Multiple Myeloma Disease Progression Research Articles

Natural killer cell biology and therapy in multiple myeloma: challenges and opportunities.

Despite therapeutic advancements, multiple myeloma (MM) remains incurable. NK cells have emerged as a promising option for the treatment of MM. NK cells are heterogenous and typically classified based on the relative expression of their surface markers (e.g., CD56 and CD16a). These cells elicit an antitumor response in the presence of low mutational burden and without neoantigen presentation via germline-encoded activating and inhibitory receptors that identify the markers of transformation present on the MM cells. Higher NK cell activity is associated with improved survival and prognosis, whereas lower activity is associated with advanced clinical stage and disease progression in MM. Moreover, not all NK cell phenotypes contribute equally toward the anti-MM effect; higher proportions of certain NK cell phenotypes result in better outcomes. In MM, the proportion, phenotype, and function of NK cells are drastically varied between different disease stages; this is further influenced by the bone marrow microenvironment, proportion of activating and inhibitory receptors on NK cells, expression of homing receptors, and bone marrow hypoxia. Antimyeloma therapies, such as autologous stem cell transplant, immunomodulation, proteasome inhibition, and checkpoint inhibition, further modulate the NK cell landscape in the patients. Thus, NK cells can naturally work in tandem with anti-MM therapies and be strategically modulated for improved anti-MM effect. This review article describes immunotypic and phenotypic differences in NK cells along with the functional changes in homeostatic and malignant states and provides expert insights on strategies to harness the potential of NK cells for improving outcomes in MM.

Allogeneic hematopoietic cell transplantation for therapy-related myeloid neoplasms arising following treatment for multiple myeloma: a retrospective study on behalf of the Chronic Malignancies Working Party of the EBMT.

Therapy-related myeloid neoplasms (t-MN) are a complication of multiple myeloma (MM) treatment. Our retrospective, EBMT registry study included 157 such patients allografted (allo-HCT) between 2006 and 2018. Most patients (130) had a prior autologous HCT. Fifty-seven (36.4%) were transplanted for t-AML and 100 (63.6%) for t-MDS. Median times from MM and t-MN diagnoses to allo-HCT were 72.6 (interquartile range (IQR), 46.1-102.9) and 6.4 (IQR, 3.9-9.4) months. Fifty-eight (38.4%) t-MN patients were in complete remission (CR) at allo-HCT predominantly conditioned with reduced intensity (70.3%). With a median follow-up of 64.9 (95% CI: 39-76) months, relapse incidence (RI) from MM at 1 and 5 years was 4% (0-10%) and 12% (2-22%), respectively, with few deaths (n = 3) only due to MM disease progression, whereas t-MN RI and non-relapse mortality (NRM) at 1 and 5 years were 35% (95% CI 28-43%) and 45% (95% CI: 36-53%) and 20% (95% CI 13-26%) and 31% (95% CI: 23-39%). Overall survival (OS) and progression-free survival (PFS) estimates at 1 and 5 years were 55% (95% CI: 47-63%) and 27% (95% CI: 19-35%) and 45% (95% CI 36-53%) and 24% (95% CI 16-32%). Older (>65 years) t-MN patients with high-risk cytogenetics do not benefit from allo-HCT.

A single-cell transcriptomic map of the murine and human multiple myeloma immune microenvironment across disease stages

BackgroundThe long-term effectiveness of immunotherapies against Multiple Myeloma (MM) remains elusive, demonstrated by the inevitable relapse in patients. This underscores the urgent need for an in-depth analysis of the MM tumor-immune microenvironment (TME). Hereto, a representative immunocompetent MM mouse model can offer a valuable approach to study the dynamic changes within the MM-TME and to uncover potential resistance mechanisms hampering effective and durable therapeutic strategies in MM.MethodsWe generated a comprehensive single-cell RNA-sequencing atlas of the MM-TME in bone marrow and spleen encompassing different stages of disease, using the immunocompetent 5T33MM mouse model. Through comparative analysis, we correlated our murine dataset with the pathogenesis in MM patients by reanalyzing publicly available datasets of human bone marrow samples across various disease stages. Using flow cytometry, we validated the dynamic changes upon disease progression in the 5T33MM model. Furthermore, interesting target populations, as well as the immune-boosting anti-CD40 agonist (αCD40) therapy were tested ex vivo on murine and human primary samples and in vivo using the 5T33MM model.ResultsIn this study, we identified the heterogenous and dynamic changes within the TME of murine and human MM. We found that the MM-TME was characterized by an increase in T cells, accompanied with an exhausted phenotype. Although neutrophils appeared to be rather innocuous at early disease stages, they acquired a pro-tumorigenic phenotype during MM progression. Moreover, conventional dendritic cells (cDCs) showed a less activated phenotype in MM, underscoring the potential of immune-boosting therapies such as αCD40 therapy. Importantly, we provided the first pre-clinical evaluation of αCD40 therapy and demonstrated successful induction of cDC- and T-cell activation, accompanied by a significant short-term anti-tumor response.ConclusionsThis resource provides a comprehensive and detailed immune atlas of the evolution in human and murine MM disease progression. Our findings can contribute to immune-based patient stratification and facilitate the development of novel and durable (immune) therapeutic strategies in MM.

Association of ADAM family members with proliferation signaling and disease progression in multiple myeloma

Multiple myeloma (MM) is a hematological malignancy whose curability is greatly challenged by recurrent patient relapses and therapy resistance. We have previously proposed the high expression of ADAM8, ADAM9 and ADAM15 (A Disintegrin And Metalloproteinase 8/9/15) as adverse prognostic markers in MM. This study focused on the so far scarcely researched role of ADAM8/9/15 in MM using two patient cohorts and seven human MM cell lines (HMCL). High ADAM8/9/15 expression was associated with high-risk cytogenetic abnormalities and extramedullary disease. Furthermore, ADAM8/15 expression increased with MM progression and in relapsed/refractory MM compared to untreated patient samples. RNA sequencing and gene set enrichment analysis comparing ADAM8/9/15high/low patient samples revealed an upregulation of proliferation markers and proliferation-associated gene sets in ADAM8/9/15high patient samples. High ADAM8/9/15 expression correlated with high Ki67 and high ADAM8/15 expression with high MYC protein expression in immunohistochemical stainings of patient tissue. Conversely, siRNA-mediated knockdown of ADAM8/9/15 in HMCL downregulated proliferation-related gene sets. Western blotting revealed that ADAM8 knockdown regulated IGF1R/AKT signaling and ADAM9 knockdown decreased mTOR activation. Lastly, high ADAM8/9/15 expression levels were verified as prognostic markers independent of Ki67/MYC expression and/or high-risk abnormalities. Overall, these findings suggest that ADAM8/9/15 play a role in MM progression and proliferation signaling.

Prognostic factors and clinical characteristics of patients with newly diagnosed non-secretory multiple myeloma in the era of new drugs in "real-world" study: Experiences of the Polish Myeloma Group.

Non-secretory multiple myeloma (NSMM) accounts for approx. 2-3% of multiple myeloma (MM) cases. Due to the rare occurrence and ineligibility of patients with NSMM to participate in clinical trials, we have limited data on treatment efficacy and the clinical course in these patients. Most of the literature consists of case reports and small retrospective studies. The study aimed to analyze patient characteristics, prognostic factors and treatment outcomes in newly diagnosed (ND) NSMM. This is a multicenter, retrospective analysis of 43 patients with NSMM diagnosed between June 2010 and September 2021, conducted in 8 Polish hematology centers. The median overall survival (OS) was 103 months (95% confidence interval (95% CI): 20-72). The most common cause of death was MM disease progression. The overall response rate (ORR) was 84.6%; complete response (CR), very good partial response (VGPR), partial response (PR), and no response (NR) rates were 20.5%, 46.2%, 17.9%, and 15.4%, respectively. In multivariable analysis, factors contributing to worse OS included International Staging System stage 3 (ISS-3) (p = 0.0277), anemia (Hb <10 g/dL or >2 below upper limit of normal value (ULN), p = 0.0270), renal insufficiency (RI, serum creatinine >2 mg/dL, p = 0.0476), and serum albumin <5.5 mg/L (0.0408). Non-secretory multiple myeloma is a rare subtype of MM. This small study demonstrates that outcomes are comparable to secretory MM. However, the inclusion of this subset of patients in clinical trials is essential to assess prognosis, treatment efficacy and clinical outcomes.

Fluorescence in situ hybridization reveals the evolutionary biology of minor clone of gain/amp(1q) in multiple myeloma

Growing evidence suggests that gain or amplification [gain/amp(1q)] accumulates during disease progression of multiple myeloma (MM). Previous investigations have indicated that small gain/amp(1q) subclones present at the time of diagnosis may evolve into dominant clones upon MM relapse. However, the influence of a minor clone of gain/amp(1q) on MM survival, as well as the correlation between different clonal sizes of gain/amp(1q) and the chromosomal instability (CIN) of MM, remains poorly understood. In this study, we analyzed fluorescence in situ hybridization (FISH) results of 998 newly diagnosed MM (NDMM) patients. 513 patients were detected with gain/amp(1q) at diagnosis. Among these 513 patients, 55 had a minor clone (≤20%) of gain/amp(1q). Patients with a minor clone of gain/amp(1q) displayed similar survival outcomes compared to those without gain/amp(1q). Further analysis demonstrated patients with a minor clone of gain/amp(1q) exhibited a clonal architecture similar to those without gain/amp(1q). Lastly, our results showed a significant increase in the clonal size of the minor clone of gain/amp(1q), frequently observed in MM. These findings suggested that a minor clone of gain/amp(1q) might represent an earlier stage in the pathogenesis of gain/amp(1q) and propose a “two-step” process in the clonal size changes of gain/amp(1q) in MM.

The numerous facets of 1q21+ in multiple myeloma: Pathogenesis, clinicopathological features, prognosis and clinical progress (Review).

Multiple myeloma (MM) is a malignant neoplasm characterized by the clonal proliferation of abnormal plasma cells (PCs) in the bone marrow and recurrent cytogenetic abnormalities. The incidence of MM worldwide is on the rise. 1q21+ has been found in ~30-40% of newly diagnosed MM (NDMM) patients.1q21+ is associated with the pathophysiological mechanisms of disease progression and drug resistance in MM. In the present review, the pathogenesis and clinicopathological features of MM patients with 1q21+ were studied, the key data of 1q21+ on the prognosis of MM patients were summarized, and the clinical treatment significance of MM patients with 1q21+ was clarified, in order to provide reference for clinicians to develop treatment strategies targeting 1q21+.

CARving a Path Forward: Unveiling T Cell Phenotypes Across the Spectrum of Multiple Myeloma Disease Progression to Inform Future Strategies for Overcoming Resistance to CAR T Therapy

CARving a Path Forward: Unveiling T Cell Phenotypes Across the Spectrum of Multiple Myeloma Disease Progression to Inform Future Strategies for Overcoming Resistance to CAR T Therapy

Elucidating the Role of Tolerogenic Dendritic Cells in Immune Tolerance and Disease Progression in Multiple Myeloma

Introduction: Previous research has demonstrated that programmed death-ligand 1 (PD-L1) in multiple myeloma (MM) cells not only suppresses anti-tumor immune responses but also enhances malignant potential through the PD-L1-PD-1 interaction [Ishibashi M, et al. Cancer Immunol Res. 2016]. However, it has not yet been shown that monotherapy and combination therapies utilizing PD-1/PD-L1 antibodies are sufficiently effective for MM. One reason for this is the absence of myeloma-specific cytotoxic T lymphocytes induction in the MM tumor microenvironment. In certain cancers, tumor-infiltrating dendritic cells (DCs) undergo conversion into tolerogenic DCs, which are crucial for inducing and maintaining T-cell tolerance, although this remains unclear in MM. Thus, this study aimed to investigate the function and induction mechanisms of tolerogenic DCs in the MM microenvironment. Materials and Methods: (1) Flow cytometry (FCM) was employed to assess the two conventional DC (cDC; lineage negative, HLA-DR +CD11c high) subsets, cDC1 (CD141 + cDC) and cDC2 (CD1c + cDC), in bone marrow (BM) samples obtained from MM patients (n=17) and healthy controls (n=13). (2) CD14 + monocytes were cultured with IL-4 and GM-CSF to generate monocyte-derived DCs (moDCs). Simultaneously, moDCs were co-cultured with MM cell lines using a trans-well system. The characteristics and functions of moDCs were analyzed using real-time PCR, RNA-sequencing (RNA-seq), western blotting, ELISA, and FCM after lipopolysaccharide stimulation. Results: The frequency of cDC1 subsets was significantly lower in MM patients compared to controls, while the major cDC2 subset did not differ. Similarly, when moDCs were co-cultured with MM cells, moDC numbers were markedly reduced. These co-cultured moDCs exhibited downregulated expression of activation markers CD80, CD86, and CD83, along with decreased phosphorylation of ERK and S6K. Conversely, these moDCs upregulated the expression of immunosuppressive factors IL-10, ARG1, and NOS2 through increased transcription factor C/EBPβ. Moreover, these moDCs inhibited T-cell proliferation, activation, and interferon-γ production in comparison to control moDCs, indicating the presence of immature and tolerogenic phenotypes. However, these phenotypes were not observed in moDCs co-cultured with soluble factors secreted from MM and stromal cells. Treatment with AZD3965 improved the phenotype of moDCs co-cultured with MM cells, reversing their suppressive effect on T-cell proliferation. AZD3965 targets monocarboxylate transporter 1 (MCT1) in MM cells, thereby suppressing extracellular lactate accumulation and acidic pH in the MM microenvironment. Furthermore, RNA-seq analysis comparing moDCs co-cultured with and without MM cells revealed downregulation of gene sets associated with activated DCs in moDCs co-cultured with MM. Notably, moDCs co-cultured with MM showed high enrichment of gene sets related to lysosome biogenesis and NOD-like receptor signaling. This suggests that inflammasome activation in these moDCs leads to caspase-1 activation, resulting in increased levels of mature IL-1β and IL-18 in their cell culture supernatants. Additionally, co-culturing CD38-positive MM cells with CD203a/CD73-positive HS-5 stromal cells and NAD + resulted in the generation of adenosine in the cell culture supernatant. Adenosine promotes the immaturation of mature DCs; however, inflammasome activation does not occur in these DCs. Conclusions: Patients with MM exhibit a reduced frequency of cDC1 subsets, which are crucial for cross-presentation to CD8 + T cells. moDCs in acid pH conditions derived from MM cells display an immature phenotype and impaired function due to decreased DC activation signals. Furthermore, inflammasome activation promotes the secretion of IL-1β and IL-18, contributing to pro-tumorigenic inflammation in the MM microenvironment. Moreover, adenosine generated via the CD38/CD203a/CD73 adenosinergic pathway promotes the immaturation and dysfunction of moDCs. Thus, the characteristics of tolerogenic DCs may vary depending on the MM microenvironment. These findings highlight the induction of tolerogenic DCs by the metabolic microenvironment in MM, leading to immune tolerance and disease progression.

Disruption of DNA-PK-Mediated Cgas Retention on Damaged Chromatin Potentiates Doxorubicin-Induced Cgas/Sting-Dependent Anti-Multiple Myeloma Activity

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM with multidrug resistance or extensive extramedullary plasmacytoma are difficult to manage and often treated with DNA damage-inducing chemotherapies, including topoisomerase II inhibitor doxorubicin. However, the efficacy of this treatment is still limited and requires further improvement. Targeting DNA damage response (DDR) may offer an opportunity for effective treatment of MM. In particular, in combination with DNA damage-inducing agents, this strategy has shown to improve chemo-radiotherapies in part by cGAS-STING-mediated innate immune response, which could be directly activated by an elevated level of cytosolic DNA due to DNA repair inhibition. However, as cGAS is primarily sequestered by chromatin in the nucleus, it remains unclear how cGAS is released from chromatin and translocated to the cytoplasm upon DNA damage, leading to cGAS-STING activation. Here, after testing the potential of several DDR inhibitors in sensitizing MM cells (MMCs) to doxorubicin, we found that inhibition of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key component of classical non-homologous end joining (c-NHEJ) repair, using DNA-PKcs inhibitor NU7441, synergistically enhanced doxorubicin-induced anti-MM effect by activation of cGAS-STING pathway, the combination index values at different combination doses were all less than 0.85. Knocking out either cGAS or STING in ARP-1 cells significantly reduced not only the transcription of IFNB1 and multiple ISGs but also the proportion of NU7441/doxorubicin-induced apoptotic cells. Stably overexpressing STING in U266 and RPMI-8226 cells significant upregulated the IFNB1 and ISGs levels, and made MMCs more susceptible to NU7441/doxorubicin-induced inhibition of cell proliferation. Despite a strong type I interferon (IFN) response induced by activation of cGAS-STING, apoptosis of MMCs caused by activated cGAS-STING was mediated in part by IRF3-NOXA-BCL2 axis, which is independent of type I IFN response. In addition to inducing intrinsic apoptosis of MMCs, activation of cGAS-STING signaling by NU7441/doxorubicin in MMCs also induced M1 polarization of macrophages (Mφs), which increased M1 marker CD86 of Mφs but decreased M2 marker CD163 and CD206, and reduced the IL-10 concentration secreted by Mφs, thus suppressing tumor-protecting potential of M2-like Mφs and improving bortezomib-induced apoptosis of MMCs. Mechanistic studies showed that the activation of cGAS-STING signaling was not due to the defective c-NHEJ following continuous DNA-PKcs inhibition, because NU7441 did not enhance the damaged DNA accumulation after doxorubicin induction detected by γH2AX immunoblot analysis and alkaline comet assay. Besides, deletion of XRCC4 in ARP-1 cells to directly impair c-NHEJ did not enhance doxorubicin-induced activation of cGAS-STING signaling, further excluding a role for impaired c-NHEJ. Instead, we found that cGAS was mainly localized in the nucleus and chromatin-bound in MMCs under mock treatment, and doxorubicin induced DNA damage and mobilized inactive cGAS into cytoplasm. Both mass spectrometry data and immunoblotting of cGAS immunocoprecipitates showed that cytoplasmic cGAS was associated with DNA-PK complex and nucleosomal histone H2A-H2B heterodimer, and DNA-PKcs inhibition promoted cGAS release from cytoplasmic chromatin fragments and increased the amount of free cytosolic cGAS and DNA, activating cGAS-STING. Disruption of cytoplasmic cGAS-nucleosome binding by mutating cGAS R236 or R255 sites to E abolished the effect of NU7441 on doxorubicin-induced activation of cGAS-STING signaling. Analysis of Datasets MMRF CoMMpass and GSE2658 showed that MM patients with higher DNA-PKcs expression showed poorer prognosis than those with lower expression. Additionally, the DNA-PKcs expression was elevated with MM disease progression according to Datasets GSE5900, GSE13591 and MMRF CoMMpass, these indicated a possible involvement of DNA-PKcs in MM progression and provided a basis for targeting DNA-PKcs in MM treatment. Taken together, our study suggests that DNA-PKcs may help maintain the cGAS sequestration in damaged chromatin. Inhibition of DNA-PKcs may consequently disrupt this sequestration, inducing activation of cGAS-STING signaling and improving the efficacy of doxorubicin in treating MM.

Transcriptional Co-Activator P300 Promotes Extra-Medullary Disease Formation and Myeloma Disease Progression

The transcription co-activator P300 regulates gene expression by histone H3 lysine 27 acetylation (H3K27ac) of chromatin and transcriptional regulators. P300 has been suggested to control a variety of biological functions, including differentiation, cell-cycle control, proliferation, apoptosis, and DNA damage response. The clinicopathologic/prognostic importance of P300 in multiple myeloma (MM) is unknown to date. P300 has been linked to the induction of epithelial-to-mesenchymal transition (EMT) and metastasis in a variety of solid tumor malignancies. Like EMT in solid tumors, extra-medullary disease (EMD) in MM relates to a poor prognosis and resistance to treatment. Similarly, the molecular changes concerning EMD in MM are poorly understood, and the role of P300 in MM EMD is yet to be investigated. We have employed chemical and functional genomics to unravel the role of P300 in MM disease progression and EMD formation. Six human MM cell lines-MM1S, KMS-18, H929, U266, OPM-2, and AMO-1-were used to construct lentiviral shRNA-mediated P300 knockdown (K/D) cells. In addition, to analyzing the effect of time-dependent P300 degradation, we have used a proteolysis-targeting chimera (PROTAC) compound termed “JQAD1” that selectively targets P300 for degradation. Treatment with JQAD1 effectively reduced the survival with an IC 50 of 50 nM in MM1S cells treated for 72 h. Furthermore, we wanted to understand the mechanism of P300 degradation-mediated inhibition of cell proliferation. Cell cycle analysis revealed a 40% increase in G0/G1 phase cell cycle arrest following treatment with 50 nM JQAD1 for 72 h. Blocking CD138 receptors is known to enhance MM sensitivity to proteasome inhibitors and significantly reduce tumor size. Immuno-flowcytometry analysis revealed that loss of P300 reduces CD138 expression in MM cells, suggesting their susceptibility to proteasome inhibitors. Next, we wanted to understand if P300 plays a role in developing EMD in MM. Boyden chamber-based migration and invasion assays exhibited reduced migration and invasion of P300 K/D cells with respect to control cells. Furthermore, to understand the distribution and localization of P300 silenced MM cells in vivo, we have used immune deficient NSG mice and studied MM progression after intravenous tumoral injection. In vivo studies revealed that P300 silencing led to reduced MM liver, kidney homing, and EMD formation, as observed through bioluminescence imaging, histology, and flow cytometry analysis, respectively. Further investigations are currently underway to find novel interaction partners and potential targets of P300 in MM and to delineate its mechanism of action. In conclusion, our research indicates that P300 is critical for the development of EMD and the progression of MM. This could potentially serve as a new therapeutic target for MM relapse and dissemination.

Dysregulation of Cellular Immunity in High-Risk Multiple Myeloma

Introduction: Multiple myeloma (MM) is the second most prevalent hematological malignancy, characterized by the abnormal proliferation of plasma cells in the bone marrow. Approximately 20% of MM patients present with high-risk (HR) clinical and genetic features that are associated with aggressive disease course and poor prognosis. Novel immune therapies are changing the paradigm of MM treatment. A comprehensive understanding of immune dysregulation within the tumor microenvironment (TME) of HR vs standard risk (SR) MM patients could inform the development of targeted immunotherapeutic strategies for HR MM. Methods: 55 HR and 25 SR patients were selected from the Mt. Sinai Hematologic Malignancies Tissue Bank (IRB# STUDY-11-01978). HR included one or more cytogenic abnormalities (HRCA) such as translocations t(4;14), deletion of chromosome 17 (del17p), and gain/amplification of 1q (1q+/++) based on the Second Revised International Staging System (R2-ISS). We also included nine patients with t(14;16) translocation alone or combined with one or two of the other HRCA. We performed comprehensive immune phenotyping of the bone marrow micro-environment on specimens from HR and SR patients using mass cytometry (CyTOF) and single cell RNA sequencing (scRNASeq) of bone marrow mononuclear cells (BMMC) depleted of CD138 positive MM cells. Results: ScRNAseq of BMMC from HR MM patients revealed a robust cytotoxic and cytolytic signature on CD4+ T cells relative to SR, characterized by the upregulation of genes such as GNLY, NKG7, PRF1, and IFNG, and a higher frequency of Granzyme K-positive (GZMK+) CD8+ T cells, previously described as progenitors of exhausted cells. Both CD4 and CD8 T cells, along with monocytes, exhibited high expression of a group of Interferon-stimulated genes (ISGs) including IFI27, IFIT3, IFI44L, IFITM1/3, and OASL. Notably, high expression of this group of ISGs was associated with T cell exhaustion and poor prognosis in the setting of immune checkpoint inhibitor therapy for solid tumors. Mass cytometry analysis confirmed the activated state of T cells in HR MM patients, with increased expression of immune activation markers HLA-DR, CD38, and the senescence marker CD57. Furthermore, HR patients exhibited higher frequencies of mast cells, CD14+ Monocytes and plasmacytoid dendritic cells compared to SR, suggesting an inflammatory and immunosuppressive environment that may dampen effective antitumor immune responses. Conclusions: These results indicate distinct bone marrow microenvironments of HR and SR MM. CD4 and CD8 T cells appear to be more activated in HR patients compared to SR. However, the higher frequencies of mast cells and plasmacytoid dendritic cells, and the prominent expression of ISGs associated with inhibition of anti-tumor immunity suggest dysregulation and exhaustion of T cell activity in HR patients. Collectively, these findings highlight the complex immune dynamics in HR MM and provide insights into the underlying immune dysfunction which may contribute to disease progression and therapy resistance in HR MM. Such insights have the potential to inform the design of targeted therapeutic interventions aimed at modulating the immune response and improving treatment outcomes for HR MM patients.

Exposure to Fine Particulate Matter (PM2.5) Is Associated with Worse Multiple Myeloma Outcomes in a Large, Urban, Multi-Ethnic Cohort

Introduction Outdoor air pollution (OAP) is a complex mixture, primarily consisting of black carbon (BC), nitrogen dioxide (NO2), sulfur dioxide (SO2), ozone (O3), and particulate matter with an aerodynamic diameter of less than 2.5 microns (PM2.5). OAP is typically generated by the combustion process from motor vehicles, fossil fuel burning and industrial waste. The potential impact of air pollutants on cancer outcomes has not been systematically studied in communities of color. Multiple Myeloma (MM) is a hematologic malignancy defined by the clonal expansion of plasma cells leading to subsequent monoclonal protein expression resulting in multiple complications including anemia, renal disease, significant bone pain and increased fracture risk. MM is the second most common hematologic malignancy. The US Surveillance, Epidemiology, and End Results (SEER) registry estimates 34,000 new cases diagnosed and 13,000 deaths annually from MM. Risk factors for multiple myeloma include race, BMI, and occasionally genetic predisposition. Blacks and Hispanics were shown to have a higher incidence of MM that of non-Hispanic whites (Kaur G et al Clin Lymphoma Myeloma Leuk. 2021). There is recent evidence to suggest that environmental exposures, OAP in particular, may play a role in propagating disease progression in multiple myeloma (Landgren O JAMA Oncol 2015). A recent study demonstrated that first responders to the World Trade Center disaster had a higher rate of developing myeloma precursor disease (MGUS) and an earlier age of developing multiple myeloma (Landgren O JAMA Oncol. 2018). Our study seeks to further characterize patterns of exposure to OAP in patients with MM. Methods A retrospective cohort was developed of patients diagnosed with Multiple Myeloma at Montefiore Health System (Bronx, NY) between 1997-2018 (n=1608), using the Montefiore Electronic Data Warehouse. Of those patients, 1525 were identified as having 1 st cancer diagnosis of MM. Patient addresses (n=1514) were then used to geospatially overlay with OAP and Organic Matter from 2000-2018 using land use regression models. Ground-level PM2.5 was estimated by combining Aerosol Optical Depth (AOD) retrievals from the NASA MODIS, MISR, and SeaWIFS instruments with the GEOS-Chem chemical transport model, and subsequently calibrated to regional ground-based observations of both total and compositional mass using Geographically Weighted Regression (Hammer M.S et al Environ. Sci. Technol. 2020). This initial analysis focuses specifically on PM2.5, which was summarized as the number of years each patient was exposed to levels higher than the EPA standard of 12ug/m 3 and if the patient experienced an average PM2.5 &amp;gt;12ug/m 3 across their entire follow-up in the cohort. Additional information regarding demographics, dates of diagnosis and certain clinical parameters (Hgb, Cr, albumin, etc) were extracted. Differences in PM2.5 exposures, and clinical parameters, were compared by race and ethnicity using t-tests and chi-squared tests. Cox proportional hazards regression estimated the risk of death during follow-up associated with PM2.5 exposure, adjusting for confounders. Results Our patient population was highly diverse. Patients were 21% White, 50% Black with 27% having Race classified as Other. Thirty percent were Hispanic. Age at mean diagnosis was 66.24 years. On average, Bronx MM patients had higher exposure to PM2.5 than the EPA safe standard of 12 µg/m 3 from years 2000-2007 and the WHO safe standard of 5 µg/m 3 in all years of follow-up. Improved overall survival among our Multiple Myeloma patients was associated with fewer number of years exposed to PM2.5 &amp;gt;12 µg/m 3 (Hazard Ratio (HR)=1.12, 95% Confidence Interval (CI)=0.99- 1.27), when adjusting for sex and race. Further adjustment for smoking status yielded similar results (HR = 1.13, 95%CI = 0.98-1.30). Conclusion In this large urban, multi-ethnic cohort of MM patients, many patients experienced annual exposures to PM2.5 that were higher than the EPA and WHO safe standards of 12 µg/m 3 and 5 µg/m 3. Overall survival in MM patients was associated with fewer years of exposure to PM2.5, suggesting that reducing exposure to environmental pollutants, particularly OAP for our patients may lead to better outcomes. Additional analyses are needed to better understand the impact of treatment and clinical factors, as well as the other air pollutants in this patient population.

Comprehensive Characterization of the Bone Marrow Microenvironment Transcriptional Remodeling in the Progression from MGUS to Smoldering and Multiple Myeloma

Multiple Myeloma (MM) is a hematological malignancy characterized by the abnormal proliferation and accumulation of clonal plasma cells (PCs) in the bone marrow (BM). Despite significant advances in treating MM patients, most patients eventually relapse and succumb to the disease. Understanding the mechanisms driving progression from precursor conditions into clinically active MM may contribute to implementing early curative approaches. Accumulating evidence indicates that the tumor formation and progression process is influenced not only by the malignant cells but also by the microenvironment components and interconnectedness between them. However, the understanding of the contribution of the non-immune BM microenvironment (BME) to disease progression and relapse remains largely unknown. Our aim was to identify the transcriptional changes in the endothelial cells (ECs) and mesenchymal stromal cells (MSCs) during the transition from premalignant stages to MM to identify potential mechanisms of transformation and therapeutic targets. To this end, ECs and MSCs were immunophenotypically isolated from BM samples of healthy aged donors (n=19) and patients with (MGUS, n=10), Smoldering Multiple Myeloma (SMM, n=13), and MM (n=36). Bulk RNA sequencing analysis of ECs revealed enrichment in terms related to cell cycle and fatty acid metabolism in MGUS, SMM, and MM patients compared to old adults. Indeed, a selective upregulation of pathways associated with calcium signaling, cytokines, and angiogenesis was detected in ECs from MM patients. Regarding MSCs, our results revealed an increase in bone remodeling, inflammation, oxidative defense, and DNA damage repair in MM patients. Interestingly, during the transition from MGUS to SMM, MSCs exhibited enrichment in functions associated with cell morphogenesis and epithelial transition, while lipid-related functions were decreased. To gain a deeper understanding, single-cell RNA sequencing analysis of BME and PCs was performed in unique immunocompetent murine models recapitulating the principal clinical, genetic, and immunological characteristics of MGUS and MM patients (Larrayoz et al., 2023). Using as a reference a previous BME characterization in mice we conducted (Ye et al., 2022) and in line with findings in human patients, our results unveiled a compromised BME characterized by the angiogenic profile of ECs and the increase in the immune-related state of MSCs associated with the progression of the disease. Furthermore, MGUS ECs displayed enrichment in ion homeostasis, immune response, and cell signaling-related pathways compared to healthy controls. While MM ECs exhibited a significant increase in immune terms, migration, and vascular-related pathways when compared to MGUS ECs, indicating a more angiogenic functional profile. Moreover, MSCs in the MGUS stage depicted significant activation of pathways related to immune defense, detoxification, lipid metabolism, and bone remodeling. Likewise, genes upregulated in MM MSCs were associated with calcium signaling, cytokines, and interferon response-related pathways, demonstrating the inflammatory signature of MSCs that may be in relation to the immune exhaustion underlying MM transformation. Finally, subsequent single-cell derived cell-to-cell interaction analysis using Liana was conducted to characterize the interactions between malignant PCs and their niche and delineate the communication dynamics of the tumor-BM ecosystem. Our results showed that PCs interact with ECs mainly through Itga1 and Cd44, while they communicate with MSCs mainly via Egfr and Sdc1. Remarkably, the interactions initiated during the MGUS stage between ECs and PCs persisted, and MM-specific interactions were involved in adhesion, migration, and angiogenesis pathways. In the crosstalk between PCs and MSCs, MGUS-specific interactions were associated with fat and bone-related processes. In contrast, MM-specific interactions were related to the extracellular matrix, collagen organization, and immune-related processes. In conclusion, our findings provide several novel insights into the transcriptional profiles of the non-immune BME during MM disease progression as well as the cellular interactions established with malignant PCs that can drive a pro-MM environment. Additional analyses are ongoing to further comprehend the BME's remodeling through MM development.

Multiplex immunohistochemistry elucidates increased distance between cytotoxic T cells and plasma cells in relapsed myeloma, and identifies Lag-3 as the most common checkpoint receptor on cytotoxic T cells of myeloma patients.

A dysfunctional immune tumor microenvironment facilitates disease progression in multiple myeloma (MM). Using multiplex immunohistochemistry (mIHC), we describe the quantitative and qualitative changes in CD3+CD8+ cytotoxic T cells and assess their proximity to malignant plasma cells (PC) in patients with monoclonal gammopathy of undetermined significance (MGUS), and newly diagnosed (ND) and relapsed and/or refractory (RR) MM. Formalin-fixed, paraffin-embedded trephine sections from patients with MGUS (N=32), NDMM (N=65), and RRMM (N=59) were sequentially stained for CD138, CD3, CD8, and checkpoint receptors (CPR) Tim-3, Lag-3, and PD-1. The Halo® image analysis platform was used for cell segmentation and phenotyping, facilitating enumeration of cytotoxic T cells and analysis of proximity to PC. The percentage of CD8+ cytotoxic T cells in proximity to PC is greater in patients with NDMM than patients with RRMM (at 50 μm distance, 90.8% vs. 81.5%; P=0.038). There is a trend for more CD3+ T cells in MGUS (P=0.08) but no difference was observed in the prevalence of CD8+ cytotoxic T cells (P=0.48). Lag-3 is the most common CPR expressed on cytotoxic T cells in myeloma (P<0.0001), while PD-1 is the most common CPR on CD8- T cells of patients with MGUS and RRMM. Our study is the first to report on the spatial relationship between T cells and PC using mIHC on FFPE bone marrow trephine sections from patients with PC dyscrasia. The proximity of T cells to PC during early stages of MM, and overexpression of Lag-3, validate the move of immune therapeutic strategies, including T-cell engagers and checkpoint inhibitors, to upfront treatment or in early-line treatment of MM.

Myeloperoxidase creates a permissive microenvironmental niche for the progression of multiple myeloma.

Expression of myeloperoxidase (MPO), a key inflammatory enzyme restricted to myeloid cells, is negatively associated with the development of solid tumours. Activated myeloid cell populations are increased in multiple myeloma (MM); however, the functional consequences of myeloid-derived MPO within the myeloma microenvironment are unknown. Here, the role of MPO in MM pathogenesis was investigated, and the capacity for pharmacological inhibition of MPO to impede MM progression was evaluated. In the 5TGM1-KaLwRij mouse model of myeloma, the early stages of tumour development were associated with an increase in CD11b+ myeloid cell populations and an increase in Mpo expression within the bone marrow (BM). Interestingly, MM tumour cell homing was increased towards sites of elevated myeloid cell numbers and MPO activity within the BM. Mechanistically, MPO induced the expression of key MM growth factors, resulting in tumour cell proliferation and suppressed cytotoxic T-cell activity. Notably, tumour growth studies in mice treated with a small-molecule irreversible inhibitor of MPO (4-ABAH) demonstrated a significant reduction in overall MM tumour burden. Taken together, our data demonstrate that MPO contributes to MM tumour growth, and that MPO-specific inhibitors may provide a new therapeutic strategy to limit MM disease progression.

P-218 TLR-activation may promote drug resistance and disease progression in multiple myeloma

P-218 TLR-activation may promote drug resistance and disease progression in multiple myeloma

P-409 Elevated LAG-3 expression in plasma cells and lymphoid subpopulations at distinct stages of multiple myeloma disease progression

P-409 Elevated LAG-3 expression in plasma cells and lymphoid subpopulations at distinct stages of multiple myeloma disease progression

Understanding DNA Damage Response and DNA Repair in Multiple Myeloma.

Multiple myeloma (MM) is a plasma cell malignancy characterized by several genetic abnormalities, including chromosomal translocations, genomic deletions and gains, and point mutations. DNA damage response (DDR) and DNA repair mechanisms are altered in MM to allow for tumor development, progression, and resistance to therapies. Damaged DNA rarely induces an apoptotic response, given the presence of ataxia-telangiectasia mutated (ATM) loss-of-function or mutations, as well as deletions, mutations, or downregulation of tumor protein p53 (TP53) and tumor protein p73 (TP73). Moreover, DNA repair mechanisms are either hyperactive or defective to allow for rapid correction of the damage or permissive survival. Medications used to treat patients with MM can induce DNA damage, by either direct effects (mono-adducts induced by melphalan), or as a result of reactive oxygen species (ROS) production by proteasome inhibitors such as bortezomib. In this review, we will describe the mechanisms of DDR and DNA repair in normal tissues, the contribution of these pathways to MM disease progression and other phenotypes, and the potential therapeutic opportunities for patients with MM.

CXCL10 Recruitment of γδ T Cells into the Hypoxic Bone Marrow Environment Leads to IL17 Expression and Multiple Myeloma Progression.

In multiple myeloma (MM), bone marrow stromal cells (BMSC) shape a unique niche within the bone marrow, promoting T-cell dysfunction and driving MM progression; however, the precise underlying mechanisms remain elusive. Here, we show that BMSC-mediated reprogramming of MM cells led to heightened production of CXCL10. CXCL10 orchestrated the recruitment of γδ T cells into the bone marrow, and this was observed in both the Vk*MYC and 5TGM1 mouse models of MM, as well as in patients experiencing refractory or relapsed MM. Furthermore, the dysfunctional γδ T cells in the MM bone marrow niche exhibited increased PD-1 expression and IL17 production. In the Vk*MYC mouse model, MM-associated bone lesions and mortality were markedly alleviated in Tcrd-/- mice, and MM disease progression could be rescued in these mice upon transplantation of γδ T cells expanded from wild-type mice, but not from Il17-/- mice. Mechanistically, the hypoxic microenvironment prevailing in the MM bone marrow niche stimulated the expression of steroid receptor coactivator 3 (SRC-3) in γδ T cells, which in turn interacted with the transcriptional factor RORγt, promoting Il17 transcription. Pharmacologic inhibition of SRC-3 utilizing SI-2 effectively suppressed Il17A expression in γδ T cells, leading to alleviation of MM progression in the murine models and enhancing the anti-multiple myeloma efficacy of bortezomib. Our results illuminated the bone marrow microenvironment's involvement in provoking γδ T-cell dysfunction throughout MM progression and suggest SRC-3 inhibition as a promising strategy to enhance the effectiveness of immunotherapies targeting γδ T cells.