Bone Marrow Of Multiple Myeloma Patients Research Articles

Unveiling a novel NKG2A inhibitor based on monoclonal antibody to HLAE-peptide complex.

e14677 Background: HLAE is a non-classical MHC class I molecule complexed with the VMAPRTLFL nonapeptide, derived from the leader sequence of HLAG (HLAEpHLAG). HLAEpHLAG is overexpressed across a wide type of tumors prominently on Multiple Myeloma (MM). HLAEpHLAG complex is the ligand to CD94-NKG2A in the immune system which negatively regulates the activation of natural killer (NK) cells as well as NKG2A positive T cells. Harnessing of the HLAE-NKG2A axis by the cancer cells leads to evasion from the immune system attack resulting in cancer progression. Currently, several Immune Checkpoint Inhibitors (ICIs) target the NKG2A receptor which has a low specificity to cancer and high toxicity to the patient. Engineering a new ICI that targets the HLAEpHLAG complex can potentially yield improved outcomes in cancer therapy. Methods: Using the hybridoma system, a new mouse monoclonal antibody (mAb) called 4D7 that targets the HLAEpHLAG complex was developed. mAb 4D7 affinity was assessed in vitro with recombinant proteins and a panel of cell lines that expressed zero to high levels of HLAE. The functional impacts of the mAb 4D7 was examined in a system where various MM cell lines were separately co-cultured with primary NK (pNK) cells. pNK were isolated from the peripheral blood of 5 healthy donors, and their activity was examined in the presence of mAb 4D7 by CD107a degranulation assay. Also, the functional impacts of the mAb 4D7 on allogenic pNK and MM blast cells, isolated from the bone marrow of 6 MM patients were assessed. Results: mAb 4D7 was determined to be an IgG-kappa isotype showing a high specific binding to recombinant HLAEpHLAG. Cell lines with overexpression of membranal HLAEpHLAG: RPMI8226, U266, 721.221 HLAE, and 721.221 HLAG showed positive staining with the mAb 4D7. While cell lines with low to zero expression of HLA-E like 721.221 WT showed low or negative staining with the mAb 4D7. The degranulation of NKG2A+ pNK subset cells against MM target cells increased in presence of mAb 4D7. However, adding mAb 4D7 to NKG2A -pNK subset cells co-cultured with MM target cells did not affect the pNK activity. Similar results were obtained when the target and effector cells were isolated from the bone marrow of active treatment-naive MM patients. Conclusions: MAb 4D7 demonstrates a remarkably high affinity for binding to the HLAEpHLAG complex, ensuring the safety of its application. The ability of 4D7 mAb to effectively block the HLAEpHLAG complex and counteract the inhibition signal in NK cells. mAb 4D7 is a promising monoclonal antibody with substantial potential to serve as an ICI for clinical use in MM patients.

The Ratio of Serum Urea Nitrogen to Albumin Is a Better Predictor of Overall Survival in Multiple Myeloma Patients than Urea Nitrogen Alone

Introduction: Multiple myeloma (MM) is a malignant proliferative disease of plasma cells. Abnormally cloned plasma cells secrete large amounts of monoclonal immunoglobulins in the bone marrow of MM patients. Serum urea nitrogen (sUN) is a byproduct of protein metabolism, and its effect on MM patients’ prognoses remains unknown. Therefore, we analyzed MM patients’ clinical data to explore the role of sUN and sUN/serum albumin (sUAR) in the baseline tumor load and MM prognosis of MM patients. Methods: We downloaded the clinical data of 762 MM patients from the MMRF database. After excluding those without baseline sUN, 452 patients were finally included in the study. Smoothed curve fitting, threshold analysis, Tamhane’s T2 test, multivariate-adjusted Cox regression analysis, Kaplan-Meier (K-M) curves, and receiver operating characteristic (ROC) analysis were applied in the study. Results: There were 452 newly diagnosed MM patients included in this study. In most patient groups, sUN and sUAR were positively linked with β2-microglobulin (β2-MG) and lactic dehydrogenase (LDH) according to smoothing curve fitting and threshold analysis. The higher the ISS stage, the greater the values of sUN and sUAR. Furthermore, smoothed curve fitting and threshold analysis showed that sUN was correlated with overall survival (OS), although sUAR had a stronger correlation with OS and could be applied to a broader group. The results of a multivariate-adjusted Cox regression analysis demonstrated that sUN and sUAR were independent prognostic factors for OS. The K-M curve confirmed the correlation between higher sUN and sUAR levels and worse OS. β2-MG and LDH are generally recognized prognostic factors of OS. ROC analysis revealed that sUN might boost β2-MG and LDH’s predictive value and sUAR had a higher predictive value. Conclusion: This retrospective study based on the MMRF database showed that high sUN and sUAR levels were positively associated with β2-MG, LDH, and ISS staging, and sUAR exhibited a stronger correlation with OS than sUN alone.

FABP4 Suppresses the Progress of Multiple Myeloma through Enhancing Alarming Function of Bone Marrow Resident Memory T Lymphocytes

Multiple myeloma (MM) is a hematological malignancy which characterized by malignant plasma cells resident in the bone marrow (BM). The BM microenvironment plays critical roles in the invasion, proliferation and migration of myeloma cells. Despite the therapies of MM improved in recent years, this disease is still incurable and requires precise treatment. Resident memory (TRM) cells are a distinct tissue-localized T cell lineage that is crucial for protective immunity in peripheral tissues. Recent studies reveal TRM cells as a vital component of the host immune response to cancer. TRM-like tumor-infiltrating lymphocytes (TILs) can be found in a wide range of human cancers, where they portend improved prognosis. However, the precise role and mechanism of bone marrow TRM cells in MM still remain elusive. In present study we first found that the accumulation of T RM cells (CD8 +CD44 +CD69 +CD62L -) in BM newly diagnosed MM (NDMM) patients was significantly higher than that of relapse recurrent MM (RRMM) ( Figure A), which was further confirmed by the immunohistochemistry (IHC) staining analysis ( Figure B). By using Seahorse XF e analyzer we found a decrease of oxygen consumption rate (OCR) of T RM cells in RRMM comparing to that of NDMM ( Figure C). Since fatty acid metabolism is important in β oxidation, we further investigated the expression of fatty acid binding proteins (FABP4/FABP5) in T RM cells and showed that FABP4 was dominant in T RM cells of NDMM patients ( Figure D). FABP4 +/+ mice and their relative controls FABP4 -/- mice were adopted to further explore the role and mechanism of T RM cells in MM progression. The abundance of genes related fatty acids β oxidation was significantly decreased in FABP4 -/- T RM cells ( Figure E). The tumor cell viability was decreased in myeloma cells co-cultured with FABP4 -/- T RM cells in the presence of bortezomib ( Figure F). Moreover, the expression of cytotoxic cytokines including IFN γ, TNFα and CXCL10 was significantly higher in FABP4 -/- T RM cells ( Figure G). The alarming function of T RM cells was further enhanced via recruiting more effector T (CD8 +CXCR3 +) cells ( Figure H), therefore the apoptosis of tumor cells was elevated in BM of MM patients. In summary, T RM cells are necessary and sufficient for long-lived protection against tumors. T RM cells derived FABP4 exerts anti-tumor immunity through prolonging the survival of T RM cells and enhancing the recruitment of more effector T cells to suppress the tumor progression ( Figure I). This study suggests that T RM cells might serve as a novel therapeutic target in MM and treatment specific targeting FABP4 might shed light on the development of potential therapeutic strategies for treating MM especially relapse patients. Acknowledgement This project is supported by the Sun Yat-sen University Hundred Talents Program (PT19200101), Natural Science Foundation of Guangdong Province (2022A1515010290).

Metabolomics-Driven Systematic Research Reveals Different Metabolomic and Lipidomic Profiling of Bone Marrow Plasma in Patients with Multiple Myeloma

Myeloma cells resident in bone marrow (BM), therefore the microenvironment plays critical roles in the invasion, proliferation and migration. Obesity increases the morbidity and drug resistance in patient of multiple myeloma (MM) through changing the bone marrow environment. Extracellular environments such as the BM plasma likely have unique lipidomic metabolite profiles that predicts the prognosis in patients with multiple myeloma. However, the study of multiple omics on metabolic changes in BM of MM patients are still very limited. The present study utilized an untargeted metabolite and targeted complex lipid profiling (Liquid chromatography-mass spectrometry, LC-MS) analysis to identify significant differences in the metabolite levels of BM plasma between newly diagnosed MM patients with different BMI (≤24 kg/m 2, n = 10; ≥25 kg/m 2, n = 10). This was followed by verification of some of the metabolite differences of interest by targeted quantification of the metabolites using isotopic internal standards in the exploratory cohort as well as an independent validation cohort. Significant differences were noted in the amino acid profiles, lipid metabolism and amino sugar metabolism, such as increased L-Glutamate, L-Proline, D-Proline, palmitoleic acid and D-Fructose, etc between MM patients with different BMI ( Figure A-C). Glycerol phosphate shuttle, glycerolipid metabolism and de novo triacylglycerol biosynthesis were the top 3 enriched signaling pathways in BM of obese MM patients ( Figure D). Targeted complex lipid profiling analysis further showed that triglyceride (TG)，diglyceride (DG) were increased notably in obese BM plasma of MM patients and correlated significantly with BMI ( Figure E-G). Glycerolipids, triradylglycerols, diradylglycerols and fatty acyls metabolism were enriched in obese group by GSEA analysis ( Figure H). Furthermore, KEGG enrichment analysis showed differentiate expressed lipid metabolites in BM plasma of obese MM patients comparing to that of lean group, including regulation of lipolysis in adipocytes, glycerolipid metabolism, fat digestion and absorption, cholesterol metabolism and metabolic pathways, etc ( Figure I ). In summary, metabolite and complex lipid profiling of the BM plasma identifies differences in levels of metabolites and lipids between MM patients with different BMI. This may provide insight into the explanation that obesity increases the susceptibility to MM and drug resistance. Acknowledgement This project is supported by the Sun Yat-sen University Hundred Talents Program (PT19200101), Natural Science Foundation of Guangdong Province (2022A1515010290).

Multiple Myeloma B Cells and Pre-Plasma Cells Are Important Reservoirs for Myeloma Relapse Following Plasma Cell-Directed Therapy and Prevent Cure with Standard Therapies

Multiple Myeloma (MM) is a B cell neoplasm characterized by the accumulation of mature plasma cells (PCs) within the bone marrow (BM). Despite treatment advances, MM remains incurable in the vast majority of patients. Disease relapse typically occurs regardless of treatment with proteasome inhibitors (such as bortezomib and carfilzomib), immunomodulatory drugs (such as lenalidomide and pomalidomide), anti-CD38 monoclonal antibodies (such as daratumumab and isatuximab) or myeloablative melphalan and autologous stem cell transplantation, and also occurs following treatment with immunotherapeutics such as bispecific antibodies and/or CAR-T cells (targeting BCMA, FcRH5 or GPRC5D). This failure to achieve reliable cure in MM, despite the routine attainment of deep treatment responses against PCs, indicates the existence of important intra-tumor heterogeneity and the presence of rare drug-resistant MM cells with full tumorigenic capacity. To identify intra-clonal MM cell subpopulations, we examined BM samples from MM patients using a combination of FACS, single cell resolved immunoflourescence-FISH (IF-FISH), whole exome sequencing (WES), custom-capture targeted deep sequencing (CC-Seq) and single cell RNA sequencing (scRNAseq). We sought to characterize the genomic landscape and to track the dynamic interconnectedness of these intra-clonal subpopulations in patients over time. Using sequential FACS-IF-FISH studies of MM BM samples (n=140) we identified MM clone cellular subpopulations within the BM of MM patients that recapitulate the normal maturation stages between non-malignant post germinal centre B cells and mature PCs. These subpopulations include rare MM cells that resemble CD20 +CD38 -CD138 -Bcma -Irf4 - Xbp1s - B cells, CD20 -CD38 -CD138 - Bcma -Irf4 -Xbp1s - pre-plasmablasts and CD38 +CD138 -Bcma -/lowIrf4 -/+Xbp1s + pre-plasma cells, as well as predominating tumor-bulk CD38 +CD138 +Irf4 +Bcma +Xbp1s + plasma cells. Importantly, MM progenitor cells were typically found to possess all of the chromosomal translocations and copy number variations (CNV) present in MM PCs including whole chromosome ploidy changes, translocations such as t(11;14), t(4;14) and t(14;16), and secondary aberrations such as gain(1q), del(1p) and del(17p). To examine if MM progenitor cells also possess the single nucleotide variations (SNV) present within MM PCs, and are thus fully malignant, and to track MM progenitor subpopulations and their inter-relatedness within patients over time, we performed WES (n=60) plus targeted deep CC-Seq (n=210) on purified cell subpopulations from 17 patients, including 5 patients whom we sampled serially over >3 years. A median of 5 cellular subpopulations were isolated from each BM sample, enriched by factors of up to 1000-fold or more, and each subpopulation was then sequenced to a depth of 4,000-20,000x. Significantly, SNVs that were present in tumor-bulk MM PCs were also regularly detected in MM progenitor cells, although the low frequency of the clonal progenitor cells often prevented capture of complete SNV profiles. At the same time, analyses of serial BM samples from repeat donor patients taken pre- and post-treatment demonstrated that relapsing MM PCs typically did not appear to derive linearly from pre-treatment PCs, as the relapsing PCs commonly lacked a number of SNVs that had been present in pre-treatment PCs. Importantly, we instead observed that relapsed MM plasma cells appeared to consistently derive from MM progenitor cell populations, frequently B cells, as emergent relapse-specific SNVs that were exclusively detected in relapsing post-treatment PCs (but in not pre-treatment PCs) could often also be detected within pre-treatment CD138- CD38- progenitor cells isolated from BM samples that had been collected up to 3 years prior to relapse. We conclude that MM B cells and pre-plasma cell progenitors possess all of the clonal genomic aberrations required for full malignant potential. Tracking of these cells in vivo in patients over time by their SNV profile implicates them as the cellular origin of PC relapse and recurrent MM disease. We propose that cure of MM requires eradication of MM progenitor cells alongside plasma cells. We are currently conducting scRNA-seq and CITE-seq studies of primary MM samples to further characterize MM progenitor cells for gene and protein expression in order to define their optimal therapeutic targets.

Anti-BCMA Dual Epitope-Binding CAR-Marrow Infiltrating Lymphocytes (MILs) Could Offer a Potent Innovative Immunotherapeutic Tool Against Multiple Myeloma

Introduction: Chimeric antigen receptor (CAR) T cells are a promising new treatment for multiple myeloma (MM). Marrow-infiltrating lymphocytes (MILs) derived from MM patient's bone marrow are another potential innovative T cell source for CAR-based cell therapy, capable of robustly and specifically targeting myeloma cells through polyclonal targets. MILs possess polyclonal central memory T cell populations, which could be as effective MM killings via polyantigenic cytotoxic T cells with greater recognitions of broad range of tumor antigens via the polyclonal memory TCR as well as the CAR pathway with strong recognition of tumor antigens. Nanobodies are currently the smallest stable antibody molecules having high affinity for antigen, allowing them to strongly penetrate tumor tissue and even directly to the blood-brain barrier while remaining low toxicity and immunogenicity. With the peculiar properties, nanobodies have been widely used as the antigen biding domain of CAR-T cell. In this study, we investigated anti-B-cell maturation antigen (BCMA) dual epitope-binding CAR-MILs against MM. Methods: CD3 + T cells isolated from bone marrow mononuclear cells of MM patients were stimulated with Ex Vivo CD3/CD28 in the presence of recombinant human IL-2. The nanobodies VHH01 and VHH02 were provided as dual epitope-binding for BCMA (Senlang, China). After expansion and activation for 2 days, anti-BCMA dual epitope-binding CAR was transduced to the activated MILs and, they were stimulated with Ex Vivo CD3/CD28 in the presence of recombinant human IL-2, IL-7, IL-15, and IL-21. Expanded and activated BCMA dual epitope-binding CAR-MILs (BCMA CAR-MILs) were harvested on days 7, 14, and 21 for further experiments. Results: The transduction efficiency of BCMA CAR-MILs was approximately 34%, and the expansion of BCMA CAR-MILs was > 100 folds over 21-day culture period. Additionally, BCMA CAR-MILs showed a dramatically increased proportion of CD8 + T cells, while the proportion of CD4 + T cells decreased. Moreover, BCMA CAR-MILs were enriched with a higher proportion of central memory T cells (Tcm) (80%), while the proportions of regulatory T cells (Tregs) and checkpoint molecules (TIGIT, TIM3, and CD73) were not so high. The BCMA CAR-MILs showed potent cytotoxicity, particularly high levels of CD107a degranulation against BCMA-expressing target cancer cell lines, such as Daudi, Raji, RPMI8226, and U266B1 cells, and autologous CD138 + primary myeloma cells. Conclusion: This study suggested that BCMA dual epitope-binding CAR-MILs were easily expanded and activated using bone marrow mononuclear cells from MM patients with high transduction efficiency and induced a potent anti-myeloma immune response, which may offer an alternative innovative immunotherapeutic approach against MM patients in the future.

Single-cell analysis of the CD8+ T-cell compartment in multiple myeloma reveals disease specific changes are chiefly restricted to a CD69- subset suggesting potent cytotoxic effectors exist within the tumor bed.

Multiple myeloma (MM) is an incurable disease of the bone marrow (BM) characterized by the uncontrolled proliferation of neoplastic plasma cells. While CD8+ T cells have an established role in disease control, few studies have focused on these cells within the MM tumor microenvironment (TME). We analyzed CD8+ T cells in the BM and peripheral blood (PB) of untreated patients with MM and non-myeloma controls using flow cytometry, mass cytometry and single-cell RNA sequencing, using several novel bioinformatics workflows. Inter-tissue differences were most evident in the differential expression of Granzymes B and K, which were strongly associated with two distinct subsets of CD8+ T cells delineated by the expression of CD69, accounting for roughly 50% of BM-CD8+ T cells of all assessed cohorts. While few differences were observable between health and disease in the BM-restricted CD8CD69+ T-cell subset, the CD8+CD69- T-cell subset in the BM of untreated MM patients demonstrated increased representation of highly differentiated effector cells and evident compositional parallels between the PB, absent in age-matched controls, where a marked reduction of effector cells was observed. We demonstrate the transcriptional signature of BM-CD8+ T cells from patients with MM more closely resembles TCR-activated CD8+ T cells from age-matched controls than their resting counterparts.

Lactate Decreases Bortezomib Sensitivity and Predicts Poor Clinical Outcomes of Multiple Myeloma.

Metabolic disorders are regarded as hallmarks of multiple myeloma (MM) and are responsible for rapid cancer cell proliferation and tumor growth. However, the exact biological roles of metabolites in MM cells have not been fully explored. This study aimed to explore the feasibility and clinical significance of lactate for MM and investigate the molecular mechanism of lactic acid (Lac) in the proliferation of myeloma cells and cell sensitivity to bortezomib (BTZ). Metabolomic analysis of the serum was carried out to obtain metabolites expression and clinical characteristics in MM patients. The CCK8 assay and flow cytometry were used to detect cell proliferation, apoptosis, and cell cycle changes. Western blotting was used to detect the potential mechanism and apoptosis- and cycle-related protein changes. Lactate was highly expressed in both the peripheral blood and bone marrow of MM patients. It was significantly correlated with Durie-Salmon Staging (DS Staging) and the International Staging System (ISS Staging) and the serum and urinary involved/uninvolved free light chain ratios. Patients with relatively high lactate levels had a poor treatment response. Moreover, in vitro experiments showed that Lac could promote the proliferation of tumor cells and decrease the proportion of G0/G1-phase cells, which was accompanied by an increased proportion of S-phase cells. In addition, Lac could decrease tumor sensitivity to BTZ by disrupting the expression of nuclear factor kappa B subunit 2 (NFkB2) and RelB. Metabolic changes are important in MM cell proliferation and treatment response; lactate could be used as a biomarker in MM and as a therapeutic target to overcome cell resistance to BTZ.

Radiomics Analysis for Multiple Myeloma: A Systematic Review with Radiomics Quality Scoring

Multiple myeloma (MM) is one of the most common hematological malignancies affecting the bone marrow. Radiomics analysis has been employed in the literature in an attempt to evaluate the bone marrow of MM patients. This manuscript aimed to systematically review radiomics research on MM while employing a radiomics quality score (RQS) to accurately assess research quality in the field. A systematic search was performed on Web of Science, PubMed, and Scopus. The selected manuscripts were evaluated (data extraction and RQS scoring) by three independent readers (R1, R2, and R3) with experience in radiomics analysis. A total of 23 studies with 2682 patients were included, and the median RQS was 10 for R1 (IQR 5.5–12) and R3 (IQR 8.3–12) and 11 (IQR 7.5–12.5) for R2. RQS was not significantly correlated with any of the assessed bibliometric data (impact factor, quartile, year of publication, and imaging modality) (p > 0.05). Our results demonstrated the low quality of published radiomics research in MM, similarly to other fields of radiomics research, highlighting the need to tighten publication standards.

Investigation into aberrant B cell repertoire in myeloma on humoral immunity and patient survival.

8016 Background: B cells are critical to inhibiting tumor progression, as they generate tumor-reactive antibodies; promote anti-tumor killing capacity of NK cells and phagocytosis of macrophages/DCs; and enrich priming of T cells against the tumor. Multiple myeloma (MM) is a B-cell-derived malignancy, and its survival is partly driven by a deficiency in the immune response. Yet, the abnormalities of the B-cell-mediated humoral responses in MM are ill defined. Methods: We investigated B cell composition in bone marrow (BM) from patients with newly diagnosed MM (NDMM, N = 170), relapsed refractory MM (RRMM, N = 140) and healthy donors (HD, N = 21) by multi-color flow cytometry. Results: We observed that B1b cells was significantly reduced by 45% (p = 0.004) in NDMM patients compared to HD. B1b cells are important in generating long-lasting protective antibodies, particularly against vaccines, in a T cell independent fashion. Similarly, B2 cells, which can produce all antibody types, were significantly reduced in the periphery of NDMM and RRMM patients. Within the B2 cell population, germinal center (GC) matured B cells were significantly reduced in both BM and periphery of MM patients, as shown by significantly higher expression of Fas receptor. Because of deficiencies in the GC-maturation of these B cells, their ability to switch from IgM to IgG was also significantly decreased. On the other hand, the population of regulatory B cells (Bregs) were significantly elevated by 2.3-fold (p < 0.05) in the BM and periphery of NDMM and periphery of RRMM patients. However, the ability of Breg cells to produce suppressive cytokines, like IL-10, was significantly reduced (p = 0.03), indicating impaired suppressive functional capacity. We investigated whether this dysregulated B cell homeostasis impacts myeloma patient survival following treatment. We found that a higher proportion of Bregs and a lower percent of B1a in BM and periphery prior to treatment was associated with favorable progression-free survival (PFS) in both NDMM and RRMM patients. We hypothesized that reduction of B2 cells and their inability to have GC-based maturation could impair antibody generation against acute infections and tumor neoantigens. Therefore, we evaluated MM patients’ ability to produce antibodies against hepatitis B surface antigen-based vaccination. We observed that only 31% of MM patients (N = 32) respond to the vaccine compared to 90% of HD. Furthermore, MM patients’ antibody titers were significantly lower ( < 120 IU/L) compared to > 5000IU/L in HD. Conclusions: All together, these results show that in MM patients, vaccine-responsive populations of B cells are reduced, with fewer mature B cells with limited repertoires in GC to fight against acute infections, and with a high number of dysregulated regulatory B cells. These results indicate that compromised B cell functionality in MM patients could impact therapeutic outcome.

Unraveling the Role of Peroxisome Proliferator-Activated Receptor β/Δ (PPAR β/Δ) in Angiogenesis Associated with Multiple Myeloma.

Growing evidence suggests a role for peroxisome proliferator-activated receptor β/δ (PPAR β/δ) in the angiogenesis, growth, and metastasis of solid tumors, but little is known about its role in multiple myeloma (MM). Angiogenesis in the bone marrow (BM) is characteristic of disease transition from monoclonal gammopathy of undetermined significance (MGUS) to MM. We examined the expression and function of PPAR β/δ in endothelial cells (EC) from the BM of MGUS (MGEC) and MM (MMEC) patients and showed that PPAR β/δ was expressed at higher levels in MMEC than in MGEC and that the overexpression depended on myeloma plasma cells. The interaction between myeloma plasma cells and MMEC promoted the release of the PPAR β/δ ligand prostaglandin I2 (PGI2) by MMEC, leading to the activation of PPAR β/δ. We also demonstrated that PPAR β/δ was a strong stimulator of angiogenesis in vitro and that PPAR β/δ inhibition by a specific antagonist greatly impaired the angiogenic functions of MMEC. These findings define PGI2-PPAR β/δ signaling in EC as a potential target of anti-angiogenic therapy. They also sustain the use of PPAR β/δ inhibitors in association with conventional drugs as a new therapeutic approach in MM.

High prevalence of Wilms tumor 1 expression in multiple myeloma and plasmacytoma: A cohort of 142 Asian patients' samples.

Wilms tumor 1 (WT1) is a promising target antigen for cancer immunotherapy. However, WT1 protein expression and its clinical correlation in multiple myeloma (MM) patients are still limited. We, therefore, investigated WT1 expression in 142 bone marrow and plasmacytoma samples of MM patients at different stages of the disease by immunohistochemistry. The correlations between WT1 expression and clinical parameters or treatment outcomes were evaluated. The overall positive rate of WT1 expression was 91.5%; this high prevalence was found in both bone marrow and plasmacytoma samples, regardless of the disease status. Cytoplasmic WT1 expression was correlated with high serum free light chain ratio at presentation. However, no significant association between WT1 expression and treatment outcome was observed. This study confirms the high prevalence of WT1 expression in an Asian cohort of MM, encouraging the development of immunotherapy targeting WT1 in MM patients, particularly in those with extramedullary plasmacytoma or relapsed disease.

Igm-2644, a Novel CD38xCD3 Bispecific IgM T Cell Engager Demonstrates Potent Efficacy on Myeloma Cells with an Improved Preclinical Safety Profile

Multiple myeloma (MM) is a cancer of plasma cells affecting > 30,000 new patients every year in the United States. Despite improved treatment options, including anti-CD38 monospecific IgG antibodies such as daratumumab and isatuximab in combination with chemotherapies, resistance eventually develops in most patients. Several bispecific (CD38xCD3) or trispecific (CD38xCD3xCD28) T cell engager antibody therapies are currently in development to further improve upon the efficacy of CD38 targeted therapies by leveraging T cell dependent cellular cytotoxicity (TDCC) of MM cells. However, a unique challenge for anti-CD38 T cell engagers is to potently kill myeloma cells without depleting CD38+ immune cells, including activated cytotoxic T cells (i.e., fratricide), while also avoiding dangerous cytokine release syndrome associated with T cell engaging therapies. Encouraged by the favorable safety and tolerability profile of imvotamab (IGM-2323), our CD20xCD3 IgM T cell engager, we have developed a novel CD38xCD3 bispecific IgM T cell engager, IGM-2644. It has 10 binding sites for human CD38, and a single anti-CD3 scFv fused to the joining (J) chain. Here, we have evaluated the potency and potential safety profile of IGM-2644 in a panel of in vitro immunoassays and in vivo models. IGM-2644 demonstrated significantly improved complement-dependent cytotoxicity (CDC) activity compared to daratumumab and isatuximab, with over 30-fold increased potency on MM cell lines, and depletion of these cells was further enhanced when PBMCs were used as effector cells. IGM-2644 was able to achieve TDCC activities similar to bispecific CD38xCD3 IgGs on low CD38 expressing cell lines that are resistant to daratumumab, while showing much lower levels of cytokine release than bispecific IgGs. In a short-term in vitro colony forming unit (CFU) assay, IGM-2644 was able to reduce MM colonies using primary MM patient bone marrow samples containing autologous T cells and myeloma cells, while no effect was observed on the colony formation of erythroid, granulocyte and macrophage using bone marrow samples from normal donors. IGM-2644 was able to show in vivo efficacy and suppress CD38+ NCI-H929 (myeloma) and Raji (lymphoma) xenograft tumor growth in humanized mouse models. Importantly, IGM-2644 also demonstrated reduced T cell fratricide compared to bispecific IgGs both in vitro and in vivo. Taken together, our data demonstrates IGM-2644 is a potent molecule with both CDC and TDCC activities, and the potential to be active in daratumumab resistant tumors. It has an improved preclinical safety profile compared to other CD38xCD3 bispecific T cell engagers due to lower cytokine release and reduced T cell fratricide. A Phase I clinical study evaluating the activity and safety of IGM-2644 is planned.

Analysis of COVID-19-Associated Proteins Identifies TANK-Binding Kinase 1 As an Immunotherapeutic Target in Multiple Myeloma

Background and Rationale. Patients with multiple myeloma (MM) are at a higher risk of viral infection due to immune deficiency, as recently highlighted by the severity of COVID-19 in MM patients. However, the mechanism(s) underlying the lack of anti-viral immune response in MM are unclear. We have previously shown that there are more plasmacytoid dendritic cells (pDCs) in the bone marrow of MM patients than in the bone marrow of healthy individuals and that these dysfunctional patient pDCs suppress T and NK cell-mediated immunity. Importantly, pDCs play a key role in both recognizing viral nucleic acid and initiating anti-viral immunity via type 1 interferon (IFN) response signaling. Therefore, we analyzed the immune-pathway proteins implicated in COVID-19-host interactions [COVID-19 protein interaction map, Gordon et al, Nature 2020; 583, 459-568] to assess whether pDC-MM interactions modulate these pathways to confer immune suppression and susceptibility to COVID-19. Results. We first assessed whether the expression of human genes involved in the COVID-19-host interaction changes when ex vivo MM cells are co-cultured with patient pDCs (compared to MM cells alone). We found that 41 of these genes were differentially expressed in MM cells when exposed to pDCs (log2Fold change: ± 3.5-fold; p < 0.00001), and this expression varied widely (median (log10): -0.13 to 4.5; adj p < 0.001). Three of these genes are linked to innate immunity and are essential for IFN production in MM cells - TBK1, IRF3 (Interferon regulatory factor 3), and RAE1 (interferon-inducible mRNA nuclear export factor). Importantly, pDCs downregulated the expression of TBK1 (Log2FC: -0.5) and RAE1 (Log2FC: -0.3) and upregulated the expression of IRF3 (Log2FC: 1.5) in MM cells after coculture (compared to MM cells alone, p < 0.0001). Concordantly, analysis of publicly available gene profiling datasets on relapsed MM patients showed low levels of TBK1 and RAE1 and higher levels of IRF3 (n = 50) (Log2FC: TBK1: -0.208; RAE1: -0.286; IRF3: 0.273; vs normal; p < 0.05). Of note, TBK1 expression was positively correlated with prognosis (n = 350, p = 0.026). We found that most of the 41 genes were also expressed in pDCs (NCBI GEO Datasets based on pDC cell line CAL1 and human peripheral blood pDCs). In unstimulated pDCs, TBK1 expression was significantly lower than IRF3 and RAE1 expression (2-3-fold, p < 0.05). Yet treating pDCs with CpG-ODN type-A, which induces IFN-α expression, increased both TBK1 (adj P = 0.004) and RAE1 (adj P = 0.043) expression, without significantly altering IRF3 expression. Finally, COVID-19 relies on the host ubiquitin-proteasome system for propagation, and we found that targeting ubiquitin receptor Rpn13 with the specific inhibitor RA190 restored TBK1 expression in MM cell lines, indicating the potential clinical use of targeting Rpn13 in restoring anti-viral immune responses in MM. Conclusion. Taken together, we show that TBK1 is downregulated in pDCs, and pDCs interacting with MM cells further decrease TBK1 expression in MM cells, thereby attenuating IFN-mediated anti-viral immune response signaling. Our findings suggest that: 1) low levels of TBK1 may confer increased susceptibility of MM patients to COVID-19 and 2) targeting TBK1 may restore the anti-viral immune response and reduce COVID-19 severity in MM.

The Pre-Existing T Cell Landscape Is Associated with Response to High Dose Melphalan and Autologous Stem Cell Transplant in Multiple Myeloma

Introduction: High dose melphalan and autologous stem cell transplant (ASCT) remain a cornerstone of therapy for transplant-eligible newly diagnosed multiple myeloma (MM) patients. The bone marrow microenvironment may affect the response of MM cells to high dose melphalan and ASCT. The changes in the immune landscape and associations with response to ASCT remain largely unknown. Here we investigate the role of the microenvironment in response to high dose melphalan using scRNA and TCR sequencing in paired pre- and post-ASCT bone marrow samples from the Indiana Myeloma Registry. Methods: Paired bone marrow (BM) aspirates were collected from MM patients (n=40) after induction therapy but before ASCT and again after ASCT but prior to maintenance therapy (range 58-142 days post-ASCT). All patients had measurable disease after induction therapy. After ASCT, patients were evaluated based on their response to ASCT and divided into those who achieved a stringent complete response (n=17) and were negative for measurable residual disease (MRD) at 10-6 by flow cytometry (responders), or those who achieved a partial response or less (n=23) (non-responders). BM mononuclear cells were isolated by Ficoll and CD138 magnetic bead selection into CD138+ve and CD138-ve subsets. Paired pre- and post-ASCT CD138-ve samples underwent scRNA and TCR sequencing (10X Genomics), resulting in 488,479 high quality cells. Samples were independently normalized, cells integrated, and clusters annotated for cell type. Significant gene expression differences between clusters were calculated. Results: Significant differences in the proportions of different immune cells in pre- vs. post-ASCT samples were identified with increases in B cells (2.6% vs 19.6%; P<.001) and B-cell progenitors (1.5% vs 8.9%; P<0.001) and decreases in CD4 T cells (24.8% vs 10.2%; P<0.001) and monocytes/myeloid cells (27.8% vs 21.0%; P=0.005) in post-ASCT samples. The overall proportions of CD8 T cells remained stable (27.1% vs 25.1%; P=0.2), but there were significant differences in the proportion of cells within the CD8 T cell clusters, including an increase in effector CD8 T cells (8.6% vs 10.7%; P=0.01) that expressed IFNG, KLRG1, TBX21, and PRF1 as well as a decrease in second cluster of CD8 T cells (2.3% vs 0.6%; P<0.01) expressing IL7R and TCF7. When comparing responder and non-responder pre-ASCT samples, we found a higher percentage of a CD4 T cell subset (10.0% vs 7.6%; P=0.04) in responders, which expressed SELL, CCR7, and ICOS indicating they are likely non-effector memory cells or naïve cells. There was also a higher proportion of myeloid dendritic cells (1.47% vs 1.08%; P=0.05) and granulocyte progenitors (0.98% vs 0.62%; P=0.04) in responders in post-ASCT samples. TCR sequencing revealed expanded CD8 T cells but not CD4 T cells, suggesting the CD4 T cell subset of interest may have a modulating effect of MM cells response to high dose melphalan rather than a specific anti-myeloma immune response. Conclusions: We comprehensively describe the immune microenvironment in the BM of MM patients around ASCT and identify a pre-existing CD4 T cell subset that is associated with depth of response to ASCT. Further studies are required to better define the role of this population in modulating the response to melphalan. Prospective follow up of patients is ongoing to assess which clusters are associated with progression-free and overall survival.

High TIM-3 Expression May Contribute to the Functional Impairment of Bone Marrow and Circulating Gamma Delta T Lymphocytes during the Progression of Multiple Myeloma

Introduction: The tumor microenvironment (TME) has a significant impact on the development and evolution of multiple myeloma (MM). Understanding the reciprocal interactions between aberrant clonal plasma cells and the TME is required to shed light on the mechanisms behind disease progression from early (monoclonal gammopathy of undetermined significance (MGUS)/smoldering MM (SMM)) to active disease. γδ T cells are among the bone marrow infiltrating cells that have both innate and adaptive immune cell features and play a significant role in cancer immunosurveillance. To date, however, their contribution to the evolution of MM remains unknown. To this aim, we investigated the role of γδ T cells in MM and its early stages, developing the first single-cell (sc) atlas of this population in this disease. Methods: CD3+ cells from the bone marrow of healthy donors (HD; n=3), MGUS/SMM patients (n=5), and active MM patients (n=9) were sorted and seeded into single drops using a Chromium instrument (10X genomics), and 12527 γδ T cells were analyzed using scRNAseq. To validate transcriptomic findings, we next isolated peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) from healthy donors (n=4; PBMCs exclusively), MGUS/SMM (n=8), and MM patients (n=14) by density gradient centrifugation and performed flow cytometric analysis to evaluate the frequency, differentiation/exhaustion profile, and functionality of the two main γδ T cell subsets known as Vδ1+ and Vδ2+ T cells. Results: Bioinformatics workflow enabled us to identify 7 γδ T clusters: 2 naïve subpopulations (CD4-/CD8- and CD4+ γδ T cells), 2 GZMB effector/terminally differentiated subpopulations (CD8+/TIGIT+/LAG3+, TIM3+/CD27- and GNLY+/FTH1+ γδ T cells) and 2 GZMK memory subpopulations (GZMK+ and CXCR3+ γδ T cells). Surprisingly, we observed a decrease in naive γδ T cells (p<0.05), which was coupled with an increase in TIM3 expression as MM evolved (Figure 1). Notably, we found a decrease in Vδ2+ T cells compared to healthy donors (mean 2,3% HD vs 1% MGUS/SMM vs 1,3% MM; p<0.05), as well as an increase in the Vδ1+/Vδ2+ ratio throughout MM progression (mean 0,4 HD vs 4,4 MGUS/SMM vs 4,4 MM). The frequency of Vδ2+ T cells in MM patients was positively correlated with free kappa light chain serum levels (PB: r= 0.8303, p<0.01; BM: r= 0.7455, p<0.05), instead, Vδ1+/Vδ2+ ratio was inversely correlated (PB: r= -0.7455, p<0.05; BM: r= -0.8909, p<0.01). Furthermore, we confirmed by flow cytometry the predominance of effector memory (Vδ1+ T cells: mean 1,4% HD vs 2.2% MGUS/SMM vs 14,4% MM; Vδ2+ T cells: mean 22,1% HD vs 21,4% MGUS/SMM vs 41,7% MM; p<0.05) and terminally differentiated γδ T cells (Vδ1+ T cells: mean 28,2% HD vs 79,2% MGUS/SMM 72,8% MM; Vδ2+ T cells: mean 19,2% HD vs 42,8% MGUS/SMM vs 32,2% MM; p<0.05) in MM patients as compared to healthy donors, where naïve (Vδ1+ T cells: mean 53,7% HD vs 15% MGUS/SMM vs 5,8% MM; Vδ2+ T cells: mean 10,5% HD vs 14,1% MGUS/SMM vs 8,1% MM; p<0.05) and central memory populations predominated (Vδ1+ T cells: mean 16,5% HD vs 3.4% MGUS/SMM vs 6,8% MM; Vδ2+ T cells: mean 48,7% HD vs 42,8% MGUS/SMM vs 17,2% MM; p<0.05). Accordingly, the analysis of exhaustion markers corroborated the progressive increase of TIM3 expression (but not PD1) in both Vδ1+ (mean 5,65% HD vs 34,5% MGUS/SMM vs 36,4% MM; p<0.05) and Vδ2+ T cell subsets (mean 2,1% HD vs 22,8% MGUS/SMM vs 32,6% MM; p<0.05). Finally, we observed that the acquisition of this exhausted phenotype along MM evolution was associated with impaired functions of both Vδ1+ and Vδ2+ T cells with reduced TNF-α (mean 75,1% HD vs 14,4% MGUS/SMM vs 14,5% MM; p<0.01) and IFN-γ (mean 28,8% HD vs 7,02% MGUS/SMM vs 7,24% MM; p<0.05) expression after in vitro stimulation. Conclusions: These findings suggested that the upregulation of the immunosuppressive receptor TIM3 may play a role in destabilizing γδ T cells and inducing their dysfunction throughout the progression of MM. Furthermore, because PB-derived T cells mirrored the bone marrow landscape, monitoring them could provide a potential minimally invasive approach for identifying patients at high risk of progressing to symptomatic MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

S170: DYNAMIC INTERPLAY BETWEEN TUMOR AND MICRO-ENVIRONMENT DURING MYELOMA DISEASE PROGRESSION

Background: Multiple Myeloma (MM) is an incurable plasma cell (PC) malignancy that evolves from two premalignant stages: Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). The disease progression has been characterized to be driven by intrinsic genomic events in the myeloma cells and by gradual dysregulation of the immune system. Aims: We investigated how the interplay between tumor cells with their microenvironment and the underlying complex and dynamic immune biology evolve during this process. Methods: Single cell multi-omics profiling, including RNA, B-cell receptor (BCR) and antibody barcode-tagged 10x sequencing, was conducted on human bone marrow (BM) aspirates collected at 6 Belgian centers from 4 cohorts: 31 healthy elderly and 28 MGUS, 32 SMM and 32 newly diagnosed MM. Mononuclear cell isolation, freezing and transport to central facilities was optimizedand data were integrated and filtered using Scanpy and Scirpy. The main immune cell types were identified from the RNA and antibody data using SingleR. Further functional subtyping was done using Leiden clustering. Differential pathway expression analysis was performed with Muscat and FGSEA. Results: From the tumor cell transcriptomes, our analyses confirmed the previously documented myeloma molecular hallmarks, such as MYC and IFN-a signaling, cell proliferation, energy metabolism and oxidative phosphorylation. Evidence was found for transcriptomic similarities and within-and between-patient malignant PC transcriptomic heterogeneity, as well as the existence of multiple transcriptomic clones in several patients. We observed a positive correlation between the antigen processing mechanism in the PCs with IFN response, suggesting that this mechanism associates with initiation of the immune recognition and activation against the tumor.The gradually increasing differential gene expression was also observed in the immune microenvironment: dysregulation of signaling pathways initiates early in MGUS and spreads throughout the various cell types surrounding the tumor cells. Cell population shifts were also found. In the CD1C+ DCs, that play a role in cancer immune control, a functional shift was observed that correlated with disease progression towards a more mature and antigen presenting phenotype with higher levels of CD83, HBEGF, MCL1 and CXCL16 as well as increased TNF-a pathway. Similarly, a shift was observed in the macrophage population, toward M1 state showing high IFN response along with expression of MS4A4A, STAT1, TNFSF13B and TRAIL in more severe disease. Interestingly, in the CD8+ T cells, we detected a pre-dysfunctional subpopulation with high expression of GZMK, activation markers CD69, CCL4, CXCR4 and genes associated with T cell pre-dysfunctionality NR4A2, RGS1, TOX and TIGIT, that was found to be associated with progression (Figure). In the CD4+ cytotoxic T cells, a proportion change was observed with more severe disease. Image:Summary/Conclusion: With the atlas of healthy, precursor and active MM patient BM samples, we generated a comprehensive and granular view of the various cell types involved in disease progression and provide evidence for early and gradually increasing immune dysregulation and activation of oncogenic driver pathways. Our data evidence the co-divergence and reciprocal stimulation of transcriptomes of tumor and microenvironment and support the postulation of microenvironment as a central modulator of cancer cell growth, survival and metastasis.

Abstract 580: MM3 CAR T: A novel tumor targeting and T cell redirection platform for multiple myeloma

Abstract Novel strategies to target tumors are needed to enhance the efficacy of immune redirection therapies. The detection of tumors based on mammalian immunoglobulin scFvs has been a significant innovation in immunotherapy but is impacted by limits of immune tolerance and immune system evolution. The discovery of unique variable lymphocyte receptors (VLRs) - analogues to antibodies in evolutionarily distant jawless vertebrates - in the Cooper lab (Pancer, Nature) provides an opportunity to target antigens beyond those recognized by conventional antibodies. A particular VLR, termed MM3, was found to identify a distinct epitope specific to human plasma cells (PCs) and plasmablasts (PBs), created by CD38 dimerization with activity as an NAD+ glycohydrolase (Yu, JCI Insight). CD38 is an extensively explored target for treating multiple myeloma (MM); however, specific scFv-based targeting of CD38 on malignant plasma cells is difficult, as the protein is expressed on a variety of other immune cell lineages. Therefore, MM3 provides a unique, novel mechanism to target CD38 in a tumor-specific manner in MM and other plasma cell dyscrasias. Here, we present a CAR T cell with an MM3 binding domain replacing the standard scFv. Our CAR construct consists of the MM3 binding domain, a CD8 hinge and transmembrane domain, and 4-1BB and CD3zeta intracellular domains. Staining of MM patient bone marrow specimens revealed that the strong specificity for PCs and PBs previously observed in healthy donors was maintained in MM bone marrow, illustrating the ability of MM3 to target the malignant PC population. To assess in vitro function of the MM3 CAR T cells, flow cytometry analysis of co-cultures using healthy donor-derived MM3 CAR T cells and target cell lines revealed specific increased degranulation (CD107a expression), activation (CD69 expression), and killing (target cell Annexin V binding) over untransduced control T cells only in cell lines sensitive to MM3 binding, with no differences in negative control cell lines, demonstrating specific recognition and killing of target cells. Preliminary in vivo studies in the MISTRG6 myeloma xenograft mouse model were performed using intrafemorally injected INA-6, an IL-6-dependent MM cell line, which MM3 binds after growth in vivo. After treatment with MM3 CAR T cells via intravenous injection, we observed that the MM3 CAR T cells specifically homed to and were retained in the injected femur along with the tumor cells, suggesting tumor recognition and potential for in vivo efficacy. In summary, these data demonstrate that VLR-based targeting may allow design of novel CAR constructs. Specifically, the VLR MM3 is a promising novel, non-scFv mechanism for targeting CD38 in a malignant PC-specific manner, and the MM3 CAR T cells have shown promising preliminary activity. Therefore, the VLR-based MM3 CAR T cell platform may provide a novel strategy to improve T cell redirection to target tumors in MM. Citation Format: Samuel S. McCachren, Julia Manalo, Katherine Pendleton, Mary E. Ballestas, Kavita M. Dhodapkar, Melissa L. Kemp, Max D. Cooper, Madhav V. Dhodapkar. MM3 CAR T: A novel tumor targeting and T cell redirection platform for 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 580.

Abstract 2074: ORIC-533, a small molecule CD73 inhibitor with best-in-class properties, reversesimmunosuppression and has potential as an immunomodulatory therapy in patients with multiple myeloma

Abstract Adenosine accumulation within the tumor microenvironment can severely limit antitumor immunity by promoting the expansion of immunosuppressive cell types and impairing immune cell function, including T cells, natural killer cells and dendritic cells. The generation of adenosine from adenosine monophosphate (AMP) requires the activity of a cell surface ecto-5’-nucleotidase, CD73, expressed on the surface of various cell types including immune cells. CD73 overexpression is observed in many tumor types and correlates with unfavorable clinical outcomes. Given the essential role of CD73 in generating adenosine, inhibition of CD73 is a promising therapeutic strategy. Recent clinical proof of concept data demonstrated a significant improvement in progression free survival for non-small cell lung cancer patients upon targeting CD73 with the oleclumab anti-CD73 antibody in combination with anti-PDL1, relative to anti-PDL1 checkpoint inhibitor treatment alone.We developed ORIC-533, a potent, orally bioavailable, AMP-competitive, small molecule inhibitor of CD73, that is highly selective for CD73 and exhibits picomolar potency in biochemical assays, completely blocking adenosine production from AMP. In ex vivo assays, ORIC-533 displayed greater potency in restoring immunosuppressed CD8+ T cell proliferation and activation compared to oleclumab, as well as greater potency versus small molecule inhibitors of CD73 and the adenosine receptor. In multiple myeloma (MM) patients, elevated adenosine levels in the bone marrow (BM) niche correlate with progression of multiple myeloma through a CD73-mediated pathway. Moreover, we recently reported a functional role of CD73 signaling in BM of MM patients, indicating that CD73 inhibition may represent a unique vulnerability and treatment strategy for MM (Ray et al., abstract #2675, ASH 2021). We tested the impact of CD73 inhibition in bone marrow aspirates from patients with relapsed or refractory MM, using an autologous ex vivo assay system comprised of the multiple myeloma BM microenvironment. CD73 inhibition stimulated the activation of plasmacytoid dendritic cells and T cell activation in the context of the MM BM milieu. Moreover, ORIC CD73 inhibitor as a single agent overcame immune suppression and triggered significant lysis and cell death of multiple myeloma cells by autologous T-cells in the bone marrow microenvironment. Taken together, these results demonstrate that the ORIC CD73 inhibitor potently inhibits the adenosine pathway, which restores anti-tumor immunity and therefore holds potential for patients with MM. Based upon these data, ORIC-533 is being studied as a single agent in a Phase 1 clinical trial in patients with relapsed or refractory multiple myeloma. Citation Format: Melissa R. Junttila, Arghya Ray, Robert Warne, Xi Chen, Ting Du, Dena Sutimantanapi, Jae H. Chang, Brian Blank, Jared Moore, Chudi O. Ndubaku, Tatiana Zavorotinskaya, Pratik Multani, Omar Nadeem, Dharminder Chauhan, Kenneth C. Anderson, Lori S. Friedman. ORIC-533, a small molecule CD73 inhibitor with best-in-class properties, reversesimmunosuppression and has potential as an immunomodulatory therapy in patients with 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 2074.

Extracellular vesicles mediate the communication between multiple myeloma and bone marrow microenvironment in a NOTCH dependent way.

Multiple myeloma (MM) is an incurable hematologic neoplasm, whose poor prognosis is deeply affected by the propensity of tumor cells to localize in the bone marrow (BM) and induce the protumorigenic activity of normal BM cells, leading to events associated with tumor progression, including tumor angiogenesis, osteoclastogenesis, and the spread of osteolytic bone lesions. The interplay between MM cells and the BM niche does not only rely on direct cell-cell interaction, but a crucial role is also played by MM-derived extracellular vesicles (MM-EV). Here, we demonstrated that the oncogenic NOTCH receptors are part of MM-EV cargo and play a key role in EV protumorigenic ability. We used in vitro and in vivo models to investigate the role of EV-derived NOTCH2 in stimulating the protumorigenic behavior of endothelial cells and osteoclast progenitors. Importantly, MM-EV can transfer NOTCH2 between distant cells and increase NOTCH signaling in target cells. MM-EV stimulation increases endothelial cell angiogenic ability and osteoclast differentiation in a NOTCH2-dependent way. Indeed, interfering with NOTCH2 expression in MM cells may decrease the amount of NOTCH2 also in MM-EV and affect their angiogenic and osteoclastogenic potential. Finally, we demonstrated that the pharmacologic blockade of NOTCH activation by γ-secretase inhibitors may hamper the biological effect of EV derived by MM cell lines and by the BM of MM patients. These results provide the first evidence that targeting the NOTCH pathway may be a valid therapeutic strategy to hamper the protumorigenic role of EV in MM as well as other tumors.