Thrombocytopenia is a major complication in a subset of patients with multiple myeloma (MM). However, little is known about its development and significance during MM. Here, we show thrombocytopenia is linked to poor prognosis in MM. In addition, we identify serine, which is released from MM cells into the bone marrow microenvironment, as a key metabolic factor that suppresses megakaryopoiesis and thrombopoiesis. The impact of excessive serine on thrombocytopenia is mainly mediated through the suppression of megakaryocyte (MK) differentiation. Extrinsic serine is transported into MKs through SLC38A1 and downregulates SVIL via SAM-mediated tri-methylation of H3K9, ultimately leading to the impairment of megakaryopoiesis. Inhibition of serine utilization or treatment with TPO enhances megakaryopoiesis and thrombopoiesis and suppresses MM progression. Together, we identify serine as a key metabolic regulator of thrombocytopenia, unveil molecular mechanisms governing MM progression, and provide potential therapeutic strategies for treating MM patients by targeting thrombocytopenia.

Amino acids in the bone marrow microenvironment (BMME) are a critical factor for multiple myeloma (MM) progression. Here, we have determined that proline is elevated in BMME of MM patients and links to poor prognosis in MM. Moreover, exogenous proline regulates MM cell proliferation and drug resistance. Elevated proline in BMME is due to bone collagen degradation and abnormal expression of the key enzyme of proline catabolism, proline dehydrogenase (PRODH). PRODH is downregulated in MM patients, mainly as a result of promoter hypermethylation with high expression of DNMT3b. Thus, overexpression of PRODH suppresses cell proliferation and drug resistance of MM and exhibits therapeutic potential for treatment of MM. Altogether, we identify proline as a key metabolic regulator of MM, unveil PRODH governing MM progression and provide a promising therapeutic strategy for MM treatment.

Metabolites in the tumor microenvironment are a critical factor for tumor progression. However, the lack of knowledge about the metabolic profile in the bone marrow (BM) microenvironment of multiple myeloma (MM) limits our understanding of MM progression. Here, we show that the glycine concentration in the BM microenvironment is elevated due to bone collagen degradation mediated by MM cell-secreted matrix metallopeptidase 13 (MMP13), while the elevated glycine level is linked to MM progression. MM cells utilize the channel protein solute carrier family 6 member 9 (SLC6A9) to absorb extrinsic glycine subsequently involved in the synthesis of glutathione (GSH) and purines. Inhibiting glycine utilization via SLC6A9 knockdown or the treatment with betaine suppresses MM cell proliferation and enhances the effects of bortezomib on MM cells. Together, we identify glycine as a key metabolic regulator of MM, unveil molecular mechanisms governing MM progression, and provide a promising therapeutic strategy for MM treatment.

Purpose Bone marrow stromal cells (BMSCs) have been implicated in multiple myeloma (MM) progression. However, the underlying mechanisms remain largely elusive. Therefore, we aimed to explore key factors in BMSCs that contribute to MM development. Methods RNA-sequencing was used to perform gene expression profiling in BMSCs. Enzyme-linked immunosorbent assays (ELISAs) were performed to determine the concentrations of PGE2 and TNFα in sera and conditioned media (CM). Western blotting, qRT-PCR and IHC were used to examine the expression of cyclooxygenase 2 (COX2) in BMSCs and to analyze the regulation of TNFα by COX2. Cell growth and adhesion assays were employed to explore the function of COX2 in vitro. A 5T33MMvt-KaLwRij mouse model was used to study the effects of COX2 inhibition in vivo. Results COX2 was found to be upregulated in MM patient-derived BMSCs and to play a critical role in BMSC-induced MM cell proliferation and adhesion. Administration of PGE2 to CM derived from BMSCs promoted MM cell proliferation and adhesion. Conversely, inhibition of COX2 in BMSCs greatly compromised BMSC-induced MM cell proliferation and adhesion. PCR array-based analysis of inflammatory cytokines indicated that COX2 upregulates the expression of TNFα. Subsequent rescue assays showed that an anti-TNFα monoclonal antibody could antagonize COX2-mediated MM cell proliferation and adhesion. Administration of NS398, a specific COX2 inhibitor, inhibited in vivo tumor growth and improved the survival of 5TMM mice. Conclusions Our results indicate that COX2 contributes to BMSC-induced MM proliferation and adhesion by increasing the secretion of PGE2 and TNFα. Targeting COX2 in BMSCs may serve as a potential therapeutic approach of treating MM.

NEK2 is associated with drug resistance in multiple cancers. Our previous studies indicated that high NEK2 confers inferior survival in multiple myeloma (MM); thus, a better understanding of the mechanisms by which NEK2 induces drug resistance in MM is required. In this study, we discovered that NEK2 enhances MM cell autophagy, and a combination of autophagy inhibitor chloroquine (CQ) and chemotherapeutic bortezomib (BTZ) significantly prevents NEK2‐induced drug resistance in MM cells. Interestingly, NEK2 was found to bind and stabilize Beclin‐1 protein but did not affect its mRNA expression and phosphorylation. Moreover, autophagy enhanced by NEK2 was significantly prevented by knockdown of Beclin‐1 in MM cells, suggesting that Beclin‐1 mediates NEK2‐induced autophagy. Further studies demonstrated that Beclin‐1 ubiquitination is decreased through NEK2 interaction with USP7. Importantly, knockdown of Beclin‐1 sensitized NEK2‐overexpressing MM cells to BTZ in vitro and in vivo. In conclusion, we identify a novel mechanism whereby autophagy is activated by the complex of NEK2/USP7/Beclin‐1 in MM cells. Targeting the autophagy signaling pathway may provide a promising therapeutic strategy to overcome NEK2‐induced drug resistance in MM.

Nasopharyngeal carcinoma (NPC) has the highest rate of metastasis among head and neck cancers, and distant metastasis is the major reason for treatment failure. We have previously shown that high cyclooxygenase‐2 (COX‐2) expression is associated with a poor prognosis of patients with NPC and inhibits chemotherapy‐induced senescence in NPC cells. In this study, we found that COX‐2 was upregulated in cancer‐associated fibroblasts (CAFs) derived from NPC by RNA‐Seq. Furthermore, elevated COX‐2 expression in CAF was detected in NPC patients with poor survival and distant metastasis by using immunohistochemistry. Then, we identified that COX‐2 is highly expressed in CAF at the distant metastasis site in seven paired NPC patients. High expression of COX‐2 and secretion of prostaglandin E2, a major product catalyzed by COX‐2 in fibroblasts, promotes migration and invasiveness of NPC cells in vitro. On the contrary, inhibition of COX‐2 has the opposite effect in vitro as well as in the COX‐2−/− mouse with the lung metastasis model in vivo. Mechanistically, we discovered that COX‐2 elevates tumor necrosis factor‐α expression in CAF to promote NPC cell migration and invasiveness. Overall, our results identified a novel target in CAF promoting NPC metastasis. Our findings suggested that high expression of COX‐2 in CAF may serve as a new prognostic indicator for NPC metastasis and provide the possibility of targeting CAF for treating advanced NPC.

The serine synthesis pathway (SSP) is active in multiple cancers. Previous study has shown that bortezomib (BTZ) resistance is associated with an increase in the SSP in multiple myeloma (MM) cells; however, the underlying mechanisms of SSP-induced BTZ resistance remain unclear. In this study, we found that phosphoglycerate dehydrogenase (PHGDH), the first rate-limiting enzyme in the SSP, was significantly elevated in CD138+ cells derived from patients with relapsed MM. Moreover, high PHGDH conferred inferior survival in MM. We also found that overexpression of PHDGH in MM cells led to increased cell growth, tumour formation, and resistance to BTZ in vitro and in vivo, while inhibition of PHGDH by short hairpin RNA or NCT-503, a specific inhibitor of PHGDH, inhibited cell growth and BTZ resistance in MM cells. Subsequent mechanistic studies demonstrated PHGDH decreased reactive oxygen species (ROS) through increasing reduced glutathione (GSH) synthesis, thereby promoting cell growth and BTZ resistance in MM cells. Furthermore, adding GSH to PHGDH silenced MM cells reversed S phase arrest and BTZ-induced cell death. These findings support a mechanism in which PHGDH promotes proliferation and BTZ resistance through increasing GSH synthesis in MM cells. Therefore, targeting PHGDH is a promising strategy for MM therapy.

Background: Thrombocytopenia is major complication in a subset of patients with multiple myeloma (MM). However, a lack of detailed studies about the megakaryopoiesis, thrombopoiesis as well as their connections with the survival of patients limits us to explore whether thrombocytopenia could be used as a reliable prognostic factor for MM. Materials and Methods: In this study, 1393 newly diagnosed MM patients were selected for investigating the potential connection between PLT counts and clinical characteristics including ISS stage, overall survival as well as progression free survival. Besides, 5T33MMvt-KaLwRij mouse model were also used to examine the megakaryopoiesis and thrombopoiesis during disease progression. The proportion and function of different subpopulations of cells including megakaryocytes, megakaryocytic-erythroid progenitors (MEPs), common myeloid progenitors (CMPs) and Lin-Sca-1+c-kit+ (LSK) cells were measured both in MM patients and mouse models. Gas chromatography-time-of-flight mass spectrometry (GC-TOFMS)-based metabolomics was used to analyze the metabolites. Results: Of the 1393 studied patients, 298 cases of MM patients are found with thrombocytopenia at the time of diagnosis. PLT counts were lower both in stage Ⅱ (P<0.01) and Ⅲ patients (P<0.001) than stageⅠpatients. Interesting, we found MM patients with thrombocytopenia had a significantly lower OS (P<0.001) and PFS (P<0.001). In mouse model, we also found PLT counts gradually decreased in peripheral blood during the disease progression (P<0.001). Further analysis demonstrated the proportion and the absolute numbers of megakaryocytes and MEPs were diminished both in mouse and MM patients with thrombocytopenia. PLT counts were negative correlated with the percentage of plasma cells or IgG2b levels (P<0.001), suggesting a potential connection of malignant cells infiltration and thrombocytopenia. In mechanism, metabolomics analysis with BM plasma identified 16 differential metabolites in MM patients with thrombopoiesis (VIP > 1.2, P < 0.05). Among them, serine was observed significantly be elevated in MM mouse and suffice to inhibit the megakaryopoieis and thrombopoiesis in vitro. Conclusion: PLT counts might be used as a reliable prognostic factor for MM patients since the thrombocytopenia was associated with poor survival in MM patients. The thrombocytopenia in MM might attribute to, at least partially, the inhibition by serine from the microenvironment. Our findings revealed novel mechanism of MM and might eventually shed light on the treatment of MM patients. Disclosures No relevant conflicts of interest to declare.

Cervical cancer is a common malignant tumour of the female reproductive system that seriously threatens the health of women. The aims of this study were to identify key genes and pathways and to illuminate new molecular mechanisms underlying cervical cancer. Altogether, 1829 DEGs were identified, including 794 significantly down-regulated DEGs and 1035 significantly up-regulated DEGs. GO analysis suggested that the up-regulated DEGs were mainly enriched in mitotic cell cycle processes, including DNA replication, organelle fission, chromosome segregation and cell cycle phase transition, and that the down-regulated DEGs were primarily enriched in development and differentiation processes, such as tissue development, epidermis development, skin development, keratinocyte differentiation, epidermal cell differentiation and epithelial cell differentiation. KEGG pathway analysis showed that the DEGs were significantly enriched in cell cycle, DNA replication, the p53 signalling pathway, pathways in cancer and oocyte meiosis. The top 9 hub genes with a high degree of connectivity (over 72 in the PPI network) were down-regulated TSPO, CCND1, and FOS and up-regulated CDK1, TOP2A, CCNB1, PCNA, BIRC5 and MAD2L1. Module analysis indicated that the top 3 modules were significantly enriched in mitotic cell cycle, DNA replication and regulation of cell cycle (P < 0.01). The heat map based on TCGA database preliminarily demonstrated the expression change of the key genes in cervical cancer. GSEA results were basically coincident with the front enrichment analysis results. By comprehensive analysis, we confirmed that cell cycle was a key biological process and a critical driver in cervical cancer. In conclusion, this study identified DEGs and screened the key genes and pathways closely related to cervical cancer by bioinformatics analysis, simultaneously deepening our understanding of the molecular mechanisms underlying the occurrence and progression of cervical cancer. These results might hold promise for finding potential therapeutic targets of cervical cancer.

Background: Metabolites in tumor microenvironment have been confirmed to contribute to cancer progression. Our previous untargeted metabolomics study has indicated that glycine was significantly increased in bone marrow and peripheral blood derived from Multiple Myeloma(MM) patients compared with health donors(HD). However, the role of glycine in MM progression and its underling mechanisms remain unclear. Materials and Methods: Liquid chromatography-mass spectrometry (LC-MS) was used to detect the concentration of glycine in peripheral blood derived from (25) MM patients and (21) HD. Metabolic flux experiment was performed to explore the distribution of exogenous glycine in MM cell lines ARP1 and 5TGM1. Soft agar colony formation and cell cycle assay were performed to detect MM cells proliferation. 5TGM1 MM mouse models were prepared to examined the effect of glycine on MM in vivo. The unpaired t test was used to evaluate the difference between two different groups. Two-sided Fisher's exact tests were used to assess the associations between glycine abundance and clinical characteristics in MM patients, with a confidence coefficient (confidence interval, CI) of 95%. Results: Targeted metabolic assay of glycine in peripheral blood confirmed that glycine was significantly higher in MM patients than HD(HD vs. MM patients, 14000 vs. 15200, p=0.047). To explore the role of high glycine in MM progression, the associations between glycine abundance and clinical characteristics were investigated. We found that MM patients with high glycine had significantly higher plasma cells percentage(High glycine vs. Low glycine, 11.00% vs. 27.95, p=0.039) and lower hemoglobin concentration(High glycine vs. Low glycine, 96g/l vs. 77g/l, p=0.016). Moreover, high glycine was found to associate with bone damage(p=0.031). Additionally, colony formation and cell cycle assay results showed Glycine-free RPMI 1640 media inhibited MM cells proliferation. Furthermore, 5TMG1 MM mouse fed with glycine-deficiency fodder had slower progression as compared with 5TMG1 MM mouse fed with normal fodder(p=0.0007). These data suggested that exogenous glycine contributes to MM progression. To characterize how exogenous glycine is metabolized in MM cells, MM cell lines ARP1 and 5TGM1 were cultured in the presence of uniformly labeled 13C-glycine for 2, 4, and 6 hours, then the concentration of glycine metabolism related metabolites in conditional media and MM cells were tested by using LC-MS. As a result, 13C-glycine derived GSH was observed in ARP1 as well as 5TGM1, accounting for 37.2% and 52.7% of total GSH after 6 hours of culture, respectively, alternatively, the levels of 13C-GSH in both cell lines were up-regulated with the extension of culture time, indicating that exogenous glycine was involved in GSH synthesis in MM cells. Furthermore, addition of GSH(10 uM) to glycine-free RPMI 1640 media recover the proliferation ability of ARP1 and 5TGM1. Interestingly, betaine, a competitive similar of glycine, was found to suppress MM cell proliferation, and addition of GSH partially counteracted the effect of betaine on MM cells. Conclusion: These findings thus indicate that glycine promotes MM proliferation in vivo and in vitro, and GSH synthesis is the main metabolic pathway contributing to proliferation. Pharmacological blockage of glycine uptake and utilization shows therapeutic potential in MM treatment. Disclosures No relevant conflicts of interest to declare.

Background: Multiple Myeloma(MM) is a neoplastic plasma-cell disorder that is characterized by clonal proliferation of malignant of plasma cells in the bone microenvironment, and monoclonal protein in the blood or urine. Drug resistance is one of the main causes of cancer relapse, thus exploring the molecular mechanisms of drug resistance is important for improving therapeutic effect in MM. Our previous publications have demonstrated NEK2 mediated drug resistance via up-regulation of multidrug resistance proteins. In this study, we found autophagy was involved in NEK2 induced Bortezomib resistance in MM cells. Materials and Methods: Primary CD138 positive cells derived from healthy donors(n=6), newly diagnosed MM patients(n=9) and relapsed MM patients(n=7) were isolated by using beads conjugated with human CD138 antibody. Immunofluorescence was performed to detect the expression of NEK2 and LC3B(LC3B-Ⅰand LC3B-Ⅱ) in primary CD138 positive cells. DALGreen was used to detect autolysosome in MM cells. TAP-MS and CO-IP were used to analyze NEK2 interacting proteins. Cell proliferation were examined with soft agar colony formation and cell count. Cell apoptosis was tested through detecting cleaved Caspase3 and cleaved PARP by western blot. Xenograft mouse model of MM were prepared by subcutaneous injection of MM cells(KMS11 NEK2 OE+Scramble, KMS11 NEK2 OE+Beclin1 sh) into immunodeficient B-NDG mouse(1×106 cell/mice). Results: Firstly, immunofluorescence results showed NEK2 expression and LC3B-Ⅱ labeled autophagosome were significantly increased in CD138 positive cells derived from relapsed MM patients as compared with newly diagnosed MM patients and HD. Moreover, over-expression of NEK2 enhanced antophagy, while knockdown of NEK2 suppressed autophagy in MM cells. To explore the underling mechanisms of NEK2 induces autophagy in MM cells, TAP-MS was performed. As a result, Beclin1, an important regulatory protein of autophagy, was identified as NEK2 interacting protein, which was further confirmed by CO-IP in MM cell lines KMS11 and RPMI 8226. Additionally, we found that NEK2 regulated the expression of Beclin1 at protein level, but not at transcriptional level. Subsequent mechanism study indicated NEK2 increased the stability of Beclin1 protein through USP7 mediated deubiquitination, thereby promoting the formation of the Beclin1-Vps15-Vps34 complex, and finally, enhancing autophagy in MM cells. Furthermore, knockdown of Beclin1 significantly suppressed NEK2 induced autophagy in MM cells. Alternatively, down-regulation of Beclin1 overcame NEK2 mediated Bortezomib resistance in xenograft mouse model of MM , suggesting that targeting Beclin1 is a promising approach to therapy MM patients with high NEK2 expression. Conclusion: Our findings revealed NEK2 induced autophagy through up-regulation of Beclin1 at protein level, and confirmed autophagy was involved in NEK2 mediated Bortezomib resistance in MM. Taken together, this study provided novel insight into treating relapsed MM patients. Disclosures No relevant conflicts of interest to declare.

Abstract E-mail: wenzhou@csu.edu.cn Background: Metabolism reprogramming is one of ten features in cancer. It is well known that metabolites in tumor microenvironment contribute to the survival and proliferation of cancer cells. Currently, a lack of detailed information about the metabolites profiling in bone marrow microenvironment limits us to understand the roles of metabolites associated with multiple myeloma(MM) and its diagnosis and treatment. Here we report a serum untargeted metabolomics study of MM patients, together with healthy donors(HD), with the aim of discovering metabolite markers associated with MM. Materials and Methods: Gas chromatography-time-of-flight mass spectrometry (GC-TOFMS)-based metabolomics was used to analyze 140 serum subjects, including 81 bone marrow subjects(22 HD, 59 MM patients) and 59 peripheral blood subjects(27 HD, 32 MM patients). The bone marrow subjects were divided into training set(11 HD, 32 MM patients) and testing set(11 HD, 27 MM patients). SIMCA-14.1 software package was used to visualize the metabolite alterations between MM patient and HD through Principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). Both the T-test and the receiver operating characteristic curve(ROC) analysis were performed by SPSS software. Metabolites in serum with higher fold change(FC) and variable importance in the projection(VIP) value(VIP > 1.5, P < 0.05 and FC > 1.5, P < 0.05, FDR < 0.05) were considered as biomarker candidates. Results: A total of 117 and 123 metabolites were annotated from the detected spectral features in bone marrow serum subjects derived from training set and testing set, respectively. Based on multivariate statistical analysis(PCA and OPLS-DA) and univariate statistical analysis(T-test), a panel of 6 and 10 metabolites were identified as differential metabolites(VIP > 1.5, P < 0.05 and FC > 1.5, P < 0.05, FDR < 0.05) between MM patients and HD in training set and testing set, respectively, among of which 5 metabolites were found significantly altered in both sets. Creatinine and glycine were significantly elevated in MM patients compared with HD, while fatty acid consists of palmitic acid, petroselinic acid and stearic acida were found decreased in MM patients compared with HD. ROC analysis of these 5 metabolites resulted in an area under the receiver operating characteristic curve (AUC) of 0.922(95% confidence interval=0.748-1) in the training set and 0.923(95% confidence interval=0.853-1) in the testing set. Furthermore, the diagnostic potential of the metabolite signatures was assessed in peripheral blood subjects. Consistent with bone marrow subjects, metabolite signatures were significantly changed(VIP > 1.5, P < 0.05 and FC > 1.5, P < 0.05, FDR < 0.05) in peripheral blood subjects derived from MM patients compared with HD. The AUC of this metabolites signatures was 0.901(95% confidence interval=0.748-1) in peripheral blood subjects, implying that this panel of metabolites could be of potential clinical significance for the diagnosis of MM. Conclusion: We conclude that a panel of 5 metabolites, including creatinine, glycine, palmitic acid, petroselinic acid and stearic acid, in serum has great potential in discriminating MM patient from HD. This metabolite signatures provides a novel and promising molecular diagnostic approach for the detection of MM. Disclosures No relevant conflicts of interest to declare.