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Mutations in RAS isoforms (KRAS, NRAS, and HRAS) are among the most frequent oncogenic alterations in many cancers, making these proteins high priority therapeutic targets. Effectively targeting RAS isoforms requires an exact understanding of their active, inactive, and druggable conformations. However, there is no structural catalog of RAS conformations to guide therapeutic targeting or examining the structural impact of RAS mutations. Here we present an expanded classification of RAS conformations based on analyses of the catalytic switch 1 (SW1) and switch 2 (SW2) loops. From 721 human KRAS, NRAS, and HRAS structures available in the Protein Data Bank (206 RAS–protein cocomplexes, 190 inhibitor-bound, and 325 unbound, including 204 WT and 517 mutated structures), we created a broad conformational classification based on the spatial positions of Y32 in SW1 and Y71 in SW2. Clustering all well-modeled SW1 and SW2 loops using a density-based machine learning algorithm defined additional conformational subsets, some previously undescribed. Three SW1 conformations and nine SW2 conformations were identified, each associated with different nucleotide states (GTP-bound, nucleotide-free, and GDP-bound) and specific bound proteins or inhibitor sites. The GTP-bound SW1 conformation could be further subdivided on the basis of the hydrogen bond type made between Y32 and the GTP γ-phosphate. Further analysis clarified the catalytic impact of G12D and G12V mutations and the inhibitor chemistries that bind to each druggable RAS conformation. Overall, this study has expanded our understanding of RAS structural biology, which could facilitate future RAS drug discovery.Significance:Analysis of >700 RAS structures helps define an expanded landscape of active, inactive, and druggable RAS conformations, the structural impact of common RAS mutations, and previously uncharacterized RAS inhibitor–binding modes.

Supplementary Data from Delineating the RAS Conformational Landscape

Supplementary Data from Association of TP53 and CDKN2A Mutation Profile with Tumor Mutation Burden in Head and Neck Cancer

Purpose:Head and neck squamous cell carcinoma (HNSCC) is a frequently devastating cancer that affects more than a half million people annually worldwide. Although some cases arise from infection with human papillomavirus (HPV), HPV-negative HNSCC is more common, and associated with worse outcome. Advanced HPV-negative HNSCC may be treated with surgery, chemoradiation, targeted therapy, or immune checkpoint inhibition (ICI). There is considerable need for predictive biomarkers for these treatments. Defects in DNA repair capacity and loss of cell-cycle checkpoints sensitize tumors to cytotoxic therapies, and can contribute to phenotypes such as elevated tumor mutation burden (TMB), associated with response to ICI. Mutation of the tumor suppressors and checkpoint mediators TP53 and CDKN2A is common in HPV-negative HNSCC.Experimental Design:To gain insight into the relation of the interaction of TP53 and CDKN2A mutations with TMB in HNSCC, we have analyzed genomic data from 1,669 HPV-negative HNSCC tumors with multiple criteria proposed for assessing the damaging effect of TP53 mutations.Results:Data analysis established the TP53 and CDKN2A mutation profiles in specific anatomic subsites and suggested that specific categories of TP53 mutations are more likely to associate with CDKN2A mutation or high TMB based on tumor subsite. Intriguingly, the pattern of hotspot mutations in TP53 differed depending on the presence or absence of a cooccurring CDKN2A mutation.Conclusions:These data emphasize the role of tumor subsite in evaluation of mutational profiles in HNSCC, and link defects in TP53 and CDKN2A to elevated TMB levels in some tumor subgroups.

Oncogenic KRAS mutations occur in approximately 30% of lung adenocarcinoma. Despite several decades of effort, oncogenic KRAS-driven lung cancer remains difficult to treat, and our understanding of the positive and negative regulators of RAS signaling is incomplete. To uncover the functional impact of diverse KRAS-interacting proteins on lung cancer growth in vivo, we used multiplexed somatic CRISPR/Cas9-based genome editing in genetically engineered mouse models with tumor barcoding and high-throughput barcode sequencing. Through a series of CRISPR/Cas9 screens in autochthonous lung tumors, we identified HRAS and NRAS as key suppressors of KRASG12D-driven tumor growth in vivo and confirmed these effects in oncogenic KRAS-driven human lung cancer cell lines. Mechanistically, RAS paralogs interact with oncogenic KRAS, suppress KRAS-KRAS interactions, and reduce downstream ERK signaling. HRAS mutations identified in KRAS-driven human tumors partially abolished this effect. Comparison of the tumor-suppressive effects of HRAS and NRAS in KRAS- and BRAF-driven lung cancer models confirmed that RAS paralogs are specific suppressors of oncogenic KRAS-driven lung cancer in vivo. Our study outlines a technological avenue to uncover positive and negative regulators of oncogenic KRAS-driven cancer in a multiplexed manner in vivo and highlights the role of RAS paralog imbalance in oncogenic KRAS-driven lung cancer.

Cryo-EM structure of TFIIK provides insight into CTD phosphorylation of RNA polymerase II in the preinitiation-Mediator complex., During transcription initiation, the general transcription factor TFIIH marks RNA polymerase II by phosphorylating Ser5 of the carboxyl-terminal domain (CTD) of Rpb1, which is followed by extensive modifications coupled to transcription elongation, mRNA processing, and histone dynamics. We have determined a 3.5-Å resolution cryo–electron microscopy (cryo-EM) structure of the TFIIH kinase module (TFIIK in yeast), which is composed of Kin28, Ccl1, and Tfb3, yeast homologs of CDK7, cyclin H, and MAT1, respectively. The carboxyl-terminal region of Tfb3 was lying at the edge of catalytic cleft of Kin28, where a conserved Tfb3 helix served to stabilize the activation loop in its active conformation. By combining the structure of TFIIK with the previous cryo-EM structure of the preinitiation complex, we extend the previously proposed model of the CTD path to the active site of TFIIK.

A primary pathological feature of polycystic kidney disease (PKD) is the hyperproliferation of epithelial cells in renal tubules, resulting in formation of fluid-filled cysts. The proliferative aspects of the two major forms of PKD—autosomal dominant PKD (ADPKD), which arises from mutations in the polycystins PKD1 and PKD2, and autosomal recessive PKD (ARPKD), which arises from mutations in PKHD1—has encouraged investigation into protein components of the core cell proliferative machinery as potential drivers of PKD pathogenesis. In this review, we examine the role of signaling by ERBB proteins and their effectors, with a primary focus on ADPKD. The ERBB family of receptor tyrosine kinases (EGFR/ERBB1, HER2/ERBB2, ERBB3, and ERBB4) are activated by extracellular ligands, inducing multiple pro-growth signaling cascades; among these, activation of signaling through the RAS GTPase, and the RAF, MEK1/2, and ERK1/2 kinases enhance cell proliferation and restrict apoptosis during renal tubuloepithelial cyst formation. Characteristics of PKD include overexpression and mislocalization of the ERBB receptors and ligands, leading to enhanced activation and increased activity of downstream signaling proteins. The altered regulation of ERBBs and their effectors in PKD is influenced by enhanced activity of SRC kinase, which is promoted by the loss of cytoplasmic Ca2+ and an increase in cAMP-dependent PKA kinase activity that stimulates CFTR, driving the secretory phenotype of ADPKD. We discuss the interplay between ERBB/SRC signaling, and polycystins and their depending signaling, with emphasis on thes changes that affect cell proliferation in cyst expansion, as well as the inflammation-associated fibrogenesis, which characterizes progressive disease. We summarize the current progress of preclinical and clinical trials directed at inhibiting this signaling axis, and discuss potential future strategies that may be productive for controlling PKD.

6552 Background: The tumor suppressors TP53 and CDKN2A are commonly mutated or lost in HNSCC, impairing G1 checkpoints. This reduces ability to repair DNA damage arising from hypoxia, replication stress, and mutagen exposure, thus increasing TMB, a potential predictive biomarker for immunotherapy benefit. TP53 mutations can be classified as loss-of-function (LOF) with or without dominant negative (DNE) activity, gain-of-function (GOF) and benign. We investigated whether specific categories of TP53 mutation were associated with increased TMB, and whether these cooperated with CDKN2A mutation to elevate TMB. Methods: We analyzed 1010 HPV- HNSCC tumor samples (246 female) profiled with a 592-gene panel by Caris Life Sciences from 2015 to 2019. Predominant subsites were oral cavity (285), oropharynx (225) and larynx (153). TMB reflected all somatic nonsynonymous missense mutations detected. We report mean TMB per megabase (MB). Pathogenicity of TP53 and CDKN2A mutations was determined according to American College of Medical Genetics (ACMG) guidelines. We also used four alternative methods of characterizing TP53 mutations based on analysis of protein structure, public databases (IARC, ClinVar, InterVar), and publications (PMID: 25108461 and others) assessing structure-function relations. Results: 60% of cases had TP53 mutations ( TP53mut) designated pathogenic by ACMG guidelines. Estimates of frequency of LOF/DNE mutations ranged from 30-42.8% of cases among the alternative classification methods. Damaging CDKN2A mutations were present in 20%. Average TMB per MB varied from 8.2/8.6 (females/males) in oral cavity cancers to 26.5/27.7 (females/males) in cancer of the lip. Mean TMB was typically higher in the presence of damaging LOF/DNE TP53 mutations or CDKN2A mutations, but not TP53 GOF mutations. Based on ACMG, for tumors with TP53 and CDKN2A wild type (WT) TMB was 8.03, for those with CDKN2Amut-only 9.82, for TP53mut-only 10.56, and TP53 mut/CDKN2A mut 17.6 (p < 0.001). For disruptive TP53mut (Poeta algorithm), mean TMB for WT/WT was 8.67, for TP53mut 11.31, CDKN2Amut 17.9 and TP53mut/CDKN2A mut 15.83 (p < 0.001). Conclusions: Mutation of TP53 and/or CDKN2A is associated with increased mean TMB relative to WT; mean TMB was highest for tumors bearing damaging mutations in both genes. GOF TP53 mutation was not clearly associated with increased TMB. As TMB is evaluated as a predictive biomarker in the immunotherapy of HNSCC, specific TP53/CDKN2A mutational status should also be evaluated.

Epithelioid angiosarcoma is a rare high-grade tumor with irregular vascular morphology. We report an unusual case of intra-abdominal epithelioid angiosarcoma affecting the omentum and peritoneal surfaces resulting in significant hemorrhagic and inflammatory changes. As in other cases of this tumor this patient had previously undergone radiation treatment for a history of cervical cancer.

PROBLEM Little is known about how infection and the response to inflammation affect the microRNA (miRNA) profile of the male reproductive tract. We hypothesized that expression of inflammatory-related miRNAs would be altered following immune activation of rat testis. METHOD OF STUDY Testis total RNA was purified from Sprague-Dawley rats 3 or 6 hours after receiving a 5 mg/kg intraperitoneal injection of bacterial lipopolysaccharide (LPS) and examined by qPCR using an 84-panel miRNA array. RESULTS Five inflammatory-related miRNAs showed a greater than twofold downregulation (P

Schwannomas are benign, often asymptomatic, slow-growing tumors that originate from Schwann cells of the neural sheath. Although H. Pylori has been associated with gastric adenocarcinoma, there has never been a recorded association with schwannoma formation. We present a 64-year-old woman who underwent a laparoscopic partial wedge gastrectomy for an incidentally discovered gastric mass. Histologic examination was consistent with schwannoma; however, chronic inflammation with microorganisms morphologically consistent with H. Pylori was also present. This case suggests the first recorded case of H. Pylori in an immunohistochemically confirmed gastric schwannoma.