<p>PDF file, 109K, Clonogenic survival of lung SqCC after radiation (S1); High-throughput profiling of lung SqCC after radiation (S2); Mutations in NFE2L2 and KEAP1 in lung SqCC cell lines (S3); MSigDB Gene Sets from Fig S5 (S4); TP53 alterations, LOH, and mRNA levels in lung SqCC cell lines (S5).</p>
<p>Supplementary Table S2 - XLSX file 1063K, Gene list derived from a gene expression based comparative marker selection between cell lines with normal or amplified levels of 3q26</p>
Abstract Cells residing in mesenchymal state are often associated with stem cell properties. The phenotypic changes from epithelial to mesenchymal cell state, or from non-stem-like to stem-like cell state contribute to tumor heterogeneity and play important roles in tumor initiation, progression and metastasis. To systematically interrogate the modulators of epithelial-to-mesenchymal transition, we performed a genome-scale ORF screen to identify regulators of mesenchymal and stem-like cell states using a barcoded human ORFome expression library in human mammary epithelial cells. In the screen, we used flow cytometric analysis of the CD44 cell surface marker and identified 68 ORFs that can switch cells from CD44 low state to CD44 high state in 7 days. Among these genes, the RNA splicing factors were highly enriched as analyzed by GO terms and Gene set enrichment analysis (GSEA).We employed six different assays for candidate validation: 1) Induction of CD44 cell surface markers; 2) Evaluation of the expression of EMT markers; 3) Test of the ability to form mammospheres; 4) Investigation of the expression during EMT induction; 5) Test of the necessity of these splicing factors for EMT and stem-like states; 6) Examination of the ability to promote tumor formation in vivo. We discovered that QKI and RBFOX1 were both necessary and sufficient to promote EMT and stem-like states. MBNL1, MBNL2 and CELF4 were sufficient to induce some mesenchymal markers. We further investigated the downstream targets of these splicing factors by RNA-sequencing analysis. We found that QKI and RBFOX1 regulated the alternative splicing of genes in 5 functional modules: 1) Cell motility and ECM/cytoskeleton organization; 2) Stem cell fate determination; 3) Oncogenic signaling; 4) Epigenetic targets; 5) cell polarity. Strikingly, using molecular and biochemical assays, we found that QKI and RBFOX1/2 interacted and cooperatively regulated the alternative splicing of a large number of overlapping transcripts, including Filamin B (FLNB). QKI and RBFOX1 induced a shorter isoform via exon skipping. which plays a key functional role in the regulation of EMT. Importantly, the expression of QKI, RBFOX1/2 and the short isoform of FLNB are elevated in basal B type of breast cancer cell lines and in basal-like breast cancer patient samples, the subtype of breast cancer that displays higher degree of mesenchymal and stem-like traits. In conclusion, alternative RNA splicing plays a key role in the regulation of EMT and stem-like cell states. QKI and RBFOX1/2 are both necessary and sufficient to promote EMT and stem-like traits. Alternative splicing of FLNB controlled by QKI and RBFOX1/2 is one of the key downstream targets that regulates EMT. Thus, the molecular targets and mechanism identified in this study may aid in the development of new diagnostic and therapeutic approaches for breast tumors, especially for basal-like breast cancer. Citation Format: Ji Li, Peter Choi, Christine Chaffer, Katherine Labella, Jong Wook Kim, John Doench, Chao Dai, Andrew Giacomelli, Seav Huong Ly, Justin Hwang, Andrew Hong, Nina Ilic, Ole Gjoerup, Matthew Meyerson, Angela Brooks, Robert Weinberg, William Hahn. A genome-scale ORF screen reveals an alternative splicing program that regulates mesenchymal and stem-like cell states in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5020. doi:10.1158/1538-7445.AM2017-5020
Abstract Malignant rhabdoid tumors (MRT) are aggressive cancers of early childhood that are largely resistant to traditional therapies. MRT exhibit a remarkably low mutation rate, with no recurrent mutations beyond the defining biallelic inactivating mutation in SMARCB1, a core subunit of the SWI/SNF (BAF) chromatin-remodeling complex. Thus, MRT do not display traditional oncogenic mutations that are amenable to targeted therapies, limiting their use for this disease. In order to nominate new drug targets for MRT, we screened MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries. The most significant vulnerabilities consistent across all three screens were MDM2 and MDM4, the major negative regulators of p53. We found that MRT cell lines are more sensitive than other p53 wild-type cancer cell lines to both idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual) in vitro. Both inhibitors induced substantial activation of the p53 pathway in MRT cell lines, which responded with permanent apoptotic or senescent cell fate decisions. CRISPR-Cas9-mediated inactivation of TP53 caused a significant resistance to these compounds, confirming that on-target mechanisms were responsible for MRT sensitivity. We found that loss of SMARCB1 sensitizes MRT cells to idasanutlin by shifting the p53 response towards apoptosis. In MRT xenograft studies, both idasanutlin and ATSP-7041 slowed tumor growth. Most strikingly, an idasanutlin pulse of only five days was sufficient to induce sizable regression of all tumors, which remained complete and durable in 50% of mice. Finally, gene expression analysis of primary MRT predicts that, like cell lines and xenografts, MRT in patients are likely to be sensitive to MDM2 inhibition. Collectively, these studies describe a genetic link between SWI/SNF complex mutations and p53, while providing evidence to support the use of MDM2 and MDM2/4 inhibitors that have already entered clinical trials for the treatment of this devastating pediatric cancer. Citation Format: Thomas P. Howard, Taylor E. Arnoff, Melinda R. Song, Andrew O. Giacomelli, Xiaofeng Wang, Andrew L. Hong, Neekesh V. Dharia, Su Wang, Francisca Vazquez, Minh-Tam Pham, Ann M. Morgan, Franziska Wachter, Gregory H. Bird, Guillaume Kugener, Elaine M. Oberlick, Matthew G. Rees, Hong Tiv, Justin H. Hwang, Katherine H. Walsh, April Cook, John M. Krill-Burger, Aviad Tsherniak, Prafulla C. Gokhale, Peter J. Park, Kimberly Stegmaier, Loren D. Walensky, William C. Hahn, Charles W. Roberts. MDM2 and MDM4 are therapeutic vulnerabilities in malignant rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2867.
<div>Abstract<p>Malignant rhabdoid tumors (MRT) are highly aggressive pediatric cancers that respond poorly to current therapies. In this study, we screened several MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries to identify potential drug targets specific for these cancers. We discovered <i>MDM2</i> and <i>MDM4</i>, the canonical negative regulators of p53, as significant vulnerabilities. Using two compounds currently in clinical development, idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual), we show that MRT cells were more sensitive than other p53 wild-type cancer cell lines to inhibition of MDM2 alone as well as dual inhibition of MDM2/4. These compounds caused significant upregulation of the p53 pathway in MRT cells, and sensitivity was ablated by CRISPR-Cas9–mediated inactivation of <i>TP53</i>. We show that loss of SMARCB1, a subunit of the SWI/SNF (BAF) complex mutated in nearly all MRTs, sensitized cells to MDM2 and MDM2/4 inhibition by enhancing p53-mediated apoptosis. Both MDM2 and MDM2/4 inhibition slowed MRT xenograft growth <i>in vivo</i>, with a 5-day idasanutlin pulse causing marked regression of all xenografts, including durable complete responses in 50% of mice. Together, these studies identify a genetic connection between mutations in the SWI/SNF chromatin-remodeling complex and the tumor suppressor gene <i>TP53</i> and provide preclinical evidence to support the targeting of MDM2 and MDM4 in this often-fatal pediatric cancer.</p>Significance:<p>This study identifies two targets, MDM2 and MDM4, as vulnerabilities in a deadly pediatric cancer and provides preclinical evidence that compounds inhibiting these proteins have therapeutic potential.</p></div>
Malignant rhabdoid tumors (MRT) are highly aggressive pediatric cancers that respond poorly to current therapies. In this study, we screened several MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries to identify potential drug targets specific for these cancers. We discovered MDM2 and MDM4, the canonical negative regulators of p53, as significant vulnerabilities. Using two compounds currently in clinical development, idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual), we show that MRT cells were more sensitive than other p53 wild-type cancer cell lines to inhibition of MDM2 alone as well as dual inhibition of MDM2/4. These compounds caused significant upregulation of the p53 pathway in MRT cells, and sensitivity was ablated by CRISPR-Cas9-mediated inactivation of TP53. We show that loss of SMARCB1, a subunit of the SWI/SNF (BAF) complex mutated in nearly all MRTs, sensitized cells to MDM2 and MDM2/4 inhibition by enhancing p53-mediated apoptosis. Both MDM2 and MDM2/4 inhibition slowed MRT xenograft growth in vivo, with a 5-day idasanutlin pulse causing marked regression of all xenografts, including durable complete responses in 50% of mice. Together, these studies identify a genetic connection between mutations in the SWI/SNF chromatin-remodeling complex and the tumor suppressor gene TP53 and provide preclinical evidence to support the targeting of MDM2 and MDM4 in this often-fatal pediatric cancer. SIGNIFICANCE: This study identifies two targets, MDM2 and MDM4, as vulnerabilities in a deadly pediatric cancer and provides preclinical evidence that compounds inhibiting these proteins have therapeutic potential.
3q26 is frequently amplified in several cancer types with a common amplified region containing 20 genes. To identify cancer driver genes in this region, we interrogated the function of each of these genes by loss- and gain-of-function genetic screens. Specifically, we found that TLOC1 (SEC62) was selectively required for the proliferation of cell lines with 3q26 amplification. Increased TLOC1 expression induced anchorage-independent growth, and a second 3q26 gene, SKIL (SNON), facilitated cell invasion in immortalized human mammary epithelial cells. Expression of both TLOC1 and SKIL induced subcutaneous tumor growth. Proteomic studies showed that TLOC1 binds to DDX3X, which is essential for TLOC1-induced transformation and affected protein translation. SKIL induced invasion through upregulation of SLUG (SNAI2) expression. Together, these studies identify TLOC1 and SKIL as driver genes at 3q26 and more broadly suggest that cooperating genes may be coamplified in other regions with somatic copy number gain.These studies identify TLOC1 and SKIL as driver genes in 3q26. These observations provide evidence that regions of somatic copy number gain may harbor cooperating genes of different but complementary functions.
