A widespread approach to modern cancer therapy is to identify a single oncogenic driver gene and target its mutant protein product (e.g. EGFR inhibitor treatment in EGFR-mutant lung cancers). However, genetically-driven resistance to targeted therapy limits patient survival. Through genomic analysis of 1122 EGFR-mutant lung cancer cell-free DNA samples and whole exome analysis of seven longitudinally collected tumor samples from an EGFR-mutant lung cancer patient, we identify critical co-occurring oncogenic events present in most advanced-stage EGFR-mutant lung cancers. We define new pathways limiting EGFR inhibitor response, including WNT/β-catenin and cell cycle gene (e.g. CDK4, CDK6) alterations. Tumor genomic complexity increases with EGFR inhibitor treatment and co-occurring alterations in CTNNB1, and PIK3CA exhibit non-redundant functions that cooperatively promote tumor metastasis or limit EGFR inhibitor response. This study challenges the prevailing single-gene driver oncogene view and links clinical outcomes to co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancer patients.

Here we report that the transcription factor CREB-H is required for the maintenance of normal plasma triglyceride (TG) levels. CREB-H deficient mice displayed hypertriglyceridemia (HTG) secondary to inefficient TG clearance catalyzed by lipoprotein lipase (Lpl), partly due to defective expression of the Lpl coactivators, Apoc2, Apoa4, and Apoa5 and concurrent augmentation of the Lpl inhibitor, Apoc3. Multiple nonsynonymous mutations in CREB3L3 that produced hypomorphic or nonfunctional CREB-H protein were identified in patients with extreme HTG, implicating a critical role for CREB-H in human TG metabolism.

Purpose: Concomitant genetic alterations could account for transient clinical responses to tyrosine kinase inhibitors of the EGF receptor (EGFR) in patients harboring activating EGFR mutations.Experimental Design: We have evaluated the impact of pretreatment somatic EGFR T790M mutations, TP53 mutations, and Bcl-2 interacting mediator of cell death (BCL2L11, also known as BIM) mRNA expression in 95 patients with EGFR-mutant non-small-cell lung cancer (NSCLC) included in the EURTAC trial (trial registration: NCT00446225).Results: T790M mutations were detected in 65.26% of patients using our highly sensitive method based on laser microdissection and peptide-nucleic acid-clamping PCR, which can detect the mutation at an allelic dilution of 1 in 5,000. Progression-free survival (PFS) to erlotinib was 9.7 months for those with T790M mutations and 15.8 months for those without, whereas among patients receiving chemotherapy, it was 6 and 5.1 months, respectively (P < 0.0001). PFS to erlotinib was 12.9 months for those with high and 7.2 months for those with low/intermediate BCL2L11 expression levels, whereas among chemotherapy-treated patients, it was 5.8 and 5.5 months, respectively (P ¼ 0.0003). Overall survival was 28.6 months for patients with high BCL2L11 expression and 22.1 months for those with low/intermediate BCL2L11 expression (P ¼ 0.0364). Multivariate analyses showed that erlotinib was a marker of longer PFS (HR ¼ 0.35; P ¼ 0.0003), whereas high BCL2L11 expression was a marker of longer PFS (HR ¼ 0.49; P ¼ 0.0122) and overall survival (HR ¼ 0.53; P ¼ 0.0323).Conclusions: Low-level pretreatment T790M mutations can frequently be detected and can be used for customizing treatment with T790M-specific inhibitors. BCL2L11 mRNA expression is a biomarker of survival in EGFR-mutant NSCLC and can potentially be used for synthetic lethality therapies.

Pleuropulmonary blastoma is a rare childhood malignancy of lung mesenchymal cells that can remain dormant as epithelial cysts or progress to high-grade sarcoma. Predisposing germline loss-of-function DICER1 variants have been described. We sought to uncover additional contributors through whole exome sequencing of 15 tumor/normal pairs, followed by targeted resequencing, miRNA analysis and immunohistochemical analysis of additional tumors. In addition to frequent biallelic loss  of TP53 and mutations of NRAS or BRAF in some cases, each case had compound disruption of DICER1: a germline (12 cases) or somatic (3 cases) loss-of-function variant plus a somatic missense mutation in the RNase IIIb domain. 5p-Derived microRNA (miRNA) transcripts retained abnormal precursor miRNA loop sequences normally removed by DICER1. This work both defines a genetic interaction landscape with DICER1 mutation and provides evidence for alteration in miRNA transcripts as a consequence of DICER1 disruption in cancer.

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. Here, we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT-PCR)-derived cycle threshold (C t ) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.

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Nhp6A is an abundant non-histone chromatin-associated protein in Saccharomyces cerevisiae that contains a minor groove DNA binding motif called the HMG box. In this report, we show that Nhp6Ap binds to cisplatin intrastrand cross-links on duplex DNA with a 40-fold greater affinity than to unmodified DNA with the same sequence. Nevertheless, Nhp6Ap bound to cisplatinated DNA readily exchanges onto unmodified DNA. Phenanthroline-copper footprinting and two-dimensional NMR on complexes of wild-type and mutant Nhp6Ap with DNA were employed to probe the mode of binding to the cisplatin lesion. Recognition of the cisplatin adduct requires a surface-exposed phenylalanine on Nhp6Ap that promotes bending of DNA by inserting into the helix from the minor groove. We propose that Nhp6Ap targets the cisplatin adduct by means of intercalation by the phenylalanine and that it can bind in either orientation with respect to the DNA lesion. A methionine, which also inserts between base pairs and functions in target selection on unmodified DNA, plays no apparent role in recognition of the cisplatin lesion. Basic amino acids within the N-terminal arm of Nhp6Ap are required for high-affinity binding to the cisplatin adduct as well as to unmodified DNA. Cisplatin mediates its cytotoxicity by forming covalent adducts on DNA, and we find that Deltanhp6a/b mutants are hypersensitive to cisplatin in comparison with the wild-type strain. In contrast, Deltanhp6a/b mutants are slightly more resistant to hydrogen peroxide and ultraviolet irradiation. Therefore, Nhp6A/Bp appears to directly or indirectly function in yeast to enhance cellular resistance to cisplatin.

Activation of the phosphoinositide 3-kinase (PI3K) pathway occurs widely in human cancers. Although somatic mutations in the PI3K pathway genes PIK3CA and PTEN are known to drive PI3K pathway activation and cancer growth, the significance of somatic mutations in other PI3K pathway genes is less clear. Here, we establish the signaling and oncogenic properties of a recurrent somatic mutation in the PI3K p110β isoform that resides within its kinase domain (PIK3Cβ(D1067V)). We initially observed PIK3Cβ(D1067V) by exome sequencing analysis of an EGFR-mutant non-small cell lung cancer (NSCLC) tumor biopsy from a patient with acquired erlotinib resistance. On the basis of this finding, we hypothesized that PIK3Cβ(D1067V) might function as a novel tumor-promoting genetic alteration, and potentially an oncogene, in certain cancers. Consistent with this hypothesis, analysis of additional tumor exome data sets revealed the presence of PIK3Cβ(D1067V) at low frequency in other patient tumor samples (including renal cell carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, melanoma, thyroid carcinoma and endometrial carcinoma). Functional studies revealed that PIK3Cβ(D1067V) promoted PI3K pathway signaling, enhanced cell growth in vitro, and was sufficient for tumor formation in vivo. Pharmacologic inhibition of PIK3Cβ with TGX-221 (isoform-selective p110β inhibitor) specifically suppressed growth in patient-derived renal-cell carcinoma cells with endogenous PIK3Cβ(D1067V) and in NIH-3T3 and human EGFR-mutant lung adenocarcinoma cells engineered to express this mutant PI3K. In the EGFR-mutant lung adenocarcinoma cells, expression of PIK3Cβ(D1067V) also promoted erlotinib resistance. Our data establish a novel oncogenic form of PI3K, revealing the signaling and oncogenic properties of PIK3Cβ(D1067V) and its potential therapeutic relevance in cancer. Our findings provide new insight into the genetic mechanisms underlying PI3K pathway activation in human tumors and indicate that PIK3Cβ(D1067V) is a rational therapeutic target in certain cancers.