Neurological Applications of Belzutifan in von Hippel Lindau Disease.

Von Hippel Lindau (VHL) disease is a tumor predisposition syndrome caused by mutations in the VHL gene that presents with visceral neoplasms and growths, including clear cell renal cell carcinoma, and central nervous system manifestations, such as hemangioblastomas of the brain and spine. The pathophysiology involves a dysregulation of oxygen sensing caused by inability to degrade HIFα, leading to overactivation of hypoxic pathways. Hemangioblastomas are the most common tumors in patients with VHL and cause significant morbidity. Until recently, there were no systemic therapies available for patients that could effectively reduce the size of these lesions. Belzutifan, the first approved HIF-2α inhibitor, has demonstrated benefit in VHL-associated tumors, with a 30% response rate in hemangioblastomas and ~30-50% reduction in their sizes over the course of treatment. Anemia is the most prominent adverse effect, affecting 76-90% of participants and sometimes requiring dose reduction or transfusion. Other significant adverse events include hypoxia and fatigue. Overall, belzutifan is well tolerated; however, long term data on dosing regimens, safety, and fertility are not yet available. Belzutifan holds promise for treatment of neurological manifestations of VHL and its utility may influence the clinical management paradigms for this patient population.

Functional succinate dehydrogenase deficiency is a common adverse feature of clear cell renal cancer.

Reduced succinate dehydrogenase (SDH) activity resulting in adverse succinate accumulation was previously considered relevant only in 0.05 to 0.5% of kidney cancers associated with germline SDH mutations. Here, we sought to examine a broader role for SDH loss in kidney cancer pathogenesis/progression. We report that underexpression of SDH subunits resulting in accumulation of oncogenic succinate is a common feature in clear cell renal cell carcinoma (ccRCC) (∼80% of all kidney cancers), with a marked adverse impact on survival in ccRCC patients (n = 516). We show that SDH down-regulation is a critical brake in the TCA cycle during ccRCC pathogenesis and progression. In exploring mechanisms of SDH down-regulation in ccRCC, we report that Von Hippel-Lindau loss-induced hypoxia-inducible factor-dependent up-regulation of miR-210 causes direct inhibition of the SDHD transcript. Moreover, shallow deletion of SDHB occurs in ∼20% of ccRCC. We then demonstrate that SDH loss-induced succinate accumulation contributes to adverse loss of 5-hydroxymethylcytosine, gain of 5-methylcytosine, and enhanced invasiveness in ccRCC via inhibition of ten-eleven translocation (TET)-2 activity. Intriguingly, binding affinity between the catalytic domain of recombinant TET-2 and succinate was found to be very low, suggesting that the mechanism of succinate-induced attenuation of TET-2 activity is likely via product inhibition rather than competitive inhibition. Finally, exogenous ascorbic acid, a TET-activating demethylating agent, led to reversal of the above oncogenic effects of succinate in ccRCC cells. Collectively, our study demonstrates that functional SDH deficiency is a common adverse feature of ccRCC and not just limited to the kidney cancers associated with germline SDH mutations.

HIF1α is not a target of 14q deletion in clear cell renal cancer.

HIF1α has been termed a tumor-suppressor in clear cell renal cell carcinoma (ccRCC), primarily based on functional proliferation studies in cell lines (in vitro and in vivo) with genetic manipulation, and the adverse prognosis of 14q-deleted ccRCC patients. In other malignancies, however, HIF1α has an established tumor-promoting role. Therefore, this study sought to further examine the role of HIF1α in ccRCC using bioinformatic analyses of 530 ccRCC patients from The Cancer Genome Atlas (TCGA) and The Cancer Proteome Atlas (TCPA) registries. Although lower copy numbers of HIF1A (encoding HIF1α, located at 14q23.2) was associated with worse survival, there was no survival difference based on either HIF1A mRNA or HIF1α protein expression. Interestingly, L2HGDH (L-2-Hydroxyglutarate Dehydrogenase), a recently characterized epigenetic modulating ccRCC tumor-suppressor with a marked impact on survival, was found to be located only ~ 11.5Mbp from HIF1A on 14q (at 14q21.3). L2HGDH was therefore co-deleted in ~ 95% of 14q deletions involving HIF1A locus. Remarkably, HIF1A CNV had a markedly stronger correlation with L2HGDH expression (Rho = 0.55) than its own gene expression (Rho = 0.27), indicating high preserved-allele compensation of HIF1A. Genetic loss of HIF1A was therefore associated with a much greater reduction of L2HGDH gene expression than its own gene expression, providing a possible explanation for survival differences based on HIF1A CNV and mRNA expression. Furthermore, in 14q-deleted ccRCC patients with complete (uncensored) survival data, in the relatively rare cases where genetic loss of HIF1A occurred without genetic loss of L2HGDH (n = 5), the survival was significantly greater than where there was simultaneous genetic loss of both (n = 87) (mean survival 1670.8 ± 183.5 days vs 885.1 ± 78.4 days; p = 0.007). In addition, there was no correlation between HIF1A mRNA and HIF1α protein expression in ccRCC (R = 0.02), reflecting the primarily post-translational regulation of HIF1α. Lastly, even between L2HGDH and HIF1A loci, 14q was found to have several other yet-to-be-characterized potential ccRCC tumor-suppressors. Taken together, the data indicate that HIF1α is not a target of 14q deletion in ccRCC and that it is not a tumor-suppressor in this malignancy.

Immune evasion in renal cell carcinoma: biology, clinical translation, future directions.

Targeted therapies and immune checkpoint inhibitors have advanced the treatment landscape of Renal Cell Carcinoma (RCC) over the last decade. While checkpoint inhibitors have demonstrated survival benefit and are currently approved in the front-line and second-line settings, primary and secondary resistance is common. A comprehensive understanding of the mechanisms of immune evasion in RCC is therefore critical to the development of effective combination treatment strategies. This article reviews the current understanding of the different, yet coordinated, mechanisms adopted by RCC cells to evade immune killing; summarizes various aspects of clinical translation thus far, including the currently registered RCC clinical trials exploring agents in combination with checkpoint inhibitors; and provides perspectives on the current landscape and future directions for the field.

Considerations for target oxygen saturation in COVID-19 patients: are we under-shooting?

BACKGROUND: The current target oxygen saturation range for patients with COVID-19 recommended by the National Institutes of Health is 92-96%. MAIN BODY: This article critically examines the evidence guiding current target oxygen saturation recommendation for COVID-19 patients, and raises important concerns in the extrapolation of data from the two studies stated to be guiding the recommendation. Next, it examines the influence of hypoxia on upregulation of ACE2 (target receptor for SARS-CoV-2 entry) expression, with supporting transcriptomic analysis of a publicly available gene expression profile dataset of human renal proximal tubular epithelial cells cultured in normoxic or hypoxic conditions. Finally, it discusses potential implications of specific clinical observations and considerations in COVID-19 patients on target oxygen saturation, such as diffuse systemic endothelitis and microthrombi playing an important pathogenic role in the wide range of systemic manifestations, exacerbation of hypoxic pulmonary vasoconstriction in the setting of pulmonary vascular endothelitis/microthrombi, the phenomenon of "silent hypoxemia" with some patients presenting to the hospital with severe hypoxemia disproportional to symptoms, and overburdened health systems and public health resources in many parts of the world with adverse implications on outpatient monitoring and early institution of oxygen supplementation. CONCLUSIONS: The above factors and analyses, put together, call for an urgent exploration and re-evaluation of target oxygen saturation in COVID-19 patients, both in the inpatient and outpatient settings. Until data from such trials become available, where possible, it may be prudent to target an oxygen saturation at least at the upper end of the recommended 92-96% range in COVID-19 patients both in the inpatient and outpatient settings (in patients that are normoxemic at pre-COVID baseline). Home pulse oximetry, tele-monitoring, and earlier institution of oxygen supplementation for hypoxemic COVID-19 outpatients could be beneficial, where public health resources allow for their implementation.

High-dose ascorbic acid synergizes with anti-PD1 in a lymphoma mouse model.

Major efforts are underway to identify agents that can potentiate effects of immune checkpoint inhibition. Here, we show that ascorbic acid (AA) treatment caused genomewide demethylation and enhanced expression of endogenous retroviral elements in lymphoma cells. AA also increased 5-hydroxymethylcytosine (5hmC) levels of CD8+ T cells and enhanced their cytotoxic activity in a lymphoma coculture system. High-dose AA treatment synergized with anti-PD1 therapy in a syngeneic lymphoma mouse model, resulting in marked inhibition of tumor growth compared with either agent alone. Analysis of the intratumoral epigenome revealed increased 5hmC with AA treatment, consistent with in vitro findings. Analysis of the tumor immune microenvironment revealed that AA strikingly increased intratumoral infiltration of CD8+ T cells and macrophages, suggesting enhanced tumor immune recognition. The combination treatment markedly enhanced intratumoral infiltration of macrophages and CD8+ T lymphocytes, granzyme B production by cytotoxic cells (cytotoxic T cells and natural killer cells), and interleukin 12 production by antigen-presenting cells compared with single-agent anti-PD1. These data indicate that AA potentiates anti-PD1 checkpoint inhibition through synergistic mechanisms. The study provides compelling rationale for testing combinations of high-dose AA and anti-PD1 agents in patients with aggressive B cell lymphoma as well as in preclinical models of other malignancies.

Epigenetic dysregulation by aberrant metabolism in renal cell carcinoma can be reversed with Ascorbic acid.

Our recently published study uncovered mechanisms and prognostic impact of aberrant DNA methylation/hydroxymethylation in clear cell renal cell carcinoma, and comprehensively explored the potential of Ascorbic acid in reversing the epigenetic aberrancy. This article provides a summary of the findings and their translational significance, and important considerations while testing Ascorbic acid as an anti-cancer agent. Abbreviations- ccRCC: clear cell renal cell carcinoma; TET: Ten-Eleven Translocation; 5mC: 5-methylcytosine; 5hmC: 5-hydroxymethylcytosine; L2HG: l-2-hydroxyglutarate; L2HGDH: l-2-hydroxyglutarate dehydrogenase; 2-OG: 2-Oxoglutarate; AA: Ascorbic acid.

Ascorbic acid-induced TET activation mitigates adverse hydroxymethylcytosine loss in renal cell carcinoma.

Although clear cell renal cell carcinoma (ccRCC) has been shown to result in widespread aberrant cytosine methylation and loss of 5-hydroxymethylcytosine (5hmC), the prognostic impact and therapeutic targeting of this epigenetic aberrancy has not been fully explored. Analysis of 576 primary ccRCC samples demonstrated that loss of 5hmC was strongly associated with aggressive clinicopathologic features and was an independent adverse prognostic factor. Loss of 5hmC also predicted reduced progression-free survival after resection of nonmetastatic disease. The loss of 5hmC in ccRCC was not due to mutational or transcriptional inactivation of ten eleven translocation (TET) enzymes, but to their functional inactivation by l-2-hydroxyglutarate (L2HG), which was overexpressed due to the deletion and underexpression of L2HG dehydrogenase (L2HGDH). Ascorbic acid (AA) reduced methylation and restored genome-wide 5hmC levels via TET activation. Fluorescence quenching of the recombinant TET-2 protein was unaffected by L2HG in the presence of AA. Pharmacologic AA treatment led to reduced growth of ccRCC in vitro and reduced tumor growth in vivo, with increased intratumoral 5hmC. These data demonstrate that reduced 5hmC is associated with reduced survival in ccRCC and provide a preclinical rationale for exploring the therapeutic potential of high-dose AA in ccRCC.

Ascorbic Acid in Cancer Treatment: Let the Phoenix Fly.

Vitamin C (ascorbic acid, ascorbate), despite controversy, has re-emerged as a promising anti-cancer agent. Recent knowledge of intravenous ascorbate pharmacokinetics and discovery of unexpected mechanisms of ascorbate action have spawned many investigations. Two mechanisms of anti-cancer activity with ascorbate have gained prominence: hydrogen peroxide-induced oxidative stress and DNA demethylation mediated by ten-eleven translocation enzyme activation. Here, we highlight salient aspects of the evolution of ascorbate in cancer treatment, provide insights into the pharmacokinetics of ascorbate, describe mechanisms of its anti-cancer activity in relation to the pharmacokinetics, outline promising preclinical and clinical evidence, and recommend future directions.

Association Between Renal Cell Carcinoma and Myelodysplastic Syndromes: Epigenetic Underpinning?

BACKGROUND: Renal cell carcinoma (RCC) and certain myeloid malignancies are both characterized by widespread aberrant DNA hypermethylation. After clinical observations of patients with a personal history of both malignancies, we sought to explore a potential association, and to describe the clinical characteristics of these patients. PATIENTS AND METHODS: Mayo Clinic's 'Advanced Cohort Explorer' database was used to identify patients with a history of both malignancies. Clinical features and long-term outcome were abstracted. Prevalence of myelodysplastic syndromes (MDSs) in patients ≥ 65 years with a personal history of nephrectomy for RCC was then compared with the prevalence of MDSs in the Dusseldorf MDS registry and the general patient population at Mayo Clinic, using 1-sample test of proportions. RESULTS: A total of 59 patients with a diagnosis of both RCC and myeloid malignancy were identified. The myeloid malignancies included 38 MDSs, 12 acute myelogenous leukemia, and 9 myeloproliferative neoplasms. The cohort was characterized by marked male predominance (4.4:1). The median age at RCC diagnosis was 64 years (range, 37-87 years), and for myeloid malignancy was 75 years (range, 44-90 years). Prevalence of MDS in patients > 65 years with a personal history of nephrectomy for RCC was ≈ 8.4 times that of the age-concordant general population based on the Dusseldorf registry (28/6490 or 395/100,000 vs. ≈ 47/100,000; P < .001), and 3.07 times that of the age-concordant patient population at Mayo Clinic (28/6490 or 395/100,000 vs. 128.4/100,000; P < .001). CONCLUSIONS: We observed a strong association between RCC and MDS. Patients with a history of RCC appear to have a substantially increased risk of developing MDS compared with the general population.

Low hypoxia inducible factor-1α (HIF-1α) expression in testicular germ cell tumors - a major reason for enhanced chemosensitivity?

The molecular basis for enhanced chemosensitivity of testicular germ cell tumors (GCT) has been an area of great interest, as it could potentially give us therapeutic leads in other resistant malignancies. Thus far, however, the increased sensitivity of GCT has been variously attributed to multiple factors - an inability to detoxify cisplatin, a lack of export pumps, an inability to repair the DNA damage, an intact apoptotic cascade and lack of p53 mutation; but a unifying underlying etiology leading to the aforementioned processes and having a translational implication has so far been elusive. Herein, we offer evidence to support a potential significant role for the previously demonstrated low hypoxia inducible factor-1α (HIF-1α) expression in mediating the general exquisite chemosensitivity of testicular GCT, through the aforementioned processes. This molecular mechanism based hypothesis could have a significant translational implication in platinum refractory GCT as well as other platinum resistant malignancies.

Scleroderma Induced by Pembrolizumab: A Case Series.

Immune checkpoint inhibitors are approved for select cancer treatment and have shown survival benefit in patients with advanced melanoma. Adverse events, including immune-related adverse events, are common and potentially life-threatening. We describe cases of 2 patients with scleroderma (patient 1 had diffuse scleroderma, and patient 2 had limited scleroderma) that developed while they were receiving pembrolizumab therapy for metastatic melanoma. Prompt recognition and treatment of immune-related adverse events may improve tolerance to immune checkpoint inhibitors and contribute to an understanding of the manifesting autoimmune disease.

Drugs with anti-oxidant properties can interfere with cell viability measurements by assays that rely on the reducing property of viable cells.

Cell viability assays such as Cell Titer Blue and Alamar Blue rely on the reducing property of viable cells to reduce the reagent dye to a product which gives a fluorescent signal. The current manufacture-recommended protocols do not take into account the possibility of the reagent substrate being reduced directly to the fluorescent product by drugs with an anti-oxidant property. After suspecting spurious results while determining the cytotoxic potential of a drug of interest (DOI) with known anti-oxidant property against a renal cell cancer (RCC) cell line, we aimed to establish that drugs with anti-oxidant property can indeed cause false-negative results with the current protocols of these assays by direct reduction of the reagent substrate. We also aimed to counter the same with a simple modification added to the protocol. Through our experiments, we conclusively demonstrate that drugs with anti-oxidant properties can indeed interfere with cell viability measurements by assays that rely on the reducing property of viable cells. A simple modification in the protocol, as elaborated in the manuscript, can prevent spurious results with these otherwise convenient assays.Laboratory Investigation advance online publication, 27 February 2017; doi:10.1038/labinvest.2017.18.

Notch Pathway Is Activated via Genetic and Epigenetic Alterations and Is a Therapeutic Target in Clear Cell Renal Cancer.

Clear cell renal cell carcinoma (CCRCC) is an incurable malignancy in advanced stages and needs newer therapeutic targets. Transcriptomic analysis of CCRCCs and matched microdissected renal tubular controls revealed overexpression of NOTCH ligands and receptors in tumor tissues. Examination of the TCGA RNA-seq data set also revealed widespread activation of NOTCH pathway in a large cohort of CCRCC samples. Samples with NOTCH pathway activation were also clinically distinct and were associated with better overall survival. Parallel DNA methylation and copy number analysis demonstrated that both genetic and epigenetic alterations led to NOTCH pathway activation in CCRCC. NOTCH ligand JAGGED1 was overexpressed and associated with loss of CpG methylation of H3K4me1-associated enhancer regions. JAGGED2 was also overexpressed and associated with gene amplification in distinct CCRCC samples. Transgenic expression of intracellular NOTCH1 in mice with tubule-specific deletion of VHL led to dysplastic hyperproliferation of tubular epithelial cells, confirming the procarcinogenic role of NOTCH in vivo Alteration of cell cycle pathways was seen in murine renal tubular cells with NOTCH overexpression, and molecular similarity to human tumors was observed, demonstrating that human CCRCC recapitulates features and gene expression changes observed in mice with transgenic overexpression of the Notch intracellular domain. Treatment with the γ-secretase inhibitor LY3039478 led to inhibition of CCRCC cells in vitro and in vivo In summary, these data reveal the mechanistic basis of NOTCH pathway activation in CCRCC and demonstrate this pathway to a potential therapeutic target.

Role of systemic chemotherapy in metastatic hormone-sensitive prostate cancer.

INTRODUCTION: Patients with metastatic hormone sensitive prostate cancer (mHSPC) have traditionally been treated with androgen deprivation therapy (ADT). Recently, there has been a demonstration of a survival benefit with the addition of docetaxel to ADT from three large randomized controlled trials. This review summarizes these trials, draws comparisons between the trials, and attempts to provide critical evidence-based recommendation on the role of docetaxel in mHSPC. METHODS: Of the two published (GETUG-AFU, Chemo-Hormonal therapy vs. Androgen Ablation Randomized Trial for Extensive Disease in prostate cancer [CHAARTED]) and one presented trial (STAMPEDE) an analysis of the study design, patient characteristics, outcomes, variables, and a critical comparison between the trials was performed for making practice recommendations. RESULTS: All the three trials demonstrated statistically significant progression free survival with the addition of docetaxel to ADT in mHSPC. However, while CHAARTED trial demonstrated a significant survival benefit with addition of docetaxel to ADT in patients with high volume mHSPC, GETUG-AFU failed to demonstrate statistically significant survival benefit although there was an absolute difference in survival between the two arms, with lower sample size and statistical power compared to CHAARTED. The largest study, STAMPEDE, reported a 22 month survival benefit in patients with M1 disease with statistical significance; with subgroup analysis of high volume and low volume disease patients yet to be reported. CONCLUSION: After a careful comparison between the trials, we conclude that systemic docetaxel chemotherapy within 4 months of initiating ADT for metastatic, high-volume HSPC should be considered the standard of care for patients with good performance status.

miR-375 induces docetaxel resistance in prostate cancer by targeting SEC23A and YAP1.

BACKGROUND: Treatment options for metastatic castrate-resistant prostate cancer (mCRPC) are limited and typically are centered on docetaxel-based chemotherapy. We previously reported that elevated miR-375 levels were significantly associated with poor overall survival of mCRPC patients. In this study, we evaluated if miR-375 induced chemo-resistance to docetaxel through regulating target genes associated with drug resistance. METHODS: We first compared miR-375 expression level between prostate cancer tissues and normal prostate tissues using data from The Cancer Genome Atlas (TCGA). To examine the role of miR-375 in docetaxel resistance, we transfected miR-375 using a pre-miRNA lentiviral vector and examined the effects of exogenously overexpressed miR-375 on cell growth in two prostate cancer cell lines, DU145 and PC-3. To determine the effect of overexpressed miR-375 on tumor growth and chemo-resistance in vivo, we injected prostate cancer cells overexpressing miR-375 into nude mice subcutaneously and evaluated tumor growth rate during docetaxel treatment. Lastly, we utilized qRT-PCR and Western blot assay to examine two miR-375 target genes, SEC23A and YAP1, for their expression changes after miR-375 transfection. RESULTS: By examining 495 tumor tissues and 52 normal tissues from TCGA data, we found that compared to normal prostate, miR-375 was significantly overexpressed in prostate cancer tissues (8.45-fold increase, p value = 1.98E-23). Docetaxel treatment induced higher expression of miR-375 with 5.83- and 3.02-fold increases in DU145 and PC-3 cells, respectively. Interestingly, miR-375 appeared to play a dual role in prostate cancer proliferation. While miR-375 overexpression caused cell growth inhibition and cell apoptosis, elevated miR-375 also significantly reduced cell sensitivity to docetaxel treatment in vitro, as evidenced by decreased apoptotic cells. In vivo xenograft mouse study showed that tumors with increased miR-375 expression were more tolerant to docetaxel treatment, demonstrated by greater tumor weight and less apoptotic cells in miR-375 transfected group when compared to empty vector control group. In addition, we examined expression levels of the two miR-375 target genes (SEC23A and YAP1) and observed significant reduction in the expression at both protein and mRNA levels in miR-375 transfected prostate cancer cell lines. TCGA dataset analysis further confirmed the negative correlations between miR-375 and the two target genes (r = -0.62 and -0.56 for SEC23A and YAP1, respectively; p < 0.0001). CONCLUSIONS: miR-375 is involved in development of chemo-resistance to docetaxel through regulating SEC23A and YAP1 expression. Our results suggest that miR-375 or its target genes, SEC23A or YAP1, might serve as potential predictive biomarkers to docetaxel-based chemotherapy and/or therapeutic targets to overcome chemo-resistance in mCRPC stage.

Copy number variations in urine cell free DNA as biomarkers in advanced prostate cancer.

Genetic profiling of urine cell free DNA (cfDNA) has not been evaluated in advanced prostate cancer. We performed whole genome sequencing of urine cfDNAs to identify tumor-associated copy number variations in urine before and after initiating androgen deprivation therapy in HSPC stage and docetaxel chemotherapy in CRPC stage. A log2 ratio-based copy number analysis detected common genomic abnormalities in prostate cancer including AR amplification in 5/10 CRPC patients. Other abnormalities identified included TMPRSS2-ERG fusion, PTEN gene deletion, NOTCH1 locus amplification along with genomic amplifications at 8q24.3, 9q34.3, 11p15.5 and 14q11.2, and deletions at 4q35.2, 5q31.3, 7q36.3, 12q24.33, and 16p11.2. By comparing copy number between pre- and post-treatment, we found significant copy number changes in 34 genomic loci. To estimate the somatic tumor DNA fraction in urine cfDNAs, we developed a Urine Genomic Abnormality (UGA) score algorithm that summed the top ten most significant segments with copy number changes. The UGA scores correlated with tumor burden and the change in UGA score after stage-specific therapies reflected disease progression status and overall survival. The study demonstrates the potential clinical utility of urine cfDNAs in predicting treatment response and monitoring disease progression.