KRAS Drives Immune Evasion in a Genetic Model of Pancreatic Cancer
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ARTICLE https://doi.org/10.1038/s41467-021-21736-w OPEN KRAS drives immune evasion in a genetic model of pancreatic cancer Irene Ischenko1, Stephen D’Amico1, Manisha Rao2, Jinyu Li2, Michael J. Hayman1, Scott Powers 2, ✉ ✉ Oleksi Petrenko 1,3 & Nancy C. Reich 1,3 Immune evasion is a hallmark of KRAS-driven cancers, but the underlying causes remain unresolved. Here, we use a mouse model of pancreatic ductal adenocarcinoma to inactivate 1234567890():,; KRAS by CRISPR-mediated genome editing. We demonstrate that at an advanced tumor stage, dependence on KRAS for tumor growth is reduced and is manifested in the sup- pression of antitumor immunity. KRAS-deficient cells retain the ability to form tumors in immunodeficient mice. However, they fail to evade the host immune system in syngeneic wild-type mice, triggering strong antitumor response. We uncover changes both in tumor cells and host immune cells attributable to oncogenic KRAS expression. We identify BRAF and MYC as key mediators of KRAS-driven tumor immune suppression and show that loss of BRAF effectively blocks tumor growth in mice. Applying our results to human PDAC we show that lowering KRAS activity is likewise associated with a more vigorous immune environment. 1 Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, USA. 2 Department of Pathology, Stony Brook University, ✉ Stony Brook, NY, USA. 3These authors jointly supervised this work: Oleksi Petrenko, Nancy C. Reich. email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2021) 12:1482 | https://doi.org/10.1038/s41467-021-21736-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21736-w RAS is frequently associated with some of the deadliest and characterization of KRASG12D p53KO mouse cell lines forms of cancer. The prevailing tenet is that activating (termed KC) representing different stages of pancreatic cancer K 19 KRAS mutations underpin both establishment and main- progression . These cells have stable tumorigenic phenotypes tenance of the transformed state, and therefore they are logical and were chosen to model pharmacological inhibition of KRAS drug targets. Genetically engineered mouse models of KRAS (Supplementary Fig. 1a, b). To that end, we eliminated oncogenic mutant cancer have confirmed that tumor regression can be KRASG12D by CRISPR/Cas9-mediated genome editing in clonal achieved via KRAS extinction1–4. The results have supported the precancerous cell lines and their cancer-derived derivatives. We view that inactivation of mutant KRAS is critically important for used sgRNA targeting KRAS which has been validated to have no successful cancer treatment, in accordance with the oncogene off-target activity14,15. The effect of Kras gene editing was eval- addiction concept5,6. Significant efforts have centered on devel- uated by Western blotting (Fig. 1a). Sequencing analysis of opment of drugs that target RAS itself or its downstream sig- independent clones revealed deletions in the mutant Kras gene naling pathways, with the expectation of killing cancer cells while locus leading to a premature stop codon or an unstable and sparing normal cells. However, these targeted approaches have virtually undetectable KRAS protein (Supplementary Fig. 1c, d). not been as successful as was hoped as today they benefit only a The majority of precancerous KC cells treated with Kras sgRNA small minority of cancer patients (www.cancer.gov). differentiated into non-proliferative colonies based on changes in The past failures in developing anti-RAS therapies have been cell morphology and proliferative rate (Fig. 1b). In contrast, cell attributed to the difficulty of targeting RAS directly and to both lines established from the resected tumors formed viable colonies intrinsic and acquired resistance mechanisms. The discovery with higher frequencies, as determined from the analysis of 150 of direct KRASG12C inhibitors highlights the challenges of randomly picked clones (Fig. 1b). The increase in viability of this therapeutic strategy and potential need for combinatorial KRAS-ablated cancer cells relative to precancerous cells was strategies7–9. The key question from the perspective of cancer therefore considered to be due to various degrees of KRAS treatment is the extent to which KRAS mutant cancers retain dependence for survival. Using these data, we selected four KRAS dependence on KRAS. Although inhibition of KRAS expression intact and four KRAS KO KC cell lines for molecular and func- in mice causes tumor regression, tumors relapse and become tional studies (Supplementary Fig. 1e). A similar approach was KRAS-independent4,10–12. Human and mouse mutant KRAS cell used to inactivate endogenous Kras expression in KRASG12D lines have been identified whose growth and tumorigenicity p53R172H (KPC) PDAC cell lines21 (Supplementary Fig. 1b, e). do not depend on oncogenic KRAS13–15. However, no clear The KRAS KO clones showed reduced proliferation and biomarkers of escape pathways currently exist8,9. These findings colony-forming ability compared with parental KRAS intact cells call into question the degree to which cancers depend on con- when grown in serum-free epithelial cell medium. However, the tinuous KRAS activity. They lend support to the concept that the growth rate was increased in serum-containing culture, support- initiation of oncogenic transformation and maintenance of the ing the role of oncogenic KRAS in growth factor-independence transformed state are separable, and that KRAS dependency is (Supplementary Fig. 2a). Likewise, KRAS knockout had no not a fundamental trait of KRAS-induced tumors16–18. While detrimental effect on cell viability in 3D non-adherent conditions these studies support the initiating role of KRAS in cancer (Supplementary Fig. 2a). To determine whether KRAS-ablated development, they underscore the need for a comprehensive view cells could form tumors in vivo, we implanted them into nude of stage-specific and cell type-specific cancer dependencies and mice. When injected subcutaneously or into the pancreas, both novel rationale-based therapies. KRAS intact and KRAS KO cells formed tumors, although KRAS In this work, we have addressed these questions by using a mouse KO tumors grew more slowly than those from KRAS intact cells model of KRAS-driven pancreatic ductal adenocarcinoma (Fig. 1c and Supplementary Fig. 2b). When injected into the tail (PDAC)19. PDAC is a highly aggressive malignancy characterized vein, both KRAS intact and KRAS KO clones formed lung and by rapid progression, exceptional resistance to all forms of antic- lymph node metastases. We observed that KRAS KO cells ancer treatment, and a high propensity for metastatic spread. A displayed reduced capacity for lung colonization but unabated striking feature of pancreatic cancer is that activating KRAS capacity for lymph node metastases, indicating aggressive mutations are found in ∼90% of cases. Mutations in other pre- behavior (Supplementary Fig. 2c). Using limiting dilution assays sumptive and validated driver oncogenes are remarkably rare20. in nude mice, we estimated that the frequency of tumor-initiating Our objective was to confirm that PDAC cells surviving genetic cells (TIC) ranged from 0.7% in KRAS intact KC/KPC cells to ablation of KRAS retain their tumorigenic capacity, to identify ~0.35% in KRAS KO cells (Fig. 1d). The cell lines derived from stages of tumor progression when KRAS is essential, and to reveal KRAS KO tumors exhibited stable loss of KRASG12D expression, signaling nodes in the KRAS pathway that are responsible for thus demonstrating that the malignant phenotype of KRAS- tumor maintenance. To accomplish these goals, here we use ablated cells is also stable (Supplementary Fig. 2d). CRISPR-mediated gene editing to inactivate mutant KRAS in The morphology of tumors formed by KRAS intact KC/KPC PDAC-derived cell lines. We show that KRAS-ablated cancer cells cells resembled moderately differentiated adenocarcinomas, retain substantial tumorigenic capacity; however, they fail to evade whereas loss of KRAS resulted in poorly differentiated neoplasms the host immune system, triggering strong antitumor effects. Single- (Fig. 1e). The predominant sarcomatous elements in KRAS KO cell RNA sequencing of tumors reveals that KRAS ablation causes tumors maintained expression of pancreatic ductal markers, such changes both in tumor cells and host immune cells. Our data as KRT19 and SOX9, but expression of mesenchymal genes such indicate that the ability of mutant KRAS to modulate tumor as ACTA2 (smooth muscle actin) was increased (Fig. 1e and immunity appears to be an essential component of its oncogenicity. Supplementary Fig. 2e). We used reverse-phase protein arrays The data imply that treatment of PDAC and, by extension, of other (RPPA) of multiple clones to validate these findings. We KRAS mutant cancers will require inhibition of KRAS and con- calculated pathway scores from the expression data of ~300 current activation of immune pathways suppressed by cancer. cancer-associated proteins22, and identified two pathways whose scores were significantly altered in KRAS knockouts: EMT and DNA damage response (Supplementary Fig. 2f). In contrast, there Results was no effect of KRAS loss on MAPK/ERK pathway activation, Loss of KRAS reduces, but does not abolish, the tumorigenic corroborating previously published data on KRAS knockouts15,23. capacity of PDAC cells. We previously described the isolation In