Gain of function (GOF) DNA binding domain (DBD) mutations of TP53 upregulate chromatin regulatory genes that promote genome-wide histone methylation and acetylation. Here, we therapeutically exploit the oncogenic GOF mechanisms of p53 codon 158 (Arg 158) mutation, a DBD mutant found to be prevalent in lung carcinomas. Using high throughput compound screening and combination analyses, we uncover that acetylating mutp53 R158G could render cancers susceptible to cisplatin-induced DNA stress. Acetylation of mutp53 R158G alters DNA binding motifs and upregulates TRAIP, a RING domain-containing E3 ubiquitin ligase which dephosphorylates IĸB and impedes nuclear translocation of RelA (p65), thus repressing oncogenic nuclear factor kappa-B (NF-ĸB) signaling and inducing apoptosis. Given that this mechanism of cytotoxic vulnerability appears inapt in p53 wild-type (WT) or other hotspot GOF mutp53 cells, our work provides a therapeutic opportunity specific to Arg 158-mutp53 tumors utilizing a regimen consisting of DNA-damaging agents and mutp53 acetylators, which is currently being pursued clinically.

MET receptors are activated in cancers through genomic events like tyrosine kinase domain mutations, juxtamembrane splicing mutation and amplified copy numbers, which can be inhibited by c-MET small molecule inhibitors. Here, we discover that the most common polymorphism known to affect MET gene (N375S), involving the semaphorin domain, confers exquisite binding affinity for HER2 and enables MET N375S to interact with HER2 in a ligandindependent fashion. The resultant MET N375S /HER2 dimer transduces potent proliferative, pro-invasive and pro-metastatic cues through the HER2 signaling axis to drive aggressive squamous cell carcinomas of the head and neck (HNSCC) and lung (LUSC), and is associated with poor prognosis. Accordingly, HER2 blockers, but not c-MET inhibitors, are paradoxically effective at restraining in vivo and in vitro models expressing MET N375S . These results establish MET N375S as a biologically distinct and clinically actionable molecular subset of SCCs that are uniquely amenable to HER2 blocking therapies.

as well as from patients who have undergone surgical resection at NUH. Germline DNA was obtained retrospectively from Pharmacogenetics DNA Bank and prospectively from patients with metastatic LUSC, HNSCC and NPC. All samples were processed with approval from National Healthcare Group's Domain Specific Review Board (DSRB) Committee, and under the guideline of Institutional Review Board (IRB) at National University of Singapore (NUS, Singapore). Written informed consent was obtained in all cases from patients at time of enrolment. Mass spectrometry analysis.Samples were treated by in-gel digestion prior to MS analysis. In brief, samples were reduced in 10 mM DTT for 1h at 56°C followed by alkylation with 55mM iodoacetamide (Sigma) for 45 min in the dark. Tryptic digest was performed in 50 mM ammonium bicarbonate buffer with 2 μg trypsin at 37°C overnight. Peptides were desalted on StageTips and analysed by nanoflow liquid chromatography on an EASY-nLC 1200 system coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Peptides were separated on a C18-reversed phase column (25 cm long, 75 μm inner diameter) packed in-house with ReproSil-Pur C18-QAQ 1.9 μm resin (Dr Maisch). The column was mounted on an Easy Flex Nano Source and temperature controlled by a column oven (Sonation) at 40°C. A 105-min gradient from 2 to 40% acetonitrile in 0.5% formic acid at a flow of 225 nl/min was used. Spray voltage was set to 2.4 kV. The Q Exactive HF was operated with a TOP20 MS/MS spectra acquisition method per MS full scan. MS scans were conducted with 60,000 and MS/MS scans with 15,000 resolution. The raw files were processed with MaxQuant version 1.5.2.8 1 with preset standard settings for SILAC labelled samples and the re-quantify option was activated.Carbamidomethylation was set as fixed modification while methionine oxidation and protein N-acetylation were considered as variable modifications. Search results were filtered with a false discovery rate of 0.01. Cell viability assay.Approximately 3,000 cells/well in complete medium were seeded into clear-bottom black 96well plates 24 hours prior to drug treatment. Indicated compounds or rSema were added in serial dilution for 72 hours. The viability of cells were assayed using CellTiter-Glo luminescent reagent (Promega, #G7572). The luminescence signals were detected using TECAN Infinite M1000 pro multi-mode plate reader using an integration time of 1000 ms.The relative luminescence units from treated wells were normalized against the readings from DMSO control and expressed as percentage cell viability and presented as mean ± SD relative to DMSO-treated controls. IC50 values were calculated by curve-fitting using GraphPad Prism software, and expressed as mean value ± SD. RNAseq, EMT scoring, hierarchical clustering and pathway analysis.RNAseq and gene expression analyses were performed by Novogene. In brief, isogenic H2170 cell lines of wild type (WT) and MET N375S genotypes were subjected to Illumina HiSeqTM2500 for RNA-seq. CASAVA v1.8 was used to convert the...