Abstract Clinical resistance to epidermal growth factor receptor (EGFR) inhibition in lung cancer has been linked to the emergence of the EGFR T790M resistance mutation or amplification of MET. Additional mechanisms contributing to EGFR inhibitor resistance remain elusive. By applying combined analyses of gene expression, copy number, and biochemical analyses of EGFR inhibitor responsiveness, we identified homozygous loss of PTEN to segregate EGFR-dependent and EGFR-independent cells. We show that in EGFR-dependent cells, PTEN loss partially uncouples mutant EGFR from downstream signaling and activates EGFR, thereby contributing to erlotinib resistance. The clinical relevance of our findings is supported by the observation of PTEN loss in 1 out of 24 primary EGFR-mutant non–small cell lung cancer (NSCLC) tumors. These results suggest a novel resistance mechanism in EGFR-mutant NSCLC involving PTEN loss. [Cancer Res 2009;69(8):3256–61]

While the effect of amplification-induced oncogene expression in cancer is known, the impact of copy-number gains on "bystander" genes is less understood. We create a comprehensive map of dosage compensation in cancer by integrating expression and copy number profiles from over 8000 tumors in The Cancer Genome Atlas and cell lines from the Cancer Cell Line Encyclopedia. Additionally, we analyze 17 cancer open reading frame screens to identify genes toxic to cancer cells when overexpressed. Combining these approaches, we propose a class of 'Amplification-Related Gain Of Sensitivity' (ARGOS) genes located in commonly amplified regions, yet expressed at lower levels than expected by their copy number, and toxic when overexpressed. We validate RBM14 as an ARGOS gene in lung and breast cancer cells, and suggest a toxicity mechanism involving altered DNA damage response and STING signaling. We additionally observe increased patient survival in a radiation-treated cancer cohort with RBM14 amplification.

The time required to conduct clinical trials limits the rate at which we can evaluate and deliver new treatment options to patients with cancer. New approaches to increase trial efficiency while maintaining rigor would benefit patients, especially in oncology, in which adjuvant trials hold promise for intercepting metastatic disease, but typically require large numbers of patients and many years to complete. We envision a standing platform - an infrastructure to support ongoing identification and trial enrolment of patients with cancer with early molecular evidence of disease (MED) after curative-intent therapy for early-stage cancer, based on the presence of circulating tumour DNA. MED strongly predicts subsequent recurrence, with the vast majority of patients showing radiographic evidence of disease within 18 months. Such a platform would allow efficient testing of many treatments, from small exploratory studies to larger pivotal trials. Trials enrolling patients with MED but without radiographic evidence of disease have the potential to advance drug evaluation because they can be smaller (given high probability of recurrence) and faster (given short time to recurrence) than conventional adjuvant trials. Circulating tumour DNA may also provide a valuable early biomarker of treatment effect, which would allow small signal-finding trials. In this Perspective, we discuss how such a platform could be established.

ABSTRACTOptical pooled screening (OPS) is a highly scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture, due to limitations associated within situsequencing (ISS) of perturbation barcodes. Here, we developed PerturbView, an OPS technology that leveragesin vitrotranscription (IVT) to amplify barcodes prior to ISS, enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues. We demonstrate PerturbView in iPSC-derived neurons, primary immune cells, and tumor tissue sections from animal models. In a screen of immune signaling pathways in primary bone marrow-derived macrophages, PerturbView uncovered both known and novel regulators of NFκB signaling. Furthermore, we combined PerturbView with spatial transcriptomics in tissue sections from a mouse xenograft model, paving the way toin vivoscreens with rich optical and transcriptomic phenotypes. PerturbView broadens the scope of OPS to a wide range of models and applications.

Supplementary Information from Modeling Genomic Diversity and Tumor Dependency in Malignant Melanoma

Significantly altered genes in the combined cohort. MutSigCV ranked list of significantly mutated genes across the entire cohort (n = 50 patients). Entries are sorted by q values. Entries with significant q values that failed manual review in Integrated Genomics Viewer (IGV) were marked as "Blacklist" in the Notes column and excluded from the final results (see Methods).

Supplementary Experimental Methods

Supplementary Tables 2 and 3

Chromosomal gains are among the most frequent somatic genetic alterations occurring in cancer. While the effect of sustained oncogene expression has been characterized, the impact of copy-number gains affecting collaterally-amplified “bystander” genes on cellular fitness remains less understood. To investigate this, we built a comprehensive map of dosage compensations across human cancers by integrating expression and copy number profiles from over 8,000 TCGA tumors and CCLE cell lines. Further, we analyzed the effect of gene overexpression across 17 human cancer ORF screens to provide an overview of genes that prove toxic to cancer cells when overexpressed. Combining these two independent approaches we propose a class of ‘Amplification-Related Gain Of Sensitivity’ (ARGOS) genes. These genes are located in commonly amplified regions of the genome, have lower expression levels than expected by their copy-number status, and are toxic to cancer cells when overexpressed. We experimentally validated CDKN1A and RBM14 as high-confidence pan-cancer ARGOS genes in lung and breast cancer cell line models. We additionally suggest that RBM14’s mechanism of toxicity involves altered DNA damage response and innate immune signaling processes following gene overexpression. Finally, we provide a comprehensive catalog of compensated, toxic, and ARGOS genes as a community resource.### Competing Interest StatementKS is on the SAB and has stock options with Auron Therapeutics. KS receives grant funding from Novartis and KronosBio on topics unrelated to this manuscript. WCH is a consultant for Thermo Fischer Scientific, Solasta Ventures, MPM capital, KSQ Therapeutics, Tyra Biosciences, Frontier Medicines, Jubilant Therapeutics, RAPPTA Therapeutics, Serinus Biosciences, Hexagon Biosciences, Kestral Therapeutics, Function Oncology, and Calyx. RB consults for Scorpion Therapeutics and receives grant funding from Novartis.

Supplementary Table S2. Summary of variant calls from WES analysis of matched pre-treatment and post-progression biopsies.

SF1: NRAS mutant melanoma sensitivity to trametinib and palbociclib treatment; SF2: MELJUSO ORF Screen Optimization and Validation; SF3: MELJUSO CRISPR Screen Optimization and Validation; SF4: EGFR-PI3K-AKT signaling modulates resistance to trametinib/palbociclib treatment in NRAS mutant melanoma cell lines; SF5: RAS signaling modulates resistance to trametinib/palbociclib treatment in NRAS mutant melanoma cell lines; SF6: EGFR-PI3K-AKT signaling cascade differentially replaces loss of genetic NRAS in NRAS mutant melanoma cell lines; SF7: Active KRAS can replace loss of genetic NRAS

Supplementary Tables - PDF file 207K, Five supplementary tables denoting clinical characteristics of tumors, statistical correlations between PI3K mutational status, HPV status, cancer gene mutation status, and significance of pathway and gene mutation rates compared to background mutation rates, normalized to bases covered by exome sequencing

Supplementary Figure S6. Representative example of complex amplified locus showing a CRISPR-CN correlation. Supplementary Figure S7. Comparative analysis of the relationship of CRISPR-Cas9 guide scores to the predicted number of CRISPR-Cas9-induced DNA cuts based on either copy number or total predicted perfect-match on- and off-target alignments. Supplementary Figure S8. Comprehensive analysis of CRISPR-Cas9 sensitivity correlated to the total predicted number of DNA cuts conferred by each sgRNA. Supplementary Figure S9. (A) PANC-1 infection efficiency corresponding to Figure 6 in vitro validation experiment measuring short-term proliferation and viability response of PANC-1 cells transduced at high multiplicity of infection. (B) Immunoblot of protein from PANC-1 cells harvested 48 hours after infection at high MOI with the indicated sgRNAs targeting inside (19q8) or outside (19q4) the PANC-1 19q13 amplicon shown in Figure 6A. Supplementary Figure S10. CRISPR-Cas9 targeting of amplified regions or multiple genomic loci induces DNA damage and a G2 cell cycle arrest in CAL120 cells.

Supplementary Legends - PDF file 122K, Legends for all Supplementary information

XLSX file 12K, Candidate Resistance Genes from RNAi and ORF Functional Screens

Figure S1: Following BRAF inhibition, recovery of ERK phosphorylation is observed and RKO cells continue to proliferate in the presence of PLX4720. Figure S2: DAPPLE analysis of the top 300 candidate genes from the primary screen identifies potential protein interaction networks. Figure S3: Knockdown of candidate genes sensitises cells to BRAF inhibition. Figure S4: Inhibition of MET is synergistic with PLX4720. Figure S5: Inhibition of SHP2 sensitises to PLX4720. Figure S6: Combinations of PLX4720 and AZD6244 are not synergistic. Figure S7: Cells displaying acquired resistance to PLX4720 are sensitive to combined pan-RAF and MEK inhibition. Figure S8: The combination of AZD6244 and RAF265 is synergistic. Figure S9: The combination of AZD6244 and MLN2480 is synergistic. Figure S10: The combination of GSK1120212 and AZ628 is synergistic. Figure S11: AZ628 shows greater synergy when combined with a MEK inhibitor than GSK2118436. Figure S12: Synergy between pan-RAF and MEK inhibition is rescued by a resistant allele of MEK. Figure S13: Pan-RAF and MEK inhibition results in decreased cell viability.

Supplementary Figure 4 PDF file - 38K, Identification of human melanoma and colorectal cancer cell lines with coincident B-RAF and NF1 mutations is associated with resistance to PLX4720. Human melanoma and colorectal cancer cell lines from the Cancer Cell Line Encyclopedia with B-RAFV600E and NF1 mutation were identified and compared to NF1 wild-type melanoma cell lines in terms of sensitivity to PLX4720, NF1 mRNA expression, NF1 copy number alterations and NF1 mutation status. In the heat map, blue represents lower values and red represents higher values, whilst NF1 mutation status is represented as wild-type: blue, point mutation: white, potential loss-of-function mutation: red