Genetic disruption of oncogenic Kras sensitizes lung cancer cells to Fas receptor-mediated apoptosis Haiwei Moua,1, Jill Mooreb,1, Sunil K. Maloniac,1, Yingxiang Lib,d, Deniz M. Ozataa, Soren Hougha, Chun-Qing Songa, Jordan L. Smitha, Andrew Fischere, Zhiping Wengb, Michael R. Greenc,f,2, and Wen Xuea,c,g,2 aRNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605; bProgram in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA 01605; cDepartment of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605; dDepartment of Bioinformatics, School of Life Science and Technology, Tongji University, Shanghai 200092, People’s Republic of China; eDepartment of Pathology, University of Massachusetts Memorial Medical Center, University of Massachusetts Medical School, Worcester, MA 01605; fHoward Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01604; and gProgram in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605 Contributed by Michael R. Green, December 28, 2016 (sent for review July 14, 2016; reviewed by Michael T. Hemann and Andrea Ventura) Genetic lesions that activate KRAS account for ∼30% of the 1.6 million Because shRNAs targeting Kras do not completely eliminate annual cases of lung cancer. Despite clinical need, KRAS is still undrug- Kras expression, residual Kras in cells could contribute to Kras- gable using traditional small-molecule drugs/inhibitors. When onco- independent tumor growth. The best way to rule out this possibility genic Kras is suppressed by RNA interference, tumors initially regress is to genetically delete Kras altogether. Unfortunately, the Kras Kras but eventually recur and proliferate despite suppression of . Here, knockout mouse is embryonically lethal (17), and genetic disruption Kras we show that tumor cells can survive knockout of oncogenic , of KRAS—or other oncogenes—in human cells remains a chal- indicating the existence of Kras-independent survival pathways. Thus, lenge due to the low efficiency of homologous recombination using even if clinical KRAS inhibitors were available, resistance would re- main an obstacle to treatment. Kras-independent cancer cells exhibit traditional gene-targeting technology. We recently showed that decreased colony formation in vitro but retain the ability to form CRISPR (18) can be used to efficiently and specifically edit cancer tumors in mice. Comparing the transcriptomes of oncogenic Kras cells genes in adult mice in a fraction of the time and cost of traditional and Kras knockout cells, we identified 603 genes that were specifi- mouse models (19–21). CRISPR therefore provides a flexible ge- CELL BIOLOGY cally up-regulated in Kras knockout cells, including the Fas gene, netic system to manipulate the function of cancer genes (22, 23). which encodes a cell surface death receptor involved in physiological Previous work has shown that oncogenic KRAS epigenetically regulation of apoptosis. Antibodies recognizing Fas receptor effi- silences Fas expression (24, 25). In addition, RAS directs epige- ciently induced apoptosis of Kras knockout cells but not oncogenic netic silencing of Fas through a highly ordered pathway that cul- Kras-expressing cells. Increased Fas expression in Kras knockout cells minates in methylation of the Fas promoter (26, 27). It remains was attributed to decreased association of repressive epigenetic unexplored whether Fas can be restored by genetic inactivation of Fas marks at the promoter. Concordant with this observation, treat- oncogenic RAS. ing oncogenic Kras cells with histone deacetylase inhibitor and Fas- activating antibody efficiently induced apoptosis, thus bypassing the need to inhibit Kras. Our results suggest that activation of Significance Fas could be exploited as an Achilles’ heel in tumors initiated by oncogenic Kras. Oncogenic KRAS underlies 30–90% of lung, colon, and pancreatic cancers, but despite more than 30 y of research, clinical inhibitors Kras | lung cancer | Fas | apoptosis of KRAS—and potential resistance mechanisms—remain elusive. Using CRISPR-mediated genome editing of oncogenic Kras,we show that some lung cancer cells can survive Kras knockout, ung cancer is a leading cause of cancer death, accounting for indicating the existence of mechanisms that allow tumors to ∼1.3 million deaths worldwide each year (1). Non–small-cell L escape Kras oncogene addiction. We identify genes highly lung cancer (NSCLC), the most common subtype, is associated expressed in Kras knockout cells, including the Fas receptor with frequent mutations in KRAS (∼30%). KRAS is also frequently gene. Antibodies that activate Fas receptor selectively induced mutated in other tumor types, including pancreatic (>90%) and apoptosis in Kras-independent lung cancer cells, suggesting a colon (∼30%) cancer (2). Although various pharmacological in- potential strategy for combinatorial therapies against Kras- hibitors are being developed for RAS, especially for the mutant driven tumors. These findings have direct translational implica- KRASG12C (3–5), these small molecules have not been tested in tions for the treatment of lung cancer and other KRAS mutant the clinic (6, 7). As a result, advanced oncogenic KRAS lung cancer types. cancers are usually treated with conventional therapy such as ra- diation and chemotherapy, often with limited success (1, 8). Author contributions: H.M., J.M., S.K.M., Z.W., M.R.G., and W.X. designed research; H.M., Controlled expression of oncogenic RAS cDNA in mouse S.K.M., D.M.O., S.H., C.-Q.S., and J.L.S. performed research; H.M., S.K.M., Y.L., D.M.O., S.H., models of melanoma, lung, breast, and pancreatic cancer has C.-Q.S., J.L.S., and A.F. analyzed data; J.M., Y.L., and Z.W. analyzed RNA-seq and TCGA shown that the withdrawal of oncogenic RAS results in complete data; A.F. analyzed tumor pathology; and H.M., J.M., S.K.M., Z.W., M.R.G., and W.X. tumor regression (9–11). This phenomenon, known as “oncogene wrote the paper. – addiction,” suggests that oncogenic KRAS alleles (e.g., KRASG12D) Reviewers: M.T.H., Massachusetts Institute of Technology; and A.V., Memorial Sloan Kettering Cancer Center. not only initiate tumorigenesis but also play a crucial role in tumor The authors declare no conflict of interest. maintenance. To recapitulate KRAS oncogene addiction in a mouse model of lung cancer, we developed a conditional Kras Data deposition: The deep-sequencing data reported in this paper have been deposited in G12D/+ −/− the National Center for Biotechnology Information BioProject database (accession no. shRNA system (shKras) to knock down Kras in Kras ;p53 PRJNA356881). – (KP) cell lines derived from a mouse lung tumor (12 14). When 1H.M., J.M., and S.K.M. contributed equally to this work. we orthotopically transplanted shKras KP cells into immuno- 2To whom correspondence may be addressed. Email: [email protected] or compromised mice, we found that Kras-driven lung tumors can [email protected]. escape oncogene addiction and become independent of Kras This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. signaling (termed Kras independence) (15, 16). 1073/pnas.1620861114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1620861114 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 − − Here, we use CRISPR to establish viable Kras knockout (Kras / ) G12D/+ lung cancer cell lines from parental oncogenic Kras (Kras ) cells. While investigating the mechanism of Kras-independent tu- morigenesis in this model, we identified Fas among the genes most highly regulated by Kras. Fas (also known as CD95, APO-1, and TNFRSF6) encodes a cell surface death receptor that triggers apoptosis upon binding by its cognate ligand, Fas ligand (FasL) (or CD95L), and plays critical roles in the immune elimination of cancer cells (28, 29). In both mouse and human lung cancer cells, genetic disruption of Kras elevated Fas expression on the cell sur- face and increased sensitivity to Fas-mediated apoptosis, thereby demonstrating a selective vulnerability of Kras-independent cells. Consistent with previous work showing that oncogenic KRAS epi- genetically silences Fas expression (24, 25), we show that Fas is − − activated in Kras / cells by loss of both Dnmt1 and Ezh2 re- cruitment and repressive epigenetic marks of the Fas promoter. + Remarkably, treatment of parental KrasG12D/ cells with pharma- cological histone deacetylase (HDAC) inhibitors not only increased Fas levels but also sensitized cells to Fas-mediated apoptosis. These results suggest a combinatorial strategies for targeted elimination of Kras-independent and oncogenic Kras lung cancer cells. Results Kras Knockout Murine Lung Cancer Cells Are Viable and Can Form Fig. 1. CRISPR-mediated Kras knockout in Kras-driven mouse lung adeno- Tumors in Mice. Our previous study showed that shRNAs target- carcinoma cells. (A) Schematic diagram of CRISPR sgRNA design targeting exon G12D/+ “ ” ing Kras do not completely eliminate Kras in cells (16), hence the 2 of the mutant Kras allele (Kras ). Codon 12 is underlined. GAT encodes G12D mutation. Red arrowhead indicates the Cas9 cutting site. (B) residual Kras might contribute to Kras independence. We there- Immunoblot demonstrating total Kras protein levels in KP1 and KP2 clone fore used CRISPR-based method to genetically disrupt oncogenic pairs. Each pair included two clones: one parental (KP1/KP2) and one Kras G12D/+ −/− Kras in two independent mouse Kras ;p53 lung cancer knockout (KP1 clone/KP2 clone). Hsp90 was used as a loading control. (C) cell lines (termed KP1 and KP2) (30–32). Lentiviral vector Deep-sequencing analysis showing small deletions induced by CRISPR in a (lentiCRISPR) with puromycin selection marker, as described representative clone. (Upper) Representative IGV plots. Black bars denote previously (33), was used to deliver Cas9 and a sgRNA targeting deletions (purple circle). The red “T” is the G12D mutation. The HindIII site G12D G12D/+ Kras (sgKras) into the target cells (Fig. 1A). The puromycin- (“C” SNP) was engineered in the original Kras mouse model.