloma

Abstract Multiple myeloma (MM), an incurable plasma cell malignancy, is addicted to oncogenic transcription factor (TF) IRF4 signaling. Lineage TFs like IRF4 are difficult to drug but represent high value oncology targets owing to the depth and context selectivity of their genetic dependency. Using a transcription regulatory network (TRN) based approach to integrate a high dimensional multi-platform dataset, we defined the key regulatory activity of IRF4 and its druggable co-factors. This unbiased analysis identified p300 lysine acetyl transferase (KAT) function as the nearest selective neighbor for therapeutic intervention, with more potent and selective effects than historically observed with p300/CBP bromodomain inhibitors. To further probe this biology, we developed a highly selective inhibitor of the KAT domain of p300 and its close homolog CBP. p300 KAT inhibition led to selective downregulation of IRF4 at the mRNA and protein level as well as reduction in acetylation at highly specific sites co-localized with IRF4, in contrast to more general agents like BET inhibitors. Gene expression reduction was most pronounced at IRF4/p300 co-bound genes, in particular IRF4 itself and MYC. IRF4 and p300 also directly bind to each other and other key MM nodes regulating them at the chromatin, mRNA, and protein levels. p300 KAT inhibition exhibits strong antiproliferative activity across in vitro MM models regardless of cytogenetics or resistance to other therapies. Moreover, this relationship is mediated by the KAT domain and not its acetyl-lysine recognizing bromodomain. Comparative pharmacology studies found that a p300 KAT inhibitor downregulated the IRF4 TRN and induced apoptosis in MM cells more effectively than existing standard of care agents, including IMiDs and glucocorticoid receptor agonists, or p300/CBP bromodomain inhibitors. Combinations of a p300 KAT inhibitor with clinically active molecules such as IMiDs and glucocorticoid agonists showed synergistic impacts on reducing key MM TFs and cell line viability. Ex vivo treatment of primary patient samples demonstrated broad anti-proliferative activity with loss of IRF4 and induction of apoptosis in relapsed-refractory MM patient samples, independent of prior lines of therapy. Importantly, the induction of MM cell apoptosis occurs in the milieu of CD138 negative bone marrow stromal cells while also sparing them from toxicity, supporting a potential therapeutic index. Finally, p300 KAT inhibition demonstrated activity in in vivo MM models with robust tumor growth inhibition at tolerated doses and exposures correlated to reduced p300-dependent substrate acetylation. IRF4 dependency is a hallmark of MM that has been challenging to target with existing therapies, motivating further development of p300 KAT targeting therapeutics. Citation Format: Michael R. McKeown, Giulia Giorgetti, Walter F. Lenoir, Marek J. Kobylarz, Tamara D. Hopkins, Wayne L. Glore, Michelle G. Shum, Yare Calderon, Kameron R. Mori, Hua Gao, Luis A. Carvajal, Nikolaus D. Obholzer, Benjamin W. Trotter, Christopher D. Dinsmore, Nikhil C. Munshi, Peter B. Rahl, Mariateresa Fulciniti, Charles Y. Lin. p300 catalytic inhibition selectively targets IRF4 oncogenic activity in 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 1691.

Abstract 1906: UNO-A001 (A001), a clinical candidate CD38 x CD3 bispecific with a novel structure, potently kills multiple myeloma cells in vitro and is highly effective in vivo

Abstract CD38 is a validated target for the treatment of multiple myeloma (MM) as evidenced by the important clinical activity of CD38 targeting monoclonal antibodies. Bispecific T Cell Engagers (TCEs) kill cancer cells by distinct mechanisms as compared to standard format antibody therapeutics and several TCEs have demonstrated impressive clinical activity, and have been approved for clinical use, in hematologic malignancies. Here we describe a novel bispecific leveraging the potent killing mechanism of TCEs to target CD38 bearing cancer cells. UNO-A001 is a TCE binding the cell surface receptor CD38 on target human cancer cells and CD3 on T cells. The molecule has a differentiated format, binding CD38 with a high affinity humanized camelid VHH moiety and CD3 on T cells through a unique antibody Fab arm. The affinities of each arm were optimized so that the CD38 arm bound with high affinity and the CD3 arm with much lower relative binding affinity. This configuration was designed to engage CD38 bearing target cells with multiple TCE molecules at lower doses and concentrations while minimizing T cell binding in the periphery, and in the absence of cell surface CD38. A001 potently and specifically killed human CD38 bearing multiple myeloma cell lines in T cell killing assays in vitro with EC50s in the 2.7-6.6 pM range. This potency, despite the designed lower affinity to CD3, may be due to the unique structure of A001 enabling a productive immunologic synapse between T cells and the target cancer cells, and an optimal TAA/CD3 binding affinity ratio. A001 was also highly effective in inhibiting tumor growth in H929 xenograft models. In a flank model of H929 tumor growth A001 dose dependently inhibited tumor growth with complete growth inhibition of established tumors at the highest dose level tested. In a systemic disease model with NCI-H929-luc cells A001 was also highly effective and improved survival relative to control treated animals. In preparation for clinical studies a robust and high yield production cell line was created and characterized. It achieved high expression levels of quality monodisperse UNO-A001 product that was stable in all relevant storage conditions. A001 has similar affinities for human and cynomolgus macaque CD38 and CD3. Dose ranging studies in cynomolgus macaques demonstrated tolerability consistent with clinically approved TCEs, and the expected dose dependent pharmacodynamic effects on CD38 expressing myeloid and lymphoid cell populations. These data support planned clinical testing of A001 in multiple myeloma and other CD38 bearing hematologic malignancies, including mantle cell lymphoma, T cell lymphoma, and acute myelogenous leukemia. Citation Format: Mark Anderson, Yan Ling, Mark L. Chiu, Peter Cheung. UNO-A001 (A001), a clinical candidate CD38 x CD3 bispecific with a novel structure, potently kills multiple myeloma cells in vitro and is highly effective in vivo [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 1906.

Abstract 521: Anticancer mechanisms of epigallocatechin gallate revealed via cellular and molecular profiling

Abstract Introduction: Epigallocatechin gallate (EGCG) is a polyphenol present in green tea that is known for its anti-oxidant, anti-inflammatory, anti-angiogenic, pro-apoptotic, and anti-cancer properties. However, EGCG has not been systematically evaluated on a large panel of cancer cell lines. We hypothesized that EGCG exhibits varying effects on specific cancer cell genetic backgrounds and types. This study aimed to provide a comprehensive analysis of EGCG on both cell viability and gene expression profiles. Methods: 750 cancer cell lines were cultured and seeded in 1536-well plates for cell viability studies (by the CTD2 Center at the Broad Institute). After 24 h, cells were treated with 16 two-fold serial dilutions of EGCG. After 72 h, cell viability was assayed using CellTiter-Glo. For gene expression profiling (with the Broad Institute and NIH), 8 cell lines were treated with 10 µM EGCG for 6 and 24 h, and transcriptomes were assessed using Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. The CMap large-scale transcriptome dataset was used for comparisons. Results: EGCG had the highest potency in the non-Hodgkins B-cell lymphoma cell line DoHH2 (IC50=0.42 µM), the myeloid leukemia cell line Ku812 (IC50=3.76 µM), and the multiple myeloma cell line KMS-28BM (IC50=4.57 µM). Tissue-of-origin analysis showed that EGCG was most potent against lymphoid cancers, and least potent against prostate cancers. Bioinformatics-based analyses found that EGCG-treated cells resembled cells overexpressing CHEK2, DDP4, CBLC, ZNF350, and TRAF6, and cells underexpressing TRIM16. EGCG-induced gene expression resembled lapatinib and sulfasalazine treatment. Conclusions: This study provides a comprehensive examination of the differential impact of EGCG treatment on cancer cells. Subsequent research on the timing and duration of the changes, along with pathway enrichment analyses, will potentially guide the translational potential of EGCG and its chemical analogs in personalized cancer treatment approaches. Citation Format: Aria Panchal, Jacqueline H. Law, Clement Lo, Kenneth W. Yip. Anticancer mechanisms of epigallocatechin gallate revealed via cellular and molecular profiling [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 521.

Abstract 4517: Novel chemical degraders targeting NSD2 in head and neck squamous cell carcinoma (HNSCC)

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Despite advances in treatments and significant efficacy of immunotherapy drugs in HNSCC, no effective targeted therapies have been approved in this disease yet. NSD2 is a histone lysine methyltransferase, which is overexpressed in HNSCC and is implicated in tumor growth, cell cycle regulation, epithelial-mesenchymal transition (EMT) and metastasis not only in HNSCC, but also in many other solid tumors. Development of inhibitors to the catalytic activity of NSD2 has been rather limited to date, which necessitates an alternative approach. To target NSD2, we have recently developed a series of novel proteolysis targeting chimeras (PROTACs), which chemically degrade NSD2 utilizing native cellular machinery - ubiquitin-proteasome system - with VHL as an E3 ligase. A series of >40 compounds were tested in HNSCC cell lines (JHU-011 and FaDu) and NSD2-dependent multiple myeloma cell line (KMS11) for degradation activity against NSD2 protein, and H3K36me2 chromatin mark reduction, using Western blot. We also tested their anti-proliferative activity in cell viability assay (CTB) and clonogenic assay. Compounds NUCC-0227381, NUCC-0227384, NUCC-0227414 and NUCC-0227383 show promising results by reducing NSD2 and H3K36me2 levels. NUCC-0227383 compound was highly promising, as it was the most potent in reducing NSD2/K36me2 levels without affecting NSD1/NSD3 levels and had profound effects on HNSCC cell growth. Lead compound NUCC-0227383 was chosen for further testing and in preliminary testing, showed a dose-dependent, VHL-mediated, and proteasome dependent function. Proteomics analysis, testing for specificity of NUCC-0227383 against a panel of histone methyltransferases, and in vivo testing of this promising compound against HNSCC is planned. Citation Format: Amr Ismail, Ekaterina Roshchina, Iuliia Topchu, Gary Schiltz, Yanis Boumber. Novel chemical degraders targeting NSD2 in head and neck squamous cell carcinoma (HNSCC) [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 4517.

Steroid-free combination of 5-azacytidine and venetoclax for the treatment of multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy that, despite an unprecedented increase in overall survival, lacks truly risk-adapted or targeted treatments. A proportion of patients with MM depend on BCL-2 for survival, and, recently, the BCL-2 antagonist venetoclax has shown clinical efficacy and safety in t(11;14) and BCL-2 overexpressing MM. However, only a small proportion of MM patients rely on BCL-2 (approx. 20%), and there is a need to broaden the patient population outside of t(11;14) that can be treated with venetoclax. Therefore, we took an unbiased screening approach and screened epigenetic modifiers to enhance venetoclax sensitivity in 2 non-BCL-2 dependent MM cell lines. The demethylase inhibitor 5-azacytidine was one of the lead hits from the screen, and the enhanced cell killing of the combination was confirmed in additional MM cell lines. Using dynamic BH3 profiling and immunoprecipitations, we identified the potential mechanism of synergy is due to increased NOXA expression, through the integrated stress response. Knockdown of PMAIP1 or PKR partially rescues cell death of the venetoclax and 5-azacytidine combination treatment. The addition of a steroid to the combination treatment did not enhance the cell death, and, interestingly, we found enhanced death of the immune cells with steroid addition, suggesting that a steroid-sparing regimen may be more beneficial in MM. Lastly, we show for the first time in primary MM patient samples that 5-azacytidine enhances the response to venetoclax ex vivo across diverse anti-apoptotic dependencies (BCL-2 or MCL-1) and diverse cytogenetic backgrounds. Overall, our data identify 5-azacytidine and venetoclax as an effective treatment combination, which could be a tolerable steroid-sparing regimen, particularly for elderly MM patients.

Mezigdomide-A Novel Cereblon E3 Ligase Modulator under Investigation in Relapsed/Refractory Multiple Myeloma.

Mezigomide is an oral cereblon E3 ligase modulator (CELMoD) that is under clinical investigation in patients with relapsed/refractory (RR) multiple myeloma (MM). Like other CELMoD compounds, mezigdomide acts by altering the conformation of cereblon within the cullin 4A ring ligase-cereblon (CRL4CRBN) E3 ubiquitin ligase complex, thereby recruiting novel protein substrates for selective proteasomal degradation. These include two critical lymphoid transcription factors, Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3), also known as Ikaros and Aiolos, which have important roles in the development and differentiation of hematopoietic cells, in MM pathobiology, and in suppressing the expression of interferon-stimulating genes and T-cell stimulation. Among the CELMoDs, mezigdomide has the greatest cereblon-binding potency, plus the greatest potency for the degradation of Ikaros and Aiolos and subsequent downstream antimyeloma effects. Preclinical studies of mezigdomide have demonstrated its anti-proliferative and apoptotic effects in MM, along with its immune-stimulatory effects and its synergistic activity with other antimyeloma agents, including in lenalidomide-/pomalidomide-resistant MM cell lines and mouse xenograft models. Early-phase clinical trial data indicate notable activity in heavily pretreated patients with RRMM, including those with triple-class-refractory disease, together with a tolerable and manageable safety profile. This review summarizes current preclinical and clinical findings with mezigdomide and its potential future roles in the treatment of MM.

Discovery and characterization of potent spiro-isoxazole-based cereblon ligands with a novel binding mode

The vast majority of current cereblon (CRBN) ligands is based on the thalidomide scaffold, relying on glutarimide as the core binding moiety. With this architecture, most of these ligands inherit the overall binding mode, interactions with neo-substrates, and thereby potentially also the cytotoxic and teratogenic properties of the parent thalidomide. In this work, by incorporating a spiro-linker to the glutarimide moiety, we have generated a new chemotype that exhibits an unprecedented binding mode for glutarimide-based CRBN ligands. In total, 16 spirocyclic glutarimide derivatives incorporating an isoxazole moiety were synthesized and tested for different criteria. In particular, all ligands showed a favorable lipophilicity, and several were able to outperform the binding affinity of thalidomide as a reference. In addition, all compounds showed favorable cytotoxicity profiles in myeloma cell lines and human peripheral blood mononuclear cells. The novel binding mode, which we determined in co-crystal structures, provides explanations for these improved properties: The incorporation of the spiro-isoxazole changes both the conformation of the glutarimide moiety within the canonical tri-trp pocket and the orientation of the protruding moiety. In this new orientation it forms additional hydrophobic interactions and is not available for direct interactions with the canonical neo-substrates. We therefore propose this chemotype as an attractive building block for the design of PROTACs.

Retraction: Cytokines Modulate Telomerase Activity in a Human Multiple Myeloma Cell Line.

Retraction: Cytokines Modulate Telomerase Activity in a Human Multiple Myeloma Cell Line.

Expression of the chemokine receptor CCR1 decreases sensitivity to bortezomib in multiple myeloma cell lines

BackgroundThe proteasome inhibitor bortezomib is one of the primary therapies used for the haematological malignancy multiple myeloma (MM). However, intrinsic or acquired resistance to bortezomib, via mechanisms that are not fully elucidated, is a barrier to successful treatment in many patients. Our previous studies have shown that elevated expression of the chemokine receptor CCR1 in MM plasma cells in newly diagnosed MM patients is associated with poor prognosis. Here, we hypothesised that the poor prognosis conferred by CCR1 expression is, in part, due to a CCR1-mediated decrease in MM plasma cell sensitivity to bortezomib. MethodsIn order to investigate the role of CCR1 in MM cells, CCR1 was knocked out in human myeloma cell lines OPM2 and U266 using CRISPR-Cas9. Additionally, CCR1 was overexpressed in the mouse MM cell line 5TGM1. The effect of bortezomib on CCR1 knockout or CCR1-overexpressing cells was then assessed by WST-1 assay, with or without CCL3 siRNA knockdown or addition of recombinant human CCL3. NSG mice were inoculated intratibially with OPM2-CCR1KO cells and were treated with 0.7 mg/kg bortezomib or vehicle twice per week for 3 weeks and GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. The effect of CCR1 overexpression or knockout on unfolded protein response pathways was assessed using qPCR for ATF4, HSPA5, XBP1, ERN1 and CHOP and Western blot for IRE1α and p-Jnk. ResultsUsing CCR1 overexpression or CRIPSR-Cas9-mediated CCR1 knockout in MM cell lines, we found that CCR1 expression significantly decreases sensitivity to bortezomib in vitro, independent of the CCR1 ligand CCL3. In addition, CCR1 knockout rendered the human MM cell line OPM2 more sensitive to bortezomib in an intratibial MM model in NSG mice in vivo. Moreover, CCR1 expression negatively regulated the expression of the unfolded protein response receptor IRE1 and downstream target gene XBP1, suggesting this pathway may be responsible for the decreased bortezomib sensitivity of CCR1-expressing cells. ConclusionsTaken together, these studies suggest that CCR1 expression may be associated with decreased response to bortezomib in MM cell lines.

Efbalropendekin Alfa enhances human natural killer cell cytotoxicity against tumor cell lines in vitro.

IL-15 has shown preclinical activity by enhancing the functional maturation of natural killer (NK) cells. Clinical evaluation of the potential anticancer activity of most cytokines, including IL-15, has been limited by low tolerability and rapid in vivo clearance. Efbalropendekin Alfa (XmAb24306) is a soluble IL15/IL15-receptor alpha heterodimer complex fused to a half-life extended Fc domain (IL15/IL15Rα-Fc), engineered with mutations to reduce IL-15 affinity for CD122. Reduced affinity drives lower potency, leading to prolonged pharmacodynamic response in cynomolgus monkeys. We show that in vitro, human NK cells treated with XmAb24306 demonstrate enhanced cytotoxicity against various tumor cell lines. XmAb24306-treated NK cells also exhibit enhanced killing of 3D colorectal cancer spheroids. Daratumumab (dara), a monoclonal antibody (mAb) that targets CD38 results in antibody-dependent cellular cytotoxicity (ADCC) of both multiple myeloma (MM) cells and NK cells. Addition of XmAb24306 increases dara-mediated NK cell ADCC against various MM cell lines in vitro. Because NK cells express CD38, XmAb24306 increases dara-mediated NK cell fratricide, but overall does not negatively impact the ADCC activity against a MM cell line likely due to increased NK cell activity of the surviving cells. These data show that XmAb24306 increases direct and ADCC-mediated human NK cell cytotoxicity in vitro.

FHND004 inhibits malignant proliferation of multiple myeloma by targeting PDZ-binding kinase in MAPK pathway.

Inhibitors of Epidermal growth factor receptor tyrosine kinase (EGFR-TKIs) are producing impressive benefits to responsive types of cancers but challenged with drug resistances. FHND drugs are newly modified small molecule inhibitors based on the third-generation EGFR-TKI AZD9291 (Osimertinib) that are mainly for targeting the mutant-selective EGFR, particularly for the non-small cell lung cancer (NSCLC). Successful applications of EGFR-TKIs to other cancers are less certain, thus the present pre-clinical study aims to explore the anticancer effect and downstream targets of FHND in multiple myeloma (MM), which is an incurable hematological malignancy and reported to be insensitive to first/second generation EGFR-TKIs (Gefitinib/Afatinib). Cell-based assays revealed that FHND004 and FHND008 significantly inhibited MM cell proliferation and promoted apoptosis. The RNA-seq identified the involvement of the MAPK signaling pathway. The protein chip screened PDZ-binding kinase (PBK) as a potential drug target. The interaction between PBK and FHND004 was verified by molecular docking and microscale thermophoresis (MST) assay with site mutation (N124/D125). Moreover, the public clinical datasets showed high expression of PBK was associated with poor clinical outcomes. PBK overexpression evidently promoted the proliferation of two MM cell lines, whereas the FHND004 treatment significantly inhibited survival of 5TMM3VT cell-derived model mice and growth of patient-derived xenograft (PDX) tumors. The mechanistic study showed that FHND004 downregulated PBK expression, thus mediating ERK1/2 phosphorylation in the MAPK pathway. Our study not only demonstrates PBK as a promising novel target of FHND004 to inhibit MM cell proliferation, but also expands the EGFR kinase-independent direction for developing anti-myeloma therapy.

Daptomycin Inhibits Multiple Myeloma Progression through Downregulating the Expression of RPS19.

This study aimed to explore new therapeutic drugs for multiple myeloma (MM). MM is a common plasma cell malignant proliferative disease, accounting for 15% of hematological malignancies. The role of daptomycin (DAP), a potential anti-tumor drug, remains unclear in MM. In the present research, we investigated the anticancer effect of DAP in MM cell line RPMI 8226. RPMI 8226 cells were treated with DAP (20 μM, 40 μM, and 80 μM) with 20 nM bortezomib (BZ) as a positive control. Cell function was detected using CCK8, flow cytometry, and transwell assay. In MM cells, DAP inhibited proliferation and induced apoptosis. The cell cycle was arrested at the G1 phase after the treatment of DAP. The migration and invasion abilities were also inhibited by DAP treatment in RPMI 8226 cells. Importantly, the mRNA and protein levels of RPS19 were downregulated in DAP-treated RPMI 8226 cells. DAP inhibited the proliferation, migration, and invasion and promoted the apoptosis of MM cells. Mechanistically, the RPS19 expression was significantly decreased in DAPtreated cells. This research provides a potential therapeutic drug for MM therapy.

MUC20 regulated by extrachromosomal circular DNA attenuates proteasome inhibitor resistance of multiple myeloma by modulating cuproptosis

BackgroundProteasome inhibitors (PIs) are one of the most important classes of drugs for the treatment of multiple myeloma (MM). However, almost all patients with MM develop PI resistance, resulting in therapeutic failure. Therefore, the mechanisms underlying PI resistance in MM require further investigation.MethodsWe used several MM cell lines to establish PI-resistant MM cell lines. We performed RNA microarray and EccDNA-seq in MM cell lines and collected human primary MM samples to explore gene profiles. We evaluated the effect of MUC20 on cuproptosis of PI-resistant MM cells using Co-immunoprecipitation (Co-IP), Seahorse bioenergetic profiling and in vivo assay.ResultsThis study revealed that the downregulation of Mucin 20 (MUC20) could predict PI sensitivity and outcomes in MM patients. Besides, MUC20 attenuated PI resistance in MM cells by inducing cuproptosis via the inhibition of cyclin-dependent kinase inhibitor 2 A expression (CDKN2A), which was achieved by hindering MET proto-oncogene, receptor tyrosine kinase (MET) activation. Moreover, MUC20 suppressed MET activation by repressing insulin-like growth factor receptor-1 (IGF-1R) lactylation in PI-resistant MM cells. This study is the first to perform extrachromosomal circular DNA (eccDNA) sequencing for MM, and it revealed that eccDNA induced PI resistance by amplifying kinesin family member 3 C (KIF3C) to reduce MUC20 expression in MM.ConclusionOur findings indicated that MUC20 regulated by eccDNA alleviates PI resistance of MM by modulating cuproptosis, which would provide novel strategies for the treatment of PI-resistant MM.