Anirban Kundu

Clear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer, exhibits notable metabolic reprogramming. We previously reported elevated HDAC7, a class II histone deacetylase, in ccRCC. Here, we demonstrate that HDAC7 promotes aggressive phenotypes and in vivo tumor progression in RCC. HDAC7 suppresses the expression of genes mediating branched-chain amino acid (BCAA) catabolism. Notably, lower expression of BCAA catabolism genes is strongly associated with worsened survival in ccRCC. Suppression of BCAA catabolism promotes expression of SNAIL1, a central mediator of aggressive phenotypes including migration and invasion. HDAC7-mediated suppression of the BCAA catabolic program promotes SNAI1 messenger RNA transcription via NOTCH signaling activation. Collectively, our findings provide innovative insights into the role of metabolic remodeling in ccRCC tumor progression.

Iron is an important micronutrient required for a vast array of physiological functions including oxygen transport. Iron metabolism and its regulations are specifically important for athletics as exercise leads to physiological changes, which induce iron deficiency. Iron deficiency could lead to reduced aerobic capacity and physical ability to perform. Athletes suffer iron deficiency due to expansion of plasma volume, iron loss through sweat, injury, bleeding in the gastrointestinal tract, foot strike hemolysis, etc. Female athletes are at the highest risk of iron deficiency; lack of proper intervention may lead to poor performance and long-term deterioration of overall health. Hepcidin plays a major role in iron homeostasis and its synthesis in hepatocytes is regulated by multiple factors. However, exercise-induced inflammation and the associated increase in interleukin 6 (IL6) induce hepcidin synthesis that impairs iron availability. Therefore, iron deficiency in athletes depends on the nature of exercise, intensity, duration, and recovery periods.

Tumor cells are known to undergo considerable metabolic reprogramming to meet their unique demands and drive tumor growth. At the same time, this reprogramming may come at a cost with resultant metabolic vulnerabilities. The small molecule l-2-hydroxyglutarate (l-2HG) is elevated in the most common histology of renal cancer. Similarly to other oncometabolites, l-2HG has the potential to profoundly impact gene expression. Here, we demonstrate that l-2HG remodels amino acid metabolism in renal cancer cells through combined effects on histone methylation and RNA N ⁶ -methyladenosine. The combined effects of l-2HG result in a metabolic liability that renders tumors cells reliant on exogenous serine to support proliferation, redox homeostasis, and tumor growth. In concert with these data, high–l-2HG kidney cancers demonstrate reduced expression of multiple serine biosynthetic enzymes. Collectively, our data indicate that high–l-2HG renal tumors could be specifically targeted by strategies that limit serine availability to tumors.

Mounting evidence points to alterations in mitochondrial metabolism in renal cell carcinoma (RCC). However, the mechanisms that regulate the TCA cycle in RCC remain uncharacterized. Here, we demonstrate that loss of TCA cycle enzyme expression is retained in RCC metastatic tissues. Moreover, proteomic analysis demonstrates that reduced TCA cycle enzyme expression is far more pronounced in RCC relative to other tumor types. Loss of TCA cycle enzyme expression is correlated with reduced expression of the transcription factor PGC-1 α , which is also lost in RCC tissues. PGC-1 α reexpression in RCC cells restores the expression of TCA cycle enzymes in vitro and in vivo and leads to enhanced glucose carbon incorporation into TCA cycle intermediates. Mechanistically, TGF- β signaling, in concert with histone deacetylase 7 (HDAC7), suppresses TCA cycle enzyme expression. Our studies show that pharmacologic inhibition of TGF- β restores the expression of TCA cycle enzymes and suppresses tumor growth in an orthotopic model of RCC. Taken together, this investigation reveals a potentially novel role for the TGF- β /HDAC7 axis in global suppression of TCA cycle enzymes in RCC and provides insight into the molecular basis of altered mitochondrial metabolism in this malignancy.

The Warburg effect is one of most-well studied metabolic phenomenon in cancer cells. For the most part, these studies have focused on enhanced rates of glycolysis observed in various models. The presumption has been that mitochondrial metabolism is suppressed. However, recent studies indicate that the extent of mitochondrial metabolism is far more heterogeneous in tumors than originally presumed. One tumor type with suppression of mitochondrial metabolism is renal cell carcinoma (RCC). Prior studies indicate that suppressed TCA cycle enzyme mRNA expression is associated with aggressive RCC. Yet, the mechanisms that regulate the TCA cycle in RCC remain uncharacterized. Here, we demonstrate that loss of TCA cycle enzyme expression is retained in RCC metastatic tissues. Moreover, proteomic analysis demonstrates that reduced TCA cycle enzyme expression is far more pronounced in RCC relative to other tumor types. Loss of TCA cycle enzyme expression is correlated with reduced expression of the transcription factor peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) which is also lost in RCC tissues. PGC-1α re-expression in RCC cells restores the expression of TCA cycle enzymes in vitro and in vivo and leads to enhanced glucose carbon incorporation into TCA cycle intermediates. Mechanistically, TGF-β signaling, in concert with histone deacetylase 7 (HDAC7), suppresses TCA cycle enzyme expression. In turn, pharmacologic inhibition of TGF-β restores expression of TCA cycle enzyme expression and suppresses tumor growth in an orthotopic model of RCC. Taken together, our findings reveal a novel role for the TGF-β /HDAC7 axis in global suppression of TCA cycle enzymes in RCC and provide novel insight into the molecular basis of altered mitochondrial metabolism in this malignancy.

L-2-hydroxyglutarate (L-2HG) is an oncometabolite found elevated in renal tumors. However, this molecule might have physiological roles that extend beyond its association with cancer, as L-2HG levels are elevated in response to hypoxia and during Drosophila larval development. L-2HG is known to be metabolized by L-2HG dehydrogenase (L2HGDH), and loss of L2HGDH leads to elevated L-2HG levels. Despite L2HGDH being highly expressed in the kidney, its role in renal metabolism has not been explored. Here, we report our findings utilizing a novel CRISPR/Cas9 murine knockout model, with a specific focus on the role of L2HGDH in the kidney. Histologically, L2hgdh knockout kidneys have no demonstrable histologic abnormalities. However, GC-MS metabolomics demonstrates significantly reduced levels of the TCA cycle intermediate succinate in multiple tissues. Isotope labeling studies with [U-¹³C] glucose demonstrate that restoration of L2HGDH in renal cancer cells (which lowers L-2HG) leads to enhanced incorporation of label into TCA cycle intermediates. Subsequent biochemical studies demonstrate that L-2HG can inhibit the TCA cycle enzyme α-ketoglutarate dehydrogenase. Bioinformatic analysis of mRNA expression data from renal tumors demonstrates that L2HGDH is co-expressed with genes encoding TCA cycle enzymes as well as the gene encoding the transcription factor PGC-1α, which is known to regulate mitochondrial metabolism. Restoration of PGC-1α in renal tumor cells results in increased L2HGDH expression with a concomitant reduction in L-2HG levels. Collectively, our analyses provide new insight into the physiological role of L2HGDH as well as mechanisms that promote L-2HG accumulation in disease states.

Analysis of transcriptomic data demonstrates extensive epigenetic gene silencing of the transcription factor PRDM16 in renal cancer. We show that restoration of PRDM16 in RCC cells suppresses in vivo tumor growth. RNaseq analysis reveals that PRDM16 imparts a predominantly repressive effect on the RCC transcriptome including suppression of the gene encoding semaphorin 5B (SEMA5B). SEMA5B is a HIF target gene highly expressed in RCC that promotes in vivo tumor growth. Functional studies demonstrate that PRDM16’s repressive properties, mediated by physical interaction with the transcriptional corepressors C-terminal binding proteins (CtBP1/2), are required for suppression of both SEMA5B expression and in vivo tumor growth. Finally, we show that reconstitution of RCC cells with a PRDM16 mutant unable to bind CtBPs nullifies PRDM16’s effects on both SEMA5B repression and tumor growth suppression. Collectively, our data uncover a novel epigenetic basis by which HIF target gene expression is amplified in kidney cancer and a new mechanism by which PRDM16 exerts its tumor suppressive effects.

Emergence of multidrug resistant strains and extremely drug resistant strains of Mycobacterium tuberculosis is due to its ability to form persister cells. The formation of persister cells is assumed to be triggered due to the presence of large number of toxin–antitoxin (TA) systems in its genome. Mtb genome encodes 47 VapBC TA systems. In this work, we aim to biochemically characterize VapC46 toxin of the VapBC46 TA operon from Mycobacterium tuberculosis. Heterologous expression of VapC46 in E. coli is shown to exhibit bacteriostasis and toxicity alters the surface morphology of the E. coli cells. VapC46 is shown to possess ribonuclease activity in a magnesium-dependent manner. Using FRET and pull down assay, VapC46 is shown to interact with VapB46 antitoxin. A model of VapC46 is shown to resemble PIN domain family of proteins and reveals the putative active site required for its ribonuclease activity.