Multiple Myeloma Bone Marrow Research Articles

Expression and Function of FAM72A Gene in Multiple myelomaFAM72A.

This study aims to comprehensively investigate the role of Family Member A with sequence similarity 72-A (FAM72A) in multiple myeloma. Multiple myeloma poses significant challenges. This study delves into FAM72A's impact on key cellular processes, shedding light on potential therapeutic targets and enhancing our understanding of multiple myeloma progression. Investigate the impact of FAM72A on the proliferation, apoptosis, and bortezomib sensitivity of multiple myeloma cell line U266. qRT-PCR analyzed FAM72A expression levels in bone marrow samples from 30 patients with multiple myeloma and 10 healthy donors at the Second Hospital of Shanxi Medical University. Cell lines overexpressing FAM72A were constructed, and Cell Counting Kit 8 (CCK-8) and flow cytometry were used to assess U266 cell proliferation, apoptosis, and sensitivity to bortezomib. Biological predictions for FAM72A were performed to find transcription factors binding to the FAM72A promoter region, verified using a luciferase assay. U266 cells were transfected with si-POU2F2 (POU class 2 homeobox 2), and the impact on cell proliferation was validated. Western blot analysis detected the expression of downstream proteins in the p53 signaling pathway. In vivo, experiments established a xenograft mouse model further to study the role of FAM72A in multiple myeloma. FAM72A was upregulated in multiple myeloma bone marrow tissues. Compared to the OE-NC group, the OE-FAM72A group showed increased Mouse Double Minute 2 homolog (MDM2) expression, decreased p53 expression, increased cell proliferation, and decreased apoptosis. POU2F2 was identified as the upstream transcription factor for FAM72A. Compared to the si-NC group, the si-POU2F2 group exhibited decreased MDM2 expression, increased p53 expression, slowed cell proliferation, and increased apoptosis. Silencing POU2F2 could reverse the pro-proliferative effect of over-expressing FAM72A in U266 cells. In vivo experiments in a xenograft mouse model further studied the role of FAM72A in multiple myeloma. Overexpression of FAM72A promotes U266 cell proliferation, inhibits apoptosis, and reduces sensitivity to bortezomib by regulating the POU2F2/FAM72A/p53 signaling pathway.

Coefficient of variation and texture analysis of 18F-FDG PET/CT images for the prediction of outcome in patients with multiple myeloma

In multiple myeloma (MM) bone marrow infiltration by monoclonal plasma cells can occur in both focal and diffuse manner, making staging and prognosis rather difficult. The aim of our study was to test whether texture analysis of 18 F-2-deoxy-d-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) images can predict survival in MM patients. Forty-six patients underwent 18 F-FDG-PET/CT before treatment. We used an automated contouring program for segmenting the hottest focal lesion (FL) and a lumbar vertebra for assessing diffuse bone marrow involvement (DI). Maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean) and texture features such as Coefficient of variation (CoV), were obtained from 46 FL and 46 DI. After a mean follow-up of 51 months, 24 patients died of myeloma and were compared to the 22 survivors. At univariate analysis, FL SUVmax (p = 0.0453), FL SUVmean (p = 0.0463), FL CoV (p = 0.0211) and DI SUVmax (p = 0.0538) predicted overall survival (OS). At multivariate analysis only FL CoV and DI SUVmax were retained in the model (p = 0.0154). By Kaplan-Meier method and log-rank testing, patients with FL CoV below the cut-off had significantly better OS than those with FL CoV above the cut-off (p = 0.0003), as well as patients with DI SUVmax below the threshold versus those with DI SUVmax above the threshold (p = 0.0006). Combining FL CoV and DI SUVmax by using their respective cut-off values, a statistically significant difference was found between the resulting four survival curves (p = 0.0001). Indeed, patients with both FL CoV and DI SUVmax below their respective cut-off values showed the best prognosis. Conventional and texture parameters derived from 18F-FDG PET/CT analysis can predict survival in MM patients by assessing the heterogeneity and aggressiveness of both focal and diffuse infiltration.

Single‐cell sequencing reveals the correlation of aggrephagy signaling and multiple myeloma immune microenvironment composition

AbstractAggrephagy, a type of autophagy, degrades the aggregation of misfolded protein in cells. However, the role of aggrephagy in multiple myeloma (MM) has not been fully demonstrated. In this study, we first investigated the correlation between aggrephagy signaling, MM immune microenvironment composition and disease prognosis. Single‐cell RNA‐seq data, including the expression profiles of 12,187 single cells from seven MM bone marrow (BM) and seven healthy BM samples, were analyzed by non‐negative matrix factorization for 44 aggrephagy‐related genes. Bulk RNA‐seq cohorts from the Gene Expression Omnibus database were used to evaluate the prognostic value of aggrephagy‐related immune cell subtypes and predict immune checkpoint blockade immunotherapeutic response in MM. Compared with healthy BM, MM BM exhibited different patterns of aggrephagy‐related gene expression. In MM BM, macrophages, CD8+ T cells, B cells and natural killer cells could be grouped into four to nine aggrephagy‐related subclusters. The signature of aggrephagy signaling molecule expression in the immune cells correlates with the patient's prognosis. Our investigation provides a novel view of aggrephagy signaling in MM tumor microenvironment cells, which might be a prognostic indicator and potential target for MM treatment.

Value of third-generation of VNCa dual-energy CT for differentiating diffuse marrow infiltration of multiple myeloma from red bone marrow.

This study aims to investigate the ability of bone marrow imaging using third-generation dual-energy computed tomography (CT) virtual noncalcium (VNCa) to differentiate between multiple myeloma (MM) with diffuse bone marrow infiltration and red bone marrow (RBM). Bone marrow aspiration or follow-up results were used as reference. We retrospectively reviewed 188 regions of interests (ROIs) from 21 patients with confirmed MM and diffuse bone marrow infiltrations who underwent VNCa bone marrow imaging between May 2019 and September 2022. At the same time, we obtained 98 ROIs from 11 subjects with RBM for comparative study, and 189 ROIs from 20 subjects with normal yellow bone marrow for the control group. The ROIs were delineated by 2 radiologists independently, the interobservers reproducibility was evaluated by interclass correlation coefficients. The correlation with MRI grade results was analyzed by Spearman correlation coefficient. Receiver operating characteristic (ROC) curve analysis was used to determine the optimal threshold for differentiating between these groups and to assess diagnostic performance. There were statistically significant differences in VNCa CT values of bone marrow among the MM, RBM, and control groups (all P < .001), with values decreasing sequentially. A strong positive rank correlation was observed between normal bone marrow, subgroup MM with moderately and severe bone marrow infiltration divided by MRI and their corresponding CT values (ρ = 0.897, 95%CI: 0.822 to 0.942, P < .001). When the CT value of VNCa bone marrow was 7.15 HU, the area under the curve (AUC) value for differentiating RBM and MM was 0.723, with a sensitivity of 50.5% and a specificity of 89.8%. When distinguishing severe bone marrow infiltration of MM from RBM, the AUC value was 0.80 with a sensitivity 70.9% and a specificity 78.9%. The AUC values for MM, RBM, and the combined group compared to the control group were all >0.99, with all diagnostic sensitivity and specificity exceeding 95%. VNCa bone marrow imaging using third-generation dual-energy CT accurately differentiates MM lesions from normal bone marrow or RBM. It demonstrates superior diagnostic performance in distinguishing RBM from MM with diffuse bone marrow infiltration.

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

Abstract Characterizing the heterogeneity in human leukemias is critical for understanding mechanisms of disease onset, uncovering biomarkers for disease diagnosis, and guiding research into novel treatment approaches. While extensive research focus has been placed on characterizing the molecular heterogeneity in leukemia by modern omics technologies, comparatively less emphasis has been placed on studying the diversity in holistic morphological changes that occur in single cells in the context of disease. Here, we report on a novel approach to understanding and characterizing different hematological malignancies using ghost cytometry, a recently developed high-content flow cytometric method that leverages high-speed, artificial intelligence (AI) driven morphological characterization and analysis of single cells. We performed morphometric profiling on human bone marrow samples from patients with T and B-cell acute lymphoblastic leukemia (ALL, n=5), acute myeloid leukemia (AML, n=5), multiple myeloma (MM, n=5) and healthy bone marrow mononuclear cell (BM-MNC) controls (n=6). Morphological profiles from individual cells were analyzed by unsupervised machine learning (universal manifold approximation and projection, UMAP) and compared within disease subsets and against controls. We identified disease-specific cell populations from a mixture of healthy control and disease samples without any labeling, based on completely label-free information. For validation of the specific cell subsets, we overlaid known cell surface markers (e.g. CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM). The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population. We also identified a unique cell subpopulation in the disease-specific cell population that could not be distinguished by existing markers. We also analyzed three human myeloma cell lines (AMO1, KMS, and L363) for comparison to the primary MM samples. In the UMAP analysis using LF-GC data, in all three types of cell lines, Bortezomib-resistant cell lines exhibited different distributions from the control parental cell lines. In addition, we observed distinct morphological profiles between primary myeloma samples and myeloma cell lines. Here we present a novel cell characterization approach for human hematological diseases that leverages AI-based, label-free, high-speed morphological characterization of single cells. We demonstrate that the approach can be used to identify subtle morphological differences in blast cells from a range of blood cancer subtypes. Application of morphological profiling and AI can be used to identify measurable, disease-related changes in these diseases that have diagnostic, drug screening, and therapeutic monitoring potential. Citation Format: Asako Tsubouchi, Juho J. Miettinen, Keisuke Wagatsuma, Satoru Akai, Yuri An, Mika Uematsu, Philipp Sergeev, Dimitrios Tsallos, Markus J. Vähä-Koskela, Sadao Ota, Caroline A. Heckman. Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2305.

In Vitro Low-Bortezomib Doses Induce Apoptosis and Independently Decrease the Activities of Glutathione S-Transferase and Glutathione Peroxidase in Multiple Myeloma, Taking into Account the GSTT1 and GSTM1 Gene Variants.

Multiple myeloma (MM) is a malignancy derived from plasma cells. Bortezomib affects the concentration of reduced glutathione (GSH) and the activity of glutathione enzymes. The aim of our study was to analyze deletion (null/present) variants of GSTT1 and GSTM1 genes and their association with the levels of glutathione and its enzymes in bortezomib-treated cell cultures derived from MM patients. This study included 180 individuals (80 MM patients and 100 healthy blood donors) who were genotyped via multiplex PCR (for the GSTT1/GSTM1 genes). Under in vitro conditions, MM bone marrow cells were treated with bortezomib (1-4 nM) to determine apoptosis (via fluorescence microscopy), GSH concentration, and activity of glutathione enzymes (via ELISA). Bortezomib increased the number of apoptotic cells and decreased the activity of S-glutathione transferase (GST) and glutathione peroxidase (GPx). We found significant differences in GST activity between 1 nM (GSTT1-null vs. GSTT1-present), 2 nM (GSTT1-null vs. GSTT1-present), and 4 nM (GSTM1-null vs. GSTM1-present) bortezomib: 0.07 vs. 0.12, p = 0.02; 0.06 vs. 0.10, p = 0.02; and 0.03 vs. 0.08, p = 0.01, respectively. Bortezomib affects the activities of GST and GPx. GST activity was associated with GSTT1 and GSTM1 variants but only at some bortezomib doses.

Recent advances in imaging and artificial intelligence (AI) for quantitative assessment of multiple myeloma.

Multiple myeloma (MM) is a malignant blood disease, but there have been significant improvements in the prognosis due to advancements in quantitative assessment and targeted therapy in recent years. The quantitative assessment of MM bone marrow infiltration and prognosis prediction is influenced by imaging and artificial intelligence (AI) quantitative parameters. At present, the primary imaging methods include computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET). These methods are now crucial for diagnosing MM and evaluating myeloma cell infiltration, extramedullary disease, treatment effectiveness, and prognosis. Furthermore, the utilization of AI, specifically incorporating machine learning and radiomics, shows great potential in the field of diagnosing MM and distinguishing between MM and lytic metastases. This review discusses the advancements in imaging methods, including CT, MRI, and PET/CT, as well as AI for quantitatively assessing MM. We have summarized the key concepts, advantages, limitations, and diagnostic performance of each technology. Finally, we discussed the challenges related to clinical implementation and presented our views on advancing this field, with the aim of providing guidance for future research.

Mitochondrial Fitness and Immune Function of Natural Killer Cells Are Impaired By the Multiple Myeloma (MM) Tumor Cells: The Role of MM Microenvironment Interactions

The immune system including Natural Killer (NK) activity was shown to be impaired in active MM. We and others have previously demonstrated the important role of the bone marrow (BM) microenvironment in MM pathophysiology, however, data on its effects on NK activity in MM is limited. We first assessed the phenotype of the BM-derived NK cells. BM-NK from MM pts (n=10) showed an exhausted phenotypic signature with significantly reduced expression of CD16 (p&amp;lt;0.01), and increased expression of exhaustion markers CD39 (p&amp;lt;0.001) and CD57 (p&amp;lt;0.001) compared to BM-NK cells from healthy controls (n=8). Furthermore, MM-BM NK cells demonstrated reduced mitochondrial membrane potential, suggesting impaired mitochondrial fitness. The dysfunction of the mitochondria led to deficiency of ATP synthesis; mitochondrial ATP levels were reduced in NK cells from MM BM samples in comparison to the normal BMs (p&amp;lt;0.001). Next, the functional response of BM NK cells to the stimulation with IL-2 (2000 IU/ml for 7 and 14 days) was evaluated in vitro. NK cells from MM BM demonstrated reduced proliferation capacity, increased CD39, reduced expression of activating receptors NKp44 and NKp30, and reduced mitochondrial membrane potential upon the activation with IL-2. The impaired mitochondrial fitness correlated with reduced levels of NKp44, indicating a potential link between the decreased mitochondrial activity and the reduced functionality of NK cells. Indeed, in vitro, the cytotoxic activity of IL-2-stimulated MM-BM NK cells against MM cell lines was significantly diminished compared to healthy IL-2-activated BM-NK cells. Glucose and fatty acids (FA) are key substrates of the mitochondrial bioenergetic pathways and essential for NK cell activation. Of note, glucose uptake was increased comparably upon IL-2 stimulation in NK cells from both MM patients and healthy donors. In contrast, MM-BM NK cells demonstrated reduced FA uptake in response to IL-2 stimulation compared to BM NK cells from healthy donors. These data indicate impaired mitochondrial and NK cell properties in the BM MM niche. Next, we established an in vitro model to interrogate the relationship between NK cell mitochondrial and immune dysfunction within the MM BM microenvironment. Specifically, we cultured MM cell lines (n=5) with the NK92 cell line or primary IL-2-activated or not activated blood-derived NK cells, and assessed NK cell mitochondrial fitness, metabolic activity, and immune functional properties. Co-culture with MM cells significantly impaired mitochondrial mass and mitochondrial membrane potential in NK cells, decreased mitochondrial ATP levels, while upregulating MM mitochondrial ROS in NK cells, indicating that MM cells induce mitochondrial damage and impair ATP synthesis in NK cells. Mitochondrial fitness and energy production are pivotal for NK cell persistence and cytotoxicity. In accordance, direct interaction with MM cells suppressed NK cell proliferation in vitro, inducing G0/G1 cell cycle arrest. Moreover, MM-induced mitochondrial impairment in NK cells correlated with reduced responsiveness of MM cells to NK-induced killing, while short-term exposure to MM cells significantly diminished the anti-myeloma cytotoxic activity of NK cells. Accordingly, CD39 expression was upregulated, while NKp44 levels significantly decreased in NK cells upon interaction with MM cells. To further characterize the molecular mechanisms associated with MM-mediated NK cell mitochondrial impairment, we assessed the gene expression profile of MM-exposed sorted NK92 cells versus unexposed cells. A distinct metabolic signature characterized by reduced expression of genes involved in the mitochondrial activity was detected in MM-exposed NK cells. Thus, genes involved in respiratory activity and mitochondrial dynamics were downregulated post-MM exposure. Furthermore, a profound increase in fructose-1,6-biphosphatase 1 (FBP1) expression was detected in MM-exposed NK cells. FBP1 is the rate-limiting enzyme for gluconeogenesis that simultaneously inhibits glycolysis, possibly interfering with NK cell metabolic activity and immune function. Altogether, our data suggest that MM BM microenvironment and direct interactions with malignant plasma cells impair NK mitochondrial fitness, diminishing energy production, increasing oxidative stress, and driving exhaustion with reduced anti-tumor capacity in NK cells.

Preliminary Results of the Oral CD73 Inhibitor, Oric-533, in Relapsed/Refractory Multiple Myeloma (RRMM)

Background: Although recently available immune-based approaches in multiple myeloma produce high response rates, curative outcomes have not yet been achieved, which highlights the need for new agents that further activate the immune system without increasing toxicity. ORIC-533 is a highly potent and selective, orally bioavailable, small molecule inhibitor of CD73, an enzyme expressed by various cell types, including immune cells and which catalyzes the generation of adenosine from adenosine monophosphate (AMP) (Allard 2019). Adenosine itself is highly immunosuppressive, diminishing activity of a range of immune cell types, including T, NK, and dendritic cells (Young 2014). By blocking adenosine production from AMP via inhibition of CD73, ORIC-533 has demonstrated the ability to restore and enhance immune function. Multiple myeloma (MM) patient bone marrow (BM) contains multiple cell types that also express CD73, including immune cells, cancer-associated fibroblasts, mesenchymal stem cells, and endothelial cells (Allard 2019). Moreover, elevated adenosine levels in the MM BM niche correlate with disease progression (Horenstein 2016; Yang 2020). We have previously observed that CD73-mediated adenosine activity suppresses the cytolytic antitumor immune function in the MM BM milieu (Ray 2022). ORIC-533 treatment reduced adenosine production, overcame this immune suppression with evidence of restored DC-mediated priming and activated cytotoxic CD8 + T-cell function, and significantly enhanced immune cell cytolytic behavior against RRMM tumor cells in autologous patient ex vivo experiments (Junttila 2022), making ORIC-533 a potential immunotherapy for patients with MM. Methods: ORIC-533-01 (NCT05227144) is an ongoing i3+3 Phase 1b dose escalation study in patients with RRMM. The primary objectives are to determine single agent safety/tolerability and pharmacokinetics (PK) of ORIC-533 to select the RP2D. Pharmacodynamic assessments include measurement of CD73 functional activity in serum and BM, as well as enumeration of immune cell subsets and activation state in blood and BM. Patients are also evaluated for clinical efficacy using IMWG criteria. Results: As of 23 June 2023, 17 patients received ORIC-533 once daily across 4 dose levels (400 mg to 1600 mg). All patients were triple-class refractory, 88% were penta-refractory, and 59% also received prior anti-BCMA/CD3 bispecific therapy or anti-BCMA CAR-T therapy. Overall, ORIC-533 was very well tolerated, with 5 patients experiencing a total of 10 treatment-related adverse events (TRAEs). Fatigue was the only G3 AE and the only TRAE seen in more than 1 patient (1 event each of G3 and G2); all other events seen in 1 patient each, were G1 or G2 in severity, and of no specific system organ class. No dose limiting toxicities, no Grade ≥4 TRAEs, and no treatment-related serious adverse events have been observed. PK showed increased exposure with dose with a plasma half-life of ~24 hrs. Soluble CD73 enzymatic activity was nearly completely inhibited by C1D15 in serum of all patients across all dose levels, while the largest relative suppression in surface CD73 activity in BM cells was observed at the highest dose level. There was also evidence for increases in the abundance of circulating NK and CD8+ T cells after 1 treatment cycle, with preliminary evidence of enhanced CD8+ T-cell activation at the highest dose levels tested in both peripheral blood and BM. In addition, early evidence of single agent clinical activity was observed. Conclusions: ORIC-533 has demonstrated an acceptable safety profile and preliminary evidence of immune activation in a heavily pretreated RRMM patient population. Enrollment is ongoing and updated data will be presented.

Tuning the Innate Immune Multiple Myeloma Microenvironment By Modulating IRF4

The tumor microenvironment (TME) has emerged as a key contributor to cancer progression in numerous solid tumors as well as blood cancers that evolve in the bone marrow (BM). In the context of multiple myeloma (MM), survival of malignant plasma cells is supported by interactions with the BM microenvironment, which is comprised of various components including cells, extracellular matrix, and soluble factors. Therefore, the development of strategies that specifically target the MM BM microenvironment is crucial for improving current disease prognosis. Within the BM niche, M2-like myeloma-associated macrophages (MAMs) play a significant role in cancer cell survival and progression and have been implicated in producing an immune-suppressive TME by generating inflammatory mediators, growth factors, cytokines, chemokines, etc. Macrophages exhibit plasticity with regard to their polarization state, which enable them to dynamically respond and adapt to different environmental stimuli in order to fulfill important immune functions. Thus, the reprogramming of M2-like, pro-tumoral MAMs toward a M1-like, anti-tumoral phenotype represents a promising therapeutic strategy. In MM, inflammatory and anti-viral pathways promote disease progression. Interferon regulatory factor 4 (IRF4) is a transcription factor that has previously been shown to promote MM progression through enhanced survival of malignant plasma cells. Under physiological conditions, IRF4 also regulates macrophage polarization, where its expression drives alternative activation of macrophages primarily to M2-like (anti-inflammatory) phenotypes. However, the extent to which IRF4 activation or direct inhibition governs the polarization status and pro-tumoral activity of macrophages in the MM microenvironment has not been fully elucidated. In this study, we sought to further explore the role of IRF4 in macrophage polarization and investigated the potential of modulating IRF4 expression to reprogram myeloma-associated macrophages (MAMs) as a novel therapeutic strategy. First, we developed myeloma-tailored macrophage polarization models using primary human peripheral blood-derived monocytes and the THP1 monocyte cell line. Our M1 and M2 polarization models utilize a customized cocktail of myeloid growth factors and cytokines, supplemented with interleukin (IL)-6 to mimic the IL-6-enriched MM microenvironment. Characterization of M1 (CD80, TNFα, and CXCL10) and M2 (MRC1, FN1, and CCL22) markers confirmed the presence of distinct macrophage subpopulations, as well as upregulated expression of IRF4 in M2-like progeny and IRF5 in M1-like macrophages. To investigate the extent to which IRF4 activation or direct inhibition governs macrophage polarization and function, overexpression and knockdown experiments on macrophages were performed in vitro, along with whole transcriptome sequencing of IRF4-overexpressing THP1 monocytes compared to vector-transduced controls. Gene expression analyses revealed that genetic down-modulation of IRF4 in M2-like macrophages shifts the polarization status toward a hybrid phenotype that displays enhanced M1-like properties, while still retaining M2-like characteristics. This shift in polarization state was further amplified when monocytes were transduced to overexpress IRF4 prior to differentiation into M2-like macrophages and subsequent IRF4 knockdown, suggesting that IRF4 plays a key regulatory role in tuning macrophage polarization. Taken together, our preclinical findings provide groundwork for future studies on MAMs and suggest that an IRF4-mediated macrophage reprogramming strategy is a promising therapeutic approach for patients with MM and a variety of other malignancies typified by an anti-inflammatory TME.

Phenotype Molding of Monocytes in the Multiple Myeloma Microenvironment

Human monocytes are mononuclear phagocytes that comprise a major population of innate immune cells. Various monocyte-derived cells, including osteoclasts, dendritic cells, and macrophages, have been implicated in immunosuppression within the multiple myeloma (MM) tumor microenvironment (TME). Despite recent developments in understanding the link between monocyte-derived cells and immunosuppression in myeloma, there are still critical knowledge gaps in the association between monocytes themselves and immunosuppression within the context of MM. Specifically, the functional and developmental alterations of monocytes in the MM TME have not been explored based on the single-cell studies. In the present study, we generated a comprehensive single-cell map of peripheral blood (PB) and bone marrow (BM) monocytes from both healthy donors (HDs) and MM patients. Specifically, Heparin-anticoagulated PB samples or BM aspirates were collected from seven newly diagnosed MM (NDMM) patients and healthy donors (HDs). Paired BM aspirates were obtained from 10 MM patients at the time of diagnosis and after induction therapy. Differential expression analysis showed that type I interferon (IFN) response was upregulated in MM monocytes. And BM monocytes derived from MM patients exhibited impaired T cell attraction abilities and demonstrated an elevated capacity for suppressing T cell function. Moreover, immune checkpoint molecules, including CD274 (encoding PD-L1) and PDCD1LG2 (encoding PD-L2), were significantly upregulated in MM monocytes. Additionally, a positive correlation was observed between type I IFN response and T cell suppression capacity of MM monocytes. Trajectory analyses revealed an IFN-focused disruption of developmental pathways of both BM and PB monocytes in MM. Lastly, we included 10 MM patients at the time of diagnosis and after induction therapy to monitor transcriptional changes over time. Our results revealed that anti-tumor therapy successfully mitigated the excessive type-I interferon response of BM monocytes in MM. We document that MM monocytes exhibited an augmented type-I IFN response compared to monocytes from HDs, while their overall heterogeneity remained unchanged. Furthermore, we extended the knowledge on immunosuppression of the MM TME that the excessive type-I IFN response in MM had a significant impact on the differentiation of both BM and PB monocytes, leading to T-cell immunosuppression, specifically in BM monocytes. In the cohort that we longitudinally followed, we observed a decrease in the type-I IFN response across nearly all monocyte subsets in MM BM monocytes after induction therapy, accompanied by a reduction in the expression of the T-cell suppression gene set. In sum, our findings indicates that monocytes in the BM promotes the immunosuppression of the MM TME and represents potential therapeutic targets for modulating the MM TME.

E3 Ubiquitin Ligase TRIM21 Enhances Macrophage-Mediated Bortezomib Resistance By Inducing M2 Polarization in Multiple Myeloma

Background: Multiple myeloma (MM) is a malignant plasma cell hematologic tumor with an increasing incidence and has become the second most common hematologic malignancy after non-Hodgkin lymphoma. Macrophage is an abundant component of the MM bone marrow microenvironment and contributes to the enhancement of MM drug resistance. Our previous study found that the expression of the E3 ubiquitin ligase TRIM21 was reduced in MM cells, and MM cells with low expression of TRIM21 showed reduced sensitivity to Bortezomib (Bort). This study aims to investigate the role and mechanism of TRIM21 in macrophage-mediated MM Bort resistance. Objective ：Explore the effect and mechanism of TRIM21 in mediating multiple myeloma Bortezomib resistance. Methods ：1. Immunofluorescence to analyze the expression of TRIM21 on macrophages in bone marrow biopsy specimens from patients with MM; 2. QPCR, western blot (WB) and flow cytometry (FCM) to detect the level of macrophage polarization markers after constructing overexpression or knockdown of TRIM21; 3. WB and FCM to detect the sensitivity of MM cells to Bort after co-culture of MM cells and macrophage; 4. Transcriptome sequencing to identify TRIM21-mediated pathway changes in macrophage; 5. QPCR and WB to verify the effect of TRIM21 on macrophage polarization after treatment with pathway inhibitors. Results:1. Immunofluorescence analysis showed increased expression of TRIM21 in macrophage from relapsed and refractory MM (RRMM) patients compared to newly diagnosed multiple myeloma (NDMM) patients;2. QPCR, WB and FCM results showed that overexpression of TRIM21 in macrophage led to decreased polarization markers of M1 phenotype and increased the polarization markers of M2 phenotype. In contrast, knockdown of TRIM21 increased M1 polarization and decreased M2 polarization; 3. WB and FCM results demonstrated that co-culture of MM cells with TRIM21-overexpressing macrophages significantly enhanced MM cell tolerance to Bortezomib and decreased MM cell apoptosis; 4. Transcriptome sequencing revealed activation of the JAK-STAT3 pathway in macrophages after TRIM21 overexpression; 5. QPCR and WB results showed that the macrophage polarization effect induced by TRIM21 overexpression was reversed after treatment with the STAT3 inhibitor C188-9. Conclusions: Our study shows that TRIM21 is overexpressed in macrophages in patients with relapsed and refractory MM. In addition, TRIM21 induces M2 polarization by activating the JAK-STAT3 pathway, thereby enhancing the protective effect of macrophage on MM cells. These findings provide a new insight into the role of TRIM21 in MM drug resistance and lay an important theoretical foundation for targeting macrophages in MM. Key words: TRIM21, Macrophage, Bortezomib, JAK-STAT3

Label-Free High Dimensional Single Cell Morphological Profiling of Different Hematological Malignancies By Ghost Cytometry

Characterizing the heterogeneity in human leukemias is critical for understanding mechanisms of disease onset, uncovering biomarkers for disease diagnosis, and guiding research into novel treatment approaches. While extensive research focus has been placed on characterizing the molecular heterogeneity in leukemia by modern 'omics technologies, comparatively less emphasis has been placed on studying the diversity in holistic morphological changes that occur in single cells in the context of disease. Here, we report on a novel approach to understanding and characterizing different hematological malignancies using ghost cytometry, a recently developed high-content flow cytometric method that leverages high-speed, artificial intelligence (AI) driven morphological characterization and analysis of single cells. We performed morphometric profiling on human bone marrow samples from patients with T and B-cell acute lymphoblastic leukemia (ALL, n=5), acute myeloid leukemia (AML, n=5), multiple myeloma (MM, n=5) and healthy bone marrow mononuclear cell (BM-MNC) controls (n=6) (Figure 1). Morphological profiles from individual cells were analyzed by unsupervised machine learning (universal manifold approximation and projection, UMAP) and compared within disease subsets and against controls. We identified disease-specific cell populations from a mixture of healthy control and disease samples without any labeling, based on completely label-free information (Figure 2). For validation of the specific cell subsets, we overlaid known cell surface markers (e.g. CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM). The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population. We also identified unique cell populations across blood cell populations that were distinct from the healthy BM-MNC controls. We also analyzed three human myeloma cell lines (AMO1, KMS, and L363) for comparison to the primary MM samples. In the UMAP analysis using LF-GC data, in all three types of cell lines, Bortezomib-resistant cell lines exhibited different distributions from the control parental cell lines. In addition, we observed distinct morphological profiles between primary myeloma samples and myeloma cell lines. Here we present a novel cell characterization approach for human hematological diseases that leverages AI-based, label-free, high-speed morphological characterization of single cells. We demonstrate that the approach can be used to identify subtle morphological differences in blast cells from a range of blood cancer subtypes. Application of morphological profiling and AI can be used to identify measurable, disease-related changes in these diseases that have diagnostic, drug screening, and therapeutic monitoring potential. Keywords: flow cytometry, acute leukemia, multiple myeloma, label-free cell analysis, biomarker, liquid biopsy, ghost cytometry, artificial intelligence, machine learning Figure 1. Research design overview. Bone marrow samples from patients and non-diseased controls will be analyzed by Ghost Cytometry to assess disease by conventional markers and identify novel morphometric subpopulations. Morphological information is acquired as label-free GMI signals without any labeling. The label-free GMI information is transformed into two-dimensional space by UMAP dimensionality reduction method to identify subpopulations of interest. Both samples are stained with known cell surface markers for validation of unsupervised classification. Figure 2. Identification of disease-specific cell subpopulations by unsupervised approach. Using an unsupervised machine learning technique called UMAP, label-free morphologically distinct cellular subpopulations can be identified in B-ALL, AML, MM when compared to healthy donor controls (left panels, inside the dotted circles). For validation of the subpopulations, known cell surface markers (CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM) and cell trace marker (CellTracker) were overlaid. The intensity increases monotonically through the colormaps. The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population.

Glutamine Addiction of Multiple Myeloma Cells Reprograms Mesenchymal Stromal Cell Enzymes and Transporters Towards a Pro-Tumor Phenotype

Metabolic alterations in cancer are not only functional to ensure malignant cell growth but also shape a pro-tumor behavior of normal cell populations in the tumor microenvironment. Multiple myeloma (MM) is the only human cancer that is both glutamine-addicted and glutamine-auxotroph, a feature that renders MM growth completely dependent upon extracellular glutamine availability. Indeed, plasma cells from most MM patients do not express Glutamine Synthetase (GS), the only enzyme able to synthetize glutamine from glutamate and ammonium, while express high levels of Glutaminase (GLS), which catalyze the first step of glutaminolysis by deamidating glutamine into glutamate and ammonium. As a consequence, the bone marrow (BM) plasma of MM patients has low-glutamine/high glutamate levels compared to patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS), as firstly demonstrated by our group. This particular metabolic microenvironment induces GS expression in mesenchymal stromal cells (MSCs) impairing osteoblast (OB) differentiation of MSCs, thus favoring MM bone lesions typical of active MM. The impact of these metabolic alterations on other BM cell populations is much less defined and it has been investigated in this study. For instance, although MM BM displays an oversized adipocyte population, which sustain MM growth by providing free fatty acids, the effects of MM metabolism on MSCs and their adipocyte differentiation remain to be characterized. First we show that 13C 5-Gln isotopomer distribution revealed that almost 50% of total glutamate in human myeloma cell lines (HMLCs) directly derives from glutamine deamidation by GLS. Furthermore, in standard culture conditions, HMLCs secreted glutamate likely through the SLC7A11 transporter, whose activity was higher in HMLCs than in MSCs. On the other hand, HMCLs were not able to take up glutamate from the extracellular space, while human primary MSCs isolated from healthy donors displayed high activity of the sodium-dependent glutamate transporters of the EAAT family. Moreover, glutamate uptake was higher in undifferentiated MSCs than in MSC incubated for 14 days in osteogenic medium (10 -8 M dexamethasone and 50 µg/ml ascorbic acid). Consistently, a public transcriptional profile of MSCs and OBs obtained from bone biopsies of healthy donors (n=7) or MM patients (n=16) revealed that the expression of the inward Glu transporter EAAT3 is higher in MSCs compared to OBs. In glutamine-free conditions, MSCs produced and secreted glutamine, a process boosted by extracellular glutamate in a dose-dependent manner. Glutamine secretion was hindered by either l-methionine sulfoximine (MSO), an irreversible inhibitor of GS, or D-aspartate (D-Asp), a high-affinity inhibitor of EAAT3 that hinders glutamate uptake. In a co-culture system, HMCLs induced the expression of SNAT5 glutamine efflux transporter in MSCs. Consistently, upon glutamine withdrawal, primary undifferentiated MSCs from healthy donors sustained HMCLs growth, while this nutritional support was markedly impaired by either inhibition or silencing of GS and EAAT3, with a substantial decrease in MM cell viability. Lastly, primary human MSCs were incubated under adipogenic conditions (0.5 mM 3-Isobutyl-1-methylxanthine, 5 µM indomethacin, 50 µM dexamethasone and 10 mg/ml human insulin) in the presence or in the absence of glutamine to mimic, respectively, normal or MM BM microenvironment. Glutamine deprivation increased lipogenesis, assessed with Oil Red O staining, as well as the expression of the adipocyte markers PPARG, LEP, and ADIPOQ. These data point to a MM-driven metabolic pro-tumor BM niche in which MSCs feed glutamine-addicted MM cells, and MSC differentiation is skewed from osteogenesis to adipogenesis. Several steps of these deranged pathways are amenable to pharmacological inhibition, pointing to possible novel therapeutic approaches to counteract MM growth and its effects on the BM niche.

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 &amp;gt;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.

High Dimensional Profiling of Patient-Derived Multiple Myeloma Bone Marrow Specimens Treated with an FcRH5-Targeted Bispecific Ex Vivo

Background: Cevostamab is an FcRH5 (Fc receptor-homolog 5) targeted T cell-dependent bispecific (TDB) currently being evaluated in the clinic, with promising activity and favorable safety profile as a monotherapy in patients (pts) with heavily pre-treated Relapsed/Refractory multiple myeloma (MM) (NCT03275103, Trudel et al. ASH 2021). Cevostamab selectively engages and activates T cells in the proximity of MM cells in the bone marrow (BM) to drive tumor cell killing. To dissect the transcriptional and functional impact of cevostamab treatment on the MM BM tumor microenvironment (TME) and identify factors linked to enhanced killing, we established an ex vivo TDB killing assay with MM pt BMMC (BM mononuclear cells) samples followed by detailed characterization of immune and tumor cells. Methods: We tested commercially procured frozen BMMC samples from 7 pts with Stage I or Stage III Newly Diagnosed Multiple Myeloma (NDMM) with no known prior treatments (except a paired, longitudinally collected sample) and median age of 67.5 years. BMMC samples containing endogenous T and MM cells were treated ex vivo with 10 ug/mL of either a non-targeted TDB (considered as baseline) or a research grade cevostamab surrogate (FcRH5 TDB) for 16 to 48 hours (hr). T cell enumeration and MM killing (CD138 + CD38 +) was evaluated at 48 hours by flow cytometry. Ex vivo response was normalized by T cell quantity and defined as % tumor cell killing per 10 3 T cells present in the assay, which was used to classify samples as Responders (R) (&amp;gt;= 10%) or Suboptimal Responders (SoR) (&amp;lt; 10%). Single cell RNA-seq (scRNA-seq) was performed at 16, 24 and 48 hr post treatment. Tumor-specific genes enriched in SoRs at baseline were investigated for prognostic value by examining their bulk gene expression profile in baseline tumor samples (CD138-enriched) from pts (N=1150) treated with immunomodulatory drug, proteasome inhibitors, or combinations thereof (NCT01454297, MMRF CoMMpass IA22). Results: We first examined the T cell compartment by scRNA-seq profiling. Upon FcRH5 TDB treatment, clustering analysis suggested that CD4 and CD8 T cells undergo extensive transcriptional changes compared to control TDB (Fig 1A). A TNF and LTA-expressing CD4 memory cluster was enriched upon treatment in both SoRs and Rs, but to a greater extent in Rs. A similar trend was observed with an IFNG and CCL4-enriched CD8 effector-like proliferating T cell cluster. Transcriptional profiles of MM cells suggested inter- and intra-pt tumor heterogeneity at baseline. MM cells in SoRs at baseline had higher expression of genes like CD44, SRGN, CCND2 and SLAMF1. Of these genes, higher expression of CD44 or SRGN was found to be significantly associated with worse outcome to 1L treatments. Exposure to FcRH5 TDB altered tumor cells from all pts (Figure 1B), resulting in significant upregulation of genes such as HLA-E, SOCS1, and TNFSF10. Increase in CXCL9 expression upon treatment was found to be higher in Rs than SoRs. Discussion: Our work provides a novel ex vivo approach for unbiased, high dimensional pharmacodynamic profiling of functional and transcriptomic changes in pt samples by combining TDB treatment with scRNA-seq. Consistent with the expected mechanism of action and preliminary biomarker analysis reported for cevostamab (Nakamura et al. ASH 2020), FcRH5 TDB led to increased expression of genes in T cells associated with infiltration, activation and effector function. It also induced transcriptional changes in MM cells regardless of the ex vivo response status, including upregulation of genes associated with T and NK cell infiltration and antigen presentation. For certain genes, a greater magnitude of these changes was observed in Rs. These identified genes, and enriched clusters will be examined in clinical RNAseq data from cevostamab-treated pts. It remains to be determined whether our results reflect treatment effects that are applicable to other targeted bispecifics in MM. Overall, our assay setup can serve as a platform to generate novel hypotheses, corroborate clinical observations, identify pt-specific features driving improved or dampened response, and test novel combinations for improved killing. Figure 1 : UMAP (Uniform Manifold Approximation and Projection) plot of all (A) T cells and (B) tumor cells shows distinct clustering of cells by treatment type for all pts (red: FcRH5 TDB; blue: non-targeted TDB)

A Highly Successful Model to Decrease Racial Disparities and Increase Access to Autologous Transplants Among African Americans with Multiple Myeloma - Outreach and Satellite Transplant Clinics

Background It is well known that racial disparities exist between White and African Americans (AA) with multiple myeloma (MM) resulting in an inferior outcome among AA. It is believed that the disparities are due to socioeconomic barriers that limit access to timely, appropriate and high quality medical care. A Veterans Administration study of MM patients showed that with equal access, AA have superior survival compared to Whites. Autologous hematopoietic stem cell transplant (AHST) is a standard treatment for MM and improves survival but only 20% of AA receive AHST compared to 39% for Whites. Possible obstacles for AA are lack of resources such as transportation, financial and family/care giver support to travel to a distant transplant center. There is extensive awareness and data regarding racial disparities in MM and recommendations for improvement. We present our experience to bring to attention a simple intervention to decrease the disparities in this retrospective IRB approved study. Methods At the Medical College of Georgia, Augusta University, Georgia, we embarked on a program of outreach beginning July 2017 to market our transplant and hematologic malignancy program with the intention of increasing patient referrals and transplant volumes. The health system and marketing/outreach program collaborated with our cancer center and provided resources to execute the project. This included personally traveling as a group and visiting community practices that were between 1 to 200 miles in our catchment area, meeting physicians, advanced practice providers (APPs), nurses, referral coordinators to apprise them of our services. The services included access to transplant faculty mobile numbers, a simple “ONE CALL ONLY” referral system and a meticulous line of communication. Beginning in early 2019, we started satellite clinics in two large community practices; practice A 65 miles and practice B 89 miles from our center. One transplant physician traveled to practice A and a second one to practice B once a month where patients with hematologic malignancies were evaluated for a consultation. Patients that required a transplant received induction treatment, pre-transplant work up including MM lab testing, organ function evaluation, scans, bone marrow exams at the satellite clinics to minimize travel and make it easier for patients and families. The transplant procedure itself including stem cell collection, conditioning regimen, stem cell reinfusion and recovery during myelo-suppression was performed at our transplant center. Upon discharge from the hospital, the subsequent post-transplant care was transitioned as early as possible back to the satellite clinics. This consisted of post-transplant discharge monitoring, restaging work up required such as MM blood work, scans and bone marrow evaluation. Recommendations regarding consolidation/maintenance treatment were made during one of the monthly transplant physician's satellite clinic visits but administered by the community oncologists. In this analysis, we only included patients with MM who received a transplant. Results Between 2013 and June, 2017 (no outreach and no satellite clinics) our transplant center did 81 MM transplants and 292 from July 2017 to July 2023. Before starting outreach and satellite clinics there were 3 transplants from practice A and 0 from practice B. From July 2017 to July 2023 (post outreach and satellite clinics) there were 64 transplants; 46 from practice A and 18 from practice B. The median age was 65.9 at time of transplant and 34 were male. The racial breakdown was 39 AA (60.9%) and 37.5% White. The median distance from the patients' homes to practice A and B was 16.9 and 34.8 miles and to the transplant center 85.7 and 99.25 miles respectively. 25 patients had Medicare and 39 private insurance. Conclusion We conclude that outreach, and personal and meticulous communication increases MM transplant referrals. Most importantly, setting up satellite clinics is an effective initiative and will not only increase transplant referrals, but most notably increases AHST access to AA. We think this is due to improving patient convenience by decreasing the number of visits to a distant transplant center, travel time and burden to care givers so the AA can be cared for closer to home. We believe that this is a simple yet very practical and effective way of decreasing disparities and making AHST more accessible to African Americans.

STMN1 promotes cell malignancy and bortezomib resistance of multiple myeloma cell lines via PI3K/AKT signaling

ABSTRACT Background This study investigates the biological functions of Stathmin1 (STMN1) involving drug resistance and cell proliferation in multiple myeloma (MM) and its related mechanisms. Methods Bone marrow aspirates were collected from 20 MM patients, and the bone marrow mononuclear cells (BMMCs) were separated by Ficoll-Hypaque density gradient centrifugation. Blood samples of 20 patients with monoclonal gammopathy of undetermined significance (MGUS) and 20 healthy donors were collected. Normal plasma cells sorted from the peripheral blood of MGUS patients and healthy subject as controls. Two bortezomib (BTZ)-resistant MM cell lines were established, namely NCI-H929/BTZ and KM3/BTZ cells, and then transfected with lentiviruses packaging sh-STMN1 to knock down STMN1 level in BTZ-resistant cells. Expression of STMN1 was assessed by RT-qPCR and western blotting. CCK-8 assays were performed to assess 50% growth inhibition (IC50) values. Green fluorescent protein in BTZ-resistant cells infected with lentiviruses was observed by fluorescence microscopy. Cell viability, proliferation, cell cycle, and apoptosis were evaluated through MTT assays, colony formation assays, flow cytometry analyses, and TUNEL staining. Results STMN1 was upregulated in MM cells and bone marrow aspirates of MM patients. Additionally, STMN1 depletion attenuated BTZ resistance in MM cells. Moreover, downregulation of STMN1 limited the malignant phenotypes of BTZ-resistant cells. Mechanistically, the PI3K/Akt signaling was inactivated by STMN1 downregulation in BTZ-resistant cells. Conclusion STMN1 silencing inhibits cell proliferation and BTZ resistance in MM by inactivating the PI3K/Akt signaling.

HAPLN1 matrikine: a bone marrow homing factor linked to poor outcomes in patients with MM

AbstractThe bone marrow (BM) microenvironment is critical for dissemination, growth, and survival of multiple myeloma (MM) cells. Homing of myeloma cells to the BM niche is a crucial step in MM dissemination, but the mechanisms involved are incompletely understood. In particular, any role of matrikines, neofunctional peptides derived from extracellular matrix proteins, remains unknown. Here, we report that a matrikine derived from hyaluronan and proteoglycan link protein 1 (HAPLN1) induces MM cell adhesion to the BM stromal components, such as fibronectin, endothelial cells, and stromal cells and, furthermore, induces their chemotactic and chemokinetic migration. In a mouse xenograft model, we show that MM cells preferentially home to HAPLN1 matrikine–conditioned BM. The transcription factor STAT1 is activated by HAPLN1 matrikine and is necessary to induce MM cell adhesion, migration, migration-related genes, and BM homing. STAT1 activation is mediated by interferon beta (IFN-β), which is induced by NF-κB after stimulation by HAPLN1 matrikine. Finally, we also provide evidence that higher levels of HAPLN1 in BM samples correlate with poorer progression-free survival of patients with newly diagnosed MM. These data reveal that a matrikine present in the BM microenvironment acts as a chemoattractant, plays an important role in BM homing of MM cells via NF-κB–IFN-β–STAT1 signaling, and may help identify patients with poor outcomes. This study also provides a mechanistic rationale for targeting HAPLN1 matrikine in MM therapy.

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.