Saurabh Mendiratta

Systematic Identification of Molecular Subtype-Selective Vulnerabilities in Non-Small-Cell Lung Cancer

Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6%-16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature. Target efficacies were validated in vivo, and mechanism-of-action studies informed generalizable principles underpinning cancer cell biology.

XPO1-dependent nuclear export is a druggable vulnerability in KRAS-mutant lung cancer

The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred persistent and intensive efforts to develop drugs that inhibit KRAS activity. However, advances have been hindered by the pervasive inter- and intra-lineage diversity in the targetable mechanisms that underlie KRAS-driven cancers, limited pharmacological accessibility of many candidate synthetic-lethal interactions and the swift emergence of unanticipated resistance mechanisms to otherwise effective targeted therapies. Here we demonstrate the acute and specific cell-autonomous addiction of KRAS-mutant non-small-cell lung cancer cells to receptor-dependent nuclear export. A multi-genomic, data-driven approach, utilizing 106 human non-small-cell lung cancer cell lines, was used to interrogate 4,725 biological processes with 39,760 short interfering RNA pools for those selectively required for the survival of KRAS-mutant cells that harbour a broad spectrum of phenotypic variation. Nuclear transport machinery was the sole process-level discriminator of statistical significance. Chemical perturbation of the nuclear export receptor XPO1 (also known as CRM1), with a clinically available drug, revealed a robust synthetic-lethal interaction with native or engineered oncogenic KRAS both in vitro and in vivo. The primary mechanism underpinning XPO1 inhibitor sensitivity was intolerance to the accumulation of nuclear IκBα (also known as NFKBIA), with consequent inhibition of NFκB transcription factor activity. Intrinsic resistance associated with concurrent FSTL5 mutations was detected and determined to be a consequence of YAP1 activation via a previously unappreciated FSTL5–Hippo pathway regulatory axis. This occurs in approximately 17% of KRAS-mutant lung cancers, and can be overcome with the co-administration of a YAP1–TEAD inhibitor. These findings indicate that clinically available XPO1 inhibitors are a promising therapeutic strategy for a considerable cohort of patients with lung cancer when coupled to genomics-guided patient selection and observation.

Using Functional Signature Ontology (FUSION) to Identify Mechanisms of Action for Natural Products

Grouping genetic perturbations and marine-derived compounds by the gene signatures they produce reveals potential therapeutics and their targets. Guilt by Association Cell-based screens routinely yield many small molecules that show promise as potential drugs. However, drug development requires identifying the targets of these molecules, a process that is often time-consuming and expensive. Potts et al. report an approach that combines the initial screening of molecules with the ability to assign putative biological functions. The expression of six genes was used as a surrogate for cell function to screen libraries of siRNAs, microRNAs, and natural products derived from marine species. Reagents were grouped together and assumed to have similar effects on cell functions if they produced similar gene signatures. This approach identified gene products and natural product compounds that regulated processes as diverse as the breakdown of intracellular components, directed cell migration, and survival signaling. Thus, this approach holds promise for facilitating future drug discovery. A challenge for biomedical research is the development of pharmaceuticals that appropriately target disease mechanisms. Natural products can be a rich source of bioactive chemicals for medicinal applications but can act through unknown mechanisms and can be difficult to produce or obtain. To address these challenges, we developed a new marine-derived, renewable natural products resource and a method for linking bioactive derivatives of this library to the proteins and biological processes that they target in cells. We used cell-based screening and computational analysis to match gene expression signatures produced by natural products to those produced by small interfering RNA (siRNA) and synthetic microRNA (miRNA) libraries. With this strategy, we matched proteins and miRNAs with diverse biological processes and also identified putative protein targets and mechanisms of action for several previously undescribed marine-derived natural products. We confirmed mechanistic relationships for selected siRNAs, miRNAs, and compounds with functional roles in autophagy, chemotaxis mediated by discoidin domain receptor 2, or activation of the kinase AKT. Thus, this approach may be an effective method for screening new drugs while simultaneously identifying their targets.

Host Modulators of H1N1 Cytopathogenicity

Influenza A virus infects 5–20% of the population annually, resulting in ∼35,000 deaths and significant morbidity. Current treatments include vaccines and drugs that target viral proteins. However, both of these approaches have limitations, as vaccines require yearly development and the rapid evolution of viral proteins gives rise to drug resistance. In consequence additional intervention strategies, that target host factors required for the viral life cycle, are under investigation. Here we employed arrayed whole-genome siRNA screening strategies to identify cell-autonomous molecular components that are subverted to support H1N1 influenza A virus infection of human bronchial epithelial cells. Integration across relevant public data sets exposed druggable gene products required for epithelial cell infection or required for viral proteins to deflect host cell suicide checkpoint activation. Pharmacological inhibition of representative targets, RGGT and CHEK1, resulted in significant protection against infection of human epithelial cells by the A/WS/33 virus. In addition, chemical inhibition of RGGT partially protected against H5N1 and the 2009 H1N1 pandemic strain. The observations reported here thus contribute to an expanding body of studies directed at decoding vulnerabilities in the command and control networks specified by influenza virulence factors.

RASSF1A inactivation unleashes a tumor suppressor/oncogene cascade with context-dependent consequences on cell cycle progression.

ABSTRACT The RASSF1A gene is one of the most frequently inactivated genes in over 30 different types of cancers (H. Donninger, M. D. Vos, and G. J. Clark, J. Cell Sci. 120:3163–3172, 2007, http://dx.doi.org/10.1242/jcs.010389). Despite the prevalence of RASSF1A silencing in human cancer, the mechanism by which RASSF1A functions as a tumor suppressor is not well understood. Characterization of the consequences of RASSF1A loss on epithelial cell proliferation revealed that RASSF1A expression suppresses both microRNA 21 (miR-21) expression and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. The mechanism of the former is through restraint of SCFβTrCP-dependent destruction of the repressor element 1 silencing transcription factor (REST) tumor suppressor and consequent inhibition of miR-21 promoter activation. The mechanism of the latter is through physical sequestration of MST2, which results in accumulation of inactivating S259 phosphorylation of RAF1. Whether or not inactivation of these RASSF1A regulatory relationships can unleash enhanced proliferative capacity is dependent upon the coupling of SCFβTrCP and miR-21 to suppression of SKP2 protein translation and stability. Airway epithelial cultures retain this coupling and therefore respond to RASSF1A inactivation by p27-dependent cell cycle arrest. In contrast, colonic crypt-derived epithelial cells have uncoupled SCFβTrCP from SKP2 and respond to RASSF1A inactivation by enhanced proliferation rates. These observations help account for context-specific molecular etiology of oncogenic transformation and suggest intervention strategies for recently developed SKP2 inhibitors.

Exploiting the CRISPR/Cas9 PAM constraint for single-nucleotide resolution interventions

CRISPR/Cas9 is an enabling RNA-guided technology for genome targeting and engineering. An acute DNA binding constraint of the Cas9 protein is the Protospacer Adjacent Motif (PAM). Here we demonstrate that the PAM requirement can be exploited to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on the wild-type alleles. Specifically, we target the heterozygous G13A activating mutation of KRAS in colorectal cancer cells and we show reversal of drug resistance to a MEK small-molecule inhibitor. Our study introduces a new paradigm in genome editing and therapeutic targeting via the use of gRNA to guide Cas9 to a desired protospacer adjacent motif.

Small-molecule TFEB pathway agonists that ameliorate metabolic syndrome in mice and extend C. elegans lifespan

Drugs that mirror the cellular effects of starvation mimics are considered promising therapeutics for common metabolic disorders, such as obesity, liver steatosis, and for ageing. Starvation, or caloric restriction, is known to activate the transcription factor EB (TFEB), a master regulator of lipid metabolism and lysosomal biogenesis and function. Here, we report a nanotechnology-enabled high-throughput screen to identify small-molecule agonists of TFEB and discover three novel compounds that promote autophagolysosomal activity. The three lead compounds include the clinically approved drug, digoxin; the marine-derived natural product, ikarugamycin; and the synthetic compound, alexidine dihydrochloride, which is known to act on a mitochondrial target. Mode of action studies reveal that these compounds activate TFEB via three distinct Ca2+-dependent mechanisms. Formulation of these compounds in liver-tropic biodegradable, biocompatible nanoparticles confers hepatoprotection against diet-induced steatosis in murine models and extends lifespan of Caenorhabditis elegans. These results support the therapeutic potential of small-molecule TFEB activators for the treatment of metabolic and age-related disorders.Activation of autophagy, via the transcription factor TFEB, is a promising strategy to treat metabolic diseases. Here, the authors report three novel classes of small molecules that promote TFEB nuclear translocation, and provide evidence for the therapeutic efficacy of these compounds in mice and worms.

Biomarker Accessible and Chemically Addressable Mechanistic Subtypes of BRAF Melanoma

Genomic diversity among melanoma tumors limits durable control with conventional and targeted therapies. Nevertheless, pathologic activation of the ERK1/2 pathway is a linchpin tumorigenic mechanism associated with the majority of primary and recurrent disease. Therefore, we sought to identify therapeutic targets that are selectively required for tumorigenicity in the presence of pathologic ERK1/2 signaling. By integration of multigenome chemical and genetic screens, recurrent architectural variants in melanoma tumor genomes, and patient outcome data, we identified two mechanistic subtypes of BRAFV600 melanoma that inform new cancer cell biology and offer new therapeutic opportunities. Subtype membership defines sensitivity to clinical MEK inhibitors versus TBK1/IKBKε inhibitors. Importantly, subtype membership can be predicted using a robust quantitative five-feature genetic biomarker. This biomarker, and the mechanistic relationships linked to it, can identify a cohort of best responders to clinical MEK inhibitors and identify a cohort of TBK1/IKBKε inhibitor-sensitive disease among nonresponders to current targeted therapy.Significance: This study identified two mechanistic subtypes of melanoma: (1) the best responders to clinical BRAF/MEK inhibitors (25%) and (2) nonresponders due to primary resistance mechanisms (9.9%). We identified robust biomarkers that can detect these subtypes in patient samples and predict clinical outcome. TBK1/IKBKε inhibitors were selectively toxic to drug-resistant melanoma. Cancer Discov; 7(8); 832-51. ©2017 AACR.See related commentary by Jenkins and Barbie, p. 799This article is highlighted in the In This Issue feature, p. 783.

Chemistry-First Approach for Nomination of Personalized Treatment in Lung Cancer

Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.

Synthesis and structure–activity studies of the V-ATPase inhibitor saliphenylhalamide (SaliPhe) and simplified analogs

An efficient total synthesis of the potent V-ATPase inhibitor saliphenylhalamide (SaliPhe), a synthetic variant of the natural product salicylihalamide A (SaliA), has been accomplished aimed at facilitating the development of SaliPhe as an anticancer and antiviral agent. This new approach enabled facile access to derivatives for structure-activity relationship studies, leading to simplified analogs that maintain SaliPhes biological properties. These studies will provide a solid foundation for the continued evaluation of SaliPhe and analogs as potential anticancer and antiviral agents.