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Kras Mutant Genetically Engineered Mouse Models of Human Cancers

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Kras Mutant Genetically Engineered Mouse Models of Human Cancers Kras mutant genetically engineered mouse models of PNAS PLUS human cancers are genomically heterogeneous Wei-Jen Chunga,1,2, Anneleen Daemena,1, Jason H. Chengb, Jason E. Longb, Jonathan E. Cooperb, Bu-er Wangb, Christopher Tranb, Mallika Singhb,3, Florian Gnada,4, Zora Modrusanc, Oded Foremand, and Melissa R. Junttilab,5 aBioinformatics & Computational Biology, Genentech, Inc., South San Francisco, CA 94080; bTranslational Oncology, Genentech, Inc., South San Francisco, CA 94080; cMolecular Biology, Genentech, Inc., South San Francisco, CA 94080; and dPathology, Genentech, Inc., South San Francisco, CA 94080 Edited by Anton Berns, The Netherlands Cancer Institute, Amsterdam, The Netherlands, and approved November 6, 2017 (received for review May 23, 2017) KRAS mutant tumors are largely recalcitrant to targeted therapies. treatment susceptibility and tumor evolution under selective pres- Genetically engineered mouse models (GEMMs) of Kras mutant sure of drug therapy. cancer recapitulate critical aspects of this disease and are widely used for preclinical validation of targets and therapies. Through Results comprehensive profiling of exomes and matched transcriptomes Spontaneous Acquisition of Genomic Aberrations in KrasG12D-Initiated of >200 KrasG12D-initiated GEMM tumors from one lung and two GEMM Tumors. To better define the genomic landscape of estab- pancreatic cancer models, we discover that significant intratumoral lished Kras mutant tumor models, we focused on three models of and intertumoral genomic heterogeneity evolves during tumorigen- Kras mutant cancer: a nonsmall cell lung cancer (NSCLC) model esis. Known oncogenes and tumor suppressor genes, beyond those with adenovirus-induced expression of KrasG12D and homozygous engineered, are mutated, amplified, and deleted. Unlike human tu- p53 targeting (KP:KrasLSL.G12D/wt;p53frt/frt) (11, 13), and two models mors, the GEMM genomic landscapes are dominated by copy num- ber alterations, while protein-altering mutations are rare. However, of spontaneous pancreatic ductal adenocarcinoma (PDAC) initi- interspecies comparative analyses of the genomic landscapes dem- ated by Pdx1-driven recombinase expression in the developing onstrate fidelity between genes altered in KRAS mutant human and pancreas to induce KrasG12D expression, with either heterozygous murine tumors. Genes that are spontaneously altered during murine loss of p16/p19 (Cdkn2a) in the presence of p53 mutation (KPR: GENETICS tumorigenesis are also among the most prevalent found in human KrasLSL.G12D/wt;p16/p19fl/wt;p53LSL.R270H/wt;Pdx1.Cre)orwithhomo- indications. Using targeted therapies, we also demonstrate that this zygous targeting of p16/p19 (Cdkn2a)intheabsenceofp53 muta- inherent tumor heterogeneity can be exploited preclinically to dis- tion (KPP: KrasLSL.G12D/wt;p16/p19fl/fl;Pdx1.Cre) (11, 14) (Table S1 cover cancer-specific and genotype-specific therapeutic vulnerabil- and Dataset S1). ities. Focusing on Kras allelic imbalance, a feature shared by all three models, we discover that MAPK pathway inhibition impinges uniquely on this event, indicating distinct susceptibility and fitness Significance advantage of Kras-mutant cells. These data reveal previously un- known genomic diversity among KrasG12D-initiated GEMM tumors, RAS mutant cancers represent a large unmet clinical need. Kras places them in context of human patients, and demonstrates how mutant genetically engineered mouse models (GEMMs) of to exploit this inherent tumor heterogeneity to discover therapeutic cancer recapitulate disease characteristics and are relied upon vulnerabilities. preclinically to validate targets and test therapies. Our integrative analysis of GEMM tumors revealed significantly evolved genetic Kras | mouse models | genomics | heterogeneity | cancer heterogeneity, a common feature of human tumors that under- mines therapeutic responses. Moreover, interspecies comparative utations in RAS have been found in >80% of cancer in- analyses showed the extent of gene-level fidelity between altered Mdications (1, 2) with the majority occurring in KRAS (3). oncogenes and tumor suppressors. The genomic diversity repre- These tumors are one of the largest unmet clinical needs in sents an unrecognized opportunity to identify therapeutically oncology. The foundation and advancement of our understand- susceptible genomic subsets preclinically. Moreover, this more- ing of this disease subset is due to the vast experimentation utilizing thorough understanding of the unappreciated complexity in Kras mutant genetically engineered mouse models (GEMMs). these model systems ultimately allows for better interpretation However, the models themselves are poorly characterized. For and translatability of preclinical GEMM data for the benefit of human tumors, defining the genomic landscape has significantly cancer patients. improved our understanding of cancer etiology and revealed Author contributions: M.R.J. designed research; J.H.C., J.E.L., J.E.C., B.-e.W., C.T., M.S., F.G., subpopulations uniquely susceptible to therapeutic intervention and O.F. performed research; W.-J.C., A.D., and Z.M. contributed new reagents/analytic (4–7). Similarly, interrogation of the genomic landscape of sev- tools; W.-J.C., A.D., and M.R.J. analyzed data; and A.D. and M.R.J. wrote the paper. eral cancer GEMMs has identified cooperating genomic events Conflict of interest statement: All authors were employees of Genentech when the work (8–10). The genomic and matched transcriptional landscape of was performed and A.D., J.H.C., J.E.L., B.-e.W., Z.M., O.F., and M.R.J. hold Roche stock. specifically Kras mutant GEMM tumors, however, remains largely This article is a PNAS Direct Submission. undefined, making it challenging to translate response data into This open access article is distributed under Creative Commons Attribution-NonCommercial- clinical application. Furthermore, substantial variability in growth NoDerivatives License 4.0 (CC BY-NC-ND). rate, survival, and drug response has been observed in Kras mutant Data deposition: All source code and genomic data for the GEMMs are available at research- models despite the shared initiating event (11, 12). Although pub.gene.com/Kras-mutant-GEMM. multiple factors, including tissue type, cell of origin, and stromal 1W.-J.C. and A.D. contributed equally to this work. cell involvement may contribute to tumor biology, we hypothe- 2Present address: 23andMe, Mountain View, CA 94041. sized that cooperating events—spontaneously acquired during 3Present address: Revolution Medicines, Redwood City, CA 94063. tumor development—may underlie these heterogeneities. In this 4Present address: Cell Signaling Technology, Danvers, MA 01923. study, we set out to more thoroughly define the genomic and 5To whom correspondence should be addressed. Email: [email protected]. transcriptional landscapes of three established Kras mutant This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. GEMMs and to assess the implication of tumor heterogeneity on 1073/pnas.1708391114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1708391114 PNAS Early Edition | 1of9 Downloaded by guest on September 25, 2021 We analyzed the profiles of exomes and matched transcriptomes type of genetic alteration, PAM vs. CNA in human and GEMM, of 221 KrasG12D-initiated GEMM adenocarcinomas (Table 1). respectively, the GEMM tumors acquire aberrations in genes Protein-altering mutations (PAMs) were found in >95% of tu- frequently observed in KRAS mutant human tumors. mors, with 1,908 total PAMs and on an average of nine PAMs per tumor (Table 1 and Fig. S1A and Dataset S2). There were no Intratumoral and Intertumoral Genomic Heterogeneity in Kras- recurrent hotspot mutations (Dataset S2). PAM load varied Mutant GEMM Tumors. Although spontaneously acquired genetic widely among tumors even when isolated from the same animal alterations were found in all Kras-initiated GEMM tumors, we (Fig. 1A), implying independent mutational susceptibility despite sought to interrogate their frequency within tumors as a means the shared host. In addition, there was no significant difference to gauge their contribution to tumorigenesis and maintenance. in PAM load between the models regardless of p53 status Most mutations observed in the GEMM tumors are clearly (ANOVA, P = 0.94). Twenty percent of PAMs are expressed subclonal (Fig. 2A and Fig. S1 C–F) and private, strongly suggesting (Dataset S2), consistent with recent work in human genomics that these are unlikely driver events required for tumor progression. studies where RNA-seq–based confirmation of protein-altering Because allelic imbalance at the Kras locus is reported to be – somatic variants ranged from 19 to 38% (15–17). When com- critical for in vivo KrasG12D-initiated tumorigenesis (8, 19 21), paring to patient tumors, we found that the mutational frequency we assessed the prevalence and clonality of alteration of the in GEMM tumors is 7- and 10-fold lower than in human KRAS driving oncogene. We observed Kras CNAs in all KrasG12D- mutant pancreatic and lung adenocarcinoma (never smokers), initiated GEMM tumors, but the extent and penetrance varied respectively (Fig. 1 B and C). widely (Fig. 2B). Ninety-one percent (58/64) of PDAC KPP tu- ≥ In contrast to PAMs, extensive and strongly recurrent patterns mors had focal gain or amplification ( 4 copies) of Kras with – > of copy number alterations (CNA) were seen in all three models 3 26 copies (38% had 6 copies). Amplification was predominantly
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