Signaling Pathways In Multiple Myeloma Research Articles

Research Progress on the Mechanism of PI3K/AKT/mTOR Signaling Pathway in Multiple Myeloma --Review

PI3K/AKT/mTOR signaling pathway is of great significance in the development and prognosis of tumors, and is closely related to the pathogenesis of multiple myeloma (MM). PI3K/AKT/mTOR signaling pathway can participate in the regulation of MM through multichannel and multitarget, such as regulating the tumor microenvironment of MM cells survival, affecting tumor development and migration, regulating the proliferation, apoptosis and autophagy of MM cells. It have shown that after the PI3K/AKT/mTOR signaling pathway is inhibited, the apoptosis and autophagy of MM cells are activated, which promote the death of MM cells and inhibit the metastasis and recurrence of MM cells. Therefore, indepth study of the mechanism of PI3K/AKT/mTOR signaling pathway in MM is helpful to elucidate the pathogenesis and prognosis of MM.

SSBP1 is a novel prognostic marker and promotes disease progression via p38MAPK signaling pathway in multiple myeloma.

Multiple myeloma (MM) remains an incurable disease. Identification of meaningful co-expressed gene clusters or representative biomarkers of MM may help to identify new pathological mechanisms and promote the development of new therapies. Here, we performed weighted sgene co-expression network analysis and a series of bioinformatics analysis to identify single stranded DNA binding protein 1 (SSBP1) as novel hub gene associated with MM development and prognosis. In vitro, CRISPR/cas9 mediated knockdown of SSBP1 can significantly inhibit the proliferation of MM cells through inducing apoptosis and cell cycle arrest in G0/G1 phase. We also found that decreased SSBP1 expression significantly increased mitochondrial reactive oxygen species (mtROS) generation and the level of phosphorylated p38MAPK. Furthermore, it was further verified that disruption of SSBP1 expression could inhibit the tumor growth via p38MAPK pathway in a human myeloma xenograft model. In summary, our study is the first to demonstrate that SSBP1 promotes MM development by regulating the p38MAPK pathway.

LGR4 promotes tumorigenesis by activating TGF-β1/Smad signaling pathway in multiple myeloma

Multiple myeloma (MM) is a common hematologic malignancy that remains incurable. Although accumulating evidence suggests that the leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) plays a biological function in a variety of cancers, its biological function and molecular mechanisms in MM are unclear. In the present study, we found that LGR4 was significantly upregulated in MM tissues and cells. In vitro and in vivo experiments showed that knockdown of LGR4 significantly inhibited proliferation of MM cells, promoted apoptosis and arrested cell cycle in G1. Overexpression showed the opposite effect. Mechanistic studies revealed that LGR4 could interact with TGF-β1 and regulate TGF-β1 expression, thereby activating the TGF-β1/Smad signaling pathway and promoting MM progression. LGR4 may be a potential new target for MM diagnosis and treatment.

Ruxolitinib Enhances Anti-MM Function of Cytotoxic T-Cells By Upregulating IFNγ and Gzmb

Introduction: Multiple myeloma (MM) tumor cells evade host immunity through the interaction of checkpoint proteins on immune cells which creates an immunosuppressive milieu in the bone marrow (BM) microenvironment. The JAK/STAT signaling pathway in MM plays a pivotal role in mediating the effects of many different molecules, including interleukins, interferons (IFNs), colony-stimulating factors, growth factors, and cytokines. Our previous study demonstrated that the JAK1/2 inhibitor ruxolitinib (RUX) reverses checkpoint inhibition by downregulating PD-L1, PD-L2, CXCL12, CXCR4, MUC1, and CD44 expression in MM cells. We have reported that RUX overcomes resistance of MM patients to lenalidomide and glucocorticosteroids in an ongoing clinical trial. In this study, we further examined IFNγ and granzyme B (GZMB) expression in MM treated with RUX alone or in combination with anti-BCMA/CD3 bispecific antibodies. IFNγ is a key moderator of cell-mediated immunity that enhances anti-tumor effects of CD8 T cells and GZMB induces cancer cell death by cytotoxic T lymphocytes and natural killer cells. Method: Two fresh bone marrow (BM) aspirates from MM patients in complete remission (CR) were collected following informed consent. BM mononuclear cells (MCs) were isolated using density gradient centrifugation with Histopaque-1077 (Sigma, St. Louis) according to a standard protocol. MM tumor cells were isolated from the human myeloma xenograft LAG- λ1 after it was established in SCID mice. LAG- λ1 MM cells were co-cultured with BMMCs and treated with or without RUX alone or in combination with anti-BCMA/CD3 bispecific antibody for 48 hours. Following incubation, supernatant was collected for ELISA, and the cells were washed three times using PBS in preparation for flow cytometric analysis. Supernatants of culture medium from each experimental group were collected, and protein levels determined according to the IFN-gamma (R&D Systems™) and Granzyme B (R&D Systems™) ELISA protocols, without dilution. Each data was the average of duplicate samples. Results: IFNγ and GZMB levels were markedly increased when LAG- λ1 MM cells were co-cultured with BMMCs without any treatment. The BMMCs maintain the normal secretory activities of IFNγ and GZMB. IFNγ and GZMB levels in the culture supernatants were increased in BM treated with RUX alone. The level of IFNγ and GZMB were further increased in BMMCs co-cultured with LAG--λ1 MM cells that were treated with RUX. We further examined BMMCs co-cultured with LAG- λ1 MM cells treated with RUX in combination with anti-BCMA/CD3 bispecific antibody. The results showed that the levels of IFNγ and GZMB in the supernatants was markedly increased. We also determined the amount of CD8+ T-cells in BMMCs co-cultured with LAG--λ1 MM cells treated with or without RUX using flow cytometric analysis. The results showed that CD8+ T-cells were increased when BMMCs co-cultured with LAG- λ1 MM cells treated with 1, 5, and 15 µM. However, CD56+ cells were only slightly increased in BMMCs co-cultured with LAG- λ1 MM cells that were treated with RUX. Summary Immune checkpoint blockade therapy has become a novel therapeutic approach to treat cancer patients. We previously showed RUX reverses checkpoint inhibition by downregulating PD-L1 in MMBM. In this study, we further demonstrated RUX upregulates IFNγ and GZMB and CD8+ T-cells in MMBM. RUX may enhance anti-MM effects of immune-based therapies including anti-BCMA/CD3 bispecific antibody treatment as shown in these preclinical studies.

G6PD promotes cell proliferation and dexamethasone resistance in multiple myeloma via increasing anti-oxidant production and activating Wnt/β-catenin pathway

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) as the rate-limiting enzyme in the pentose phosphate pathway (PPP) is well-established as an aberrantly expressed protein in numerous clinical diseases; however, its role in cancer, specifically in multiple myeloma (MM) remains elusive.MethodsIn this study, serum metabolites in 70 normal people and 70 newly diagnosed MM patients were analyzed using untargeted metabolomics and the results were verified using ELISA. The survival analysis of multiple clinical datasets was performed to identify a potential target gene in MM. The oncogenic role of G6PD was investigated using lentivirus-based overexpression or knockdown of G6PD using RNAi or an inhibitor in vitro, and in a xenograft mouse model in vivo. The mechanisms of induced Dexamethasone (Dexa)-resistance of G6PD were further explored using the above established MM cell lines in vitro.ResultsBased on the screening of potential genes, PPP was shown to be involved in the occurrence of MM, which was evidenced by the differential expression of serum metabolites of G6P and Dehydroepiandrosterone sulfate (DHEAS, the more stable sulfate ester form of an endogenously uncompetitive G6PD inhibitor known as DHEA). Elevated G6PD promoted MM cell proliferation. Mechanistically, high G6PD expression enhanced enzymatic generation of the antioxidant NADPH via the PPP and decreased the production of reactive oxygen species (ROS), thus inducing the proliferation and Dexa resistance in MM cells. Furthermore, canonical Wnt/β-catenin signaling also participated in regulating G6PD-induced drug resistance and cellular redox levels of ROS. Intriguingly, DHEA treatment could enhance the sensitivity of MM cells to Dexa primarily through augmenting cellular oxidative stress.ConclusionsOur data demonstrate that G6PD enhances the generation of the enzymatic anti-oxidant NADPH and decreases ROS generation, thereby promoting resistance to Dexa-induced apoptosis via the enzymatic PPP and non-enzymatic Wnt/β-catenin signaling pathway in MM. Targeting G6PD to harness cellular redox may serve as a promising novel strategy for the management of MM.

NCX1 disturbs calcium homeostasis and promotes RANKL-induced osteoclast differentiation by regulating JNK/c-Fos/NFATc1 signaling pathway in multiple myeloma

Although several types of calcium channels abnormalities have been shown to promote myeloma bone disease (MBD), the relationship between Na+/Ca2+ exchanger 1 (NCX1) and MBD remains unexplored. Here, we examined the role of NCX1 in the development of multiple myeloma (MM), with a special focus on the underlying effects involved osteoclast differentiation. Firstly, we detected NCX1 protein highly expressed in BM tissues of MM patients, and its expression was positively correlated with serum calcium and the percentage of BM CD138+ cells. In vitro, NCX1 suppression with the inhibitor KB-R7943 reduced cell viability of MM cells and caused apoptosis. Extracellular high Ca2+ environment increased the level of intracellular Ca2+ in MM cells through gating the calcium influx, with subsequently promoting the expression of NCX1 and osteoclastogenesis-related genes (receptor activator of nuclear factor-κB (RANKL), nuclear factor of activated T cell cytoplasmic 1 (NFATc1), and proto-oncogene Fos (c-Fos). This phenomenon could be reversed by KB-R7943 or calcium chelation. Furthermore, NCX1 overexpression in MM cells accelerated osteoclastogenesis, while NCX1 knockdown or suppression resulted in the opposite effect. Mechanistically, we further investigated the related mechanisms of NCX1 regulating osteoclast differentiation using RNA sequencing, western blotting and Enzyme linked immunosorbent assay, and found that NCX1 modulated osteoclast differentiation in MM though JNK/c-Fos/NFATc1 signaling pathway. In conclusion, the Ca2+/NCX1-mediated signaling participates in the osteoclasts-myeloma cell interactions, which represents a promising target for future therapeutic intervention in MBD.

Hedgehog signaling pathways in multiple myeloma

Multiple myeloma (MM) is a hematological disease characterized by the uncontrolled proliferation of bone marrow malignant plasma cells. Localization and survival of malignant cells relies on bone marrow niche, in turn determined by the interaction between MM cells and mesenchymal stromal cells (MSCs). Several reports suggest that Hedgehog (Hh) pathway plays an outstanding role … Continued

The role of E3 ubiquitin ligase in multiple myeloma: potential for cereblon E3 ligase modulators in the treatment of relapsed/refractory disease

Introduction Insights into the mechanisms of protein homeostasis and proteasomal degradation have led to new strategies of redirecting the ubiquitin–proteasome system (UPS) to reduce or eliminate proteins or survival factors key to malignant pathobiology, multiple myeloma (MM) in particular. These strategies have enabled researchers to target proteins that were previously considered difficult to modulate by pharmacological means. Areas covered This review provides a brief overview of UPS biology, particularly the role of the CRL4CRBN E3 ubiquitin ligase complex, and summarizes current strategies for co-opting the UPS, including CELMoD compounds, SNIPERs, PROTACs, and degronimids. A detailed discussion is provided on lead CELMoD compounds iberdomide and mezigdomide, which are currently being evaluated in clinical trials in patients with MM. Expert opinion Since a high proportion of patients develop drug resistance, it is vital to have novel therapeutic agents for treating relapsed patients with MM more effectively. It is encouraging that the expanding pathophysiological insight into cellular signaling pathways in MM increasingly translates into the development of novel therapeutic agents such as targeted protein degraders. This holds promise for improving outcomes in MM and beyond.

Multiple Myeloma: Bioinformatic Analysis for Identification of Key Genes and Pathways.

Multiple myeloma (MM) is a hematological malignancy in which monoclonal plasma cells multiply in the bone marrow and monoclonal immunoglobulins are overproduced in older people. Several molecular and cytogenetic advances allow scientists to identify several genetic and chromosomal abnormalities that cause the disease. The comprehension of the pathophysiology of MM requires an understanding of the characteristics of malignant clones and the changes in the bone marrow microenvironment. This study aims to identify the central genes and to determine the key signaling pathways in MM by in silico approaches. A list of 114 differentially expressed genes (DEGs) is important in the prognosis of MM. The DEGs are collected from scientific publications and databases (https://www.ncbi.nlm.nih.gov/). These data are analyzed by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) software (https://string-db.org/) through the construction of protein-protein interaction (PPI) networks and enrichment analysis of the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, by CytoHubba, AutoAnnotate, Bingo Apps plugins in Cytoscape software (https://cytoscape.org/) and by DAVID database (https://david.ncifcrf.gov/). The analysis of the results shows that there are 7 core genes, including TP53; MYC; CDND1; IL6; UBA52; EZH2, and MDM2. These top genes appear to play a role in the promotion and progression of MM. According to functional enrichment analysis, these genes are mainly involved in the following signaling pathways: Epstein-Barr virus infection, microRNA pathway, PI3K-Akt signaling pathway, and p53 signaling pathway. Several crucial genes, including TP53, MYC, CDND1, IL6, UBA52, EZH2, and MDM2, are significantly correlated with MM, which may exert their role in the onset and evolution of MM.

NCX1 Disturbs Calcium Homeostasis, and Promotes RANKL-Induced Osteoclast Differentiation by Regulating JMK/ C-Fos/NFATc1 Signaling Pathway in Multiple Myeloma

NCX1 Disturbs Calcium Homeostasis, and Promotes RANKL-Induced Osteoclast Differentiation by Regulating JMK/ C-Fos/NFATc1 Signaling Pathway in Multiple Myeloma

OAB-031: Circular RNA protein tyrosine kinase 2 promotes cell proliferation, migration and suppresses apoptosis via activating microRNA-638 mediated MEK/ERK, WNT/β-catenin signaling pathways in myeloma

Background Our previous study (Poster in IMW20) observed that circular RNA protein tyrosine kinase 2 (circ-PTK2) was upregulated and correlated with worse clinical features and unfavorable prognosis in multiple myeloma (MM) patients. Thus, this study aimed to further characterize the regulatory function of circ-PTK2 on cell malignant activities and its target microRNA-638 (miR-638) as well as downstream MEK/ERK, WNT/β-catenin signaling pathways in MM. Methods The effect of circ-PTK2 on MM cell proliferation, apoptosis, migration, invasion and its potential target miRNAs was assessed by transfecting circ-PTK2 overexpression plasmids into U226 cells and circ-PTK2 knock-down plasmids into LP-1 cells. Furthermore, the interaction between circ-PTK2 and miR-638 mediated MEK/ERK and WNT/β-catenin signaling pathways was validated by rescue experiments. Circ-PTK2 was overexpressed in most MM cell lines compared to normal plasma cells. Results Overexpressing circ-PTK2 promoted proliferation and migration, inhibited apoptosis in U266 cells, but did not affect cell invasion; knocking down circ-PTK2 achieved opposite effect in LP-1 cells. Besides, circ-PTK2 reversely regulated miR-638 expression but not miR-4690, miR-6724, miR-6749 or miR-6775. The following luciferase reporter assay illustrated the direct bind of circ-PTK2 towards miR-638. In rescue experiments, overexpressing miR-638 suppressed proliferation, migration, while promoted apoptosis in both wild U266 cells and circ-PTK2-overexpressed U266 cells; meanwhile, overexpressing miR-638 also suppressed MEK/ERK and WNT/β-catenin pathways in both wild U266 cells and circ-PTK2-overexpressed U266 cells. Knocking down miR-638 achieved opposite effect in both wild LP-1 cells and circ-PTK2-knocked-down LP-1 cells. Conclusions circ-PTK2 promotes cell proliferation, migration, suppresses cell apoptosis via miR-638 mediated MEK&ERK and WNT&β-catenin signaling pathways in MM. Our previous study (Poster in IMW20) observed that circular RNA protein tyrosine kinase 2 (circ-PTK2) was upregulated and correlated with worse clinical features and unfavorable prognosis in multiple myeloma (MM) patients. Thus, this study aimed to further characterize the regulatory function of circ-PTK2 on cell malignant activities and its target microRNA-638 (miR-638) as well as downstream MEK/ERK, WNT/β-catenin signaling pathways in MM. The effect of circ-PTK2 on MM cell proliferation, apoptosis, migration, invasion and its potential target miRNAs was assessed by transfecting circ-PTK2 overexpression plasmids into U226 cells and circ-PTK2 knock-down plasmids into LP-1 cells. Furthermore, the interaction between circ-PTK2 and miR-638 mediated MEK/ERK and WNT/β-catenin signaling pathways was validated by rescue experiments. Circ-PTK2 was overexpressed in most MM cell lines compared to normal plasma cells. Overexpressing circ-PTK2 promoted proliferation and migration, inhibited apoptosis in U266 cells, but did not affect cell invasion; knocking down circ-PTK2 achieved opposite effect in LP-1 cells. Besides, circ-PTK2 reversely regulated miR-638 expression but not miR-4690, miR-6724, miR-6749 or miR-6775. The following luciferase reporter assay illustrated the direct bind of circ-PTK2 towards miR-638. In rescue experiments, overexpressing miR-638 suppressed proliferation, migration, while promoted apoptosis in both wild U266 cells and circ-PTK2-overexpressed U266 cells; meanwhile, overexpressing miR-638 also suppressed MEK/ERK and WNT/β-catenin pathways in both wild U266 cells and circ-PTK2-overexpressed U266 cells. Knocking down miR-638 achieved opposite effect in both wild LP-1 cells and circ-PTK2-knocked-down LP-1 cells. circ-PTK2 promotes cell proliferation, migration, suppresses cell apoptosis via miR-638 mediated MEK&ERK and WNT&β-catenin signaling pathways in MM.

Circular RNA Protein Tyrosine Kinase 2 Promotes Cell Proliferation, Migration and Suppresses Apoptosis via Activating MicroRNA-638 Mediated MEK/ERK, WNT/β-Catenin Signaling Pathways in Multiple Myeloma.

Our previous study observed that circular RNA protein tyrosine kinase 2 (circ-PTK2) was upregulated and correlated with worse clinical features and unfavorable prognosis in multiple myeloma (MM) patients. Thus, this study aimed to further characterize the regulatory function of circ-PTK2 on cell malignant activities and its target microRNA-638 (miR-638) as well as downstream MEK/ERK, WNT/β-catenin signaling pathways in MM. The effect of circ-PTK2 on MM cell proliferation, apoptosis, migration, invasion and its potential target miRNAs was assessed by transfecting circ-PTK2 overexpression plasmids into U226 cells and circ-PTK2 knock-down plasmids into LP-1 cells. Furthermore, the interaction between circ-PTK2 and miR-638 mediated MEK/ERK and WNT/β-catenin signaling pathways was validated by rescue experiments. Circ-PTK2 was overexpressed in most MM cell lines compared to normal plasma cells. Overexpressing circ-PTK2 promoted proliferation and migration, inhibited apoptosis in U266 cells, but did not affect cell invasion; knocking down circ-PTK2 achieved opposite effect in LP-1 cells. Besides, circ-PTK2 reversely regulated miR-638 expression but not miR-4690, miR-6724, miR-6749 or miR-6775. The following luciferase reporter assay illustrated the direct bind of circ-PTK2 towards miR-638. In rescue experiments, overexpressing miR-638 suppressed proliferation, migration, while promoted apoptosis in both wild U266 cells and circ-PTK2-overexpressed U266 cells; meanwhile, overexpressing miR-638 also suppressed MEK/ERK and WNT/β-catenin pathways in both wild U266 cells and circ-PTK2-overexpressed U266 cells. Knocking down miR-638 achieved opposite effect in both wild LP-1 cells and circ-PTK2-knocked-down LP-1 cells. In conclusion, circ-PTK2 promotes cell proliferation, migration, suppresses cell apoptosis via miR-638 mediated MEK&ERK and WNT&β-catenin signaling pathways in MM.

Bioactive Compounds from Herbal Medicine Targeting Multiple Myeloma

Multiple myeloma (MM) is one of the most widespread hematological cancers. It is characterized by a clonal proliferation of malignant plasma cells in the bone marrow and by the overproduction of monoclonal proteins. In recent years, the survival rate of patients with multiple myeloma has increased significantly due to the use of transplanted stem cells and of the new therapeutic agents that have significantly increased the survival rate, but it still cannot be completely cured and therefore the development of new therapeutic products is needed. Moreover, many patients have various side effects and face the development of drug resistance to current therapies. The purpose of this review is to highlight the bioactive active compounds (flavonoids) and herbal extracts which target dysregulated signaling pathway in MM, assessed by in vitro and in vivo experiments or clinical studies, in order to explore their healing potential targeting multiple myeloma. Mechanistically, they demonstrated the ability to promote cell cycle blockage and apoptosis or autophagy in cancer cells, as well as inhibition of proliferation/migration/tumor progression, inhibition of angiogenesis in the tumor vascular network. Current research provides valuable new information about the ability of flavonoids to enhance the apoptotic effects of antineoplastic drugs, thus providing viable therapeutic options based on combining conventional and non-conventional therapies in MM therapeutic protocols.

Heparanase-induced proliferation and inhibition of apoptosis are associated with the phosphatase and tensin homologue deleted on chromosome 10/focal adhesion kinase signaling pathway in multiple myeloma

Heparanase (HPSE) has an important effect on the proliferation, invasion, metastasis, and drug resistance of tumor cells. HPSE can promote proliferation and inhibit apoptosis of various solid tumor cells. Previous studies regarding the function of HPSE in multiple myeloma (MM) have primarily focused on tumor invasion and metastasis, whereas few studies have examined the proliferation and apoptosis of MM and the mechanisms associated with HPSE. This study recruited patients with MM and isolated MM cells (RPMI8226, LP-1) were isolated to measure the expression levels of HPSE, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), and focal adhesion kinase (FAK) proteins to elucidate their roles in tumor formation. Compared with non-tumor patients, the mRNA and protein expression levels of HPSE and FAK in MM patients increased, whereas the levels of PTEN mRNA and protein decreased. Thus, the increase of HPSE coincided with an increase of FAK and a decrease of PTEN. MM cells exhibiting high HPSE expression exhibited increased proliferation and decreased AS2O3-induced apoptosis. These results indicate that changes in HPSE expression affect the proliferation and apoptosis of MM cells and this mechanism may be associated with the PTEN/FAK signaling pathway. Gene transfection needs proper vector, and proper gene transport system can improve transfection efficiency. In this paper, magnetic nanoparticles were transfected with overexpressed HPSE, and to detect the transfection efficiency and the proliferation ability of MM cells in the control group. The results showed that the cells transfected with magnetic nanoparticles had higher transfection efficiency and higher gene expression level. The results of this experiment provide a new way to explore new cancer therapy genes.

Blockage of the JAK/STAT3 signaling pathway in multiple myeloma by leelamine

BackgroundLeelamine (LEE) is a lipophilic diterpene amine phytochemical, which can be naturally extracted from pine's bark trees. It has been extensively studied recently for its promising chemopreventive and anti-cancer effects against various cancers such as that of prostate and breast. HypothesisWe examined the potential impact of LEE in affecting the activation of signal transducer and activator of transcription 3 (STAT3) and promoting apoptosis in human multiple myeloma (MM) cells. MethodsWe evaluated the effect of LEE on STAT3 signaling pathway in MM cells by using Western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Thereafter, apoptosis was evaluated using cell cycle analysis and Annexin V assay. ResultsWe noted that LEE could attenuate the phosphorylation of STAT3 and other up-stream signaling molecules such as JAK1, JAK2, and Src activation in U266 and MM.1S cells. It also diminished STAT3 translocation into the nucleus and enhanced the expression of protein-tyrosine phosphatase epsilon (PTPε). Additionally, LEE caused cell cycle arrest and synergistically augmented the apoptotic actions of bortezomib against MM cells. ConclusionsOur data indicates that LEE could block STAT3 signaling cascade linked to tumorigenesis and can be used in combination with approved anti-cancer agents in attenuating MM growth and survival.

Characterization of Rare Germline Variants in Familial Multiple Myeloma

Introduction: The risk of developing Multiple Myeloma (MM) is 2-4 fold higher in first-degree relatives of patients with MM compared to the general population, suggesting genetic predisposition to this cancer. Indeed, recent genome-wide association studies have identified common risk alleles that predispose for MM. Yet, the impact of these variants on MM risk is too low to explain familial aggregation of MM. High-impact alleles have been identified for other cancers such as ovarian and breast cancer (BRCA1,-2) and melanoma (CDKN2A) but the search for such alleles in MM is still in its infancy. In order to identify high-impact alleles in MM we have performed whole genome/exon sequencing (WGS/WES) in members of MM high risk families. Methods: We included 21 families with multiple cases of MM/MGUS. Whole genome/exome sequencing was performed on a total of 46 affected and 20 unaffected family members. Filtering and prioritization of the variants were performed in accordance with the criteria of our in-house familial cancer variant prioritization pipeline version 2 (FCVPPv2). Loss-of-function variants were further screened using MutPred-LOF, Translate tool and IntOGen/c-BioPortal in order to discriminate pathogenic and neutral variants, to translate a nucleotide sequence to a protein sequence and to visualize the domain affected by the variant and the portion of the protein lost after the newly formed stop codon. Variants were analyzed for predicted effects on splicing by using Human Splicing Finder. Results: We found a total of 148 potentially pathogenic variants, 109 non-synonymous and 39 LOF, in 18 out of 21 MM families. Among our genes, many affect protein metabolism, immune system, and other have known links to carcinogenesis. Additionally, some of them are known to interact with key signaling pathways in MM, including PI3K/Akt/mTOR, Ras/Raf/MEK/MAPK, JAK/STAT, NF-κB, Wnt/β-catenin, and RANK/RANKL/OPG, showing congruency with previously reported literature. Interestingly, we also found different missense variants in the same two genes in two unrelated families. Conclusions: We have identified potentially pathogenic gene variants in 85% of MM/MGUS families. Our results can offer a useful reference to gene finding efforts by others in order to improve screening, early diagnosis and personalized therapy of individuals at risk of developing MM. Disclosures Durie: Amgen, Celgene, Johnson & Johnson, and Takeda: Consultancy. Goldschmidt:Merck Sharp and Dohme (MSD): Research Funding; Molecular Partners: Research Funding; Incyte: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Johns Hopkins University: Other: Grants and/or provision of Investigational Medicinal Product; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Dietmar-Hopp-Foundation: Other: Grants and/or provision of Investigational Medicinal Product:; Chugai: Honoraria, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany: Current Employment; GlaxoSmithKline (GSK): Honoraria; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product, Research Funding; Novartis: Honoraria, Research Funding; Mundipharma GmbH: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Ex Vivo Drug Sensitivity and Functional Genomics Platform Identifies Novel Combinations Targeting Intrinsic and Extrinsic Apoptotic Signaling Pathways in Multiple Myeloma

Introduction: Despite some long-term remissions, eventual drug resistance in most patients remains a critical obstacle in the treatment of multiple myeloma (MM). The development of new drugs/drug combinations with novel mechanisms of action are needed for continued improvement in patient outcomes. Initiation of tumor cell death via activation of the intrinsic (mitochondrial) and/or extrinsic (death receptor) apoptotic signaling pathways has been shown to be an effective therapeutic strategy in MM. Venetoclax (Ven) is a selective, small-molecule inhibitor of BCL-2 that exhibits clinical activity in MM cells, particularly in patients harboring the t(11;14) translocation. Navitoclax (Nav) is a small-molecule that targets multiple antiapoptotic BCL-2 family proteins, including BCL-XL, BCL-2, and BCL-W to initiate the intrinsic apoptotic pathway. Eftozanermin alfa (Eftoza) is a novel, second generation TRAIL receptor agonist that induces cell death via death receptor pathways and is under investigation in multiple solid and heme malignancies. In addition, the pan-BET inhibitor mivebresib (Miv) and the BDII selective BET inhibitor ABBV-744 have shown synergistic activity with Ven in cell line models of multiple heme malignancies. Results reported here describe ex vivo drug sensitivities and functional genomic analyses of Ven, Nav, Eftoza, Miv, and ABBV-744 alone or in combination with standard-of-care agents, including bortezomib, carfilzomib, panobinostat, daratumumab, or pomalidomide. Methods: A high-throughput ex vivo drug screening assay using a coculture system of bone marrow (BM)-derived MM and stromal cells was used to assess the sensitivity of MM patient tumor cells (Figure 1A). Paired whole exome sequencing (WES) and RNA sequencing (RNA-seq) analyses were performed. Results: Primary MM patient specimens (n=52) were evaluated in the ex vivo platform, including treatment-naïve, early relapse (1-3 prior lines), and late relapse (4-8 prior lines) patients treated with proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies. As expected, t(11;14)-positive MM patient specimens were more sensitive than wildtype to Ven ex vivo (D AUC, -18.6, P=0.002), however MM cells harboring amp(1q) were more resistant than wildtype (D AUC, +5.07, P=0.032), suggesting MCL1 (1q21 gene locus) is a key resistance factor to Ven single-agent activity in MM. Gene set enrichment analysis identified B-cell receptor signaling (normalized enrichment score (NES), 1.96, adjusted P=0.010) and MYC pathway (NES, 1.95, adjusted P=0.010) overexpression as predictors of increased sensitivity to Ven ex vivo. A t(11;14) gene expression signature was also generated using a penalized regression model approach in an additional MMWG/ORIEN MM patient cohort (n=155). The t(11;14) predictive gene expression signature was confirmed by correlation with Ven AUC in the ex vivo model. Additional pathway analyses were performed to identify potential predictive markers of sensitivity/resistance for each single agent and drug combination. Although ex vivo activity of Nav was higher in t(11;14) specimens compared to non-t(11;14) (D AUC, -17.8, P=0.011), ex vivo activity in non-t(11;14) specimens was also observed, indicating additional anti-MM activity by cotargeting of BCL-XL and BCL-2. Both Miv and ABBV-744 showed single-agent activity ex vivo, however Miv demonstrated higher activity (median LD50=88.4nM), suggesting that pan-BET inhibition is more effective than BDII-specific BET inhibition in MM. Finally, a novel drug-combination effect analysis was used that identified novel synergistic ex vivo combinations including Ven and panobinostat (P=0.0013) and Eftoza with bortezomib (P=1.8E-7) or carfilzomib (P=7E-4). Additionally, single-agent induction of macrophage-mediated phagocytosis was observed in both Ven and daratumumab, which was synergistic when the 2 drugs were combined (Figure 1B). Conclusion: An ex vivo functional genomic screen of MM patient specimens demonstrated the usefulness of this approach to identify candidate drugs and potential predictive biomarkers for continued evaluation in clinical trials. This approach confirmed known mechanisms of drug sensitivity and identified new ones, including a novel characterized immune-mediated synergy between Ven and daratumumab, and potential combination strategy for Eftoza and proteasome inhibitors. Figure 1 Disclosures Siqueira Silva: Karyopharm: Research Funding; NIH/NCI: Research Funding; AbbVie: Research Funding. Kulkarni:M2GEN: Current Employment. Mitchell:AbbVie: Other: payment for bioinformatics analysis, Research Funding; M2GEN: Current Employment, Research Funding. Dai:Cygnal Therapeutics: Current Employment; M2GEN: Ended employment in the past 24 months. Hampton:M2GEN: Current Employment. Lu:AbbVie: Current Employment, Current equity holder in publicly-traded company. Modi:AbbVie: Current Employment, Other: may own stock or stock options. Motwani:AbbVie: Current Employment, Current equity holder in publicly-traded company. Harb:AbbVie: Current Employment, Other: may hold stock or stock options. Ross:AbbVie: Current Employment, Current equity holder in publicly-traded company. Shain:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GlaxoSmithKline: Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: Research Funding, Speakers Bureau; AbbVie: Research Funding; Takeda: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Amgen: Speakers Bureau; Adaptive: Consultancy, Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: While this is a preclinical study, venetoclax for treatment of multiple myeloma is not an approved indication

Induction of cell cycle arrest and apoptosis by CPUC002 through stabilization of p53 and suppression of STAT3 signaling pathway in multiple myeloma.

Multiple myeloma has always been an important health problem in human beings due to its high morbidity, high mortality, and lack of effective therapeutic drugs. This study investigated the anticancer effect and mechanism of the newly synthesized small molecule compound CPUC002 on multiple myeloma. Our results confirmed that CPUC002 inhibited proliferation and induced G0/G1 cell cycle arrest in multiple myeloma cells. Moreover, CPUC002 also induced apoptosis by mitochondrial pathway and exogenous pathway. In mechanism, CPUC002 triggered apoptosis by stabilizing p53 in NCI-H929 cells which expressed wt-p53. Knockdown of p53 partially suppressed CPUC002-induced apoptosis. This suggests that there are other molecular mechanisms underlying CPUC002's antitumor effect. Further studies showed that the CPUC002 also inhibited the STAT3 signaling pathway, while knockdown of STAT3 abolished CPUC002-induced apoptosis and cell cycle arrest. In vivo, CPUC002 has significant antitumor activity through the same mechanism as our in vitro studies, and is highly safe in xenograft models. Together these findings indicate that CPUC002 induces apoptosis and G0/G1 cell cycle arrest in multiple myeloma cells by stabilizing p53 and inhibiting the STAT3 signaling pathway.

Preliminary study on the relationship among stem cell markers, drug resistance and PI3K signaling pathway in multiple myeloma (MM) cell.

BackgroundThis study aims to explore the mechanism of drug resistance in multiple myeloma (MM).MethodsIn this study, the possible mechanism of chemotherapeutic tolerance was preliminarily explored from two aspects: (I) the changes in cell morphology, cell cycle, cell apoptosis, stem cell markers and the signaling transduction pathway after the irradiation of RPMI-8226 cells; (II) the mechanism of enhancing chemotherapeutic sensitivity through the PI3K/AKT/mTOR signaling pathway.ResultsThe results showed that the cell morphology and cell cycle of RPMI-8226 had been significantly changed after receiving a radiation dose of 6 Gy in the logarithmic growth phase, the sensitivity of cells to bortezomib had been decreased, the level of stem cell markers had been upregulated, and the PI3K/AKT/mTOR signaling pathway had been activated. However, blocking the PI3K/AKT/mTOR signaling pathway caused the expression of the stem cell markers of RPMI-8226 cells. In addition, the sensitivity of cells to bortezomib had been increased.ConclusionsBlocking the PI3K/AKT/mTOR might decrease the RPMI-8226 cells which survived the radiotherapy and increase the sensitivity of cells to bortezomib.

Targeting the cross-talk between the hedgehog and NF-κB signaling pathways in multiple myeloma

Multiple myeloma (MM) is an incurable plasma cell malignancy. Aberrant activation of the Hedgehog (Hh) and NF-κB signaling pathways is observed in MM and plays a pivotal role in the development of MM by promoting myeloma cell growth, survival, and drug resistance. In this study, we found that the Sonic Hh (SHh) ligand in the bone marrow microenvironment is responsible for the enhancement of NF-κB activity in MM cell lines NCI-H929 and U266. Notably, we discovered that Hh signaling regulates NF-κB through its classical pathway (SHh/PTCH1/SMO/GLI1) in MM cells. Meanwhile, non-classical pathway by SMO recruitment of TRAF6 to ubiquitination is also involved in it. Moreover, the SMO inhibitor cyclopamine enhances the cytotoxic effects of bortezomib in MM cell lines. Our study reveals the cross-talk between Hh members and the NF-κB pathway in the myeloma cells and provides a theoretical basis for combined utilization of Hh members and proteasome inhibition in MM.