Multiple Myeloma Proliferation Research Articles

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

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

MiR-125b targeted regulation of MKNK2 inhibits multiple myeloma proliferation and invasion.

An increasing number of studies demonstrate that abnormal miRNA expression contributes to the advancement of many tumors. Nonetheless, the potential role of miR-125b in multiple myeloma (MM) remains unknown. To explore the potential effects and mechanism of miR-125b in MM. Real-time quantitative PCR was used to measure the expression levels of miR-125b and MKNK2 in a variety of MM samples. Colony formation and cell counting Kit-8 (CCK-8) assays were used to assess cell proliferation, the transwell assay was used to evaluate the cell invasion capability, and dual luciferase reporter gene assay and Western blot were used to examine the interaction between miR-125b and MKNK2. The levels of miR-125b were higher in MM tissue samples, alongside increased expression of MKNK2. There was a negative correlation between MKNK2 and miR-125b expression in MM tissues. MKNK2 was identified as a direct target gene of miR-125b in MM cells. Overexpression of miR-125b suppressed MM cell growth, colony formation, and invasion. In addition, MKNK2 was found to mediate the effects of miR-125b on cell proliferation, colony formation, and invasion in MM. miR-125b acts as a suppressive factor in multiple myeloma and can affect the malignant behavior of MM by regulating the expression of MKNK2.

Discovery and Evaluation of C6-Substituted Pyrazolopyrimidine-Based Bisphosphonate Inhibitors of the Human Geranylgeranyl Pyrophosphate Synthase and Evaluation of Their Antitumor Efficacy in Multiple Myeloma, Pancreatic Ductal Adenocarcinoma, and Colorectal Cancer.

Novel C6-substituted pyrazolo[3,4-d]pyrimidine- and C2-substituted purine-based bisphosphonate (C6-PyraP-BP and C2-Pur-BP, respectively) inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS) were designed and evaluated for their ability to block the proliferation of multiple myeloma (MM), pancreatic ductal adenocarcinoma (PDAC), and colorectal cancer (CRC) cells. Pyrazolo[3,4-d]pyrimidine analogs were identified that induce selective intracellular target engagement leading to apoptosis and downregulate the prenylation of Rap-1A in MM, PDAC, and CRC cells. The C6-PyraP-BP inhibitor RB-07-16 was found to exhibit antitumor efficacy in xenograft mouse models of MM and PDAC, significantly reducing tumor growth without substantially increasing liver enzymes or causing significant histopathologic damage, usually associated with hepatotoxicity. RB-07-16 is a metabolically stable compound in cross-species liver microsomes, does not inhibit key CYP 450 enzymes, and exhibits good systemic circulation in rat. Collectively, the current studies provide encouraging support for further optimization of the pyrazolo[3,4-d]pyrimidine-based GGPPS inhibitors as potential human therapeutics for various cancers.

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.

Identification of small compounds that inhibit multiple myeloma proliferation by targeting c-Maf transcriptional activity

Multiple myeloma displays the clonal B cell expansion and the overproduction of monoclonal immunoglobulins. Genetic translocations at 14q32, particularly with partners like 16q23, lead to the dysregulation of oncogene expression, including the significant enhancement of c-Maf. This aberrant expression of c-Maf has prompted research into strategies for targeting this transcription factor as a potential therapeutic avenue for multiple myeloma treatment. In this study, we introduce a screening pipeline to test small compounds for their ability to inhibit c-Maf. Using a luciferase indicator driven by the Ccl8 gene promoter, we identified two small compounds that inhibit transcriptional activity of c-Maf. These molecules impede the proliferation of c-Maf-expressing myeloma cells, and repress the expression of c-Maf target genes such as ITGB7 and CCR1. Importantly, these molecules target c-Maf-expressing multiple myeloma cells, but not c-Maf-negative myeloma cells, showing potential for tailoring therapeutic intervention. In conclusion, our screening pipeline is effective to explore leads for a novel c-Maf inhibitor for multiple myeloma therapy.

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.

Correction for: CD229 interacts with RASAL3 to activate RAS/ERK pathway in multiple myeloma proliferation.

Correction for: CD229 interacts with RASAL3 to activate RAS/ERK pathway in multiple myeloma proliferation.

Inhibitory Effect of Resveratrol on the Proliferation of Multiple Myeloma Cells and the Underlying Mechanism

To investigate the effect of resveratrol (RSV) on the proliferation of multiple myeloma (MM) cells and its molecular mechanism. MM cells (MM1.S, RPMI-8226 and U266) were treated with different concentrations of RSV for 24-72 h. The effect of RSV on the proliferation of MM cells was detected by CCK-8 (cell counting kit-8) assay. RPMI-8226 cells were divided into RSV, miR-21 mimic, RSV+miR-21 mimic, miR-21 inhibitor and RSV+miR-21 inhibitor groups, and transfected with corresponding plasmids. The cell cycle distribution of each group was detected by flow cytometry with propidium iodide (PI) single staining. The cell apoptosis of each group was detected by AnnexinV-FITC/PE-PI double staining. The expression of miR-21 in MM cells treated with RSV and the expression of KLF5 mRNA in each group were detected by qRT-PCR. The expression of KLF5 protein in each group was detected by Western blot. RSV inhibited the proliferation and induced apoptosis of MM cells in a time- and dose-dependent manner. After the MM cells were treated with RSV, the number of cells in sub-G1 phase was increased, and that in G2/M phase was decreased. Moreover, RSV significantly downregulated the expression of miR-21 in MM cells, and the inhibitory effect of miR-21 mimic on KLF5 expression in MM cells was counteracted by RSV. RSV may inhibit the proliferation and induce apoptosis of MM cells by inhibiting miR-21 and up-regulating KLF5 expression.

Bone Marrow Adipocytes Contribute to Tumor Microenvironment-Driven Chemoresistance via Sequestration of Doxorubicin.

Chemoresistance is a significant problem in the effective treatment of bone metastasis. Adipocytes are a major stromal cell type in the bone marrow and may play a crucial role in developing microenvironment-driven chemoresistance. However, detailed investigation remains challenging due to the anatomical inaccessibility and intrinsic tissue complexity of the bone marrow microenvironment. In this study, we developed 2D and 3D in vitro models of bone marrow adipocytes to examine the mechanisms underlying adipocyte-induced chemoresistance. We first established a protocol for the rapid and robust differentiation of human bone marrow stromal cells (hBMSCs) into mature adipocytes in 2D tissue culture plastic using rosiglitazone (10 μM), a PPARγ agonist. Next, we created a 3D adipocyte culture model by inducing aggregation of hBMSCs and adipogenesis to create adipocyte spheroids in porous hydrogel scaffolds that mimic bone marrow sinusoids. Simulated chemotherapy treatment with doxorubicin (2.5 μM) demonstrated that mature adipocytes sequester doxorubicin in lipid droplets, resulting in reduced cytotoxicity. Lastly, we performed direct coculture of human multiple myeloma cells (MM1.S) with the established 3D adipocyte model in the presence of doxorubicin. This resulted in significantly accelerated multiple myeloma proliferation following doxorubicin treatment. Our findings suggest that the sequestration of hydrophobic chemotherapeutics by mature adipocytes represents a potent mechanism of bone marrow microenvironment-driven chemoresistance.

CUDC-101 as a dual-target inhibitor of EGFR and HDAC enhances the anti-myeloma effects of bortezomib by regulating G2/M cell cycle arrest.

CUDC-101, an effective and multi-target inhibitor of epidermal growth factor receptor (EGFR), histone deacetylase (HDAC), and human epidermal growth factor receptor 2 (HER2), has been reported to inhibit many kinds of cancers, such as acute promyelocytic leukemia and non-Hodgkin's lymphoma. However, no studies have yet investigated whether CUDC-101 is effective against myeloma. Herein, we proved that CUDC-101 effectively inhibits the proliferation of multiple myeloma (MM) cell lines and induces cell apoptosis in a time- and dose-dependent manner. Moreover, CUDC-101 markedly blocked the signaling pathway of EGFR/phosphoinositide-3-kinase (PI3K) and HDAC, and regulated the cell cycle G2/M arrest. Moreover, we revealed through in vivo experiment that CUDC-101 is a potent anti-myeloma drug. Bortezomib is one of the important drugs in MM treatment, and we investigated whether CUDC-101 has a synergistic or additive effect with bortezomib. The results showed that this drug combination had a synergistic anti-myeloma effect by inducing G2/M phase blockade. Collectively, our findings revealed that CUDC-101 could act on its own or in conjunction with bortezomib, which provides insights into exploring new strategies for MM treatment.

Abstract 5018: PROTAC degraders of CDK8/CDK19 Mediator kinases potently suppress multiple myeloma proliferation

Abstract CDK8 and CDK19 are kinase components of the CDK module that comprises CDK8 or CDK19 together with their binding partner cyclin C (CCNC) and two other proteins; the CDK module is associated with transcriptional Mediator complex. CDK8/19 Mediator kinases potentiate transcription induced by different signals, regulate protein levels of Mediator complex components and protect CCNC from proteolytic degradation; the latter activity is exerted in a kinase-independent manner. CDK8/19 kinase inhibitors have entered clinical trials for solid tumors and leukemias. To extend the effects of CDK8/19 inhibition and to suppress their kinase-independent activities, we have developed three series of PROteolysis TArgeting Chimeras (PROTACs) based on different selective inhibitors of CDK8/19 kinases, connected via different linkers to a moiety binding cereblon (CRBN) E3 ligase. The most potent PROTACs degraded both CDK8 and CDK19 in different cell types with DC50s of 10-20 nM. RNA-Seq analysis of 293 cells treated with a CDK8/19 kinase inhibitor or PROTAC showed that the bulk of the PROTAC’s transcriptomic effects matched the effects of the kinase inhibitor. In contrast to the kinase inhibitors, CDK8/19-degrading PROTACs also induced CCNC degradation. Cancer Dependency Map (DepMap) analysis revealed that multiple myelomas (MM), where CRBN is an established therapeutic target, show greater dependency on CCNC than most of the tumor cell lines. We have tested two strongly CCNC-dependent MM cell lines and one CCNC-independent MM line for sensitivity to different CDK8/19 kinase inhibitors and PROTACs. Kinase inhibitors showed low to moderate anti-proliferative activity in all three MM lines, whereas CDK8/19 PROTACs were an order of magnitude more potent than the kinase inhibitors in the two CCNC-dependent MM lines (IC50s of 20-30 nM) but not in the CCNC-independent line. The effects of CDK8/19-degrading PROTACs were stronger than those of the CRBN-targeting drug pomalidomide (approved for MM), a CRBN-binding PROTAC acting on androgen receptor or a CDK8 degradation-inactive PROTAC analog containing CDK8/19- and CRBN-binding moieties. These results suggest the potential of CDK8/CDK19-degrading PROTACs for the treatment of MM. Citation Format: Li Zhang, Jing Li, Charles E. Dowling, Eugenia V. Broude, Igor B. Roninson, Campbell McInnes, Mengqian Chen. PROTAC degraders of CDK8/CDK19 Mediator kinases potently suppress multiple myeloma proliferation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5018.

Development and characterization of three cell culture systems to investigate the relationship between primary bone marrow adipocytes and myeloma cells.

The unique properties of the bone marrow (BM) allow for migration and proliferation of multiple myeloma (MM) cells while also providing the perfect environment for development of quiescent, drug-resistant MM cell clones. BM adipocytes (BMAds) have recently been identified as important contributors to systemic adipokine levels, bone strength, hematopoiesis, and progression of metastatic and primary BM cancers, such as MM. Recent studies in myeloma suggest that BMAds can be reprogrammed by tumor cells to contribute to myeloma-induced bone disease, and, reciprocally, BMAds support MM cells in vitro. Importantly, most data investigating BMAds have been generated using adipocytes generated by differentiating BM-derived mesenchymal stromal cells (BMSCs) into adipocytes in vitro using adipogenic media, due to the extreme technical challenges associated with isolating and culturing primary adipocytes. However, if studies could be performed with primary adipocytes, then they likely will recapitulate in vivo biology better than BMSC-derived adipocytes, as the differentiation process is artificial and differs from in vivo differentiation, and progenitor cell(s) of the primary BMAd (pBMAds) may not be the same as the BMSCs precursors used for adipogenic differentiation in vitro. Therefore, we developed and refined three methods for culturing pBMAds: two-dimensional (2D) coverslips, 2D transwells, and three-dimensional (3D) silk scaffolds, all of which can be cultured alone or with MM cells to investigate bidirectional tumor-host signaling. To develop an in vitro model with a tissue-like structure to mimic the BM microenvironment, we developed the first 3D, tissue engineered model utilizing pBMAds derived from human BM. We found that pBMAds, which are extremely fragile, can be isolated and stably cultured in 2D for 10 days and in 3D for up to 4 week in vitro. To investigate the relationship between pBMAds and myeloma, MM cells can be added to investigate physical relationships through confocal imaging and soluble signaling molecules via mass spectrometry. In summary, we developed three in vitro cell culture systems to study pBMAds and myeloma cells, which could be adapted to investigate many diseases and biological processes involving the BM, including other bone-homing tumor types.

CD229 interacts with RASAL3 to activate RAS/ERK pathway in multiple myeloma proliferation.

Multiple myeloma (MM) is an incurable plasma cell malignancy, while CAR-T therapy offers a new direction for the treatment of MM. Recently, signaling lymphocytic activation molecule family 3 (CD229), a cell surface immune receptor belonging to the signaling lymphocyte activating molecule family (SLAMF), is emerging as a CAR-T therapeutic target in MM. However, a clear role of CD229 in MM remains elusive. In this study, MM patients with elevated CD229 expression achieved poor prognosis by analyzing MM clinical databases. In addition, CD229 promoted MM cell proliferation in vitro as well as in xenograft mouse model in vivo. Mechanism study revealed that CD229 promoted MM cell proliferation by regulating the RAS/ERK signaling pathway. Further exploration employed co-immunoprecipitation coupled with mass spectrometry to identify RASAL3 as an important downstream protein of CD229. Additionally, we developed a co-culture method combined with the immunofluorescence assay to confirm that intercellular tyrosine phosphorylation mediated self-activation of CD229 to activate RAS/ERK signaling pathway via interacting with RASAL3. Taken together, these findings not only demonstrate the oncogenic role of CD229 in MM cell proliferation, but also illustrate the potential of CD229 as a promising therapeutic target for MM treatment.

Investigating the Relationship between Primary Bone Marrow Adipocytes and Myeloma Cells with Three Cell Culture Systems

Introduction & Aims of the Study: The unique properties of the bone marrow allow for migration and proliferation of multiple myeloma (MM) cells, while also providing the ideal environment for development of quiescent, drug-resistant MM cell clones. Recent studies suggest that bone marrow adipocytes (BMAds) can contribute to myeloma-induced bone disease, and reciprocally, that BMAds support MM cells in vitro. Importantly, most data investigating BMAds have been generated using adipocytes differentiated from bone marrow-derived mesenchymal stromal cells (MSCs) in vitro due to the technical challenges associated with isolating and culturing primary adipocytes. To that end, we investigated the MM-BMAd relationship ex vivo utilizing primary adipocytes (pBMAds), which overcome many of the drawbacks associated with using MSC-derived adipocytes. Methods: pBMAds were isolated from the top of human bone marrow biopsies after centrifugation and seeded onto one of three in vitro systems. To develop an in vitro model with a tissue-like structure to mimic the bone marrow microenvironment, we developed the first 3D, tissue engineered model utilizing pBMAds derived from human bone marrow. Following adherence of pBMAds, MM cells were added to investigate bidirectional tumor-host signaling. Viable pBMAds were measured utilizing CellTiterGlo and RealTime Glo (bioluminescence), alone and in culture with MM cells (MM.1Sgfp+luc+ and RPMI-8226). MM.1Sgfp+luc+ cell number was determined by luciferin spike-in and measurement of bioluminescence. To investigate the effects of myeloma cells on pBMAds, we used fluorescence and confocal microscopy to image cell morphology, and mass spectrometry proteomics of conditioned media samples to specifically interrogate soluble signals. Gene expression in both cell types was investigated by qPCR. Results: Utilizing adipocytes isolated from 12 different human donors, we found that pBMAds, which are extremely fragile, can be isolated and stably cultured in 2D for 10 days and in 3D for short-terms (~2 weeks) or long-terms (1 month) in vitro (Figure 1A). We monitored stable pBMAd presence in 2D transwells via Cell TiterGlo and on 3D scaffolds via RealTime Glo up to 9 days post-seeding, and by calcein staining after 10 days in culture. Using bioluminescence in pBMAs (Cell TiterGlo) and MM.1S (luciferin spike-in) we were able to detect viability of both cell types in co-culture. Utilizing mass spectrometry proteomics, in pBMAds alone, we detected secretion of CXCL12 (SDF1; p<0.01), as well as TGFB1 (p<0.01) which has implications for bone remodeling. Interestingly, in conditioned media collected from the co-culture condition, we found increased secretion of the WNT inhibitor DKK3 (p<0.05) and detection of proteins known to be associated with extracellular exosomes including: CNTN1, ACP1, OMD, RPS18, RPL24, PGAM1, LAMA4, CTSB, PLOD2 and ARHGDIA (Figure 1B), suggesting that pBMAds and MM cells likely interact via exosomes. Conclusions: In sum, we developed three in vitro cell culture systems to study primary bone marrow adipocytes and myeloma cells, which could be adapted to investigate many diseases and biological processes involving the bone marrow, including other bone-homing tumor types. Our use of these models to examine the interaction between MM cells and pBMAds has revealed new roles for pBMAds in the MM bone marrow microenvironment, with implications for MM-induced bone disease and pBMAd-MM bidirectional signaling. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Emerging role of LINC00461 in cancer

LINC00461 is located in the intergenic region between the protein-coding genes MEF2C and TMEM161B. LINC00461 upregulation was associated with the risk of 13 tumors and was strongly associated with clinicopathologic features and poor prognosis in 11 tumors. LINC00461 is involved in resistance to four anticancer drugs, including sunitinib for renal cell carcinoma, cisplatin for head and neck squamous cell carcinoma and rectal cancer, temozolomide for glioma, and docetaxel for breast cancer. LINC00461 can sponge 18 miRNAs to form a complex ceRNA network that regulates the expression of a large number of downstream genes. LINC00461 is involved in the MAPK/ERK signaling pathway and PI3K/AKT signaling pathway, thereby promoting tumorigenesis. Notably, knockdown of LINC00461 in exosomes antagonizes tumor cell proliferation in multiple myeloma. This article summarizes the diagnostic, prognostic, and therapeutic value of LINC00461 in various tumors, and systematically describes the ceRNA network and signaling pathways associated with LINC00461, providing potential directions for future LINC00461 research.

Abstract 2956: Superenhancer-drvien SMC4 promote cell growth and proliferation in multiple myeloma

Abstract Background Multiple myeloma (MM) is an incurable malignancy characterized by complex heterogeneous cytogenetic abnormalities. Defined as large clusters of cis-acting enhancers, super-enhancers (SEs) could promote oncogenic transcription to which cancer cells highly addicted. Structural maintenance of chromosome 4 (SMC4) is a core subunit of condensin complexes, which contributes to chromosome condensation and segregation. Here, we identified SMC4 as a novel SE-associated oncogene of myeloma cells, whose functional roles in MM remain largely elusive. Methods We performed H3K27ac ChIP-seq and RNA-seq on primary MM patient samples, MM cell lines, normal CD138+ plasma cell and lymphoma cell lines as the controls, then the ROSE analysis was used to annotate SEs and their associated genes. THZ1 as a transcriptional CDK7 inhibitor was used to further filter THZ1-responsive genes. Combined analysis of THZ1-sensitive and SE-associated gene uncovered a number of promising MM oncogenes as SMC4. The public data mining, RNA interference, CRISPR/Cas9 gene editing system, overexpression and a variety of cellular functional assays were performed to determine the oncogenic effects of SMC4 on MM malignant. Transcriptome analysis with SMC4 silencing and overexpression, and the underlying molecular mechanism of SMC4 oncogenic role in MM are ongoing. Results We have demonstrated SMC4 as one of the SE-associated genes specific to MM patient samples and cell lines. SMC4 was related to myelomagenesis with increased expression from MGUS, SMM to MM cases, and overexpression of SMC4 predicted poor overall survival of MM patients, suggesting its clinical relevance. SMC4 silencing in MM cells suppressed cell proliferation and lead to cell apoptosis. Repression of the SMC4-SE region also led to impairment of cell viability and cell growth, consistent with an oncogenic function. Conclusion In summary, our integrative approached by combing H3H27ac ChIP-seq, RNA-seq and THZ1-sensitive transcripts identified the landscape of SEs and novel oncogenic transcripts as SMC4 in MM. Mapping these acquired SEs and their associated genes may provide novel insight into understanding MM biology, and SMC4 may serve as novel therapeutic targets in MM. Citation Format: Yunlu Jia, Jianbiao Zhou, Tze-King Tan, Tae-Hoon Chung, Yongxia Chen, Enfan Zhang, Zhen Cai, Takaomi Sanda, Wee-Joo Chng. Superenhancer-drvien SMC4 promote cell growth and proliferation in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2956.

Novel microgel culture system as semi-solid three-dimensional in vitro model for the study of multiple myeloma proliferation and drug resistance.

Multiple myeloma (MM) is a hematological malignancy in which the patient's drug resistance is one of the main clinical problems. As 2D cultures do not recapitulate the cellular microenvironment, which has a key role in drug resistance, there is an urgent need for better biomimetic models. Here, a novel 3D platform is used to model MM. The semi-solid culture consists of a dynamic suspension of microspheres and MM cells, termed as microgel. Microspheres are synthesized with acrylic polymers of different sizes, compositions, and functionalities (fibronectin or hyaluronic acid). Optimal conditions for the platform in terms of agitation speed and microsphere size have been determined. With these parameters the system allows good proliferation of the MM cell lines RPMI8226, U226, and MM1.S. Interestingly, when used for drug resistance studies, culture of the three MM cell lines in microgels showed close agreement in revealing the role of acrylic acid in resistance to anti-MM drugs such as dexamethasone and bortezomib. This work presents a unique platform for the in vitro modeling of non-solid tumors since it allows keeping non-adherent cells in suspension conditions but in a 3D context that can be easily tuned with different functionalizations.

Root Bark of Morus alba L. and Its Bioactive Ingredient, Ursolic Acid, Suppress the Proliferation of Multiple Myeloma Cells by Inhibiting Wnt/β-Catenin Pathway.

The root bark of Morus alba L. has cytotoxic activity against several types of cancer cells. However, little is known about its chemopreventive mechanisms and bioactive metabolites. In this study, we showed that M. alba L. root bark extracts (MRBE) suppressed β-catenin response transcription (CRT), which is aberrantly activated in various cancers, by promoting the degradation of β-catenin. In addition, MRBE repressed the expression of the β-catenin/T-cell factor (TCF)-dependent genes, cmyc and cyclin D1, thus inhibiting the proliferation of RPMI-8226 multiple myeloma (MM) cells. MRBE induced apoptosis in MM cells, as evidenced by the increase in the population of annexin VFITC- positive cells and caspase-3/7 activity. We identified ursolic acid in MRBE through LC/mass spectrum (MS) and observed that it also decreased intracellular β-catenin, c-myc, and cyclin D1 levels. Furthermore, it suppressed the proliferation of RPMI-8226 cells by stimulating cell cycle arrest and apoptosis. These findings suggest that MRBE and its active ingredient, ursolic acid, exert antiproliferative activity by promoting the degradation of β-catenin and may have significant chemopreventive potential against MM.

Mitochondria Are Transferred from Non-Malignant Stroma Cells to Malignant Plasma Cells, Via Tunnelling Nanotubules in a CD38 Dependent Mechanism, a Process That Promotes Myeloma Proliferation

BackgroundSurvival and proliferation of multiple myeloma (MM) is dependent on the bone marrow microenvironment where the malignant plasma cells rely on both glycolysis and oxidative phosphorylation to generate ATP. Recent work by our group and others have shown that malignant cells can acquire mitochondria from benign non-malignant cells in the tumor micro-environment (Marlein at al. 2017 Blood). Furthermore others have shown that CD38 controls mitochondrial transfer between astrocytes and neurons after stroke (Hayakawa et al. 2016 Nature). Therefore, since mitochondria are essential for the survival, proliferation and chemotherapy resistance of leukemia together with the knowledge that CD38 controls mitochondrial transfer in brain injury we hypothesised that mitochondria are transferred to MM from BMSC via CD38 dependent mechanism.MethodsBone marrow containing primary MM cells was obtained from patients following informed consent, in accordance with the Declaration of Helsinki and under approval from the United Kingdom National Research Ethics Service. Malignant plasma cells were purified using magnetic-activated cell sorting with CD138+ microbeads. Primary BMSC were also purified from patient bone marrow, using adherence and characterised using flow cytometry for expression of CD90+, CD73+, CD105+ and CD45-. MM-derived cell lines were obtained from the European Collection of Cell Cultures where they are authenticated by DNA fingerprinting. Mitochondrial transfer was assessed using two methods; a MitoTracker Green based staining and an in vivo MM NSG xenograft model. Visualisation of tunnelling nanotubes (TNT) was achieved using fixed cell confocal microscopy, quantification of these was carried out by quantification of TNT-anchor points [1]. CD38 expression on primary MM and cell lines was analysed using flow cytometry and qPCR. To inhibit CD38 we used a blocking antibody and lentivirus targeted shRNA. In vivo MM progression in an NSG MM xenograft model was monitored through bioluminescent live animal imaging over time and by overall survival.ResultsFirst we stained BMSC with MitoTracker Green stain and cultured these with primary MM cells. Using flow cytometry and confocal microscopy we detected MitoTracker fluorescence in the MM cells after co-culture, showing that stained mitochondria are transferred from BMSC to MM cells in-vitro. In a human MM cell line into NSG mouse xenograft model, we detect murine mitochondrial DNA in sorted human MM1S and U266 cells post-transplant. Through fixed cell confocal microscopy we found that mitochondria move through TNTs. Using our BMSC with MitoTracker Green stain MM co-culture assay, we found that CD38 knockdown (Kd) of MM1S cells reduced mitochondrial transfer in-vitro. CD38 Kd improved survival in our NSG xenograft model and moreover blocking CD38 expression reduced mitochondrial transfer between BMSC and MM cells in-vivo. Finally the number of tumor derived TNT-anchor points on BMSC is significantly reduced after co-culture with CD38 knockdown MM cells compared to control knockdown MM cells, highlighting that CD38 is important for TNT formation and binding to BMSC.ConclusionHere we show for the first time that mitochondrial transfer occurs between BMSC and malignant plasma cells which supports the growth and proliferation of MM in-vivo and in-vitro. Moreover we report that the mitochondria move through TNTs and that CD38 expression on MM is functionally important for this pro-tumoral mitochondrial transfer to occur. DisclosuresNo relevant conflicts of interest to declare.

Discovery of novel tripeptide propylene oxide proteasome inhibitors for the treatment of multiple myeloma

The ubiquitin proteasome pathway (UPP) plays a critical role in the maintenance of cell homeostasis and the development of diseases, such as cancer and neurodegenerative disease. A series of novel tripeptide propylene oxide compounds as proteasome inhibitors were designed, synthesized and biologically investigated in this manuscript. The enzymatic activities of final compounds against 20S human proteasome were investigated and structure-activity relationship (SAR) was summarized. Some potent compounds were further evaluated to inhibit the proliferation of multiple myeloma (MM) cancer cell lines RPMI8226 and U266B. The results showed that some compounds were active against MM cancer cell lines with IC50 values of less than 50 nM. The microsomal metabolic stabilities in human, rat and mice species were carried out and the results showed that compounds 30 and 31 were stable enough to be in vivo investigated. The in vivo pharmacokinetic results showed that compounds 30 and 31 had acceptable biological parameters for both ig and iv administrations. In vivo antitumor activities of compounds 30 and 31 with the doses of 100 mg/kg and 50 mg/kg BIW were performed by using RPMI8226 xenograft nude mouse model. Toxicities of compounds 30 and 31 were not observed during the experiment and dose dependent effect was obvious and the tumor volume was greatly inhibited.