Supplementary Figure 3 show schematic of diversification of the Lenti-sgTSG19-sgV1/Cre and Lenti-sgTSG19-sgV3/Cre pools and tumor initiation in Cas9-negative mice, additional explanation of adaptive sampling method used in assessing the impact of Lenti-sgRNA/Cre vectors in V1- and V3-driven models and additional data on tumor suppressor function in V1- and V3-driven lung cancer including tumor burden, tumor number and effect.

Supplementary Table 9 shows that number of sgNT1 tumors sampled in Cas9-EGFP V1 cohort for the Lenti-sgTSG19 and Lenti-sgTSG75 pools, Kras control V1 cohorts, Cas9-EGFP V1 cohort in the drug experiment and Kras control V1 cohort in the drug experiment.

Supplementary Figure 8 shows histological analysis and quantification of cancer type marker gene expression and signaling protein phosphorylation in EML4-ALK V1 and V3 tumors of different tumor suppressor genotypes

Supplementary Table 8 shows the Odds ratios (OR) and P-values from two-sided Fisher's exact tests comparing the occurrence of alterations in samples with EML4-ALK V1 relative to samples with EML4-ALK V3 for genes with at least 10 alterations across both cohorts

Diverse fusions of echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) are oncogenic drivers in lung adenocarcinoma. EML4-ALK variants have distinct breakpoints within EML4, but their functional differences remain poorly understood. In this study, we use somatic genome editing to generate autochthonous mouse models of EML4-ALK-driven lung tumors and show that variant 3 (V3) is more oncogenic than variant 1 (V1). By using multiplexed genome editing and quantifying the effects of 29 putative tumor-suppressor genes on V1- and V3-driven lung cancer growth, we show that many tumor-suppressor genes have variant-specific effects on tumorigenesis. Pharmacogenomic analyses further suggest that tumor genotype can influence therapeutic responses. Analysis of human EML4-ALK-positive lung cancers also identified variant-specific differences in their genomic landscapes. These findings suggest that EML4-ALK variants behave more like distinct oncogenes than a uniform entity and highlight the dramatic impact of oncogenic fusion partner proteins and coincident tumor-suppressor gene alterations on the biology of oncogenic fusion-driven cancers. SIGNIFICANCE:EML4-ALK-driven lung cancer is treated as a uniform disease despite the presence of distinct fusion variants in patients. Our findings show that EML4-ALK variants are functionally distinct, which may have implications for the treatment of this cancer type and highlights the need to consider differences among variants of other oncogenic fusions.

Supplementary Figure 10 shows Gene Ontology (GO) and GSEA analysis for molecular functions enriched in genes that are differentially expression between V1 and V3 tumors that are Setd2-proficient and Setd2-deficient.

Supplementary Figure 13 shows additional data on the differences in tumor suppressor effects across different oncogenic contexts including an example plot of correlation analysis with an explanation of comparisons, correlation in the rank order of tumor suppressive effects across different oncogenic contexts calculated across various values of Ni=basal,j=basal, and correlation in the rank order of tumor suppressive effects across different oncogenic contexts across various values of Ni=basal,j=basal with genes ranked by fold change in adaptively sampled 95th percentile tumor size.

Supplementary Table 12 shows differential gene expression (log2 fold change and significance) between V1 and V3 tumors as well as between Setd2-deficient and -proficient tumors.

Supplementary Table 1 shows the frequency of mutations in the selected genes in EML4-ALK patients (from AACR Project GENIE) as well as inclusion criteria and major pathway or function

Supplementary Figure 11 shows the schematic for ATAC-Seq analysis of Eml4-Alk V1 and V3 lung tumors with and without Setd2 (sgSetd2) inactivation in vivo, principal component analyses, a heatmap of region accessibility across all samples, and HOMER Motif analyses.

Supplementary Figure 9 shows a schematic of the experimental workflow to isolate Setd2-proficient and Setd2-deficient V1- and V3-driven cancer cells, quality control metrics of the sorted cells, principal components analyses, and a heatmaps of differentially expressed genes.

Supplementary Figure 14 shows the the response of EML4-ALK-driven lung tumors to lorlatinib in vivo, including tumor volume measured by µCT and histology, adaptively sampled mean tumor size of tumor suppressor knockouts, Kolmogorov–Smirnov (KS) distance between the cumulative density plots of the lorlatinib-treated and the “shrunk” vehicle-treated sgInert tumor sizes, estimated optimal shrinkage values of sgInert tumors, and the impact of each tumor suppressor targeting vector on lorlatinib response.

Supplementary Figure 1 shows the genomic loci and mouse allele, as well as additional data on lung tumorigenesis, the generation of the correct inversion at the genomic level and expression or EML4-ALK V1 and V3 fusion proteins.

Supplementary Figure 2 show the increase tumorigenesis of V3 tumors in a different mouse model, the general cloning strategy to generate the triple sgRNA vectors, and the efficiency of GFP inactivation in EML4-Alk-driven autochthonous tumors.

Supplementary Figure 15 shows additional data on the genomic landscape of human EML4-ALK-driven lung cancer, including breakpoints in EML4 and ALK for all samples in the EML4-ALK cohort, distribution of alteration counts for all genes queried in Foundation Medicine’s sequencing panel, genes with recurrent alterations that have not previously been shown to be altered in EML4-ALK-driven lung cancer, calculation of power to detect differences in mutation frequency between V1- and V3-driven tumors, and alteration counts for NOTCH genes and PTEN in samples with the indicated “long” and “short” EML4-ALK variants.

Supplementary Figure 7 shows representative lung images and quantification from mice with V1 and V3 tumors of different tumor suppressor genotypes including immunohistochemistry for target genes.

Supplementary Figure 6 show data on the adaptively sample mean size of tumors with each tumor suppressor gene inactivation at the sgRNA-level in the Lenti-sgTSG75-sgV1/Cre and Lenti-sgTSG75-sgV3/Cre pools in both Cas9EGFP and Cas9-negative control mice.

Supplementary Table 11 shows the impact of genes (Score RTN and significance) on EML4-ALK-V3 tumorigenesis in the context of lorlatinib treatment.

This file contains addition methods.

Oncogenic fusions of EML4 and ALK occur in ~5% of lung adenocarcinomas. More than 15 EML4-ALK variants with distinct breakpoints within EML4 have been identified, but the functional differences between these variants remain poorly understood. Here we use CRISPR/Cas9 somatic genome editing to generate autochthonous mouse models of the two most common EML4-ALK variants, V1 and V3, and show that V3 is more oncogenic than V1. By integrating these models with multiplexed genome editing, we quantify the effects of 29 putative tumor suppressor genes on V1- and V3-driven lung cancer growth in vivo and show that many tumor suppressor genes have dramatically variant-specific effects on tumorigenesis. Analysis of a novel dataset representing the largest human EML4-ALK lung cancer cohort to date identified alterations in the genomic landscape depending on the EML4-ALK variant. These findings demonstrate functional heterogeneity among EML4-ALK variants, suggesting that EML4-ALK variants behave more like distinct oncogenes than a uniform entity. More broadly, these findings highlight the dramatic impact of oncogenic fusions partner proteins on tumor biology. ### Competing Interest Statement M.M.W. and D.A.P. are co-founders of, and hold equity in, Guide Oncology, Inc. S.S, S.D.S, and E.S.S., are employees at Foundation Medicine, Inc., with an equity interest in Roche.