Activating mutations in the proto-oncogene KRAS are a hallmark of pancreatic ductal adenocarcinoma (PDAC), an aggressive malignancy with few effective therapeutic options. Despite efforts to develop KRAS-targeted drugs, the absolute dependence of PDAC cells on KRAS remains incompletely understood. Here we model complete KRAS inhibition using CRISPR/Cas-mediated genome editing and demonstrate that KRAS is dispensable in a subset of human and mouse PDAC cells. Remarkably, nearly all KRAS deficient cells exhibit phosphoinositide 3-kinase (PI3K)-dependent mitogen-activated protein kinase (MAPK) signaling and induced sensitivity to PI3K inhibitors. Furthermore, comparison of gene expression profiles of PDAC cells retaining or lacking KRAS reveal a role of KRAS in the suppression of metastasis-related genes. Collectively, these data underscore the potential for PDAC resistance to even the very best KRAS inhibitors and provide insights into mechanisms of response and resistance to KRAS inhibition.
Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States and a major cause of morbidity and mortality worldwide1, 2. While advances in combination chemotherapy have improved median survival3, 4, long-term survival remains poor1, 2, highlighting the need for novel therapeutic approaches.
Genomic studies have identified mutations in the proto-oncogene KRAS as a hallmark of PDAC, occurring in >90% of cases5,6,7,8. KRAS is a small GTPase that acts as a molecular switch to regulate proliferation, differentiation, metabolism, and survival9. Oncogenic forms of KRAS harboring mutations in codons 12, 13, and 61 are insensitive to GTPase activating protein (GAP)-induced GTP hydrolysis, leading to constitutive activation10. Studies in animal models have confirmed an important role of oncogenic KRAS in tumor initiation11, making KRAS an attractive therapeutic target.
Unfortunately, the development of effective KRAS inhibitors has been hindered by several features of oncogenic KRAS: (1) its high affinity for GTP, impeding the identification of GTP-competitive inhibitors; (2) the difficulty of inducing gain-of-function hydrolytic activity with small molecules; and (3) redundant pathways for membrane localization required for KRAS activity9, 10. New approaches to directly inhibit KRAS through covalent binding of specific mutant variants (e.g., G12C)12, 13, interference with guanine-exchange factor (GEF) association to prevent initial GTP loading14, 15, and destabilization of additional membrane localization complexes16 continue to be developed. Furthermore, the success of a recent effort spearheaded by the National Cancer Institute of the United States to develop novel RAS-targeted therapies17, 18 requires a better understanding of the dependency of PDAC cells on KRAS as well as predicting resistance mechanisms that could develop in response to KRAS inhibition.
Given the lack of KRAS inhibitors, genetic tools have been used to evaluate the requirement of KRAS in PDAC maintenance. Acute KRAS knockdown by RNA interference (RNAi) decreased cell proliferation and/or induced apoptosis in a series of human PDAC (hPDAC) cancer cell lines19,20,21. Variability in apoptotic response to KRAS knockdown led to the classification of some cells as “KRAS-dependent” and others as “KRAS-independent”20, 21. Based on these studies, it was unclear whether the “KRAS-independent” phenotype was a consequence of the incomplete inhibitory effects of RNAi such that residual KRAS protein was sufficient to sustain cell survival and proliferation. Recent evidence for PDAC cell survival in the absence of oncogenic KRAS expression derived from a doxycycline (DOX)-inducible oncogenic KRAS transgenic mouse model22. In this model, DOX treatment led to oncogenic KRAS expression in the pancreas to initiate tumorigenesis, while DOX withdrawal halted transgene expression and induced tumor regression. Interestingly, a subset of PDAC tumors recurred lacking KRAS transgene expression22. Despite these findings, the absolute dependence of PDAC cells on endogenous KRAS, a prerequisite for the successful clinical development of novel KRAS inhibitors, remains unknown.
In this study, we examine the consequence of KRAS knockout in PDAC cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. The bacterial CRISPR/Cas adaptive immune system, modified for genome editing in mammalian cells, utilizes a single guide RNA (sgRNA) to direct the Cas9 nuclease to cleave matching double-stranded DNA (dsDNA) sequences, resulting in insertions and deletions via error-prone non-homologous end joining repair mechanisms23. We confirm the variable dependence of hPDAC cell lines based on prior RNAi studies20, 21, and further isolate a subset of hPDAC and murine PDAC (mPDAC) cells that can survive and proliferate despite the absence of endogenous KRAS function. An unbiased chemical screen identifies sensitivity to phosphoinositide 3-kinase (PI3K) inhibition in KRAS deficient cells, offering a pharmacologically tractable method to subvert resistance to KRAS blockade. Furthermore, we gain mechanistic insight into how PI3K inhibition simultaneously blocks the mitogen-activated protein kinase (MAPK) and AKT pathways to impair cap-dependent translation and cell viability in the context of KRAS ablation. Finally, gene expression profiling defines KRAS-regulated pathways in PDAC cells and reveals KRAS-relevant gene signatures that strongly predict survival in PDAC patients.