Resistant Multiple Myeloma Cell Research Articles

Discovery of novel FGF trap small molecules endowed with anti-myeloma activity

Fibroblast growth factors (FGFs) act as proangiogenic and mitogenic cytokines in several cancers, including multiple myeloma (MM). Indeed, corrupted FGF autocrine and paracrine secretion induces an aberrant activation of the FGF receptor (FGFR) signaling sustaining cancer cell spreading and resistance to pharmacological treatments. Thus, FGF traps may represent a promising anti-cancer strategy to hamper the ligand-dependent activation of the FGF/FGFR system. We previously identified NSC12 as the first orally available small molecule FGF trap able to inhibit the growth and progression of several FGF-dependent tumor models. NSC12 is a pregnenolone derivative carrying a 1,1-bis-trifluoromethyl-1,3-propanediol chain in position 17 of the steroid nucleus. Investigation of structure-activity relationships (SARs) provided more potent and specific NSC12 steroid derivatives and highlighted that the C17-side chain is pivotal for the FGF trap activity. Here, a scaffold hopping approach allowed to obtain two FGF trap compounds (22 and 57) devoid of the steroid nucleus and able to efficiently bind FGF2 and to inhibit FGFR activation in MM cells. Accordingly, these compounds exert a potent anti-tumor activity on MM cell lines both in vitro and in vivo and on MM patient-derived primary cells, strongly affecting the survival of both proteasome-inhibitor sensitive and resistant MM cells. These results propose a new therapeutic option for relapsed/refractory MM patients and set the bases for the development of novel FGF traps prone to chemical diversification to be used in the clinic for the treatment of those tumors in which the FGF/FGFR system plays a pivotal role, including MM.

N6-methyladenosine-modified circ_0000337 sustains bortezomib resistance in multiple myeloma by regulating DNA repair.

Studies have shown that bortezomib resistance in multiple myeloma (MM) is mediated by the abnormalities of various molecules and microenvironments. Exploring these resistance mechanisms will improve the therapeutic efficacy of bortezomib. In this study, bone marrow tissues from three patients with MM, both sensitive and resistant to bortezomib, were collected for circRNA high-throughput sequencing analysis. The relationship between circ_0000337, miR-98-5p, and target gene DNA2 was analyzed by luciferase detection and verified by RT-qPCR. We first found that circ_0000337 was significantly upregulated in bortezomib-resistant MM tissues and cells, and overexpression of circ_0000337 could promote bortezomib resistance in MM cells. circ_0000337 may act as a miR-98-5p sponge to upregulate DNA2 expression, regulate DNA damage repair, and induce bortezomib resistance. Furthermore, it was determined that the increased circ_0000337 level in bortezomib-resistant cells was due to an increased N6-methyladenosine (m6A) level, resulting in enhanced RNA stability. In conclusion, the m6A level of circ_0000337 and its regulation may be a new and potential therapeutic target for overcoming bortezomib resistance in MM.

MUC20 regulated by extrachromosomal circular DNA attenuates proteasome inhibitor resistance of multiple myeloma by modulating cuproptosis

BackgroundProteasome inhibitors (PIs) are one of the most important classes of drugs for the treatment of multiple myeloma (MM). However, almost all patients with MM develop PI resistance, resulting in therapeutic failure. Therefore, the mechanisms underlying PI resistance in MM require further investigation.MethodsWe used several MM cell lines to establish PI-resistant MM cell lines. We performed RNA microarray and EccDNA-seq in MM cell lines and collected human primary MM samples to explore gene profiles. We evaluated the effect of MUC20 on cuproptosis of PI-resistant MM cells using Co-immunoprecipitation (Co-IP), Seahorse bioenergetic profiling and in vivo assay.ResultsThis study revealed that the downregulation of Mucin 20 (MUC20) could predict PI sensitivity and outcomes in MM patients. Besides, MUC20 attenuated PI resistance in MM cells by inducing cuproptosis via the inhibition of cyclin-dependent kinase inhibitor 2 A expression (CDKN2A), which was achieved by hindering MET proto-oncogene, receptor tyrosine kinase (MET) activation. Moreover, MUC20 suppressed MET activation by repressing insulin-like growth factor receptor-1 (IGF-1R) lactylation in PI-resistant MM cells. This study is the first to perform extrachromosomal circular DNA (eccDNA) sequencing for MM, and it revealed that eccDNA induced PI resistance by amplifying kinesin family member 3 C (KIF3C) to reduce MUC20 expression in MM.ConclusionOur findings indicated that MUC20 regulated by eccDNA alleviates PI resistance of MM by modulating cuproptosis, which would provide novel strategies for the treatment of PI-resistant MM.

CRIP1 involves the pathogenesis of multiple myeloma via dual-regulation of proteasome and autophagy

Multiple myeloma (MM) is an incurable hematological malignancy of the plasma cells. The maintenance of protein homeostasis is critical for MM cell survival. Elevated levels of paraproteins in MM cells are cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as a backbone agent and successfully improved the outcome of patients; however, the increasing activity of autophagy suppresses the sensitivity to PIs treatment. The transcription levels of CRIP1 were explored in plasma cells obtained from healthy donors, patients with newly diagnosed multiple myeloma (NDMM), and relapsed/refractory multiple myeloma (RRMM) using Gene expression omnibus datasets. Doxycycline-inducible CRIP1-shRNA and CRIP1 overexpressed MM cell lines were constructed to explore the role of CRIP1 in MM pathogenesis. Proliferation, invasion, migration, proteasome activity and autophagy were examined in MM cells with different CRIP1 levels. Co-immunoprecipitation (Co-IP) with Tandem affinity purification/Mass spectrum (TAP/MS) was performed to identify the binding proteins of CRIP1. The mouse xenograft model was used to determine the role of CRIP1 in the proliferation and drug-resistance of MM cells. High CRIP1 expression was associated with unfavorable clinical outcomes in patients with MM and served as a biomarker for RRMM with shorter overall survival. Invitro and invivo studies showed that CRIP1 plays a critical role in protein homeostasis via the dual regulation of the activities of proteasome and autophagy in MM cells. A combined analysis of RNA-seq, Co-IP and TAP/MS demonstrated that CRIP1 promotes proteasome inhibitors resistance in MM cells by simultaneously binding to de-ubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promoted proteasome activity and autophagosome maturation by facilitating the dequbiquitination and stabilization of PA200. Our findings clarified the pivotal roles of the CRIP1/USP7/PA200 complex in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the pathogenesis of MM. A full list of funding sources can be found in the acknowledgements section.

New Insights for Polyphenolic Compounds as Naturally Inspired Proteasome Inhibitors.

Polyphenols, an important class of natural products, are widely distributed in plant-based foods. These compounds are endowed with several biological activities and exert protective effects in various physiopathological contexts, including cancer. We herein investigated novel potential mechanisms of action of polyphenols, focusing on the proteasome, which has emerged as an attractive therapeutic target in cancers such as multiple myeloma. We carried out a structure-based virtual screening study using the DrugBank database as a repository of FDA-approved polyphenolic molecules. Starting from 86 polyphenolic compounds, based on the theoretical binding affinity and the interactions established with key residues of the chymotrypsin binding site, we selected 2 promising candidates, namely Hesperidin and Diosmin. The further assessment of the biologic activity highlighted, for the first time, the capability of these two molecules to inhibit the β5-proteasome activity and to exert anti-tumor activity against proteasome inhibitor-sensitive or resistant multiple myeloma cell lines.

Exosomal miR-182 derived from bone marrow mesenchymal stem cells drives carfilzomib resistance of multiple myeloma cells by targeting SOX6

BackgroundMultiple myeloma (MM) is a common hematological malignancy. Drug resistance remains to be a major clinical challenge in MM therapy. In this study, we aim to investigate the functional roles of bone marrow mesenchymal stem cells (BMSC)-derived exosomal miR-182 on the carfilzomib resistance of MM and its underlying mechanism.MethodsqRT-PCR and Western blot methods were utilized to confirm the gene or protein expressions. CCK-8 and transwell assays were performed to measure the capabilities of proliferation, migration, and invasion. The molecular interactions were validated through ChIP and Dual luciferase assay.ResultsOur findings indicated that miR-182 expression was upregulated in serum, BMSCs and BMSC-derived exosomes from MM patients. Hypoxia-inducible factor-1α (HIF-1α), a key transcriptional factor in tumor microenvironment, could boost miR-182 expression by directly binding to its promoter, thus favoring exosomal secretion. Moreover, exosomal miR-182 from BMSCs could be transferred to MM cells and was able to promote malignant proliferation, metastasis, and invasion, as well as decrease the sensitivity of MM cells against carfilzomib. Additionally, SOX6 was identified as a downstream target of miR-182 in MM cells, and its expression was negatively regulated by miR-182. Rescue experiments proved that loss of SOX6 in MM cells dramatically reversed the promoting roles of BMSC-secreted exosomal miR-182 on proliferation, metastasis, and carfilzomib resistance in MM cells.ConclusionCollectively, our findings indicated that exosomal miR-182 derived from BMSCs contributed to the metastasis and carfilzomib resistance of MM cells by targeting SOX6. This study sheds light on the pathogenesis of the BMSC-derived exosome containing miR-182 in the malignant behaviors of MM cells and carfzomib resistance.

CNOT4 Knockout Induces Proteasome Inhibitor Resistance in Multiple Myeloma Cells

Background: The outcomes of multiple myeloma (MM) patients have been significantly improved with novel therapies including proteasome inhibitors (PI). However, many patients develop PI resistance and becoming refractory to PI. Although several studies have identified the mechanisms of PI resistance, such as mutation to the proteasome β5 subunit, ER stress suppression, autophagy activation, and activation of efflux drug pumps, it remains unclear yet. To investigate new genes that may contribute to PI resistance, we performed a genome-wide CRISPR dropout screening in an MM cell line. Materials and Methods: To identify the genes responsible for PI resistance, we used a CRISPR library (Kosuke Yusa, V3) targeting 18,740 human genes. The knockout cells, in which an CRISPR library lentivirus was introduced into a Cas9-expressing AMO1 MM cell line, were treated with ixazomib (IXA), carfilzomib (CFZ), and DMSO for two weeks. Next, we used next generation sequencing (NGS) to count sgRNA copy numbers of the pre-drug control group and the drug-treated group. NGS analysis of the treated cells showed that inactivated genes which induced higher levels of cell proliferation in the PI group than in the DMSO group were considered candidate genes for PI resistance. Results: We identified 35 genes for PI resistance including the genes encoding subunits of the proteasome 19S complex, consistent with previous reports that shows reducing 19S subunits protects MM cells from bortezomib. Among 35 candidate genes, we chose the top hit gene, CNOT4, to further investigate. CNOT4 is a component of CCR4-NOT complex which removes the mRNA poly(A) tail for degradation of mRNA. In addition, CNOT4 has E3 ubiquitin ligase activity and is reported to control the proteasome complex formation. To further validate the effect of PI resistance with inactivated CNOT4, we generated CNOT4 KO AMO1 cells and performed an MTS assay. We observed a decrease in PI sensitivity compared with AMO1 wildtype (WT) (IXA IC 50: 24.8 nM (KO) vs 13.0 nM (WT), CFZ IC 50: 7.45 nM (KO) vs 2.76 nM (WT)). To investigate the influence of CNOT4 on proteasome complex formation and its activity, we first measured the expression of ubiquitinated proteins by Western blotting. In CNOT4 KO AMO1 cells, the expression of ubiquitinated proteins is increased with or without PI treatment, suggesting that inactivated CNOT4 can suppress the function of degrade ubiquitinated proteins in proteasome 19S complex. To elucidate the mechanism underlying the resistance effect, we next checked the expression of ER stress marker proteins. In CNOT4 KO AMO1 cells, the expression of PERK and IRE1α proteins are decreased with PI treatment, suggesting that inactivated CNOT4 can suppress the ER stress levels in spite of the increases of ubiquitinated proteins. Conclusion: Our CRISPR screen revealed that CNOT4 inactivation induced PI resistance in human MM cells. CNOT4 inactivation suppresses the function of 19S proteasome, which is reported the influence of PI resistance, and downregulates the ER stress signal leading to avoid PI induced cell death. Our findings demonstrate that CNOT4 plays an important role in PI sensitivity.

CRISPR-Based Gene-Editing Screens Revealed Modulators of Dexamethasone Sensitivity in Multiple Myeloma

Background: Multiple myeloma (MM) is an incurable plasma cell malignancy that accounts for 10% of hematologic malignancies. Despite improved prognosis due to advances in treatment, most patients ultimately develop therapy resistance and relapse. The glucocorticoid dexamethasone is a potent anti-myeloma agent used in combination with proteasome inhibitors and Immunomodulatory drugs (IMIDs) as frontline treatment. Dexamethasone binds to the glucocorticoid receptor (GR), which induces apoptosis by driving an apoptotic transcriptional program. The mechanisms of Dexamethasone resistance in MM remain poorly understood. Aim: To define cellular processes that modulate dexamethasone sensitivity in MM. Methods: We performed loss-of-function CRISPR screens in the t(4;14), NSD2 overexpressing, MM cell lines KMS11 and LP1 using the genome-wide Brunello sgRNA library. Results: The screens identified genes whose loss altered MM cell sensitivity to dexamethasone, most being cell type-specific suggesting heterogeneity in mechanisms influencing dexamethasone cytotoxicity across MM patients. In KMS11 cells, disruption of the IL10/JAK/STAT signaling pathway and glycosaminoglycans/heparan sulfate synthesis pathway increases sensitivity to dexamethasone. By contrast, loss of chromatin remodeling factors including the polycomb group member EPC1 and the SWI/SNF complex components BRD7 and BRD9, altered dexamethasone response exclusively in LP1 cells. Perturbations resulting in dexamethasone resistance in both cell lines include the GR and its co-chaperone PTGES3 in addition to members of the NFKB inhibitor family (NFKBIA, NFKBIB). By contrast, disruption of the druggable GR co-chaperone FKBP5 or the histone deacetylase HDAC8 increased sensitivity to dexamethasone. In addition, the GID/CTLH E3 ubiquitin ligase complex was also identified as a general determinant of dexamethasone resistance in MM cells as disruption of several constituents of this complex resulted in increased dexamethasone sensitivity in both cell lines. The mechanism by which the GID/CTLH complex confers dexamethasone tolerance in MM cells is currently under investigation. Conclusions: Multiple cellular processes contribute to modulating the cytotoxic effect of dexamethasone in MM. Such processes often regulate expression, stability, or localization of the GR. NFKB signaling and the GID/CTLH E3 ubiquitin ligase activity are key determinants of dexamethasone resistance. Targeting these processes can increase the therapeutic effect of dexamethasone in MM.

Good Cop, Bad Cop: Profiling the Immune Landscape in Multiple Myeloma.

Multiple myeloma (MM) is a dyscrasia of plasma cells (PCs) characterized by abnormal immunoglobulin (Ig) production. The disease remains incurable due to a multitude of mutations and structural abnormalities in MM cells, coupled with a favorable microenvironment and immune suppression that eventually contribute to the development of drug resistance. The bone marrow microenvironment (BMME) is composed of a cellular component comprising stromal cells, endothelial cells, osteoclasts, osteoblasts, and immune cells, and a non-cellular component made of the extracellular matrix (ECM) and the liquid milieu, which contains cytokines, growth factors, and chemokines. The bone marrow stromal cells (BMSCs) are involved in the adhesion of MM cells, promote the growth, proliferation, invasion, and drug resistance of MM cells, and are also crucial in angiogenesis and the formation of lytic bone lesions. Classical immunophenotyping in combination with advanced immune profiling using single-cell sequencing technologies has enabled immune cell-specific gene expression analysis in MM to further elucidate the roles of specific immune cell fractions from peripheral blood and bone marrow (BM) in myelomagenesis and progression, immune evasion and exhaustion mechanisms, and development of drug resistance and relapse. The review describes the role of BMME components in MM development and ongoing clinical trials using immunotherapeutic approaches.

Results of an Open-Label Phase 1 Study Repurposing Oral Metformin and Nelfinavir in Combination with Subcutaneous Bortezomib in Patients with Relapsed and/or Refractory Multiple Myeloma

Background: GLUT4 inhibition via the use of a protease inhibitor, such as nelfinavir (NEL), is an attractive therapeutic option in multiple myeloma (MM), given the dependence of MM cells on glucose. Furthermore, combining a GLUT4 inhibitor with an oxidative phosphorylation inhibitor, such as metformin (MET), may counter the possibility of therapeutic resistance to GLUT4 inhibition in MM cells, showing synergistic promise (Dalva-Aydemir et al., Clin Can Res 2015). In addition to GLUT4 inhibition, NEL also lowers the cytotoxicity resistance of MM cells against proteasome inhibitors such as bortezomib (BOR) (Driessen et al., Blood 2018). Thus, this study evaluated the safety and tolerability of repurposing MET and NEL, in combination with BOR for the treatment of relapsed/refractory MM (RRMM) that had progressed on all standard of care (SOC) therapies. Methods: This trial utilized a 3+3 dose escalation design with 3 dose levels planned for up to a maximum of six (21-day) cycles. MET was administered for 14/21 days and started at a dose of 500 mg BID with plans to be investigated at a maximum dose of 1000 mg BID. NEL was administered for 14/21 days and started at a dose of 1250 mg BID with plans to be investigated to a maximum dose of 2500 mg BID. SubQ BOR was administered on days 1, 8, and 15 of a 21-day cycle at a dose of 1.3 mg/m 2 to a portion of patients. Patients with RRMM were required to have had 2 prior lines of therapy and prior exposure to proteasome inhibitors (PIs), immunomodulators (IMiDs), and Anti-CD38 monoclonal antibodies. The primary objective was to determine the maximal tolerated dose (MTD) of this combination. The secondary objective was to evaluate the safety as well as the overall response rate (ORR) of this combination. This trial was funded by the Helen Diller Family Foundation. Results: From 4/2019 until 9/2022, nine patients were accrued with a median age of 65 (range: 42-81) that received a median of 7 prior lines of therapy (Range: 6-13). All but one patient had a prior autologous stem cell transplant. Only one patient was not triple-class refractory, and 2/3 rd (n = 6) also had prior anti-BCMA therapy. The first three patients received only MET (500 mg BID) and NEL (1250 mg BID) at the first dose level for up to one cycle before experiencing progressive disease and coming off the study. Given the limited activity of MET and NEL in the initial treatment of these three patients, two subsequent amendments to the protocol allowed for the upfront addition of BOR 1.3 mg/m2 to the original combination, as well as shortened the DLT evaluation period from two cycles to just one. Thus, the following six patients received MET (500 mg BID) and NEL (1250 mg BID) at the first dose level in addition to weekly BOR at 1.3 mg/m2. Four (44%) patients experienced grade 3+ hematological AEs and six (67%) experienced grade 3+ non-hematological AEs. The most common hematologic grade 3+ AE observed was anemia (44%). The nonhematologic grade 3+ AEs observed were dyspnea, fatigue, nausea, and vomiting (11% each). None of the AEs were possibly related to treatment, suggesting good overall safety of the combination. While eight of nine patients have since died of progressive disease, no deaths were attributable to the treatment. All six patients who received BOR upfront with MET and NEL were previously refractory to BOR in prior lines of therapy; however, none had received BOR as their last line of therapy prior to accrual to this study. Four of these six patients had progressive disease after one cycle of therapy and went off study. However, the remaining two were able to receive 2 or more cycles of therapy, with one experiencing an unconfirmed minor response (MR) in the first cycle and the other experiencing stable disease (SD) for all six cycles. The patient who experienced SD was safely able to transition to a subsequent clinical trial utilizing an anti-BCMA bispecific antibody therapy and remains alive at the time of this analysis. The study was closed before accrual to the next dose level was started. Conclusions: This is the first study to evaluate the safety and efficacy of this repurposed drug combination in this very difficult-to-treat population of RRMM. While no ORR was observed, identification of biomarkers used for selection of patients with no other SOC options who could experience at least SD responses may be beneficial in allowing this combination to slow or stabilize the disease progression until other novel therapies or clinical trials are available.

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.

The selective activator protein-1 inhibitor T-5224 regulates the IRF4/MYC axis and exerts cooperative antimyeloma activity with bortezomib

The activating protein-1 (AP-1) transcription factors (TFs) have been associated with many different cancer types and are promising therapeutic targets in logical malignancies. However, the mechanisms of their role in multiple myeloma (MM) remain elusive. The present study determined and compared the mRNA and protein expression levels of the AP-1 family member JunB in CD138+ mononuclear cells from MM patients and healthy donors. Herein, we investigated the effect of T-5224, an inhibitor of JUN/AP-1, on MM. We found that the cytotoxicity of T-5224 toward myeloma is due to its ability to induce cell apoptosis, inhibit proliferation, and induce cell cycle arrest by increasing the levels of cleaved caspase3/7 and concomitantly inhibiting the IRF4/MYC axis. We also noticed that siJunB-mediated deletion of JunB/AP-1 enhanced MM cell apoptosis and affected cell proliferation. The software PROMO was used in the present study to predict the AP-1 TF that may bind the promoter region of IRF4. We confirmed the correlation between JunB/AP-1 and IRF4. Given that bortezomib (BTZ) facilitates IRF4 degradation in MM cells, we applied combination treatment of BTZ with T-5224. T-5224 and BTZ exerted synergistic effects, and T-5224 reversed the effect of BTZ on CD138+ primary resistance in MM cells, in part due to suppression of the IRF4/MYC axis. Our results suggest that targeting AP-1 TFs is a promising therapeutic strategy for MM. Additionally, targeting both AP-1 and IRF4 with T-5224 may be a synergistic therapeutic strategy for this clinically challenging subset of MM.

SiRNA Lipid-Polymer Nanoparticles Targeting E-Selectin and Cyclophilin A in Bone Marrow for Combination Multiple Myeloma Therapy.

Multiple myeloma (MM) is a hematological bloodcancerof the bone marrow that remains largely incurable, in partdue to itsphysical interactions with the bone marrow microenvironment. Such interactions enhance the homing, proliferation, and drug resistance of MM cells. Specifically, adhesion receptors and homing factors, E-selectin (ES) and cyclophilin A (CyPA), respectively, expressed by bone marrow endothelial cells enhance MM colonization and dissemination. Thus, silencing of ES and CyPA presents a potentialtherapeutic strategy to evade MM spreading. However,small molecule inhibition of ES and CyPA expressed by bone marrow endothelial cells remains challenging, and blocking antibodies induce further MM propagation. Therefore, ES and CyPA are promising candidates forinhibition via RNA interference (RNAi). Here, we utilized a previously developed lipid-polymer nanoparticle for RNAi therapy, that delivers siRNA to the bone marrow perivascular niche. We utilized our platform to co-deliver ES and CyPA siRNAs to prevent MM dissemination in vivo. Lipid-polymer nanoparticles effectively downregulated ESexpression in vitro, which decreased MM cell adhesion and migration through endothelial monolayers. Additionally, in vivo delivery oflipid-polymer nanoparticlesco-encapsulating ES and CyPA siRNA extended survival in a xenograft mouse modelof MM, either alone or in combination with the proteasome inhibitor bortezomib. Our combination siRNA lipid-polymer nanoparticle therapy presents a vascular microenvironment-targeting strategy as a potentialparadigm shift for MM therapies, which could be extended to other cancers that colonize the bone marrow. The online version contains supplementary material available at 10.1007/s12195-023-00774-y.

C-FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma.

The immunomodulatory drug lenalidomide, which is now widely used for the treatment of multiple myeloma (MM), exerts pharmacological action through the ubiquitin-dependent degradation of IKZF1 and subsequent down-regulation of interferon regulatory factor 4 (IRF4), a critical factor for the survival of MM cells. IKZF1 acts principally as a tumour suppressor via transcriptional repression of oncogenes in normal lymphoid lineages. In contrast, IKZF1 activates IRF4 and other oncogenes in MM cells, suggesting the involvement of unknown co-factors in switching the IKZF1 complex from a transcriptional repressor to an activator. The transactivating components of the IKZF1 complex might promote lenalidomide resistance by residing on regulatory regions of the IRF4 gene to maintain its transcription after IKZF1 degradation. To identify unknown components of the IKZF1 complex, we analyzed the genome-wide binding of IKZF1 in MM cells using chromatin immunoprecipitation-sequencing (ChIP-seq) and screened for the co-occupancy of IKZF1 with other DNA-binding factors on the myeloma genome using the ChIP-Atlas platform. We found that c-FOS, a member of the activator protein-1 (AP-1) family, is an integral component of the IKZF1 complex and is primarily responsible for the activator function of the complex in MM cells. The genome-wide screening revealed the co-occupancy of c-FOS with IKZF1 on the regulatory regions of IKZF1-target genes, including IRF4 and SLAMF7, in MM cells but not normal bone marrow progenitors, pre-B cells or mature T-lymphocytes. c-FOS and IKZF1 bound to the same consensus sequence as the IKZF1 complex through direct protein-protein interactions. The complex also includes c-JUN and IKZF3 but not IRF4. Treatment of MM cells with short-hairpin RNA against FOS or a selective AP-1 inhibitor significantly enhanced the anti-MM activity of lenalidomide in vitro and in two murine MM models. Furthermore, an AP-1 inhibitor mitigated the lenalidomide resistance of MM cells. C-FOS determines lenalidomide sensitivity and mediates drug resistance in MM cells as a co-factor of IKZF1 and thus, could be a novel therapeutic target for further improvement of the prognosis of MM patients.

Dual targeting of protein translation and nuclear protein export results in enhanced antimyeloma effects.

Selinexor (KPT-330) is a small molecule inhibitor of XPO1, which mediates the transport of tumor suppressor proteins, oncogene messenger RNAs, and other proteins involved in governing cell growthfrom the cell nucleus to the cytoplasm. It is overexpressed in many cancer types. Because eukaryotic translation initiator factor 4E (eIF4E) plays a critical role in protein translation in cancer cells in multiple myeloma (MM), we evaluated the effectiveness of combined inhibition of protein translation and nuclear export in MM. Selinexor, an inhibitor of nuclear protein export, dose-dependently decreased eIF4E, IKZF1, and c-MYC protein levels. Using a doxycycline-inducible-pLKO-Tet-On vector, knockdown of eIF4E significantly enhanced the antiproliferative effects of selinexor, sensitized resistant MM cells to selinexor, and increased apoptosis in MM cells. Immunofluorescent analysis of MM cells showed that the combined treatment increased the localization of residual eIF4E to the nucleus compared with selinexor-only treatment. The overexpression of eIF4E at least partially rescued the effects of selinexor in MM cells by reducing G1 cell cycle arrest and increasing the selinexor-IC50 10-fold. Moreover, the combination of selinexor with pharmacologic inhibitors of protein translation showed synergistic anti-MM effects. These results suggest a synergistic anti-MM effect of selinexor combined with eIF4E inhibitors invitro. Our work provides a better understanding of the potential mechanism of resistance to selinexor and a rationale for combining selinexor with eIF4E inhibitors for the treatment of MM.

DNP73 PROMOTES DRUG RESISTANCE AND IMMUNE EVASION IN MULTIPLE MYELOMA VIA UPREGULATING MYC AND N‐MYC

Introduction: Multiple myeloma (MM) remains an incurable malignancy of plasma cells. The complex bone marrow microenvironment promotes resistance to both current anti-myeloma agents and emerging immunotherapies. Crosstalk between the NF-κB and p53 can play a pivotal role in various hematologic malignancies. DNp73, an inhibitor of the p53 tumor suppressor family, drives drug resistance and cancer progression in several solid malignancies. However, the biological functions and molecular mechanisms of DNp73 in MM 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. 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 flow cytometry and xenotransplantation model. RNA-seq and CHIP-seq were performed to detect the mechanisms of drug resistance in MM cells. 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 performed phagocytosis assays. Results: Our previous study reported that miR-15a was downregulated in MM cells and correlated with the inferior outcome of MM patients. Further analysis demonstrated that DNp73 was a direct target of p65, loss of miR-15a led to the activation of NF-κB-p65 pathway in MM cells. 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). After DNp73 was down-regulated, cell proliferation and drug resistance were effectively inhibited. Further in vivo study indicated the tumor volume in DNp73 knockdown group was reduced, and increased sensitivity to the carfilzomib, epirubicin and pomalidomide was observed (p < 0.01). Of note, RNA-seq analysis indicated that DNp73 expression was positively correlated with MYCN, MYC and CDK7 transcriptional programs in MM. GSEA analysis showed that MYC targets and DNA damage repair pathways were significantly enriched in DNp73-OE cells. Compared to control group, DNp73-OE cells were resistant to irradiation-induced cell death (p < 0.01). Meanwhile, DNp73 knockdown significantly reduced the migration and invasion of cells (p < 0.01). CHIP-seq data showed that DNp73 could bind to the promoter region of MYCN. DNp73 overexpression protects MM cells from phagocytosis, treated with anti-human CD47 antibody increased phagocytosis of macrophages (p < 0.01). Conclusions: We demonstrated that miR-15a downregulation activated NF-κB, which promoted DNp73 expression at transcription level. DNp73 promotes drug resistance and immune escape in multiple myeloma by modulating Myc and N-Myc signal pathway. The research was funded by: the CAMS Innovation Fund for Medical Sciences (2022-I2M-1-022), the Natural Science Foundation of China (82170194), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2018RC320012), the Natural Science Foundation of China (81920108006), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2018PT31006) Keywords: multiple myeloma, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

Communication between bone marrow mesenchymal stem cells and multiple myeloma cells: Impact on disease progression

Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of immunoglobulin-secreting clonal plasma cells at the bone marrow (BM). The interaction between MM cells and the BM microenvironment, and specifically BM mesenchymal stem cells (BM-MSCs), has a key role in the pathophysiology of this disease. Multiple data support the idea that BM-MSCs not only enhance the proliferation and survival of MM cells but are also involved in the resistance of MM cells to certain drugs, aiding the progression of this hematological tumor. The relation of MM cells with the resident BM-MSCs is a two-way interaction. MM modulate the behavior of BM-MSCs altering their expression profile, proliferation rate, osteogenic potential, and expression of senescence markers. In turn, modified BM-MSCs can produce a set of cytokines that would modulate the BM microenvironment to favor disease progression. The interaction between MM cells and BM-MSCs can be mediated by the secretion of a variety of soluble factors and extracellular vesicles carrying microRNAs, long non-coding RNAs or other molecules. However, the communication between these two types of cells could also involve a direct physical interaction through adhesion molecules or tunneling nanotubes. Thus, understanding the way this communication works and developing strategies to interfere in the process, would preclude the expansion of the MM cells and might offer alternative treatments for this incurable disease.

A novel alkaloid compound, DCZ0358, exerts significant antitumor activity in bortezomib-resistant multiple myeloma cells through inhibition of JAK2/STAT3 pathway.

Multiple myeloma (MM), the second most common haematological malignancy, is currently incurable because patients often develop multiple drug resistance and experience subsequent relapse of the disease. This study aims to identify a potential therapeutic agent that can counter bortezomib (BTZ) resistance in MM. DCZ0358, a novel alkaloid compound, is found to exert potent cytotoxic effects against BTZ-resistant MM cells in vivo and in vitro. The anti-myeloma activity of DCZ0358 is associated with inhibition of cell proliferation, promotion of cell apoptosis via caspase-mediated apoptotic pathways, and induction of G0/G1 phase arrest via downregulation of cyclin D1, CDK4, and CDK6. Further investigation of the molecular mechanism shows that DCZ0358 suppresses the JAK2/STAT3 signaling pathway. In conclusion, DCZ0358 can successfully counter BTZ resistance in MM cells. This study provides evidence that warrants future preclinical assessments of DCZ0358 as a therapeutic agent against BTZ resistance in MM.

Bone Marrow Adipocytes Promote the Survival of Multiple Myeloma Cells and Up-Regulate Their Chemoresistance

To investigate the effect of adipocytes in the bone marrow microenvironment of patients with multiple myeloma (MM) on the pathogenesis of MM. Bone marrow adipocytes (BMA) in bone marrow smears of health donors (HD) and newly diagnosed MM (ND-MM) patients were evaluated with oil red O staining. The mesenchymal stem cells (MSC) from HD and ND-MM patients were isolated, and in vitro co-culture assay was used to explore the effects of MM cells on the adipogenic differentiation of MSC and the role of BMA in the survival and drug resistance of MM cells. The expression of adipogenic/osteogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4, FASN and ALP both in MSC and MSC-derived adipocytes was determined with real-time quantitative PCR. The Western blot was employed to detect the expression levels of IL-6, IL-10, SDF-1α, TNF-α and IGF-1 in the supernatant with or without PPAR-γ inhibitor. The results of oil red O staining of bone marrow smears showed that BMA increased significantly in patients of ND-MM compared with the normal control group, and the BMA content was related to the disease status. The content of BMA decreased in the patients with effective chemotherapy. MM cells up-regulated the expression of MSC adipogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4 and FASN, but the expression of osteogenic differentiation-related gene ALP was significantly down-regulated. This means that the direct consequence of the interaction between MM cells and MSC in the bone marrow microenvironment is to promote the differentiation of MSC into adipocytes at the expense of osteoblasts, and the cytokines detected in supernatant changed. PPAR-γ inhibitor G3335 could partially reverse the release of cytokines by BMA. Those results confirmed that BMA regulated the release of cytokines via PPAR-γ signal, and PPAR-γ inhibitor G3335 could distort PPAR-γ mediated BMA maturation and cytokines release. The increased BMA and related cytokines effectively promoted the proliferation, migration and drug resistance of MM cells. The BMA and its associated cytokines are the promoting factors in the survival, proliferation and migration of MM cells. BMA can protect MM cells from drug-induced apoptosis and plays an important role in MM treatment failure and disease progression.

Ectopic CH60 mediates HAPLN1-induced cell survival signaling in multiple myeloma.

Multiple myeloma (MM), the second most common hematological malignancy, is generally considered incurable because of the development of drug resistance. We previously reported that hyaluronan and proteoglycan link protein 1 (HAPLN1) produced by stromal cells induces activation of NF-κB, a tumor-supportive transcription factor, and promotes drug resistance in MM cells. However, the identity of the cell surface receptor that detects HAPLN1 and thereby engenders pro-tumorigenic signaling in MM cells remains unknown. Here, we performed an unbiased cell surface biotinylation assay and identified chaperonin 60 (CH60) as the direct binding partner of HAPLN1 on MM cells. Cell surface CH60 specifically interacted with TLR4 to evoke HAPLN1-induced NF-κB signaling, transcription of anti-apoptotic genes, and drug resistance in MM cells. Collectively, our findings identify a cell surface CH60-TLR4 complex as a HAPLN1 receptor and a potential molecular target to overcome drug resistance in MM cells.