Dual Chromatin and Cytoskeletal Remodeling by SETD2.

Posttranslational modifications (PTMs) of tubulin specify microtubules for specialized cellular functions and comprise what is termed a "tubulin code." PTMs of histones comprise an analogous "histone code," although the "readers, writers, and erasers" of the cytoskeleton and epigenome have heretofore been distinct. We show that methylation is a PTM of dynamic microtubules and that the histone methyltransferase SET-domain-containing 2 (SETD2), which is responsible for H3 lysine 36 trimethylation (H3K36me3) of histones, also methylates α-tubulin at lysine 40, the same lysine that is marked by acetylation on microtubules. Methylation of microtubules occurs during mitosis and cytokinesis and can be ablated by SETD2 deletion, which causes mitotic spindle and cytokinesis defects, micronuclei, and polyploidy. These data now identify SETD2 as a dual-function methyltransferase for both chromatin and the cytoskeleton and show a requirement for methylation in maintenance of genomic stability and the integrity of both the tubulin and histone codes.

Impact of COVID-19 in patients on active melanoma therapy and with history of melanoma.

INTRODUCTION: COVID-19 particularly impacted patients with co-morbid conditions, including cancer. Patients with melanoma have not been specifically studied in large numbers. Here, we sought to identify factors that associated with COVID-19 severity among patients with melanoma, particularly assessing outcomes of patients on active targeted or immune therapy. METHODS: Using the COVID-19 and Cancer Consortium (CCC19) registry, we identified 307 patients with melanoma diagnosed with COVID-19. We used multivariable models to assess demographic, cancer-related, and treatment-related factors associated with COVID-19 severity on a 6-level ordinal severity scale. We assessed whether treatment was associated with increased cardiac or pulmonary dysfunction among hospitalized patients and assessed mortality among patients with a history of melanoma compared with other cancer survivors. RESULTS: Of 307 patients, 52 received immunotherapy (17%), and 32 targeted therapy (10%) in the previous 3 months. Using multivariable analyses, these treatments were not associated with COVID-19 severity (immunotherapy OR 0.51, 95% CI 0.19 - 1.39; targeted therapy OR 1.89, 95% CI 0.64 - 5.55). Among hospitalized patients, no signals of increased cardiac or pulmonary organ dysfunction, as measured by troponin, brain natriuretic peptide, and oxygenation were noted. Patients with a history of melanoma had similar 90-day mortality compared with other cancer survivors (OR 1.21, 95% CI 0.62 - 2.35). CONCLUSIONS: Melanoma therapies did not appear to be associated with increased severity of COVID-19 or worsening organ dysfunction. Patients with history of melanoma had similar 90-day survival following COVID-19 compared with other cancer survivors.

Metastatic Penile Squamous Cell Carcinoma Responsive to Enfortumab Vedotin.

Penile squamous cell carcinoma is a rare disease with very limited data to guide treatment decisions. In particular, there is minimal evidence for effective therapies in the metastatic setting. Here, we present a case of metastatic penile squamous cell carcinoma with response to the Nectin-4 inhibitor enfortumab-vedotin-ejfv (EV). EV was selected due to the evidence of the high expression of Nectin-4 in squamous cell carcinomas, including penile carcinoma. The patient had both radiographic and symptomatic improvement after two cycles of treatment, despite having been treated with multiple prior lines of traditional chemotherapy. This case provides support for the use of antibody-drug conjugates (ADC), including EV, in this disease with few other options in the advanced setting. Further studies examining Nectin-4 and ADCs in penile squamous cell carcinoma should be completed, as high-quality evidence is needed to guide treatment after initial progression for these patients.

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.

First-line Systemic Therapy Following Adjuvant Immunotherapy in Renal Cell Carcinoma: An International Multicenter Study.

BACKGROUND AND OBJECTIVE: Adjuvant pembrolizumab significantly improved overall survival (OS) in renal cell carcinoma (RCC), but real-world data on sequential treatment are scarce. We sought to evaluate the clinical outcomes of first-line (1L) systemic therapy following adjuvant immune oncology (IO)-based regimens. METHODS: A retrospective study including patients with recurrent RCC following adjuvant IO across 29 international institutions was conducted. The primary endpoint was progression-free survival (PFS) on 1L systemic therapy estimated using the Kaplan-Meier method. Preplanned subanalyses of clinical outcomes by type of 1L systemic therapy, recurrence timing, and International Metastatic RCC Database Consortium (IMDC) risk groups were performed. Treatment-related adverse events leading to treatment discontinuation, dose reduction, or corticosteroid use were assessed. KEY FINDINGS AND LIMITATIONS: A total of 94 patients were included. Most received adjuvant pembrolizumab (n = 37, 39%), atezolizumab (n = 28, 30%), or nivolumab + ipilimumab (n = 15, 16%). The cohort included 49 (52%) patients who had recurrence within 3 mo of the last adjuvant IO dose, whereas 45 (48%) recurred beyond 3 mo. Bone metastases were significantly higher in tumors recurring at <3 mo (10/49, 20%) than those recurring at >3 mo (1/45, 2.2%; p = 0.008). Most patients received 1L vascular endothelial growth factor-targeted therapy (VEGF-TT; n = 37, 39%), IO + VEGF-TT (n = 26, 28%), or IO + IO (n = 12, 13%). The remaining underwent local therapy. The median follow-up for the 1L systemic therapy cohort was 15 mo. The 18-mo PFS and OS rates were 45% (95% confidence interval [CI]: 34-60) and 85% (95% CI: 75-95), respectively. Treatment-related adverse events occurred in 32 (42%) patients and included skin toxicity (n = 7, 9.2%), fatigue (n = 6, 7.9%), and diarrhea/colitis (n = 4, 5.3%). Limitations included selecting patients from large academic centers and the short follow-up period. CONCLUSIONS AND CLINICAL IMPLICATIONS: A subset of patients with recurrent RCC following adjuvant IO respond to systemic therapies, including VEGF-TT and IO-based regimens. Notably, patients with favorable-risk disease may derive more benefit from VEGF-TT than from IO therapies in this setting. Future approaches utilizing radiographic tools and biomarker-based liquid biopsies are warranted to detect occult metastatic disease and identify candidate patients for adjuvant IO therapy. PATIENT SUMMARY: Adjuvant pembrolizumab significantly improved overall survival in renal cell carcinoma (RCC). There are limited data on clinical outcomes after the recurrence of RCC tumors following adjuvant immunotherapy. In this study, we find that patients respond to subsequent systemic therapies across different treatment options.

Immune checkpoint inhibitors: maximizing benefit whilst minimizing toxicity.

INTRODUCTION: The advent of immunotherapy has revolutionized the treatment of cancer; anti-tumor efficacy has been observed with immune checkpoint inhibitors (ICI) in ~20 different cancer types with durable responses in some cases. However, the risk of toxicity in the form of immune-related adverse events (irAE) partially counterbalances these benefits, and there are no FDA-approved biomarkers to categorize patients by likelihood of response or risk of irAEs. AREAS COVERED: We conducted a thorough review of the literature of clinical studies regarding ICI and their toxicities. In this review, we synthesize the current body of literature about ICI treatment and irAE by summarizing the classes and uses of ICI, how to identify patients at risk for irAE, present the current understanding of irAE development, describe ongoing research into biomarkers of irAE, examine opportunities for irAE prevention, described management of steroid refractory irAE, and highlight future directions for development of prevention and management strategies. EXPERT OPINION: While ongoing biomarker studies are promising, it is unlikely that there will be a 'one-size-fits-all' approach to categorizing irAE risk. In contrast, improved management and irAE prophylaxis are potentially in reach, and ongoing trials will help elucidate best practices.

Triplet Strategies in Metastatic Clear Cell Renal Cell Carcinoma: A Worthy Option in the First-Line Setting?

Significant strides have been made in the frontline treatment of patients with advanced clear cell renal cell carcinoma (ccRCC). There are multiple standard-of-care doublet regimens consisting of either the combined dual immune checkpoint inhibitors, ipilimumab and nivolumab, or combinations of a vascular endothelial growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor. Currently, there is an emergence of clinical trials examining triplet combinations. In COSMIC-313, a randomized phase III trial for patients with untreated advanced ccRCC, the triplet combination of ipilimumab, nivolumab, and cabozantinib was compared with a contemporary control arm of ipilimumab and nivolumab. While patients receiving the triplet regimen demonstrated improved progression-free survival, these patients also experienced greater toxicity and the overall survival data are still maturing. In this article, we discuss the role of doublet therapy as standard of care, the current data available for the promise of triplet therapy, the rationale to continue pursuing trials with triplet combinations, and factors for clinicians and patients to consider when choosing among frontline treatments. We present ongoing trials with an adaptive design that may serve as alternative methods for escalating from doublet to triplet regimens in the frontline setting and explore clinical factors and emerging predictive biomarkers (both baseline and dynamic) that may guide future trial design and frontline treatment for patients with advanced ccRCC.

Polycomb repressor complex 2 suppresses interferon-responsive MHC-II expression in melanoma cells and is associated with anti-PD-1 resistance.

BACKGROUND: Despite the remarkable success of immunotherapy in treating melanoma, understanding of the underlying mechanisms of resistance remains limited. Emerging evidence suggests that upregulation of tumor-specific major histocompatibility complex-II (tsMHC-II) serves as a predictive marker for the response to anti-programmed death-1 (PD-1)/programmed death ligand 1 (PD-L1) therapy in various cancer types. The genetic and epigenetic pathways modulating tsMHC-II expression remain incompletely characterized. Here, we provide evidence that polycomb repressive complex 2 (PRC2)/EZH2 signaling and resulting H3K27 hypermethylation suppresses tsMHC-II. METHODS: RNA sequencing data from tumor biopsies from patients with cutaneous melanoma treated with or without anti-PD-1, targeted inhibition assays, and assays for transposase-accessible chromatin with sequencing were used to observe the relationship between EZH2 inhibition and interferon (IFN)-γ inducibility within the MHC-II pathway. RESULTS: We find that increased EZH2 pathway messenger RNA (mRNA) expression correlates with reduced mRNA expression of both presentation and T-cell genes. Notably, targeted inhibition assays revealed that inhibition of EZH2 influences the expression dynamics and inducibility of the MHC-II pathway following IFN-γ stimulation. Additionally, our analysis of patients with metastatic melanoma revealed a significant inverse association between PRC2-related gene expression and response to anti-PD-1 therapy. CONCLUSIONS: Collectively, our findings demonstrate that EZH2 inhibition leads to enhanced MHC-II expression potentially resulting from improved chromatin accessibility at CIITA, the master regulator of MHC-II. These insights shed light on the molecular mechanisms involved in tsMHC-II suppression and highlight the potential of targeting EZH2 as a therapeutic strategy to improve immunotherapy efficacy.

SETD2 Haploinsufficiency for Microtubule Methylation Is an Early Driver of Genomic Instability in Renal Cell Carcinoma.

Loss of the short arm of chromosome 3 (3p) occurs early in >95% of clear cell renal cell carcinoma (ccRCC). Nearly ubiquitous 3p loss in ccRCC suggests haploinsufficiency for 3p tumor suppressors as early drivers of tumorigenesis. We previously reported methyltransferase SETD2, which trimethylates H3 histones on lysine 36 (H3K36me3) and is located in the 3p deletion, to also trimethylate microtubules on lysine 40 (αTubK40me3) during mitosis, with αTubK40me3 required for genomic stability. We now show that monoallelic, Setd2-deficient cells retaining H3K36me3, but not αTubK40me3, exhibit a dramatic increase in mitotic defects and micronuclei count, with increased viability compared with biallelic loss. In SETD2-inactivated human kidney cells, rescue with a pathogenic SETD2 mutant deficient for microtubule (αTubK40me3), but not histone (H3K36me3) methylation, replicated this phenotype. Genomic instability (micronuclei) was also a hallmark of patient-derived cells from ccRCC. These data show that the SETD2 tumor suppressor displays a haploinsufficiency phenotype disproportionately impacting microtubule methylation and serves as an early driver of genomic instability.Significance: Loss of a single allele of a chromatin modifier plays a role in promoting oncogenesis, underscoring the growing relevance of tumor suppressor haploinsufficiency in tumorigenesis. Cancer Res; 78(12); 3135-46. ©2018 AACR.

SETting the Stage for Cancer Development: SETD2 and the Consequences of Lost Methylation.

The H3 lysine 36 histone methyltransferase SETD2 is mutated across a range of human cancers. Although other enzymes can mediate mono- and dimethylation, SETD2 is the exclusive trimethylase. SETD2 associates with the phosphorylated carboxy-terminal domain of RNA polymerase and modifies histones at actively transcribed genes. The functions associated with SETD2 are mediated through multiple effector proteins that bind trimethylated H3K36. These effectors directly mediate multiple chromatin-regulated processes, including RNA splicing, DNA damage repair, and DNA methylation. Although alterations in each of these processes have been associated with SETD2 loss, the relative role of each in the development of cancer is not fully understood. Critical vulnerabilities resulting from SETD2 loss may offer a strategy for potential therapeutics.

A tale of two cancers: Complete genetic analysis of chromophobe renal cell carcinoma contrasts with clear cell renal cell carcinoma.

The Cancer Genome Atlas undertook a comprehensive genetic analysis of chromophobe renal cell carcinoma, the first of the rare tumor types to be analyzed. This analysis identified the putative region of origin as the distal nephron. Alterations in mitochondrial function, mtDNA mutations, and recurrent structural rearrangements within the TERT promoter region were also identified.

The somatic genomic landscape of chromophobe renal cell carcinoma.

We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) on the basis of multidimensional and comprehensive characterization, including mtDNA and whole-genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared with other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT upregulation in cancer distinct from previously observed amplifications and point mutations.