<div>AbstractPurpose:<p>Leiomyosarcoma (LMS) is an aggressive sarcoma for which standard chemotherapies achieve response rates under 30%. There are no effective targeted therapies against LMS. Most LMS are characterized by chromosomal instability (CIN), resulting in part from <i>TP53</i> and <i>RB1</i> co-inactivation and DNA damage repair defects. We sought to identify therapeutic targets that could exacerbate intrinsic CIN and DNA damage in LMS, inducing lethal genotoxicity.</p>Experimental Design:<p>We performed clinical targeted sequencing in 287 LMS and genome-wide loss-of-function screens in 3 patient-derived LMS cell lines, to identify LMS-specific dependencies. We validated candidate targets by biochemical and cell-response assays <i>in vitro</i> and in seven mouse models.</p>Results:<p>Clinical targeted sequencing revealed a high burden of somatic copy-number alterations (median fraction of the genome altered =0.62) and demonstrated homologous recombination deficiency signatures in 35% of LMS. Genome-wide short hairpin RNA screens demonstrated <i>PRKDC</i> (DNA-PKcs) and <i>RPA2</i> essentiality, consistent with compensatory nonhomologous end joining (NHEJ) hyper-dependence. DNA-PK inhibitor combinations with unconventionally low-dose doxorubicin had synergistic activity in LMS <i>in vitro</i> models. Combination therapy with peposertib and low-dose doxorubicin (standard or liposomal formulations) inhibited growth of 5 of 7 LMS mouse models without toxicity.</p>Conclusions:<p>Combinations of DNA-PK inhibitors with unconventionally low, sensitizing, doxorubicin dosing showed synergistic effects in LMS <i>in vitro</i> and <i>in vivo</i> models, without discernable toxicity. These findings underscore the relevance of DNA damage repair alterations in LMS pathogenesis and identify dependence on NHEJ as a clinically actionable vulnerability in LMS.</p></div>
<p>Supplementary Table S1: Cancer-associated genes interrogated by Oncopanel. Samples in this study were analyzed with three versions of the assay (v1, v2, v3 and v3.1) – the gene content of each panel is indicated</p>
<p>Supplementary Table S5: Molecular features of patient-derived LMS models from Champions Oncology used in in vivo validation studies (five LMS PDX), including clinical and morphological features of LMS tumors from which they were derived.</p>
<p>Supplementary Figure S9: Cell proliferation assay (BrdU incorporation) demonstrates that combination of DNA-PK inhibition with low-dose doxorubicin reduces LMS03 and LMS05 cell proliferation compared to either drug alone. Peposertib and AZD7648 are structurally unrelated DNA-PK inhibitors. Cells were treated for 7 days with each drug and the combinations at the indicated doses; BrdU incorporation over 24h was measured at day 7 with a luminescence-based ELISA assay (Roche)</p>
Abstract Loss-of-function genetic screens in cancer cell lines permit the systematic interrogation of genes and pathways involved in cell proliferation and viability. The CRISPR-Cas9 system enables effective genome editing for perturbation of gene function. To identify genes that are essential for cancer cell proliferation and survival, we have optimized an approach for efficient genome-scale CRISPR-Cas9 pooled screening and performed negative-selection knock-out screens in 43 human cancer cell lines with diverse genetic and phenotypic features, including 8 pancreatic cancer cell lines. We report that CRISPR-Cas9 efficacy varies depending on the cell context and propose methods to effectively identify differential cancer dependencies across cell lines. Additionally, we identify a strong correlation between genomic copy number and perceived gene dependency, and further elucidate a gene-independent effect of CRISPR-Cas9 cutting. Using these screening data, we performed a detailed interrogation of gene and pathway dependencies in pancreatic cancer. Through integrative analysis of CRISPR-Cas9 screening results with RNA-interference and small molecule screening data, as well as DNA and RNA sequencing, we identified a high-confidence set of recurrently essential genes in pancreatic cancer, including several with associated biomarkers. Moreover, through pathway analyses of CRISPR-Cas9 screening data, we have identified a number of essential biologic processes including key metabolic and cell signaling pathways that may represent potential therapeutic avenues. In particular, we characterize the sterol regulatory element binding protein (SREBP) signaling pathway as a recurrent vulnerability in pancreatic cancer and credential this pathway as a potential therapeutic target. This abstract is also being presented as Poster A13 Citation Format: Andrew J. Aguirre, Wei Shao, Han Xu, Barbara Weir, Francisca Vazquez, Robin Meyers, Cheng-Zhong Zhang, Mihir Doshi, Glenn S. Cowley, Theodore Ewachiw, Zeshaan Rasheed, Todd R. Golub, Kimberly Stegmaier, Charles W. Roberts, Levi A. Garraway, Matthew Meyerson, Aviad Tsherniak, David E. Root, Peter J. Espenshade, William C. Hahn.{Authors}. Genome-scale CRISPR-Cas9 screening to identify essential genes and pathways in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr PR05.
Abstract Mutation of the serine/threonine protein kinase BRAF is observed in approximately 50% of melanomas. This leads to enhanced activation of the kinase, stimulation of the MAPK pathway and aberrant cell proliferation and tumorigenesis. Small molecule inhibitors such as vemurafenib (PLX4032) block BRAF-mediated cell proliferation and recent clinical trials show an overall response rate of about 50%. Evidently, some patients do not respond to the drug or go on to develop resistance while on treatment. Therefore, we designed an RNA interference screen to identify loss of function events that could drive resistance to growth-inhibitory concentrations of PLX4720. We used a pooled lentiviral shRNA library consisting of approximately 90,000 hairpins targeting over 16,500 genes. shRNA-infected cells were split into two groups, one treated with DMSO, the other treated with PLX4720. Following a period in culture, the abundance of each hairpin was assessed by PCR amplification of barcoded hairpin DNA followed by massively parallel paired-end sequencing. RIGER was used to rank the individual hairpins and identify candidate genes that were required for survival of A375 cells in the presence of DMSO, compared to an early time point control. Strikingly, BRAF emerged as the most depleted and therefore most essential gene, validating our screening approach. We then identified those hairpins that were enriched in the presence of PLX4720 compared to DMSO. A small number of genes were identified as candidate ‘resistance suppressors’, which when knocked down, conferred a survival advantage to BRAF inhibitor-treated cells. Validation of these genes in secondary assays and characterization of the resistance mechanism(s) will be presented. By integrating these findings with other genomic and functional studies we aim to identify clinically relevant genetic events that cause RAF-inhibitor resistance in melanoma.
Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma.
Abstract The LKB1 tumor suppressor encodes a key metabolic sensor that integrates cell growth and metabolism. LKB1 is mutationally inactivated in multiple adult malignancies, including >20% of lung cancers, often simultaneously with activating KRAS mutations. LKB1 mutations are an important predictor of poor outcome and resistance to current therapeutic approaches. We employed an integrative approach to define novel therapeutic targets in Lkb1 mutant lung cancers. Matched cell lines from genetically engineered mouse models of cancer driven by activated Kras alone or in combination with Lkb1 deletion, were employed in high-throughput RNAi, kinase inhibitor, and metabolite screens. These screens identified knockdown of either Dtymk (deoxythymidylate kinase) or Chek1 (checkpoint kinase 1) as synthetically lethal with Lkb1 deficiency in both mouse and human lung cancer cell lines, and revealed that Lkb1 inactivation conferred marked sensitivity to treatment with CHEK1 inhibitors. Lkb1 deficient cells had a distinct metabolic profile, characterized by striking decreases in multiple nucleotide metabolites. Knockdown of DTYMK inhibited dTTP biosynthesis and, consequently, DNA synthesis, and knockdown of CHEK1 caused accumulation of DNA damage. We hypothesize that Lkb1 loss enhances dependence on these enzymes due to broad defects in nucleotide metabolism. Our studies support the development of therapies target DTYMK and CHEK1 in LKB1 mutant non-small cell lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-260. doi:1538-7445.AM2012-LB-260
Identifying therapeutic targets in rare cancers remains challenging due to the paucity of established models to perform preclinical studies. As a proof-of-concept, we developed a patient-derived cancer cell line, CLF-PED-015-T, from a paediatric patient with a rare undifferentiated sarcoma. Here, we confirm that this cell line recapitulates the histology and harbours the majority of the somatic genetic alterations found in a metastatic lesion isolated at first relapse. We then perform pooled CRISPR-Cas9 and RNAi loss-of-function screens and a small-molecule screen focused on druggable cancer targets. Integrating these three complementary and orthogonal methods, we identify CDK4 and XPO1 as potential therapeutic targets in this cancer, which has no known alterations in these genes. These observations establish an approach that integrates new patient-derived models, functional genomics and chemical screens to facilitate the discovery of targets in rare cancers.
