Genome-wide screens identify specific drivers of mutant hTERT promoters.

Cancer-specific hTERT promoter mutations reported in 19% of cancers result in enhanced telomerase activity. Understanding the distinctions between transcriptional regulation of wild-type (WT) and mutant (Mut) hTERT promoters may open up avenues for development of inhibitors which specially block hTERT expression in cancer cells. To comprehensively identify physiological regulators of WT- or Mut-hTERT promoters, we generated several isogenic reporter cells driven by endogenous hTERT loci. Genome-wide CRISPR-Cas9 and small interfering RNA screens using these isogenic reporter lines identified specific regulators of Mut-hTERT promoters. We validate and characterize one of these hits, namely, MED12, a kinase subunit of mediator complex. We demonstrate that MED12 specifically drives expression of hTERT from the Mut-hTERT promoter by mediating long-range chromatin interaction between the proximal Mut-hTERT promoter and T-INT1 distal regulatory region 260 kb upstream. Several hits identified in our screens could serve as potential therapeutic targets, inhibition of which may specifically block Mut-hTERT promoter driven telomerase reactivation in cancers.

Integrated Genomic Profiling and Drug Screening of Patient-Derived Cultures Identifies Individualized Copy Number-Dependent Susceptibilities Involving PI3K Pathway and 17q Genes in Neuroblastoma.

Neuroblastoma is the commonest extracranial pediatric malignancy. With few recurrent single nucleotide variations (SNVs), mutation-based precision oncology approaches have limited utility, but its frequent and heterogenous copy number variations (CNVs) could represent genomic dependencies that may be exploited for personalized therapy. Patient-derived cell culture (PDC) models can facilitate rapid testing of multiple agents to determine such individualized drug-responses. Thus, to study the relationship between individual genomic aberrations and therapeutic susceptibilities, we integrated comprehensive genomic profiling of neuroblastoma tumors with drug screening of corresponding PDCs against 418 targeted inhibitors. We quantified the strength of association between copy number and cytotoxicity, and validated significantly correlated gene-drug pairs in public data and using machine learning models. Somatic mutations were infrequent (3.1 per case), but copy number losses in 1p (31%) and 11q (38%), and gains in 17q (69%) were prevalent. Critically, in-vitro cytotoxicity significantly correlated only with CNVs, but not SNVs. Among 1278 significantly correlated gene-drug pairs, copy number of GNA13 and DNA damage response genes CBL, DNMT3A, and PPM1D were most significantly correlated with cytotoxicity; the drugs most commonly associated with these genes were PI3K/mTOR inhibitor PIK-75, and CDK inhibitors P276-00, SNS-032, AT7519, flavopiridol and dinaciclib. Predictive Markov random field models constructed from CNVs alone recapitulated the true z-score-weighted associations, with the strongest gene-drug functional interactions in subnetworks involving PI3K and JAK-STAT pathways. Together, our data defined individualized dose-dependent relationships between copy number gains of PI3K and STAT family genes particularly on 17q and susceptibility to PI3K and cell cycle agents in neuroblastoma. Integration of genomic profiling and drug screening of patient-derived models of neuroblastoma can quantitatively define copy number-dependent sensitivities to targeted inhibitors, which can guide personalized therapy for such mutationally quiet cancers.

A chemical genetic screen identifies Aurora kinases as a therapeutic target in EGFR T790M negative, gefitinib-resistant head and neck squamous cell carcinoma (HNSCC).

BACKGROUND: Overexpression of epidermal growth factor receptor (EGFR), and downstream pathway activation appears to be a common oncogenic driver in the majority of head and neck squamous cell cancers (HNSCCs); yet targeting EGFR for the treatment of HNSCC has met with limited success. Apart from the anti-EGFR antibody cetuximab, no small molecule EGFR/tyrosine kinase inhibitors (TKIs) have progressed to routine clinical use. The aim of this study was to determine factors contributing to the lack of response to TKIs and identify alternative therapeutic vulnerabilities. METHODS: Genomic and transcriptomic sequencing, high-throughput compound screens, overexpression and siRNA knockdown, western blot, in vivo xenograft studies. FINDINGS: We derived three pairs of isogenic gefitinib (TKI)-sensitive and resistant patient-derived HNSCC cell lines. Genomic sequencing of gefitinib-resistant cell lines identified a lack of activating and resistance-associated EGFR mutations. Instead, transcriptomic sequencing showed upregulated EMT gene signature in the gefitinib-resistant cells with a corresponding increase in their migratory phenotype. Additionally, the resistant cell displayed reduced growth rate. Surprisingly, while gefitinib-resistant cells were independent of EGFR for survival, they nonetheless displayed activation of downstream ERK and AKT signalling. High-throughput screening (HTS) of druggable, small molecule libraries revealed that the gefitinib-resistant cells were particularly sensitive to inhibitors of genes involved in cell cycle and mitosis, such as Aurora kinase inhibitors (AKIs), cyclin-dependent kinase (CDK) inhibitors, and microtubule inhibitors. Notably our results showed that in the EGFR inhibited state, Aurora kinases are essential for cell survival. INTERPRETATION: Our study demonstrates that in the absence of activating EGFR mutations, HNSCCs may gain resistance to gefitinib through decreased cell proliferation, which makes them exceptionally vulnerable to cell-cycle inhibitors. FUNDING: Agency for Science, Technology, and Research (A*STAR), National Medical Research Council (NMRC), and the National Institutes of Health (NIH)/National Cancer Institute (NCI).

Targeting codon 158 p53-mutant cancers via the induction of p53 acetylation.

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 (Arg158) mutation, a DBD mutant found to be prevalent in lung carcinomas. Using high throughput compound screening and combination analyses, we uncover that acetylating mutp53R158G could render cancers susceptible to cisplatin-induced DNA stress. Acetylation of mutp53R158G 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 Arg158-mutp53 tumors utilizing a regimen consisting of DNA-damaging agents and mutp53 acetylators, which is currently being pursued clinically.

New High-Throughput Screening Identifies Compounds That Reduce Viability Specifically in Liver Cancer Cells That Express High Levels of SALL4 by Inhibiting Oxidative Phosphorylation.

BACKGROUND & AIMS: Some oncogenes encode transcription factors, but few drugs have been successfully developed to block their activity specifically in cancer cells. The transcription factor SALL4 is aberrantly expressed in solid tumor and leukemia cells. We developed a screen to identify compounds that reduce the viability of liver cancer cells that express high levels of SALL4, and we investigated their mechanisms. METHODS: We developed a stringent high-throughput screening platform comprising unmodified SNU-387 and SNU-398 liver cancer cell lines and SNU-387 cell lines engineered to express low and high levels of SALL4. We screened 1597 pharmacologically active small molecules and 21,575 natural product extracts from plant, bacteria, and fungal sources for those that selectively reduce the viability of cells with high levels of SALL4 (SALL4hi cells). We compared gene expression patterns of SALL4hi cells vs SALL4-knockdown cells using RNA sequencing and real-time polymerase chain reaction analyses. Xenograft tumors were grown in NOD/SCID gamma mice from SALL4hi SNU-398 or HCC26.1 cells or from SALL4lo patient-derived xenograft (PDX) cells; mice were given injections of identified compounds or sorafenib, and the effects on tumor growth were measured. RESULTS: Our screening identified 1 small molecule (PI-103) and 4 natural compound analogues (oligomycin, efrapeptin, antimycin, and leucinostatin) that selectively reduced viability of SALL4hi cells. We performed validation studies, and 4 of these compounds were found to inhibit oxidative phosphorylation. The adenosine triphosphate (ATP) synthase inhibitor oligomycin reduced the viability of SALL4hi hepatocellular carcinoma and non-small-cell lung cancer cell lines with minimal effects on SALL4lo cells. Oligomycin also reduced the growth of xenograft tumors grown from SALL4hi SNU-398 or HCC26.1 cells to a greater extent than sorafenib, but oligomycin had little effect on tumors grown from SALL4lo PDX cells. Oligomycin was not toxic to mice. Analyses of chromatin immunoprecipitation sequencing data showed that SALL4 binds approximately 50% of mitochondrial genes, including many oxidative phosphorylation genes, to activate their transcription. In comparing SALL4hi and SALL4-knockdown cells, we found SALL4 to increase oxidative phosphorylation, oxygen consumption rate, mitochondrial membrane potential, and use of oxidative phosphorylation-related metabolites to generate ATP. CONCLUSIONS: In a screening for compounds that reduce the viability of cells that express high levels of the transcription factor SALL4, we identified inhibitors of oxidative phosphorylation, which slowed the growth of xenograft tumors from SALL4hi cells in mice. SALL4 activates the transcription of genes that regulate oxidative phosphorylation to increase oxygen consumption, mitochondrial membrane potential, and ATP generation in cancer cells. Inhibitors of oxidative phosphorylation might be used for the treatment of liver tumors with high levels of SALL4.

Longitudinal single-cell RNA sequencing of patient-derived primary cells reveals drug-induced infidelity in stem cell hierarchy.

Chemo-resistance is one of the major causes of cancer-related deaths. Here we used single-cell transcriptomics to investigate divergent modes of chemo-resistance in tumor cells. We observed that higher degree of phenotypic intra-tumor heterogeneity (ITH) favors selection of pre-existing drug-resistant cells, whereas phenotypically homogeneous cells engage covert epigenetic mechanisms to trans-differentiate under drug-selection. This adaptation was driven by selection-induced gain of H3K27ac marks on bivalently poised resistance-associated chromatin, and therefore not expressed in the treatment-naïve setting. Mechanistic interrogation of this phenomenon revealed that drug-induced adaptation was acquired upon the loss of stem factor SOX2, and a concomitant gain of SOX9. Strikingly we observed an enrichment of SOX9 at drug-induced H3K27ac sites, suggesting that tumor evolution could be driven by stem cell-switch-mediated epigenetic plasticity. Importantly, JQ1 mediated inhibition of BRD4 could reverse drug-induced adaptation. These results provide mechanistic insights into the modes of therapy-induced cellular plasticity and underscore the use of epigenetic inhibitors in targeting tumor evolution.

Phenotype-driven precision oncology as a guide for clinical decisions one patient at a time.

Genomics-driven cancer therapeutics has gained prominence in personalized cancer treatment. However, its utility in indications lacking biomarker-driven treatment strategies remains limited. Here we present a "phenotype-driven precision-oncology" approach, based on the notion that biological response to perturbations, chemical or genetic, in ex vivo patient-individualized models can serve as predictive biomarkers for therapeutic response in the clinic. We generated a library of "screenable" patient-derived primary cultures (PDCs) for head and neck squamous cell carcinomas that reproducibly predicted treatment response in matched patient-derived-xenograft models. Importantly, PDCs could guide clinical practice and predict tumour progression in two n = 1 co-clinical trials. Comprehensive "-omics" interrogation of PDCs derived from one of these models revealed YAP1 as a putative biomarker for treatment response and survival in ~24% of oral squamous cell carcinoma. We envision that scaling of the proposed PDC approach could uncover biomarkers for therapeutic stratification and guide real-time therapeutic decisions in the future.Treatment response in patient-derived models may serve as a biomarker for response in the clinic. Here, the authors use paired patient-derived mouse xenografts and patient-derived primary culture models from head and neck squamous cell carcinomas, including metastasis, as models for high-throughput screening of anti-cancer drugs.