Proliferation Of Myeloma Cells Research Articles

Advancements in microenvironment-based therapies: transforming the landscape of multiple myeloma treatment

Multiple myeloma (MM) is the most prevalent malignant monoclonal disease of plasma cells. There is mounting evidence that interactions with the bone marrow (BM) niche are essential for the differentiation, proliferation, survival, migration, and treatment resistance of myeloma cells. For this reason, gaining a deeper comprehension of how BM microenvironment compartments interact with myeloma cells may inspire new therapeutic ideas that enhance patient outcomes. This review will concentrate on the most recent findings regarding the mechanisms of interaction between microenvironment and MM and highlight research on treatment targeting the BM niche.

Interference with PLA2G16 promotes cell cycle arrest and apoptosis and inhibits the reprogramming of glucose metabolism in multiple myeloma cells by modulating the Hippo/YAP signaling pathway.

Multiple myeloma, which is a clonal plasma cell tumor, derives from a postmitotic lymphoid B-cell lineage and remains untreatable. Group XVI phospholipase A2 (PLA2G16) can either be a tumor suppressor or an oncogene in different types of cancer. This study was intended to explore the role of PLA2G16 in multiple myeloma and to reveal the reaction mechanism. The mRNA and protein expressions of PLA2G16 in human bone marrow stromal cell line HS-5 and multiple myeloma cells were assessed using reverse transcription-quantitative PCR and western blot. The transfection efficacy of sh-PLA2G16 and oe-YAP was examined using reverse transcription-quantitative PCR and western blot. Through cell counting kit-8 assay and 5-ethynyl-2'- deoxyuridine staining, multiple myeloma cell viability and proliferation were detected. Flow cytometry was used to measure cell apoptosis and cell cycle distribution. Oxygen consumption rate, the activities of mitochondrial respiratory chain complexes I-V, and the activity of caspase-3 were estimated with Seahorse XF24 analyzer, oxidative phosphorylation activity assay kit, and caspase-3 assay kit, respectively. Lactate production and glucose consumption were evaluated usingcorresponding assay kits. Western blot was employed to meaure proteins associated with cell cycle, glycolysis, pentose phosphate pathway as well as Hippo/YAP signaling pathway. In this study, PLA2G16 expression was greatly increased in multiple myeloma cells and PLA2G16 silence inhibited cell proliferation, promoted cell apoptosis, facilitated cell cycle arrest, and suppressed the reprogramming of glucose metabolism in multiple myeloma. It was also identified that PLA2G16 depletion inhibited the Hippo/YAP signaling pathway. Further experiments revealed that the overexpression of YAP partially reversed the inhibitory effects of PLA2G16 silence on multiple myeloma cell malignant development and the reprogramming of glucose metabolism. Collectively, PLA2G16 silence impeded multiple myeloma progression and inhibited glucose metabolism reprogramming by blocking the Hippo/YAP signaling pathway.

PRAME promotes proliferation of multiple myeloma cells through CTMP/Akt/p21/CCND3 axis by ubiquitinating CTMP and p21

Multiple myeloma (MM) is a Ubiquitin Proteasome System (UPS)-dysfunction disease. We previously reported that high PRAME transcript levels associated with unfavorable progression free survival (PFS) in patients with no bortezomib therapy, and bortezomib-containing regimen significantly improved PFS in patients with high PRAME transcript levels, which indicated that PRAME expression was prognostic for MM patients, and was related to proteasome inhibitor treatment. However, molecular mechanisms underlying the above clinical performance remain unclear. In the present study, MM cell models with PRAME knockdown and overexpression were established, and PRAME was identified to play the role of promoting proliferation in MM cells. P-Akt signaling was found to be activated as PRAME overexpressed. As a substrate recognizing subunit (SRS) of the E3 ubiquitin ligase, PRAME targets substrate proteins and mediates their degradation. CTMP and p21 were found to be the novel targets of PRAME in the Cul2-dependent substrate recognition process. PRAME interacted with and mediated ubiquitination and degradation of CTMP and p21, which led to accumulation of p-Akt and CCND3 proteins, and thus promoted cell proliferation and increased bortezomib sensitivity in MM cells.

Anti-tumor effect of dandelion flavone on multiple myeloma cells and its mechanism

BackgroundMultiple myeloma (MM) is a prevalent hematologic malignancy characterized by the uncontrolled proliferation of monoclonal plasma cells in the bone marrow and excessive monoclonal immunoglobulin production, leading to organ damage. Despite therapeutic advancements, recurrence and drug resistance remain significant challenges.ObjectiveThis study investigates the effects of dandelion flavone (DF) on MM cell proliferation, migration, and invasion, aiming to elucidate the mechanisms involved in MM metastasis and to explore the potential of traditional Chinese medicine in MM therapy.MethodsDF's impact on myeloma cell viability was evaluated using the CCK-8 and colony formation assays. Cell mobility and invasiveness were assessed through wound healing and transwell assays, respectively. RT-PCR was employed to quantify mRNA levels of MMP-2, MMP-9, TIMP-1, and TIMP-2. Apoptotic rates and molecular markers were analyzed via flow cytometry and RT-PCR. The PI3K/AKT signaling pathway was studied using Western blot and ELISA, with IGF-1 and the PI3K inhibitor LY294002 used to validate the findings.ResultsDF demonstrated dose-dependent inhibitory effects on MM cell proliferation, migration, and invasion. It reduced mRNA levels of MMP-2 and MMP-9 while increasing those of TIMP-1 and TIMP-2. Furthermore, DF enhanced the expression of pro-apoptotic proteins and inhibited M2 macrophage polarization by targeting key molecules and enzymes. The anti-myeloma activity of DF was mediated through the inhibition of the PI3K/AKT pathway, as evidenced by diminished phosphorylation and differential effects in the presence of IGF-1 and LY294002.ConclusionBy modulating the PI3K/AKT pathway, DF effectively inhibits MM cell proliferation, migration, and invasion, and induces apoptosis, establishing a novel therapeutic strategy for MM based on traditional Chinese medicine.

Human IL-6 fosters long-term engraftment of patient-derived disease-driving myeloma cells in immunodeficient mice.

Multiple myeloma is a largely incurable and life-threatening malignancy of antibody-secreting plasma cells. An effective and widely available animal model that recapitulates human myeloma and related plasma cell disorders is lacking. We show that busulfan-conditioned human IL-6-transgenic (hIL-6-transgenic) NSG (NSG+hIL6) mice reliably support the engraftment of malignant and premalignant human plasma cells, including from patients diagnosed with monoclonal gammopathy of undetermined significance, pre- and postrelapse myeloma, plasma cell leukemia, and amyloid light chain amyloidosis. Consistent with human disease, NSG+hIL6 mice engrafted with patient-derived myeloma cells developed serum M spikes, and a majority developed anemia, hypercalcemia, and/or bone lesions. Single-cell RNA sequencing showed nonmalignant and malignant cell engraftment, the latter expressing a wide array of mRNAs associated with myeloma cell survival and proliferation. Myeloma-engrafted mice given CAR T cells targeting plasma cells or bortezomib experienced reduced tumor burden. Our results establish NSG+hIL6 mice as an effective patient-derived xenograft model for study and preclinical drug development of multiple myeloma and related plasma cell disorders.

#11. Bone marrow adipocyte-induced increases in lipid content and fatty acid binding proteins may support proliferation in myeloma cells

#11. Bone marrow adipocyte-induced increases in lipid content and fatty acid binding proteins may support proliferation in myeloma cells

#43. Long-chain acyl-CoA synthetases support multiple myeloma cell proliferation, survival and mitochondrial function

#43. Long-chain acyl-CoA synthetases support multiple myeloma cell proliferation, survival and mitochondrial function

Abstract 6848: Analysis of the bone microenvironment cells by single cell transcriptomics reveals altered features in multiple myeloma patients compared to pre-malignant monoclonal gammopathies

Abstract Multiple myeloma (MM) is a malignant plasma cell dyscrasia that can be preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). How the bone microenvironment (BME) changes and contributes to tumoral progression in MM remains unresolved. Published data on BME are mainly produced in vitro and few data are on MGUS/SMM patients. Up to date, no single-cell RNA sequencing database of the BME cells has been described in MM. The aim of this project was to characterize for the first time at single cell level, the BME in patients with newly diagnosed MM (MMD) and MGUS/SMM to identify alterations involved in tumoral progression. From 15 bone biopsies of MMD, MGUS and SMM patients, we depleted CD235a+, CD45+, CD31+, and CD138+ cells to enrich the rare BME non-hemopoietic cells. The CD45-CD31-CD235a-CD138- cells were analyzed by scRNAseq. Data were generated on Chromium 10X Genomics. Cellranger and Seurat pipeline in R software has been used. Cell identities were assigned by manual curation. The trajectory analysis has been made by Monocle3. Decouple R and FindMarkers were used to compare pathways, and differential gene expression (DGE) of the samples throughout the different conditions. A total of 35723 cells were profiled. We identified 13 BME cell type clusters: 7 mesenchymal stromal cells (MSCs) clusters (proliferating MSC, MSC DDK-OPG-, MSC DKK+, MSC RUNX2-, MSC OPG+, MSC Osteogenic, Perivascular MSC) and 6 osteoblasts (OBs) clusters (OB precursor, OB precursor LEPR+, OB BGLAP+, OB SPP1+, OB NFkB+, OB late). Psdeudotime analyses highlighted a complex trajectory of differentiation from more immature cell clusters (proliferating MSC) to late OB cluster, confirming the ability of the dataset to snapshot the in vivo complexity of BME and we highlighted an alteration of the MSCs clusters trajectories starting in SMM samples till a disruption in MM. Pathway analyses, in MMD compared to MGUS/SMM, revealed an upregulation of VEGF and PI3K activity in proliferating MSC, perivascular MSC and OB clusters. TRAIL pathway activity, known to suppress MM cell proliferation, was upregulated in MSC osteogenic and OB BGLAP+ in MGUS. Finally, DGE analyses revealed the modulation of different genes, many not yet described in MM. MGUS MSCs clusters showed an upregulation of FBLN1, SERPNF1 and MMD MSCs clusters dramatically downregulated WISP2 and upregulated SRGN, involved in MM cell proliferation. Interestingly, in SMM samples, we reported an upregulation of genes involved both in the promotion of osteoblastogenesis (SCARA3, SPON2) and inhibition (GREM1), likely underling the attempt to contrast the establishment of altered BME, characteristic of MM. Our approach is able to dissect the complex organization of the BME and to highlight at single cells level the alterations of the BME in patients with MM compared to MGUS and SMM. Citation Format: Paola Storti, Tommaso Torelli, Cristina Manferdini, Denise Toscani, Alessandro Guidi, Rosanna Vescovini, Nicolas Thomas Iannozzi, Benedetta Dalla Palma, Matteo Scita, Federica Librale, Stefania Ricci, Vincenzo Raimondi, Oxana Lungu, Giannalisa Todaro, Gabriella Sammarelli, Emanuela Aleo, Gina Lisignoli, Luca Agnelli, Nicola Giuliani. Analysis of the bone microenvironment cells by single cell transcriptomics reveals altered features in multiple myeloma patients compared to pre-malignant monoclonal gammopathies [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 6848.

Abstract 4477: The development of a small molecule dual inhibitor of CXCR4 and Mcl-1 to target bortezomib resistant and stem-like cells in multiple myeloma

Abstract Multiple myeloma (MM) is a malignant plasma-cell illness defined by clonal growth of malignant plasma cells within the bone marrow (BM) microenvironment. Despite the availability of many therapeutic alternatives, MM remains consistently lethal, demanding the development of novel techniques. The intimate relationship between myeloma cells and the BM microenvironment is a distinguishing hallmark of MM. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 enhance the homing of MM cells to the BM, which has important functional implications. The BM microenvironment secretes a variety of hormones and cytokines that activate several signaling pathways, resulting in enhanced production of the anti-apoptotic Mcl-1 protein, boosting the proliferation, survival, and migration of MM cells and conferring resistance to standard chemotherapy. As a result, we created GA-A, a hybrid molecule in which we coupled an aralkyl carboxylic acid moiety, known to preferentially inhibit Mcl-1, to gambogic acid, which has previously been demonstrated to inhibit CXCR4 in MM. Our western blot has shown that this unique hybrid compound successfully suppressed CXCR4 and Mcl-1. Interestingly, using qPCR we found that CXCR4 was observed to be higher expressed in CD138- versus CD138+ cells, while GA-A inhibited CXCR4 in both subpopulations. In addition, using annexin-V flow cytometry staining we found that GA-A increased the apoptotic effect of bortezomib in MM stem-like cells while inhibiting the viability of bortezomib resistant cells. Using human cytokine array we found that GA-A activates some of proinflammatory cytokines that can activate the immune system such as GM-CSF, IFN gamma, IL-2 and IL-12. In summary, our findings indicate the successful creation of a novel dual CXCR4-Mcl1 inhibitor that demonstrates efficacy against the stem cell-like population and bortezomib resistant cells and enhances the effectiveness of bortezomib. Citation Format: Weam Othman Elbezanti, Omar S. Al-Odat, Subash C. Jonnalagadda, Tulin Budak-Alpdogan, Manoj K. Pandey. The development of a small molecule dual inhibitor of CXCR4 and Mcl-1 to target bortezomib resistant and stem-like cells in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4477.

Deguelin inhibits the proliferation of human multiple myeloma cells by inducing apoptosis and G2/M cell cycle arrest: Involvement of Akt and p38 MAPK signalling pathway.

Deguelin exhibits antiproliferative activity against various cancer cell types. Previous studies have reported that deguelin exhibits pro-apoptotic activity against human cancer cells. The current study aimed at further elaborating the anticancer effects of deguelin against multiple myeloma cells. Cell growth estimations were made through MTT assay. Phase contrast microscopy was used for the analysis of the viability of multiple myeloma cells. Colony formation from multiple myeloma cells was studied using a clonogenic assay. Antioxidative assays for determining levels of glutathione (GSH) and superoxide dismutase (SOD) were carried out after treating multiple myeloma cells with deguelin. The apoptosis of multiple myeloma cells was studied using AO/EB and Annexin V-FITC/PI staining methods. Multiple myeloma cell cycle analysis was performed through flow cytometry. mRNA expression levels were depicted using qRT-PCR. Migration and invasion of multiple myeloma cells were determined with the wound-healing and transwell assays, respectively. Deguelin specifically inhibited the multiple myeloma cell growth while the normal plasma cells were minimally affected. Multiple myeloma cells when treated with deguelin exhibited remarkably lower viability and colony-forming ability. Multiple myeloma cells treated with deguelin produced more SOD and had higher GSH levels. The multiple myeloma cell growth, migration, and invasion were significantly declined by in vitro administration of deguelin. In conclusion, deguelin treatment, when applied in vitro, induced apoptotic cell death and resulted in mitotic cessation at the G2/M phase through modulation of cell cycle regulatory mRNAs in multiple myeloma cells.

Mir-186 inhibits the proliferation and growth of multiple myeloma cells by targeting Jagged-1 expression

Purpose: To investigate the suppressive influence of mir-186 on multiplication and growth of multiple myeloma (MM), as well as the processes involved.Methods: The U266 cells were divided into control, mir-186 overexpression, and inhibition groups. The latter two were transfected with mir-186 agent and antagonist, respectively. Cell viability, colony formation potential, cell cycle ratio, and Jagged-1 mRNA and protein levels were measured using various assays.Results: Cell growth increased over time in all groups. However, mir-186 overexpression cells showed significantly decreased growth and colony formation capacity, relative to control, while the mir-186 inhibition cells showed significantly higher growth and colony formation capacity. The study revealed a higher proportion of G0/G1 stage cells and lower proportion of S-phase cells in mir-186 overexpression cells than in control cells. The opposite effect was seen in mir-186 inhibition cells. Jagged-1 protein and mRNA levels were significantly lower in mir-186 overexpression cells and higher in mir-186 inhibition cells than in control group. The Jagged-1 knockout group showed significantly higher Jagged-1 mRNA and protein levels than both the control and mir-186 overexpression groups (p < 0.05).Conclusion: When overexpressed, mir-186 inhibits the growth of multiple myeloma cells by inhibiting cell colony-formation capacity and by regulating cell cycle through mir-186-induced regulation of the expression of Jagged-1. there is need for more research to confirm the clinical benefits of therapies based on these findings.

Enhanced Expression of DNp73 Promotes Drug Resistance and Immune Evasion in Multiple Myeloma By Targeting the MYC and MYCN Pathways

Background Multiple myeloma (MM) is an incurable haematological malignancy with high chromosome instability and is highly dependent on the immunosuppressive bone marrow microenvironment. TP53 mutations and deletions are uncommon in MM patients, with other types of mechanisms involved in the abrogation of TP53 pathway activity. DNp73, an inhibitor of the p53 tumour suppressor family, drives drug resistance and cancer progression in several solid malignancies. However, the biological functions of DNp73, the molecular mechanisms underlying these actions and their correlations with aberrant TP53 expression in myelomagenesis remain unclear. In this study, we investigated the role of DNp73 in the drug resistance of MM, and disclosed the corresponding mechanism of how DNp73 promotes the immune escape of MM cells. Aims To disclose the role of DNp73 in promoting drug resistance and immune evasion in MM, and explore the therapeutic strategy based on the mechanism research. Methods We constructed DNp73 overexpression and sh-RNA interference plasmids to obtain stable cell lines. The effects of DNp73 on proliferation and drug sensitivity were determined by CCK8, flow cytometry and xenotransplantation model. To identify mechanisms of drug resistance, we here perform RNA-seq and CHIP-seq analyses in MM cell lines, while Western blot and RT-PCR were used to confirm the results. The DNA damage repair and invasion ability of MM cells were detected by immunofluorescence and transwell assay. To validate the role of DNp73 in immune escape, we also performed phagocytosis assays in vitro. Results Our previous study revealed that the miRNA-15a is a tumour suppressor involved in aggressive MM cell proliferation and drug resistance. Here, we found that miRNA-15a downregulation in MM cells activated NF-κB-p65, which upregulated the expression of DNp73 at the transcriptional level. In addition, the level of miR-15a was negatively correlated with the expression of DNp73 in MM cells of patients (r=-0.672, p<0.05). Enforced expression of DNp73 promoted aggressive proliferation and multidrug resistance in MM cells (p<0.001). Bulk RNA sequencing data showed that the levels of MYCN, MYC and CDK7 were increased in cells with enforced DNp73 expression. Furthermore, a ChIP-qPCR assay revealed that DNp73 bound to the MYCN gene promoter and upregulated MYCN expression. The MM cell immune evasion rate was notable when DNp73 expression was high because of CD47 and PD-L1 upregulation. Blockade of the CD47/SIRPα and PD-1/PD-L1 signalling pathways by the SIRPα-Fc fusion protein IMM01 and monoclonal antibody atezolizumab significantly re-established the anti-MM function of macrophages and T cells in the MM microenvironment, respectively (p<0.01). Notably, DNp73 dysregulation and its pathogenetic function in MM cells were found to be independent of TP53 expression. Conclusion Altogether, our study demonstrates that DNp73 is a potential oncogene involved in MM pathogenesis and promotes MM cell proliferation and immune evasion by targeting the MYC and MYCN pathways.

TAS0612, a Triple Inhibitor for RSK, AKT, and S6K, Is a Promising Anti-Myeloma Agent Regardless of Types of Cytogenetic/Genetic Abnormality

Introduction: Despite advancements in therapeutic approaches, treatment outcome for multiple myeloma (MM) remains unsatisfactory. One critical factor of the poor outcomes is the complex inter-patient and intra-clonal heterogeneity of cytogenetic, genetic, and molecular abnormalities. To overcome this, the exploration of therapeutic target molecules that play a universal and essential role in MM regardless of the types of molecular profile is crucial even in the post-genomic era. We have previously demonstrated that RSK2 Ser227 at the N-terminal kinase domain (NTKD), as the downstream signaling hub of the RAS/ERK pathway, plays essential roles in the proliferation and survival of myeloma cells (Shimura Y, Mol Cancer Ther 2012). We also identified that RSK2-NTKD is constitutively activated by constitutive active PDPK1, the master serine-threonine kinase of >20 AGC kinases, in myeloma cells of approximately 90% of MM patients regardless of cytogenetic abnormalities (Chinen Y, Cancer Res 2014, Tatekawa S, Br J Haematol 2017). Moreover, the combinatory blockade of RSK2 and AKT, one of the other important molecules regulated by PDPK1, exhibited synergistic anti-myeloma efficacy through the modulation of various critical gene sets involved in myeloma pathophysiology, such as MYC (Isa R, Int J Mol Sci 2022). Based upon these, this study investigates the anti-myeloma potency of TAS0612 (Taiho Oncology Inc., Princeton, NJ) that targets RSK, including RSK2, AKT, and S6K, and inhibits the bindings to their respective substrate molecules (Shibata T, Mol Cancer Ther 2020). Methods and Results: Firstly, we examined the activation status of RSK, AKT, and S6K in eight human myeloma-derived cell lines (HMCLs) with diverse genetic and molecular profiles at the protein level. RSK2 Ser227 was found to be commonly activated through phosphorylation in all HMCLs examined. In contrast, the activation patterns of p-AKT Ser473 and p-S6K Thr389 were inconsistent among eight HMCLs, and depending on cell types. Despite these cell type-dependent effects, the exposure to TAS0612 resulted in the accumulation of p-total RSK Ser380, p-AKT Ser473, and p-S6K Thr389 in the majority of HMCLs examined, and this led to the subsequent inactivation of their respective target substrates, such as p-YB1 Ser102, p-PRAS Thr246, and p-S6 Ser235/236, through dephosphorylation. In accompany with these molecular effects, the modified MTT assay showed the dose-dependent growth inhibitory effects of TAS0612 in all cell lines with various types of cytogenetic/genetic abnormalities with IC50 values mostly below 0.5 μM (48 hours). In addition, flow cytometry analyses for the cell cycle analysis and the apoptosis assay by the counterstaining of propidium iodide and Annexin-V showed that TAS0612 induced G1/S cell cycle arrest and apoptotic cell death. Furthermore, patient-derived primary myeloma cells were labeled with anti-CD138 MicroBeads, and were positively isolated from bone marrow mononuclear cells from patients with newly diagnosed MM (n=7) and relapsed/refractory MM (n=11), and, then, were treated by TAS0612. As the results, the ex vivo treatment of 2.5-5.0 μM TAS0612 for 72 hours showed robust cell reductive effects on primary-myeloma cells from all 18 patients examined irrespective of cytogenetic profile. To clarify the precise molecular effects of TAS0612 treatment in myeloma cells, we conducted a comprehensive molecular analysis with TAS0612 treatment by gene expression profiling. The results showed that TAS0612 treatment significantly modulated the gene sets involved in Myc signaling, mTOR signaling, and the core serum response (CSR). Additionally, TAS0612 caused the concomitant upregulation of several bona-fide tumor suppressor genes, including TP53INP, HBP1, FBOX32, and YPEL3, while the downregulation of oncogenes LYAR and HSPA1. Intriguingly, according to MMRF CoMMpass Study, 8 of 18 upregulated genes by TAS0612 in HMCLs were reported to be associated with favorable survival outcomes when highly expressed in myeloma cells. Conclusion: Our findings strongly support the potential of TAS0612 as an effective anti-myeloma agent. Further preclinical investigations are necessary to substantiate these results and facilitate its future clinical development.

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

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

CD70/CD27 Signaling Promotes Expansion of Clonal Plasma Cells in Multiple Myeloma and Is a Promising Therapeutic Target in Advanced Disease

Introduction: Multiple Myeloma (MM) is a hematological malignancy characterized by the clonal expansion of malignant plasma cells within the bone marrow (BM). In the last decade, substantial advances in the treatment of MM patients have been made resulting in improved progression-free and overall survival rates. However, to date, MM remains incurable. CD70 and CD27 belong to the TNF receptor family. CD70 is the only known ligand for its receptor CD27. Physiologically, the CD70/CD27 interaction is involved in the regulation of immune cell expansion. However, numerous solid and hematological cancer types overexpress CD70. Aside, CD70 and CD27 co-expression has been described on leukemic stem cells (LSC) in acute myeloid leukemia patients where an autonomous signaling activation drives LSC self-renewal and expansion. While CD70/CD27 signaling plays a key role in the differentiation process of B lymphocytes into plasma cells, only little is known about its impact on myelomagenesis and MM progression. In this study, we analyzed the role of CD70/CD27 signaling in the pathogenesis of MM and studied the possibility to target CD70 by monoclonal antibodies to treat the disease. Materials and Methods: We evaluated CD70 and CD27 gene expression levels and its correlations with overall survival (OS), MM progression and high-risk molecular alterations using different databases (CoMMpass IA15, GSE6477). CD70 and CD27 protein expression patterns were analyzed in a newly established MM tissue microarray (TMA) and a collection of matched BM samples from initial and advanced disease stages (in collaboration with the Institutes of Pathology in Bern, Basel and Tübingen). To assess the functional role of CD70/CD27 signaling on MM initiation and expansion, we generated CD70 knock-out MM cell lines and newly established patient-derived xenografts (PDX) using CRISPR/Cas9 genome editing. CD70-expressing MM cell lines or patient-derived myeloma cells were grown in NOD/SCID (NSG) mice. Moreover, different monoclonal antibodies (Abs) and Ab-derivates were tested including a CD70 blocking antibody (αCD70) and an antibody-dependent cell-mediated cytotoxicity (ADCC) optimized antibody (cusatuzumab, ArgenX) that activates FcγR-expressing effector cells to assess the therapeutic potential of targeting CD70 in MM. Results: High CD70 gene expression levels correlate with increased MKI67 gene expression and elevated lactate dehydrogenase serum levels indicating a more rapid proliferation of CD70-expressing myeloma cells. In addition, high CD70 expression in MM patients correlated with shorter OS. Longitudinal analyses of CD70 gene expression in the precursor stages monoclonal gammopathy of undetermined significance and smoldering myeloma as well as MM and relapsed MM stages showed highest CD70 gene expression levels in relapsed MM patients. Assessment of the molecular background further revealed increased frequencies of the prognostically unfavorable genomic translocations t(4;14) and t(14;16), as well as gain/amplification of the chromosomal region 1q21 (1q+) in CD70 high expressing MM patients. In line with our previous findings, a TMA analysis of over 200 MM core biopsies as well as a longitudinal analysis of matched biopsies from patients at initial diagnosis and MM relapse indicated that CD70 was overexpressed in advanced disease. Functionally, we found that CD70/CD27 signaling promotes plasma cell survival inducing the expansion of MM cell lines and primary malignant plasma cells. CD70 blockade (αCD70) or CD70 knock-out (CD70 KO) resulted in a significant inhibition or complete abrogation of growth of a CD70-expressing MM patient-derived xenograft (PDX) (EMM01 CD70+) compared to controls (Figure 1). Interestingly, in vivo limiting dilution assays revealed that CD70+CD27+ double-positive myeloma subpopulations showed significantly higher cancer stem cell frequencies compared to CD70 negative fractions suggesting that CD70 expression might be related to an immature or stem-cell like phenotype. The ADCC-optimized CD70 specific antibody custatuzumab efficiently eliminated CD70-expressing MM cells in the presence of PBMC-derived natural killer (NK) cells in vitro and in a PDX model in vivo. Conclusion: Our results indicate that CD70-expressing MM cells have a stem-cell like phenotype and propagate the disease. Targeting CD70 is a promising new therapy especially in advanced disease.

The Activation Status of RSK2, but Not PDPK1 and AKT, Associates with the Prognosis of Diffuse Large B Cell Lymphoma

B-cell lymphoma (BCL) is the most common hematologic malignancy with various genetically and molecularly distinct subtypes. Although the prognosis of most BCL subtypes has been dramatically improved by the advances of disease subtype-directed, molecularly targeted therapies, some patients remain incurable with currently available therapeutics. We previously identified that a serine/threonine kinase 3-phosphoinositide-dependent kinase-1 (PDPK1) and its major downstream substrate serine/threonine kinases, AKT and RSK2, play pivotal roles in the pathophysiology of multiple myeloma and mantle cell lymphoma (MCL) (Chinen Y, Cancer Res 2014; Shimura Y, Mol Cancer Ther 2012; Matsumura-Kimoto Y, Cancer Med 2020). In particular, we found the crucial roles of the constitutive activation of the N-terminal kinase domain (NTKD) of RSK2 in cell survival, resistance to apoptosis, and cell proliferation of myeloma cells and MCL cells. However, the activation status of PDPK1, AKT, and RSK2-NTKD and their impacts on prognosis have yet to be comprehensively investigated in various subtypes of BCLs. To answer this, we here investigated the activation status of PDPK1, AKT, and RSK2-NTKD and their prognostic impacts in two major subtypes of BCLs, diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). To evaluate the activation status of the three kinases in DLBCL and FL, we performed the immunohistochemical analyses to characterize the expression profiles of phosphorylated (p)-PDPK1, p-AKT, and p-RSK2-NTKD in tumor-biopsied samples from 277 pts with DLBCL and 121 patients (pts) with FL diagnosed between 2010 and 2022 at our institution, and the expression levels of three target kinases were classified into four categories; negative (0), weak (1), moderate (2), and strong (3). As a result, in DLBCL, p-PDPK1, p-AKT, and p-RSK2 were positive in 98%, 68%, and 100%, respectively. More precisely, p-PDPK1 was 0-1 in 160 (59%) and 2-3 in 111 (41%). p-AKT was 0-1 in 225 (82%), and 2-3 in 48 (18%). p-RSK2 was 1 in 177 (65%), and 2-3 in 96 (35%). OS was significantly shorter in patients with moderate/strong expression (2-3) of p-RSK2 than those with weak expression (1) of p-RSK2 (5-year OS rate 52% vs. 73%, P<0.01), and, importantly, p-RSK2 status was identified as the independent prognostic factor by the multivariate analysis evaluating p-RSK2 expression and elements included in the International Prognostic Index as variables (HR 1.6, P=0.03). In addition, the prognostic relevance of RSK2 activity was evident in non-GCB type DLBCL but not GCB type DLBCL. In FL, p-PDPK1, p-AKT, and p-RSK2 were positive in 76%, 53%, and 69%, respectively. More precisely, p-PDPK1 status was 0-1 in 94 (89%) and 2-3 in 11 (10%), p-AKT status was 0-1 in 81 (77%) and 2-3 in 24 (23%)), and p-RSK2 was 0-1 in 67 (64%), and 2-3 in 38 (36%). In contrast to the case with DLBCL, the OS of FL was not associated with the activation status of p-PDPK1, p-AKT, and p-RSK2. In conclusion, the present analysis suggests the functional involvement of p-RSK2 activity and the role of future therapeutic targeting, especially in DLBCL, especially in high-risk non-GCB subtype.

Multi-Level Control of Myeloma Cell Proliferation and Genomic Instability By the PDZ Binding Kinase (PBK)

Genomic instability and cell proliferation play important roles in the development and progression of cancer. We identified a six-gene kinase signature that correlated with overall genomic instability in multiple human cancers including multiple myeloma (MM). PDZ Binding Kinase (PBK) was one of the top kinases in this signature. Inhibiting PBK downregulated E2F and FOXM1-related pathways and impacted several members of the DREAM complex, which represses cell cycle genes during quiescence. We further evaluated whether PBK affects entry into mitosis by controlling FOXM1 phosphorylation and affects genome stability. Using co-immunoprecipitation and chromatin immunoprecipitation assays, we observed that PBK binds to and phosphorylates FOXM1, leading to upregulation of FOXM1 target genes. Overexpression of PBK increased the association of LIN54 (a major component of the MuvB complex) with phosphorylated-FOXM1 and inhibited LIN54 interaction with E2F4 in the DREAM complex. Consistently, PBK knockdown or inhibitor increased the LIN54-E2F4 interaction and inhibited the LIN54 and phosphorylated-FOXM1 interaction. The inhibitor also impaired tumor growth in vitro and significantly increased the efficacy of a chemotherapeutic agent ( P = 0.0011) in a subcutaneous mouse model of MM. Overall, these data indicate that PBK phosphorylates FOXM1, then FOXM1 interacts with MuvB which is associated with the start of mitosis, dissociating it from E2F4 in the DREAM complex, thus allowing entry into mitosis and subsequent tumor growth. Given that phosphorylation of FOXM1 also activates the expression of major DNA repair genes (including RAD51, EXO1), we investigated whether PBK affects genome stability. Surprisingly, we observed that PBK also directly phosphorylates DNA repair/HR genes, such as flap structure-specific nuclease 1 (FEN1), and that PBK inhibition reduces spontaneous and chemotherapy (melphalan)-induced genomic instability ( P < 0.05). A significant upregulation of PBK has also been observed in MM patient samples at relapse (after melphalan treatment) relative to paired samples collected at diagnosis (GSE19554). To further investigate the consequences of elevated PBK in vivo, we transformed the normalized PBK expression into a Z-score and used this to divide patients (in MMRF-COMPASS) into three groups based on PBK expression (i.e., high, medium and low PBK expression groups). Patients with high PBK expression had poor overall survival as compared to those with medium and low PBK expression ( P=1.127e−13). DNA replication, mismatch repair, homologous recombination, cell cycle and Fanconi anemia were the top upregulated pathways in patients with high PBK expression. Known drivers of DNA replication, including those involved in the initiation of once-per-cell cycle replication and several DNA repair and HR genes, which are part of different genomic instability signatures (such as RAD51, RAD51C, RAD54B, RAD54L, EXO1, BRCA1, FEN1, ATAD2, CENPA, MAD2L1, TTK), were significantly overexpressed in patients with high PBK expression. Interestingly, FOXM1, which can impact both replication and genome stability was also overexpressed (Log2 Fold change of 1.8; P adj = 1.27E-86). In summary, we demonstrate that PBK drives DNA replication and genomic instability in MM cells by acting at multiple levels. Therefore, PBK inhibitors investigated here have the potential to impair growth and increase the efficacy of chemotherapeutic agents while reducing spontaneous as well as chemotherapeutic agent-induced genomic instability in MM.

Multiple Myeloma Induced IL-6 Reprograms Lipid Metabolism through a CD36 Dependent Mechanism

Multiple Myeloma (MM) requires a high metabolic turnover to allow for expansion and paraprotein production. MM metabolism is complex relying on various metabolic pathways to fuel its survival including fatty acid (FA) oxidation (1, 2). The liver is a master regulator of fatty acid metabolism and distribution. It also stores FA as lipid droplets during steady state or produces ketone bodies during a starvation state (3). This study aimed to determine whether MM alters FA uptake within the liver and by what mechanism this occurs. To investigate the role of FA metabolism we used a mouse model of MM (5TGM1 (GFP) cells into KaLwRij mice). Analysis of serum from 5TGM1 (GFP) engrafted KaLwRij mice showed increased FFA when compared to control animals. RT-qPCR of RNA isolated from liver samples showed a decrease in genes involved in FA uptake and metabolism. These genes included the long chain fatty acid transporter CD36, which belongs to the class B scavenger receptors and is essential for uptake of LCFA in hepatocytes. In vitro modelling showed that primary hepatocytes cultured in conditioned media from 5TGM1 cells had significantly reduced expression of CD36. Furthermore, serum isolated from MM patients (n=6) downregulated CD36 expression in the human hepatocarcinoma cell line, HepG2. These data support the hypothesis that MM induces a factor that alters lipid metabolism in the liver. To detemine the factor that MM secretes, and which regulates FA uptake in the liver, a cytokine array of the serum from 5TGM1 (GFP) engrafted KaLwRij mice was compared to control serum. Pathway analysis revealed interleukin-6 (IL-6) as possible key factor involved in regulating liver FA uptake. In vitro gene expressions analysis of mouse primary hepatocytes revealed that addition of IL-6 to the medium inhibited expression of CD36 and also showed that primary hepatocytes had decreased reliance on FA oxidation when treated with IL-6. Furthermore. 5TGM1 (GFP) engrafted KaLwRij mice showed increased serum IL-6 levels compared to controls. Finally, using the IL-6 neutralising antibody, we observed that this blocked CD36 downregulation in HepG2 cultured with MM patient serum. Here we demonstrate that MM changes FA metabolism in the liver through upregulation of IL-6. Gene expression of FA genes identifies CD36 is down regulated in hepatocytes in response to IL-6. These data suggest that MM is able to redirect FFA away from the liver by down regulating CD36, which mediates FFA uptake in the liver. References Panaroni C, Fulzele K, Mori T, Siu KT, Onyewadume C, Maebius A, et al. Multiple myeloma cells induce lipolysis in adipocytes and uptake fatty acids through fatty acid transporter proteins. Blood. 2022;139(6):876-88.Tirado-Vélez JM, Joumady I, Sáez-Benito A, Cózar-Castellano I, Perdomo G. Inhibition of fatty acid metabolism reduces human myeloma cells proliferation. PLoS One. 2012;7(9):e46484.Hodson L, Gunn PJ. The regulation of hepatic fatty acid synthesis and partitioning: the effect of nutritional state. Nat Rev Endocrinol. 2019;15(12):689-700.

Discovery of small molecule c-Maf inhibitors using molecular docking-based virtual screening, molecular dynamics simulation, and biological evaluation.

Multiple myeloma (MM) is a prevalent plasma cell malignancy in the blood system that remains incurable. Given the abnormally high expression of c-Maf in most MM patients, targeting c-Maf presents an attractive therapeutic approach for treating MM malignancies. In this study, we employed a combined strategy involving molecular docking-based virtual screening, molecular dynamics (MD) simulation, and molecular mechanics/generalized Born surface area (MM/GBSA) free energy calculation on existing FDA-approved drugs. Six compounds were selected for further experimental assay: vemurafenib, sorafenib, sildenafil, fluvastatin, erlotinib, and glimepiride. Among these compounds, sorafenib and glimepiride exhibited significant inhibition of myeloma cell proliferation in the RPMI-8226 cell line. Moreover, both compounds simultaneously downregulated c-Maf protein expression to induce G1 phase arrest and apoptosis in myeloma cells. Collectively, sorafenib and glimepiride may be considered promising candidates for developing more potent c-Maf inhibitors in the future.

MiR-26 inhibits proliferation and promotes apoptosis of multiple myeloma cells by targeting BNIP3.

This study was carried out to investigate the molecular mechanism of microRNA-26 (miR-26) targeting BNIP3 to mediate proliferation and apoptosis of multiple myeloma (MM) cells. The expression of miR-26 and BNIP3 in MM and normal tissues was detected by qRT-PCR and Western blot. According to the average expression of miR-26 and BNIP3, the patients were divided into 12 cases with high miR-26 expression group, 18 cases with low miR-26 expression group, 20 cases with BNIP3 high expression group, and 10 cases with BNIP3 low expression group. The correlation between the expression of miR-26 and BNIP3 and the clinicopathological characteristics of MM patients was compared and analyzed. The effect of up-regulation of miR-26 expression and BNIP3 overexpression on the proliferation of multiple myeloma cells RPMI8226 was examined by MTT assay. Flow cytometry was used to detect the effect of miR-26 expression and BNIP3 overexpression on the apoptosis of RPMI8226 cells. The dual luciferase reporter assay validated the targeted regulation of miR-26 on BNIP3. The expression level of miR-26 in MM tissues was lower than that in normal tissues (P<0.05), and the expression level of BNIP3 in MM tissues was higher than that in normal tissues (P<0.05). miR-26 was closely related to clinical stage, M protein type and light chain type (P<0.05), while BNIP3 was closely related to M protein type and light chain type (P<0.05). After up-regulating miR-26 expression, cell viability was significantly decreased (P<0.05), apoptosis rate was significantly increased (P<0.05) Dual luciferase reporter experiments confirmed that miR-26 could target BNIP3 and negatively regulate the expression of BNIP3 (P<0.05). Overexpression of BNIP3 reversed the effect of up-regulation of miR-26 expression on proliferation and apoptosis of RPMI8226 cells. Up-regulation of miR-26 expression inhibits MM cell proliferation and promotes apoptosis by targeting BNIP3.