Genomic, epigenomic, and transcriptomic signatures for telomerase complex components: a pan-cancer analysis.

Telomerase activation is required for malignant transformation. Recent advances in high-throughput technologies have enabled the generation of complex datasets, thus providing alternative approaches to exploring telomerase biology more comprehensively, which has proven to be challenging due to the need for laborious assays required to test for telomerase activity. To solve these issues, several groups have analyzed TCGA pan-cancer tumor datasets by investigating telomerase reverse transcriptase (TERT), the catalytic subunit for telomerase activity, or its surrogates. However, telomerase is a multiunit complex containing not only TERT, but also numerus cofactors required for telomerase function. Here we determined genomic and molecular alterations of 10 well-characterized telomerase components in the TCGA and CCLE datasets. We calculated a telomerase score (TS) based on their expression profiles and clustered tumors into low, high, and intermediate subtypes. To validate the in silico analysis result, we used immunoblotting and telomerase assays. High TS subtypes were significantly associated with stemness, proliferation, epithelial to mesenchymal transition, hyperactivation of oncogenic signaling pathways, shorter patient survival, and infiltration of dysfunctional T-cells or poor response to immunotherapy. Copy number alterations in 10 telomerase components were widespread and associated with the level of their expression. Surprisingly, primary tumors and cancer cell lines frequently displayed a homozygous deletion of the TCAB1 gene, encoding a telomerase protein essential for telomerase trafficking, assembling, and function, as previously reported. However, tumors or cells carrying a TCAB1 deletion still exhibited telomerase activity comparable to or even higher than their wildtype counterparts. Collectively, applying telomerase component-based TS in complex datasets provided a robust tool for telomerase analyses. Our findings also reveal a tight connection between telomerase and other oncogenic signaling pathways; TCAB1 may acts as a dispensable telomerase component. Moreover, TS may serve as a useful biomarker to predict patient outcomes and response to immunotherapy.

GABPA-activated TGFBR2 transcription inhibits aggressiveness but is epigenetically erased by oncometabolites in renal cell carcinoma.

BACKGROUND: The ETS transcription factor GABPA has long been thought of as an oncogenic factor and recently suggested as a target for cancer therapy due to its critical effect on telomerase activation, but the role of GABPA in clear cell renal cell carcinoma (ccRCC) is unclear. In addition, ccRCC is characterized by metabolic reprograming with aberrant accumulation of L-2-hydroxyglurate (L-2HG), an oncometabolite that has been shown to promote ccRCC development and progression by inducing DNA methylation, however, its downstream effectors remain poorly defined. METHODS: siRNAs and expression vectors were used to manipulate the expression of GABPA and other factors and to determine cellular/molecular and phenotypic alterations. RNA sequencing and ChIP assays were performed to identify GABPA target genes. A human ccRCC xenograft model in mice was used to evaluate the effect of GABPA overexpression on in vivo tumorigenesis and metastasis. ccRCC cells were incubated with L-2-HG to analyze GABPA expression and methylation. We carried out immunohistochemistry on patient specimens and TCGA dataset analyses to assess the effect of GABPA on ccRCC survival. RESULTS: GABPA depletion, although inhibiting telomerase expression, robustly enhanced proliferation, invasion and stemness of ccRCC cells, whereas GABPA overexpression exhibited opposite effects, strongly inhibiting in vivo metastasis and carcinogenesis. TGFBR2 was identified as the GABPA target gene through which GABPA governed the TGFß signaling to dictate ccRCC phenotypes. GABPA and TGFBR2 phenocopies each other in ccRCC cells. Higher GABPA or TGFBR2 expression predicted longer survival in patients with ccRCC. Incubation of ccRCC cells with L-2-HG mimics GABPA-knockdown-mediated phenotypic alterations. L-2-HG silenced the expression of GABPA in ccRCC cells by increasing its methylation. CONCLUSIONS: GABPA acts as a tumor suppressor by stimulating TGFBR2 expression and TGFß signaling, while L-2-HG epigenetically inhibits GABPA expression, disrupting the GABPA-TGFß loop to drive ccRCC aggressiveness. These results exemplify how oncometabolites erase tumor suppressive function for cancer development/progression. Restoring GABPA expression using DNA methylation inhibitors or other approaches, rather than targeting it, may be a novel strategy for ccRCC therapy.

Downregulation and Hypermethylation of GABPB1 Is Associated with Aggressive Thyroid Cancer Features.

Promoter mutations of the telomerase reverse transcriptase (TERT) gene occur frequently in thyroid carcinoma (TC), including papillary (PTC) and anaplastic subtypes (ATC). Given that the ETS family transcription factors GABPA and GABPB1 activate the mutant TERT promoter and induce TERT expression for telomerase activation, GABPB1 has been proposed as a cancer therapeutic target to inhibit telomerase. Here, we sought to determine the role of GABPB1 in TC pathogenesis. In TC-derived cells carrying the mutated TERT promoter, GABPB1 knockdown led to diminished TERT expression but significantly increased invasive potentials in vitro and metastatic potential in a xenograft zebrafish model and altered expression of markers for epithelial-to-mesenchymal transition. GABPB1 expression was downregulated in aggressive TCs. Low GABPB1 expression correlated with its promoter hypermethylation, which in turn was also associated with shorter disease-free survival. Consistently, DNA methylation inhibitors enhanced GABPB1 expression, as observed upon reduced promoter methylation. Our results suggest that GABPB1 is required for TERT expression and telomerase activation, but itself serves as a tumor suppressor to inhibit TC progression. Furthermore, aberrant DNA methylation leads to GABPB1 silencing, thereby promoting TC aggressiveness. Thus, caution is needed if targeting GABPB1 for cancer therapy is considered.

Regulatory region mutations of TERT, PLEKHS1 and GPR126 genes as urinary biomarkers in upper tract urothelial carcinomas.

Background: The hotspot regulatory region mutations of the TERT, PLEKHS1 and GPR126 genes have been shown to occur frequently in urothelial bladder carcinoma (UBC). However, it is currently unclear whether these mutations are all present in upper tract urothelial carcinomas (UTUC) including renal pelvic carcinoma (RPC) and ureter carcinoma (UC), although TERT promoter mutations were previously observed in these malignancies. Methods: The hotspot mutations of TERT and PLEKHS1 promoters and GPR126 intron 6 (enhancer) in tumors derived from 164 patients with UTUC were determined using Sanger sequencing, and the obtained results were further compared with the mutation frequency in 106 UBCs. The mutations were also assessed in urine from patients with UTUC and UBC. Results: The mutation frequencies in UTUC tumors were 28%, 5.8% and 11% for TERT and PLEKHS1 promoters and GPR126 intron 6, respectively, which were lower than those (44.3%, 26.4%, and 31.4%, respectively) in UBCs. The total frequencies for the presence of any of these mutations were 50.8% and 34.4% for RPCs and UCs, respectively. All these mutated DNA sequences were detectable in urine from both UTUC and UBC patients and disappeared rapidly in most patients after surgery. Conclusions: This proof-of-concept study demonstrates that the hotspot mutations in the TERT, PLEKHS1 and GPR126 non-coding regions are present in UTUCs, and that urinary assays of these mutated sequences serve as potential biomarkers for UTUC diagnostics and disease monitoring.

PLEKHS1 Over-Expression is Associated with Metastases and Poor Outcomes in Papillary Thyroid Carcinoma.

Pleckstrin homology domain containing S1 (PLEKHS1) is a poorly characterized factor, although its promoter mutations were identified in human malignancies including thyroid carcinoma (TC). This study was designed to determine PLEKHS1 promoter hotspot mutations in papillary and anaplastic thyroid carcinomas (PTCs and ATCs) and to evaluate if PLEKHS1 expression influences clinical outcome. The PLEKHS1 promoter mutation was observed in 1/93 of PTCs and none of 18 ATCs in our cohort; however, PLEKHS1 expression was aberrantly up-regulated in TCs compared to adjacent non-tumorous thyroid tissues. ATC tumors, an undifferentiated TC, exhibited the highest PLEKHS1 expression. In both TCGA and present cohorts of PTCs, PLEKHS1 gene methylation density was inversely correlated with its mRNA expression and demethylation at the PLEKHS1 locus occurred at two CpGs. Higher PLEKHS1 expression was associated with lymph node and distant metastases, and shorter overall and disease-free survival in our cohort of PTC patients. Importantly, PLEKHS1 over-expression predicted shorter patient survival in PTCs lacking TERT promoter mutations. Cellular experiments showed that PLEKHS1 over-expression enhanced AKT phosphorylation and invasiveness. Collectively, the PLEKHS1 gene demethylation causes its over-expression in PTCs. PLEKHS1 promotes aggressive behavior of TCs possibly by increasing AKT activity, and its over-expression predicts poor patient outcomes.

JAK2 inhibition in JAK2V617F-bearing leukemia cells enriches CD34+ leukemic stem cells that are abolished by the telomerase inhibitor GRN163L.

The activating-mutation of JAK2V617F drives the development of myeloproliferative neoplasms (MPNs). Several JAK2 inhibitors such as ruxolitinib and gandotinib (LY2784544) currently in clinical trials and, provide improvements in MPNs including myelofibrosis. However, JAK2 inhibitors are non-curative and murine experiments show that JAK2 inhibitors don't eradicate MPN stem cells and it is currently unclear how they escape. We thus determined the effect of the specific JAK2V617F inhibitor LY2784544 on leukemic stem (CD34+) cells (LSCs) using the JAK2V617F-bearing erythroleukemia cell line HEL. The LY2784544 treatment caused a transient proliferation inhibition and apoptosis of HEL cells, but a recovery occurred within a week. Thereafter, the continuous LY2784544 exposure induced the accumulation of CD34+ LSCs, and the CD34+ cells increased from 2% to >90% by week 9, which was accompanied by increased clonogenic potentials. LY2784544 was capable of stimulating CD34 expression even in CD34- HEL cells, which indicated cellular de-differentiation. A significantly enhanced expression of the stem cell factor KLF4 was observed in LY2784544-treated HEL cells. Inhibiting KLF4 expression attenuated LY2784544-mediated accumulation of CD34+ LSCs. Moreover, the telomerase inhibitor GRN163L abolished the LY2784544-effect. JAK2 inhibitors thus cause enrichment of LSCs and are unlikely to cure MPN as a monotherapy. Simultaneously targeting JAK2V617F and KLF4 or telomerase may be a novel strategy for MPN therapy, which should be of significance both biologically and clinically.

Disulfiram/copper markedly induced myeloma cell apoptosis through activation of JNK and intrinsic and extrinsic apoptosis pathways.

Disulfiram (DSF) is an FDA approved anti-alcoholism drug in use for more than 60 years. Recently, antitumor activity of the DSF/copper (DSF/Cu) complex has been identified. Its anti-multiple myeloma activity, however, has barely been investigated. In the present study, our results demonstrated that the DSF/Cu complex induced apoptosis of MM cells and MM primary cells. The results indicated that DSF/Cu significantly induced cell cycle arrest at the G2/M phase in MM.1S and RPMI8226 cells. Moreover, JC-1 and Western blot results showed that DSF/Cu disrupted mitochondrial membrane integrity and cleaved caspase-8 in MM cells, respectively, suggesting that it induced activation of extrinsic and intrinsic apoptosis pathways. Interestingly, DSF/Cu induced caspase-3 activation was partly blocked by Z-VAD-FMK (zVAD), a pan-caspase inhibitor, indicating at caspase-dependent and -independent paths involved in DSF/Cu induced myeloma cell apoptosis machinery. Additionally, activation of the c-Jun N-terminal kinase (JNK) signaling pathway was observed in DSF/Cu treated MM cells. More importantly, our results demonstrated that DSF/Cu significantly reduced tumor volumes and prolonged overall survival of MM bearing mice when compared with the controls. Taken together, our novel findings showed that DSF/Cu has potent anti-myeloma activity in vitro and in vivo highlighting valuable clinical potential of DSF/Cu in MM treatment.

Serum Interleukin-9 Concentration Is Associated with the Hemoglobin Level and Renal Function in Patients with Multiple Myeloma.

BACKGROUND: Multiple myeloma (MM) is one of the most common types of hematological malignancies, but its pathogenesis is poorly understood. Interleukin-9 (IL-9) is a growth factor, mainly produced by helper T cells. A series of observations suggested that IL-9 might act as a factor promoting oncogenesis. This study was aimed at detecting the serum concentrations of IL-9 in patients with MM, and to investigate its potential clinical significance. METHODS: The serum IL-9 levels in 34 patients with MM and 15 normal controls were quantified by using the Enzyme Linked Immunosorbent Assay (ELISA). RESULTS: Our results showed that the serum IL-9 concentration in MM patients was significantly higher than that in the controls (p<0.0001). Interestingly, the IL-9 level in serum was found to be negatively associated with the hemoglobin concentration among the newly diagnosed MM patients (p=0.0108, r=-0.5850). Moreover, MM patients with renal dysfunction showed a significant increase in serum IL-9 concentration over those with normal renal function (p=0.0395). CONCLUSION: These findings may imply a novel role of IL-9 in anemia and/or renal dysfunction development in MM.

Bone marrow PD-1 positive T cells reflect tumor mass and prognosis in multiple myeloma.

Programmed cell death protein 1 (PD-1) negatively regulates T cell effector mechanisms and contributes to tumor cell escape from immune surveillance. To evaluate potential clinical significance of PD-1 in multiple myeloma (MM), we quantified PD-1 expressing T cells in bone marrow (BM) of MM patients using flow cytometry. Our results showed that PD-1 positive T cells in BM from relapsed/refractory patients were significantly higher than those in the newly diagnosed, partial/complete remission MM, and controls, respectively. Additionally, high-risk MM patients had more PD-1 positive T cells in BM than low-risk patients. Moreover, PD-1 positive T cells in relapsed/refractory MM patients were positively associated with myeloma cell counts in BM and clinical stages. PD-1 positive T cells in BM of all MM and relapsed/refractory MM patients were positively correlated with their serum ß2-microglobulin concentrations. Our results strongly suggest that PD-1 expressing T cells in BM may be applied as a biomarker to reflect tumor mass and prognosis.

Telomere length is positively associated with the expression of IL­6 and MIP­1α in bone marrow mesenchymal stem cells of multiple myeloma.

Potential roles of mesenchymal stem cells (MSCs) in the pathogenesis of multiple myeloma (MM) are largely unknown. In the current study, the authors analyzed telomere length and the expressions of interleukin (IL)­6 and macrophage inflammatory protein (MIP)­1α in MSCs derived from the bone marrow (BM) of MM patients and controls. The current results demonstrated that there was no significant difference in cell surface expression of CD73 and CD90, and the capacity to differentiate into bone tissue were identified between the BM MSCs derived from MM patients and controls. Interestingly, telomere length (TL) and mRNA expressions of IL­6 and MIP­1α were significantly longer or higher in BM MSCs of MM than those of controls. Moreover, TL is positively associated with the expressions of IL­6 and MIP­1α at the mRNA level in BM MSCs of MM. Additionally, IL­6 and MIP­1α expression were significantly upregulated when MSCs from MM patients were cultured in the myeloma associated condition medium. The present study indicated that myeloma­associated elongation of TL of BM MSCs may be a key element contributing to the increased IL­6 and MIP­1α expression, by which MSCs in the tumor microenvironment may facilitate MM and/or MM bone disease development.

PD1 blockade enhances cytotoxicity of in vitro expanded natural killer cells towards myeloma cells.

Aiming for an adoptive natural killer (NK) cell therapy, we have developed a novel protocol to expand NK cells from peripheral blood. With this protocol using anti-human CD16 antibody and interleukin (IL)-2, NK (CD3-CD56+) cells could be expanded about 4000-fold with over 70% purity during a 21-day culture. The expanded NK (exNK) cells were shown to be highly cytotoxic to multiple myeloma (MM) cells (RPMI8226) at low NK-target cell ratios. Furthermore, NK cells expanded in the presence of a blocking antibody (exNK+PD1-blockage) against programmed cell death protein-1 (PD1), a key counteracting molecule for NK and T cell activity, demonstrated more potent cytolytic activity against the RPMI8226 than the exNK cells without PD1 blocking. In parallel, the exNK cells showed significantly higher expression of NK activation receptors NKG2D, NKp44 and NKp30. In a murine model of MM, transfusion of exNK cells, exNK+PD1-blockage, and exNK plus intratumor injection of anti-PD-L2 antibody (exNK+PD-L2 blockage) all significantly suppressed tumor growth and prolonged survival of the myeloma mice. Importantly, exNK+PD1-blockage presented more efficient therapeutic effects. Our results suggest that the NK cell expansion protocol with PD1 blockade presented in this study has considerable potential for the clinical application of allo- and auto-NK cell-based therapies against malignancies.