ATM Deficiency Is Associated with Sensitivity to PARP1-and ATR Inhibitors in Lung Adenocarcinoma

Defects in maintaining genome integrity are a hallmark of cancer. The DNA damage response kinase ATM is frequently mutated in human cancer, but the significance of these events to chemotherapeutic efficacy has not been examined deeply in whole organism models. Here we demonstrate that bi-allelic Atm deletion in mouse models of Kras-mutant lung adenocarcinoma does not affect cisplatin responses. In marked contrast, Atm-deficient tumors displayed an enhanced response to the topoisomerase-II poison etoposide. Moreover, Atm-deficient cells and tumors were sensitive to the PARP inhibitor olaparib. This actionable molecular addiction to functional PARP1 signaling was preserved in models that were proficient or deficient in p53, resembling standard or high-risk genetic constellations, respectively. Atm deficiency also markedly enhanced sensitivity to the ATR inhibitor VE-822. Taken together, our results provide a functional rationale to profile human tumors for disabling ATM mutations, particularly given their impact on PARP1 and ATR inhibitors. (C) 2017 AACR

A Functional Cancer Genomics Screen Identifies a Druggable Synthetic Lethal Interaction between MSH3 and PRKDC

Here, we use a large-scale cell line-based approach to identify cancer cell-specific mutations that are associated with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) dependence. For this purpose, we profiled the mutational landscape across 1,319 cancer-associated genes of 67 distinct cell lines and identified numerous genes involved in homologous recombination-mediated DNA repair, including BRCA1, BRCA2, ATM, PAXIP, and RAD50, as being associated with non-oncogene addiction to DNA-PKcs. Mutations in the mismatch repair gene MSH3, which have been reported to occur recurrently in numerous human cancer entities, emerged as the most significant predictors of DNA-PKcs addiction. Concordantly, DNA-PKcs inhibition robustly induced apoptosis in MSH3 mutant cell lines in vitro and displayed remarkable single-agent efficacy against MSH3-mutant tumors in vivo. Thus, we here identify a therapeutically actionable synthetic lethal interaction between MSH3 and the non-homologous end joining kinase DNA-PKcs. Our observations recommend DNA-PKcs inhibition as a therapeutic concept for the treatment of human cancers displaying homologous recombination defects. SIGNIFICANCE: We associate mutations in the MSH3 gene, which are frequently detected in microsatellite-instable colon cancer (similar to 40%), with a therapeutic response to specific DNA-PKcs inhibitors. Because potent DNA-PKcs inhibitors are currently entering early clinical trials, we offer a novel opportunity to genetically stratify patients who may benefit from a DNA-PKcs-inhibitory therapy. (C) 2014 AACR

Label-Free Protein-RNA Interactome Analysis Identifies Khsrp Signaling Downstream of the p38/Mk2 Kinase Complex as a Critical Modulator of Cell Cycle Progression

<div><p>Growing evidence suggests a key role for RNA binding proteins (RBPs) in genome stability programs. Additionally, recent developments in RNA sequencing technologies, as well as mass-spectrometry techniques, have greatly expanded our knowledge on protein-RNA interactions. We here use full transcriptome sequencing and label-free LC/MS/MS to identify global changes in protein-RNA interactions in response to etoposide-induced genotoxic stress. We show that RBPs have distinct binding patterns in response to genotoxic stress and that inactivation of the RBP regulator module, p38/MK2, can affect the entire spectrum of protein-RNA interactions that take place in response to stress. In addition to validating the role of known RBPs like Srsf1, Srsf2, Elavl1 in the genotoxic stress response, we add a new collection of RBPs to the DNA damage response. We identify Khsrp as a highly regulated RBP in response to genotoxic stress and further validate its role as a driver of the G₁/S transition through the suppression of <i>Cdkn1a^P21</i> transcripts. Finally, we identify KHSRP as an indicator of overall survival, as well as disease free survival in glioblastoma multiforme.</p></div

Changes in protein-RNA interactions in response to etoposide treatment.

<p><b>(A)</b> Etoposide induces DNA double strand breaks after 6h of treatment as reported by the DSB marker <b>γ</b>-H2AX. <b>(B)</b> Schematics of the experimental procedure for the purification of RBPs show how UV-mediated crosslinking of RNA to interacting proteins was followed by poly-A selection to identify RBPs through LC/MS/MS. <b>(C)</b> Crosslinking followed by purification of mRNA-interacting proteins and nano LC/MS/MS identified 335 protein group hits of which 287 were known as RBPs. <b>(D)</b> Heat map of differentially abundant RBPs. <b>(E)</b> Vulcano plot representing changes in mRNA-protein interactions in response to etoposide treatment identifies Khsrp as the most significantly changed RBP in response to etoposide treatment. <b>(F)</b> Immunoblot analysis of proteins co-purified with poly-A-containing RNA validates protein-RNA interactome changes identified by label free LC/MS/MS (right panel). Whole cell lysates show no significant changes in protein levels of analyzed RBPs (left panel).</p

Inference of outlying RBP-client interactions from changes in client transcripts.

<p><b>(A)</b> Using gene expression levels, numbers of altered client mRNAs were plotted against number of known client mRNAs for each respective RBP. A linear correlation could be identified between the number of changed client mRNAs and known client mRNAs. <b>(B)</b> Studentized residuals (outlyingness), leverage (potential to influence the linear model) and influence analysis (represented by the size to point) are represented through influence plots. Data points perturbing the model were identified by high leverage and studentized residuals. Outliers representing RBPs with higher number of changed client mRNAs were identified through high absolute values of standardized residuals. The same was done by <b>(C)</b> plotting number of upregulated clients against number of changed clients, as well as using vector information on <b>(D)</b> differential promoter usage, <b>(E)</b> differential splicing, and <b>(F)</b> differential CDS. DNA damage-related RBPs—Elavl1, Tia1, Tial1, Srsf1, Srsf2 could be identified through RBP-client analysis.</p

Khsrp-dependent regulation of <i>Cdkn1a</i>^<i>P21</i>.

<p><b>(A)</b> Cell cycle analysis and <b>(B)</b> mitotic index of wildtype and <i>Khsrp</i>^{<b><i>-/-</i></b>} MEFs expose a <i>Khsrp</i>-dependent <b>(A)</b> accumulation of cells in G_<b>1</b> and <b>(B)</b> decreased cycling rates. <b>(C)</b> Transcript and <b>(D)</b> protein levels of <i>Cdkn1a</i>^{<b><i>P21</i></b>} in <i>Khsrp</i>^{<b><i>-/-</i></b>} cells measured by qPCR and immunoblotting reveals an increase of Cdkn1a^{<b><i>P21</i></b>} protein levels unrelated to <i>Cdkn1a</i>^{<b><i>P21</i></b>} mRNA levels.</p

<i>KHSRP</i> transcript levels predict survival of human glioblastoma patients.

<p><b>(A)</b> Overall survival (OS) curves and <b>(B)</b> disease free survival (DFS) curves show an increased OS and DFS of patients bearing tumors with higher <i>KHSRP</i> transcript levels. <b>(C,D)</b> Although not significant, the inverted tendency can be seen when segregating patients in agreement with their tumor <i>CDKN1A</i>^{<b><i>P21</i></b>} transcript levels. Upper and lower quartiles are shown.</p

Mk2/3-dependent regulation of Khsrp and <i>Cdkn1a</i>^<i>p21</i>.

<p><b>(A)</b><i>Mk2</i>^{<b><i>-/-</i></b>}<i>;Mk3</i>^{<b><i>-/-</i></b>} cells show a differential protein-RNA interactome in response to etoposide exposure when compared to wildtype cells (most-significant changes are highlighted). <b>(B)</b><i>Khsrp</i> RNA immunoprecipitations (RIP) followed by <i>Cdkn1a</i>^{<b><i>P21</i></b>} qPCR validates interactome changes seen in wildtype and <i>Mk2</i>^{<b><i>-/-</i></b>}<i>;Mk3</i>^{<b><i>-/-</i></b>} cells upon etoposide treatment. Upon etoposide exposure Khsrp is released from <i>Cdkn1a</i>^{<b><i>P21</i></b>} transcripts. In contrast, in <i>Mk2</i>^{<b><i>-/-</i></b>}<i>;Mk3</i>^{<b><i>-/-</i></b>} MEFs, Khsrp-bound <i>Cdkn1a</i>^{<b><i>P21</i></b>} transcripts increase upon etoposide exposure. <b>(C)</b> Despite a typical arrest in G_<b>2</b> upon etoposide treatment, <b>(D)</b><i>Mk2</i>^{<b><i>-/-</i></b>}<i>;Mk3</i>^{<b><i>-/-</i></b>} MEFs show a decreased G_<b>1</b> population in comparison to wt cells. <b>(E)</b> Increased levels of the <i>Cdkn1a</i>^{<b><i>P21</i></b>} transcript in <i>Mk2</i>^{<b><i>-/-</i></b>}<i>;Mk3</i>^{<b><i>-/-</i></b>} cells upon etoposide treatment <b>(F)</b> fail to promote the upregulation of Cdkn1a^{<b><i>P21</i></b>} protein levels seen in wildtype cells.</p

Murine embryonic fibroblasts arrest in G₂ in response to etoposide treatment.

<p><b>(A)</b> Gene expression changes identified by RNA-Seq following 6h of treatment with 20μM etoposide were analyzed for enrichments in GO terms. Cell cycle, and specifically mitotic processes, emerged in the top 10 most significant GO terms. <b>(B)</b> Protein-protein interactions-based network expansion of RBPs showing differential protein-RNA interactions upon etoposide treatment identifies enrichments for cyclin-dependent processes. <b>(C)</b> Cell cycle analysis of untreated (black line) and etoposide-treated (gray) cells reveals an accumulation of cells with 4N DNA content and decreased staining of the mitotic marker pHH3.</p