A high OXPHOS CD8 T cell subset is predictive of immunotherapy resistance in melanoma patients.

Immune checkpoint inhibitor (ICI) therapy continues to revolutionize melanoma treatment, but only a subset of patients respond. Major efforts are underway to develop minimally invasive predictive assays of ICI response. Using single-cell transcriptomics, we discovered a unique CD8 T cell blood/tumor-shared subpopulation in melanoma patients with high levels of oxidative phosphorylation (OXPHOS), the ectonucleotidases CD38 and CD39, and both exhaustion and cytotoxicity markers. We called this population with high levels of OXPHOS "CD8+ TOXPHOS cells." We validated that higher levels of OXPHOS in tumor- and peripheral blood-derived CD8+ TOXPHOS cells correlated with ICI resistance in melanoma patients. We then developed an ICI therapy response predictive model using a transcriptomic profile of CD8+ TOXPHOS cells. This model is capable of discerning responders from nonresponders using either tumor or peripheral blood CD8 T cells with high accuracy in multiple validation cohorts. In sum, CD8+ TOXPHOS cells represent a critical immune population to assess ICI response with the potential to be a new target to improve outcomes in melanoma patients.

Far Upstream Binding Protein 1 (FUBP1) participates in translational regulation of Nrf2 protein under oxidative stress.

Oxidative stress is ubiquitously involved in disease etiology or progression. While the damaging effects have been well characterized, how cells deal with oxidative stress for prevention or removal of damage remains to be fully elucidated. Works from our laboratory have revealed de novo Nrf2 protein translation when cells are encountering low to mild levels of oxidative stress. Nrf2 encodes a transcription factor controlling a myriad of genes important for antioxidation, detoxification, wound repair and tissue remodeling. Here we report a role of FUBP1 in regulating de novo Nrf2 protein translation. An increase of FUBP1 binding to Nrf2 5'UTR due to H2O2 treatment has been found by LC-MS/MS, Far Western blot and ribonucleoprotein immunoprecipitation assays. Blocking FUBP1 expression using siRNA abolished H2O2 from inducing Nrf2 protein elevation or Nrf2 5'UTR activity. While no nuclear to cytoplasmic translocation was detected, cytosolic redistribution to the ribosomal fractions was observed due to oxidant treatment. The presence of FUBP1 in 40/43S ribosomal fractions confirm its involvement in translation initiation of Nrf2 protein. When tested by co-immunoprecipitation with eIF4E, eIF2a, eIF3η and eIF1, only eIF3η was found to gain physical interaction with FUBP1 due to H2O2 treatment. Our data support a role of FUBP1 for promoting the attachment of 40S ribosomal subunit to Nrf2 mRNA and formation of 43S pre-initiation complex for translation initiation of Nrf2 protein under oxidative stress.

CK2 kinase-mediated PHF8 phosphorylation controls TopBP1 stability to regulate DNA replication.

ATR functions as a master regulator of the DNA-damage response. ATR activation requires the ATR activator, topoisomerase IIß-binding protein 1 (TopBP1). However, the underlying mechanism of TopBP1 regulation and how its regulation affects DNA replication remain unknown. Here, we report a specific interaction between TopBP1 and the histone demethylase PHF8. The TopBP1/PHF8 interaction is mediated by the BRCT 7+8 domain of TopBP1 and phosphorylation of PHF8 at Ser854. This interaction is cell-cycle regulated and phosphorylation-dependent. PHF8 is phosphorylated by CK2, which regulates binding of PHF8 to TopBP1. Importantly, PHF8 regulates TopBP1 protein level by preventing its ubiquitination and degradation mediated by the E3 ligase UBR5. Interestingly, PHF8pS854 is likely to contribute to regulation of TopBP1 stability and DNA replication checkpoint. Further, both TopBP1 and PHF8 are required for efficient replication fork restart. Together, these data identify PHF8 as a TopBP1-binding protein and provide mechanistic insight into how PHF8 regulates TopBP1 stability to maintain DNA replication.

Resveratrol for protection against statin toxicity in C2C12 and H9c2 cells.

Statins are among the most commonly prescribed drugs for the treatment of high blood cholesterol. Myotoxicity of statins in certain individuals is often a severe side effect leading to withdrawal. Using C2C12 and H9c2 cells, both exhibiting characteristics of skeletal muscle cells, we addressed whether resveratrol (RSV) can prevent statin toxicity. Statins decreased cell viability in a dose and time-dependent manner. Among the five statins tested, atorvastatin, simvastatin, lovastatin, pravastatin, and fluvastatin, simvastatin is the most toxic one. Simvastatin at 10 µM caused about 65% loss of metabolic activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays in C2C12 cells or H9c2 cells. Inhibition of metabolic activity correlates with an increase in caspase activity. RSV was found to protect H9c2 cells from simvastatin-induced activation of caspase-3/7. However, such protection was not found in C2C12 cells. This cell type-dependent effect of RSV adds to the complexity in muscle cell toxicity of statins.

Autophagic Mechanism in Anti-Cancer Immunity: Its Pros and Cons for Cancer Therapy.

Autophagy, a self-eating machinery, has been reported as an adaptive response to maintain metabolic homeostasis when cancer cells encounter stress. It has been appreciated that autophagy acts as a double-edge sword to decide the fate of cancer cells upon stress factors, molecular subtypes, and microenvironmental conditions. Currently, the majority of evidence support that autophagy in cancer cells is a vital mechanism bringing on resistance to current and prospective treatments, yet whether autophagy affects the anticancer immune response remains unclear and controversial. Accumulated studies have demonstrated that triggering autophagy is able to facilitate anticancer immunity due to an increase in immunogenicity, whereas other studies suggested that autophagy is likely to disarm anticancer immunity mediated by cytotoxic T cells and nature killer (NK) cells. Hence, this contradiction needs to be elucidated. In this review, we discuss the role of autophagy in cancer cells per se and in cancer microenvironment as well as its dual regulatory roles in immune surveillance through modulating presentation of tumor antigens, development of immune cells, and expression of immune checkpoints. We further focus on emerging roles of autophagy induced by current treatments and its impact on anticancer immune response, and illustrate the pros and cons of utilizing autophagy in cancer immunotherapy based on preclinical references.

The Combination of Arginine Deprivation and 5-Fluorouracil Improves Therapeutic Efficacy in Argininosuccinate Synthetase Negative Hepatocellular Carcinoma.

Argininosuccinate synthetase (ASS), a key enzyme to synthesize arginine is down regulated in many tumors including hepatocellular carcinoma (HCC). Similar to previous reports, we have found the decrease in ASS expression in poorly differentiated HCC. These ASS(-) tumors are auxotrophic for arginine. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine, has shown activity in these tumors, but the antitumor effect is not robust and hence combination treatment is needed. Herein, we have elucidated the effectiveness of ADI-PEG20 combined with 5-Fluorouracil (5-FU) in ASS(-)HCC by targeting urea cycle and pyrimidine metabolism using four HCC cell lines as model. SNU398 and SNU387 express very low levels of ASS or ASS(-) while Huh-1, and HepG2 express high ASS similar to normal cells. Our results showed that the augmented cytotoxic effect of combination treatment only occurs in SNU398 and SNU387, and not in HepG2 and Huh-1 (ASS(+)) cells, and is partly due to reduced anti-apoptotic proteins X-linked inhibitor of apoptosis protein (XIAP), myeloid leukemia cell differentiation protein (Mcl-1) and B-cell lymphoma-2 (Bcl-2). Importantly, lack of ASS also influences essential enzymes in pyrimidine synthesis (carbamoyl-phosphate synthetase2, aspartate transcarbamylase and dihydrooratase (CAD) and thymidylate synthase (TS)) and malate dehydrogenase-1 (MDH-1) in TCA cycle. ADI-PEG20 treatment decreased these enzymes and made them more vulnerable to 5-FU. Transfection of ASS restored these enzymes and abolished the sensitivity to ADI-PEG20 and combination treatment. Overall, our data suggest that ASS influences multiple enzymes involved in 5-FU sensitivity. Combining ADI-PEG20 and 5-FU may be effective to treat ASS(-)hepatoma and warrants further clinical investigation.