Metabolic reprogramming of the immune response in the tumor microenvironment

A Division of Cancer Biology, NCI sponsored workshop, Metabolic Reprogramming of the Immune Response in the Tumor Microenvironment, was held October 2nd in Bethesda, MD. The purpose of the workshop was to bring together cancer cell biologists and immunologists to explore the mechanistic relationships between the metabolic pathways used by cancer cells and anti-tumor immune cells and how this information could be used to improve cancer immunotherapy. At the conclusion of the workshop a general discussion focused on defining the major challenges and opportunities concerning the impact of metabolism on anti-tumor immunity and cancer immunotherapy as well as what tools, technologies, resources or community efforts are required to accelerate research in this area. Overall, future studies need to consider how cancer cell metabolic pathways differ from activated lymphocytes in order to define a therapeutic window for cancer therapy. Further, studies aimed at reprogramming the metabolic qualities of T cells with the goal of improving immunotherapy were considered a promising avenue

Identifying mRNA targets of microRNA dysregulated in cancer: with application to clear cell Renal Cell Carcinoma

BACKGROUND. MicroRNA regulate mRNA levels in a tissue specific way, either by inducing degradation of the transcript or by inhibiting translation or transcription. Putative mRNA targets of microRNA identified from seed sequence matches are available in many databases. However, such matches have a high false positive rate and cannot identify tissue specificity of regulation. RESULTS. We describe a simple method to identify direct mRNA targets of microRNA dysregulated in cancers from expression level measurements in patient matched tumor/normal samples. The word "direct" is used here in a strict sense to: a) represent mRNA which have an exact seed sequence match to the microRNA in their 3'UTR, b) the seed sequence match is strictly conserved across mouse, human, rat and dog genomes, c) the mRNA and microRNA expression levels can distinguish tumor from normal with high significance and d) the microRNA/mRNA expression levels are strongly and significantly anti-correlated in tumor and/or normal samples. We apply and validate the method using clear cell Renal Cell Carcinoma (ccRCC) and matched normal kidney samples, limiting our analysis to mRNA targets which undergo degradation of the mRNA transcript because of a perfect seed sequence match. Dysregulated microRNA and mRNA are first identified by comparing their expression levels in tumor vs normal samples. Putative dysregulated microRNA/mRNA pairs are identified from these using seed sequence matches, requiring that the seed sequence be conserved in human/dog/rat/mouse genomes. These are further pruned by requiring a strong anti-correlation signature in tumor and/or normal samples. The method revealed many new regulations in ccRCC. For instance, loss of miR-149, miR-200c and mir-141 causes gain of function of oncogenes (KCNMA1, LOX), VEGFA and SEMA6A respectively and increased levels of miR-142-3p, miR-185, mir-34a, miR-224, miR-21 cause loss of function of tumor suppressors LRRC2, PTPN13, SFRP1, ERBB4, and (SLC12A1, TCF21) respectively. We also found strong anti-correlation between VEGFA and the miR-200 family of microRNA: miR-200a*, 200b, 200c and miR-141. Several identified microRNA/mRNA pairs were validated on an independent set of matched ccRCC/normal samples. The regulation of SEMA6A by miR-141 was verified by a transfection assay. CONCLUSIONS. We describe a simple and reliable method to identify direct gene targets of microRNA in any cancer. The constraints we impose (strong dysregulation signature for microRNA and mRNA levels between tumor/normal samples, evolutionary conservation of seed sequence and strong anti-correlation of expression levels) remove spurious matches and identify a subset of robust, tissue specific, functional mRNA targets of dysregulated microRNA.Cancer Institute of New Jersy; New Jersey Commission for Cacner Research; Lineberger Comprehensive Cancer Center Tissue Procurement and Genomics Core Facility; Crawford Fun

Stimulating TAM-mediated anti-tumor immunity with mannose-decorated nanoparticles in ovarian cancer

BACKGROUND: Current cancer immunotherapies have made tremendous impacts but generally lack high response rates, especially in ovarian cancer. New therapies are needed to provide increased benefits. One understudied approach is to target the large population of immunosuppressive tumor-associated macrophages (TAMs). Using inducible transgenic mice, we recently reported that upregulating nuclear factor-kappaB (NF-κB) signaling in TAMs promotes the M1, anti-tumor phenotype and limits ovarian cancer progression. We also developed a mannose-decorated polymeric nanoparticle system (MnNPs) to preferentially deliver siRNA payloads to M2, pro-tumor macrophages in vitro. In this study, we tested a translational strategy to repolarize ovarian TAMs via MnNPs loaded with siRNA targeting the inhibitor of NF-κB alpha (IκBα) using mouse models of ovarian cancer. METHODS: We evaluated treatment with MnNPs loaded with IκBα siRNA (IκBα-MnNPs) or scrambled siRNA in syngeneic ovarian cancer models. ID8 tumors in C57Bl/6 mice were used to evaluate consecutive-day treatment of late-stage disease while TBR5 tumors in FVB mice were used to evaluate repetitive treatments in a faster-developing disease model. MnNPs were evaluated for biodistribution and therapeutic efficacy in both models. RESULTS: Stimulation of NF-κB activity and repolarization to an M1 phenotype via IκBα-MnNP treatment was confirmed using cultured luciferase-reporter macrophages. Delivery of MnNPs with fluorescent payloads (Cy5-MnNPs) to macrophages in the solid tumors and ascites was confirmed in both tumor models. A three consecutive-day treatment of IκBα-MnNPs in the ID8 model validated a shift towards M1 macrophage polarization in vivo. A clear therapeutic effect was observed with biweekly treatments over 2-3 weeks in the TBR5 model where significantly reduced tumor burden was accompanied by changes in immune cell composition, indicative of reduced immunosuppressive tumor microenvironment. No evidence of toxicity associated with MnNP treatment was observed in either model. CONCLUSIONS: In mouse models of ovarian cancer, MnNPs were preferentially associated with macrophages in ascites fluid and solid tumors. Evidence of macrophage repolarization, increased inflammatory cues, and reduced tumor burden in IκBα-MnNP-treated mice indicate beneficial outcomes in models of established disease. We have provided evidence of a targeted, TAM-directed approach to increase anti-tumor immunity in ovarian cancer with strong translational potential for future clinical studies

HIF Transcription Factor Expression and Induction of Hypoxic Response Genes in a Retroperitoneal Angiosarcoma

Angiosarcoma is a rare and highly aggressive tumor of endothelial origin. The molecular mechanisms driving angiosarcoma growth have not been fully elucidated, although autocrine stimulation by vascular endothelial growth factor (VEGF) secretion may play a role in the pathogenesis of this tumor. We identified a patient with a very rare form of angiosarcoma arising from the retroperitoneum. Immunohistochemical analysis demonstrated widespread up-regulation of the hypoxic response pathway as a mechanism of enhanced VEGF expression. Disordered regulation of the hypoxic response pathway can result in the expression of factors such as VEGF and erythropoietin, which may promote autocrine tumor growth in angiosarcoma

IgG-Immune Complexes Promote B Cell Memory by Inducing BAFF

Memory B cell responses are vital for protection against infections, but must also be regulated to prevent autoimmunity. Cognate T cell help, somatic hypermutation, and affinity maturation within germinal centers (GCs) are required for high affinity memory B cell formation; however, the signals that commit GC B cells to the memory pool remain unclear. In this study, we identify a role for IgG immune complexes (ICs), FcγRs, and BAFF during the formation of memory B cells in mice. We found that early secretion of IgG in response to immunization with a T-dependent antigen leads to IC-FcγR interactions that induce DCs to secrete BAFF which acts at or upstream of Bcl-6 in activated B cells. Loss of CD16, hematopoietic cell-derived BAFF, or blocking IC:FcγR regions in vivo diminished the expression of Bcl-6, the frequency of GC and memory B cells, and secondary antibody responses. BAFF also contributed to the maintenance and/or expansion of the Tfh population, although it was dispensable for their formation. Thus, early antibody responses contribute to the optimal formation of B cell memory through IgG-ICs and BAFF. Our work defines a new role for FcγRs in GC and memory B cell responses

AMPK Is Essential to Balance Glycolysis and Mitochondrial Metabolism to Control T-ALL Cell Stress and Survival

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with Notch pathway mutations. While both normal activated and leukemic T cells can utilize aerobic glycolysis to support proliferation, it is unclear to what extent these cell populations are metabolically similar and if differences reveal T-ALL vulnerabilities. Here we show that aerobic glycolysis is surprisingly less active in T-ALL cells than proliferating normal T cells and that T-ALL cells are metabolically distinct. Oncogenic Notch promoted glycolysis but also induced metabolic stress that activated 5' AMP-activated kinase (AMPK). Unlike stimulated T cells, AMPK actively restrained aerobic glycolysis in T-ALL cells through inhibition of mTORC1 while promoting oxidative metabolism and mitochondrial Complex I activity. Importantly, AMPK deficiency or inhibition of Complex I led to T-ALL cell death and reduced disease burden. Thus, AMPK simultaneously inhibits anabolic growth signaling and is essential to promote mitochondrial pathways that mitigate metabolic stress and apoptosis in T-ALL

Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation

The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumor-suppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer