SUMMARY Intratumor genetic heterogeneity underlies the ability of tumors to evolve and adapt to different environmental conditions. Using CRISPR/Cas9 technology and specific DNA barcodes, we devised a strategy to recapitulate and trace the emergence of subpopulations of cancer cells containing a mutation of interest. We used this approach to model different mechanisms of lung cancer cell resistance to EGFR inhibitors and to assess effects of combined drug therapies. By overcoming intrinsic limitations of current approaches, CRISPR-barcoding also enables investigation of most types of genetic modifications, including repair of oncogenic driver mutations. Finally, we used highly complex barcodes inserted at a specific genome location as a means of simultaneously tracing the fates of many thousands of genetically labeled cancer cells. CRISPR-barcoding is a straightforward and highly flexible method that should greatly facilitate the functional investigation of specific mutations, in a context that closely mimics the complexity of cancer.

◥Osimertinib, a mutant-specific third-generation EGFR tyrosine kinase inhibitor, is emerging as the preferred first-line therapy for EGFR-mutant lung cancer, yet resistance inevitably develops in patients. We modeled acquired resistance to osimertinib in transgenic mouse models of EGFR L858R -induced lung adenocarcinoma and found that it is mediated largely through secondary mutations in EGFR-either C797S or L718V/Q. Analysis of circulating free DNA data from patients revealed that L718Q/V mutations almost always occur in the context of an L858R driver mutation. Therapeutic testing in mice revealed that both erlotinib and afatinib caused regression of osimertinib-resistant C797S-containing tumors, whereas only afatinib was effective on L718Q mutant tumors. Combination first-line osimertinib plus erlotinib treatment prevented the emergence of secondary mutations in EGFR. These findings highlight how knowledge of the specific characteristics of resistance mutations is important for determining potential subsequent treatment approaches and suggest strategies to overcome or prevent osimertinib resistance in vivo.Significance: This study provides insight into the biological and molecular properties of osimertinib resistance EGFR mutations and evaluates therapeutic strategies to overcome resistance.

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Osimertinib, a mutant‐specific third generation EGFR TKI, is emerging as the preferred first‐line therapy for EGFR mutant lung cancer. Despite initial responses in patients, however, resistance inevitably develops over time. In order to investigate mechanisms of resistance to first‐line osimertinib, we modeled acquired resistance to this drug in transgenic mouse models of EGFRL858R‐induced lung adenocarcinoma and found that it is mediated largely through secondary mutations in EGFR – either C797S or L718V/Q (Figure 1A and 1B). Analysis of circulating free DNA data from patients with EGFR mutant lung cancer revealed that L718Q/V mutations almost always arise in the context of an L858R driver mutation. Therapeutic testing in mice revealed that both erlotinib and afatinib caused regression of osimertinib‐resistant C797S‐containing tumors, whereas only afatinib was effective in L718Q mutant tumors (Figure 1C and 1D). Combination first‐line osimertinib plus erlotinib treatment prevented the emergence of secondary mutations in EGFR. Our data identify specific secondary EGFR mutations as a major mechanism of acquired resistance to first‐line osimertinib treatment and highlight potential strategies to overcome or prevent osimertinib resistance in vivo. Furthermore, these findings emphasize how knowledge of the specific characteristics of resistance mutations are important for determining potential subsequent treatment approaches. Support or Funding Information This work was supported by ‐‐‐‐‐Yale’s Specialized Program of Research Excellence in Lung Cancer grant (to K. Politi, S.B. Goldberg and M.A. Lemmon) and funding from AstraZeneca (to K. Politi). Additional support came from the NIH/NCI‐funded Yale Cancer Biology Training Program T32 CA193200‐01A1 and F31 CA228268‐01A1 (to J.H. Starrett), R01 CA198164 (M.A. Lemmon), the Ginny and Kenneth Grunley Fund for Lung Cancer Research, and the Canadian Institutes of Health Research Project Grant PJT‐148725 (to W.W. Lockwood). W.W. Lockwood is supported by a Michael Smith Foundation for Health Research Scholar and NIHR New Investigator Awards, A. Guernet is a fellow funded by the IMED AstraZeneca postdoc program, A. Nagelberg is supported by a scholarship from the CIHR, and K.D. Ashtekar is an Arnold and Mabel Beckman Foundation Postdoctoral Fellow. Yale Cancer Center Shared Resources used for this work were in part supported by NIH/NCI Cancer Center Support Grant P30 CA016359. Acquired resistance to first‐line osimertinib arises partially due to the emergence of secondary mutations in EGFR, which are differentially sensitive to other EGFR TKIs. A. Schema of the experiment. CCSP‐rtTA;TetO‐EGFRL858R mice were administered doxycycline (dox) for the duration of the experiment and developed tumors after ~6 weeks on dox. When tumors were detected by MRI (see pre‐treatment image), osimertinib treatment was initiated (25 mg/kg QD M‐F) which elicited a response (see representative response MRI) and treated until the emergence of resistant tumors by MRI. Coronal MR images are shown...

We have devised a barcoding strategy to recapitulate cancer evolution through the emergence of subclonal mutations of interest, whose effects can be monitored in a dynamic manner. This approach can be easily adapted for a variety of applications, including combined modeling of multiple mechanisms of drug resistance or repair of oncogenic driver mutations in addicted cancer cells. KEYWORDS ALK; APC; CRISPR/Cas9; EGFR; genetic barcoding; non-small cell lung cancer; resistance; TP53; tumor heterogeneityCancer is an evolutionary process in which the stepwise accumulation of genetic alterations is shaped by Darwinian selection. As a result, each tumor is composed of a complex mixture of clonal cell subpopulations containing a partially overlapping, but distinct, pattern of driver and passenger mutations. Such intratumor heterogeneity has dramatic consequences, not only for cancer progression and metastatic spread, but also for resistance to therapy. 1,2 While snapshots of the clonal architecture of a particular tumor at a given stage can now be obtained through deep sequencing and mathematical modeling, such complexity is generally not taken into account when investigating the effects of a particular oncogenic mutation. We recently devised a novel approach based on new technologies for DNA editing to recapitulate and trace the emergence of a new mutation in a subset of cancer cells, thus enabling functional studies on a gene of interest in a context that mimics intratumor heterogeneity. 3 Originally an adaptive immune system in prokaryotes, CRISPR (clustered regularly interspaced short palindromic repeats) has been engineered into a new powerful tool for genome editing. 4,5 This system is composed of the Cas9 nuclease from S. Pyogenes and a short RNA sequence, the singleguide RNA (sgRNA). When co-expressed in cells, Cas9 and the sgRNA form a complex that specifically recognizes a particular DNA sequence through Watson-Crick pairing and promotes its cleavage. The double-strand DNA break induced by CRISPR/ Cas9 can trigger two distinct cellular mechanisms for DNA repair that can be exploited for DNA editing: error-prone nonhomologous end-joining (NHEJ) and high-fidelity homologydirected repair (HDR). DNA repair through NHEJ frequently generates insertions or deletions (indels), which can alter the frame of a coding sequence and result in gene inactivation. In HDR, a donor DNA co-introduced into the cells functions as a template for precise repair; through appropriate design of the donor DNA this mechanism can be used to generate a wide range of genetic modifications, including specific point mutations or the insertion of an entire gene. Depending on the extent of the desired modification, a single-stranded DNA oligonucleotide (ssODN) or a double-stranded DNA targeting construct can be used as donor DNA for HDR. 4,5 Despite the undeniable potential of this new technology, two major intrinsic limitations must be considered when applying CRISPR/Cas9. First, in certain contexts this system can tolerate a few mismat...

mechanism that emerges. Using RNA-seq data, we searched for epigenetic regulators that might be mediating the differentially expressed genes in the resistant cells. This analysis revealed that the chromatin remodeling protein SMARCA4/BRG1 is required for maintenance of the resistant phenotype in one of the models as knockdown of BRG1 sensitized cells to osimertinib. Further analysis revealed that SMARCA4 is stabilized in TKI-resistant cells, thus leading to TKI resistance. Finally, immunohistochemistry (IHC) examination of a collection of TKI-resistant patient-derived xenografts (PDXs) revealed higher levels of SMARCA4 expression in TKI-resistant tumors without on-target EGFR-dependent resistant mechanisms. To further elucidate the role of SMARCA4, we are currently performing ATAC-seq experiments that will offer insights into chromatin accessibility mediated by the protein in the resistant cells. In addition, we are assessing the protein levels of SMARCA4 in clinical specimens obtained before treatment and at the time of resistance by IHC. As new and better targeted therapies are developed, complex resistance mechanisms that involve epigenetic changes in tumors are likely to be increasingly observed. Our studies offer insights into the mechanisms that underlie such resistance that could lead to new therapeutic possibilities for these tumors.

<p>Cell-free DNA Mutation Dataset</p>

<p>Supplementary Methods, Supplementary Tables S1-5, and Supplementary Figures S1-9</p>