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Legends for the supplementary figures.

This file shows all the supplementary figures as follows: Supplementary Figure 1: Clonal variability introduced by the TP53, KRAS, and MYC manipulations Supplementary Figure 2: Quantitation of signaling alterations for beta-CATENIN, P65, FOXO1 and p-ERK1/2 Supplementary Figure 3: Comparison between parental HBEC and HBECPKM cells for their sensitivity to the MEK inhibitor AZD6244 Supplementary Figure 4: Similar distributions of SMAD2/3 signaling with total or phospho antibodies Supplementary Figure 5: Alterations of p-STAT3 signaling in high EGF growth condition Supplementary Figure 6: Identification of lower EGF concentration (

Oncogene-specific changes in cellular signaling have been widely observed in lung cancer. Here, we investigated how these alterations could affect signaling heterogeneity and suggest novel therapeutic strategies. We compared signaling changes across six human bronchial epithelial cell (HBEC) strains that were systematically transformed with various combinations of TP53, KRAS, and MYC—oncogenic alterations commonly found in non–small cell lung cancer (NSCLC). We interrogated at single-cell resolution how these alterations could affect classic readouts (β-CATENIN, SMAD2/3, phospho-STAT3, P65, FOXO1, and phospho-ERK1/2) of key pathways commonly affected in NSCLC. All three oncogenic alterations were required concurrently to observe significant signaling changes, and significant heterogeneity arose in this condition. Unexpectedly, we found two mutually exclusive altered subpopulations: one with STAT3 upregulation and another with SMAD2/3 downregulation. Treatment with a STAT3 inhibitor eliminated the upregulated STAT3 subpopulation, but left a large surviving subpopulation with downregulated SMAD2/3. A bioinformatics search identified BCL6, a gene downstream of SMAD2/3, as a novel pharmacologically accessible target of our transformed HBECs. Combination treatment with STAT3 and BCL6 inhibitors across a panel of NSCLC cell lines and in xenografted tumors significantly reduced tumor cell growth. We conclude that BCL6 is a new therapeutic target in NSCLC and combination therapy that targets multiple vulnerabilities (STAT3 and BCL6) downstream of common oncogenes, and tumor suppressors may provide a potent way to defeat intratumor heterogeneity. Cancer Res; 77(11); 3070–81. ©2017 AACR.

Results of LentiMutate on KRAS-G12C cDNA and AMG 510 in the H358 cell line. Values shown are the averages of two biological replicates. The fold enrichment of "All nucleotides" is the sum of substitution rates for all nucleotides in the with doxycycline replicates divided by the sum of substitution rates for all nucleotides without doxycycline (parental) at one nucleotide position. Nucleotide w/ max error refers to the highest substitution rate for a single nucleotide at one nucleotide position.

Expression of RUVBL1 variants identified by LentiMutate confer resistance to compound B Individual cDNAs were expressed in the H1993 (left) or H2009 (right) cell lines under a doxycycline-inducible promoter. Values shown are averages of two biological replicates {plus minus} the standard deviation.

Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). Here we optimized and leveraged this property to identify drug resistance mutations, developing a technique we term LentiMutate. This technique was validated by identifying clinically relevant EGFR resistance mutations, then applied to two additional clinical anticancer drugs: imatinib, a BCR-ABL inhibitor, and AMG 510, a KRAS G12C inhibitor. Novel deletions in BCR-ABL1 conferred resistance to imatinib. In KRAS-G12C or wild-type KRAS, point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations conferred resistance to AMG 510. LentiMutate should prove highly valuable for clinical and preclinical cancer-drug development.Significance:LentiMutate can evaluate a drug's on-target activity and can nominate resistance mutations before they occur in patients, which could accelerate and refine drug development to increase the survival of patients with cancer.

Purpose: To identify druggable gene targets that would sensitize NSCLCs to available chemotherapy agents. Experimental Procedures: We developed a CRISPR-Cas9 lentivirus library of 12,474 sgRNAs targeting 660 FDA proved ‘druggable’ putative proteins (18 guides/gene) and investigated their potential to chemosensitize (“drop out” in the presence of taxane) the NSCLC lung adenocarcinoma (LUAD) line NCI-H2009 (TP53 and KRAS mutant) in parallel studies in vitro (tissue culture) & in vivo (xenografts). Key elements of our experiments were: the use low doses of paclitaxel (IC10 values for in vitro and in vivo doses that barely affected tumor growth) compared to control treatment; treatment schedules in vitro and in vivo that mirrored those used in patients; multiple biologic replicates for each transfection and drug selection; and large representation (2,000 cells/sgRNA) for each guide. At the end time point (1 month) we harvested multiple replicates of the taxane and control treatments and sequenced each to identify each guide that “remained” or “dropped out.” Data Summary: We were looking for guides that selectively dropped out in the setting of low dose taxane exposure comparing taxane to control treatment and in vitro to in vivo results. Of importance, we found little overlap between guides that dropped out during taxane exposure in tissue culture vs those that dropped out in xenografts. From the top 10 gene “sensitizers” that selectively dropped out only with taxane treatment and selectively dropped out in xenografts compared to 2D mass tissue culture, we focused on SOAT1 (Sterol O-Acyltransferase 1). SOAT1 is a key enzyme for lipid metabolism which mediates conversion of intracellular free cholesterol to cholesteryl esters which are then stored as lipid droplets. SOAT1 is a potential cancer therapeutic target with a clinically available inhibitor, avasimibe. H2009 cells with SOAT1 hemizygously removed (CRISPR) grew well in vitro and in vivo in the absence of chemotherapy treatment, but were dramatically sensitized to taxanes compared to parental H2009 cells. While avasimibe treatment, at concentrations achievable in patients, sensitized NSCLC to taxanes, this sensitization was not as dramatic as hemizygous removal by CRISPR. Of equal importance, SOAT1 H2009 hemizygous knockout (KO) cells were also dramatically sensitized to etoposide, pemetrexed, and gemcitabine, other chemotherapy agents used in NSCLC treatment. Mechanistically, RNAseq analysis identified G2/M cell cycle related gene sets as enriched in the H2009 SOAT1 KO xenografts compared to SOAT1 wildtype xenografts with paclitaxel treatment. Conclusions: Our results using CRISPR screening in vivo, identified SOAT1 as a critical therapeutic chemosensitizing target requiring only 50% inhibition of activity for sensitizing NSCLC to several chemotherapy agents routinely used in the clinic. Citation Format: Long Shan Li, Kenneth Huffman, Huiyu Li, Michael Peyton, Hyunsil Park, Kimberley Avila, Luc Girard, Mathew Augustine, Joshua T. Mendell, John D. Minna. In vivo CRISPR screen identifies SOAT1 as a chemotherapy chemosensitizing target for non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2690.

Mutations in STK11/LKB1 in non-small cell lung cancer (NSCLC) are associated with poor patient responses to immune checkpoint blockade (ICB), and introduction of a Stk11/Lkb1 (L) mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 loss (KP) resulted in an ICB refractory syngeneic KPL tumor. Mechanistically this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype recapitulated in human STK11/LKB1 mutant NSCLCs. Systemic inhibition of Axl results in increased type I interferon secretion from dendritic cells that expanded tumor-associated TCF1+PD-1+CD8 T cells, restoring therapeutic response to PD-1 ICB in KPL tumors. This was observed in syngeneic immunocompetent mouse models and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts. NSCLC-affected individuals with identified STK11/LKB1 mutations receiving bemcentinib and pembrolizumab demonstrated objective clinical response to combination therapy. We conclude that AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC. publishedVersion

Background: SARS-CoV-2 infection is the cause of the respiratory illness COVID-19, which presents most frequently with respiratory symptoms SARS-CoV-2 cell entry requires interactions with ACE2 and TMPRSS2 on the surface of the host cell Cancer patients and, specifically, those with thoracic malignancies seem to experience poorer clinical outcomes Methods: We utilized bulk and single-cell transcriptional data from a combination of normal and malignant tissues and cells from aerodigestive and respiratory tracts to explore mechanisms governing the expression of ACE2 and TMPRSS2 Additionally, we determined the effect of EMT induction, ZEB1 modulation, and SARS-CoV-2 infection on ACE2 expression Results: Our bulk data suggests that aerodigestive and lung cancer models express a broad range of ACE2 and TMRPSS2, particularly in epithelial cells, and would serve as good models for studying SARS-CoV-2 infection We assessed the relationship between ACE2 and epithelial differentiation in numerous datasets, and found consistent positive correlations with transcriptional and microRNA signifiers of epithelial differentiation The miR-200 family – zinc finger E-box-binding homeobox 1 (ZEB1) pathway, which is an established regulator of EMT, also directly regulates ACE2 expression, likely via putative ZEB1 repressor sites located in the ACE2 promoter Furthermore, SARS-CoV-2 infection reduces ACE2 expression and shifts cells to a more mesenchymal phenotype with loss of EPCAM and upregulation of ZEB1 and other EMT-associated genes Conclusions: ACE2-positive cells are almost exclusively epithelial and unexpectedly rare, considering the devastating impact of this infection Following viral entry, SARS-CoV-2 infection induces molecular changes within the cells that are reminiscent of EMT, including increased ZEB1 ZEB1, in turn, appears to directly repress the expression of ACE2 This SARS-CoV-2-induced ACE2 deficiency, compounded by the downregulation of genes, including claudins, which play a critical role in restricting epithelial and endothelial permeability, exposes respiratory cells to increased risk of edema and acute respiratory distress syndrome (ARDS) Legal entity responsible for the study: The authors Funding: NIH/NCI R01-CA207295 (L A B ), NIH/NCI U01-CA213273 (L A B , J V H ), CCSG P30-CA01667 (L A B ), University of Texas SPORE in Lung Cancer P5-CA070907 (L A B , D L G , J V H , C M G ), the Department of Defense (LC170171;L A B ), Khalifa Bin Zayed Al Nahyan Foundation (C M G ), RP170067 (EMP), through generous philanthropic contributions to The University of Texas MD Anderson Lung Cancer Moon Shot Program and Andrew Sabin Family Fellowship, and The Rexanna Foundation for Fighting Lung Cancer Disclosure: C Gay: Research grant/Funding (self): Astra Zeneca J V Heymach: Advisory/Consultancy: AstraZeneca;Advisory/Consultancy: Boehringer Ingelheim;Advisory/Consultancy: Exelixis;Advisory/Consultancy: Genentech;Advisory/Consultancy: GlaxoSmithKline;Advisory/Consultancy: Guardant Health;Advisory/Consultancy: Hengrui;Advisory/Consultancy: Spectrum L A Byers: Advisory/Consultancy, Research grant/Funding (self): AstraZeneca;Advisory/Consultancy, Research grant/Funding (self): AbbVie;Advisory/Consultancy, Research grant/Funding (self): GenMab;Advisory/Consultancy: BergenBio;Advisory/Consultancy: Pharma Mar SA;Advisory/Consultancy, Research grant/Funding (self): Sierra Oncology;Advisory/Consultancy: Merck;Advisory/Consultancy: Bristol Myers Squibb;Advisory/Consultancy: Genentech;Advisory/Consultancy: Pfizer;Research grant/Funding (self): Tolero Pharmaceuticals All other authors have declared no conflicts of interest

Mutations in

Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). Here we optimized and leveraged this property to identify drug resistance mutations, developing a technique we term LentiMutate. This technique was validated by identifying clinically relevant EGFR resistance mutations, then applied to two additional clinical anticancer drugs: imatinib, a BCR-ABL inhibitor, and AMG 510, a KRAS G12C inhibitor. Novel deletions in BCR-ABL1 conferred resistance to imatinib. In KRAS-G12C or wild-type KRAS, point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations conferred resistance to AMG 510. LentiMutate should prove highly valuable for clinical and preclinical cancer-drug development. Significance: LentiMutate can evaluate a drug's on-target activity and can nominate resistance mutations before they occur in patients, which could accelerate and refine drug development to increase the survival of patients with cancer.

Oncogene-specific changes in cellular signaling have been widely observed in lung cancer. Here, we investigated how these alterations could affect signaling heterogeneity and suggest novel therapeutic strategies. We compared signaling changes across six human bronchial epithelial cell (HBEC) strains that were systematically transformed with various combinations of TP53, KRAS, and MYC—oncogenic alterations commonly found in non–small cell lung cancer (NSCLC). We interrogated at single-cell resolution how these alterations could affect classic readouts (β-CATENIN, SMAD2/3, phospho-STAT3, P65, FOXO1, and phospho-ERK1/2) of key pathways commonly affected in NSCLC. All three oncogenic alterations were required concurrently to observe significant signaling changes, and significant heterogeneity arose in this condition. Unexpectedly, we found two mutually exclusive altered subpopulations: one with STAT3 upregulation and another with SMAD2/3 downregulation. Treatment with a STAT3 inhibitor eliminated the upregulated STAT3 subpopulation, but left a large surviving subpopulation with downregulated SMAD2/3. A bioinformatics search identified BCL6, a gene downstream of SMAD2/3, as a novel pharmacologically accessible target of our transformed HBECs. Combination treatment with STAT3 and BCL6 inhibitors across a panel of NSCLC cell lines and in xenografted tumors significantly reduced tumor cell growth. We conclude that BCL6 is a new therapeutic target in NSCLC and combination therapy that targets multiple vulnerabilities (STAT3 and BCL6) downstream of common oncogenes, and tumor suppressors may provide a potent way to defeat intratumor heterogeneity. Cancer Res; 77(11); 3070–81. ©2017 AACR.

Small Cell Lung Cancer (SCLC) is an aggressive, highly metastatic cancer with an overall 5-year survival rate of Citation Format: Allison A. Mootz, Michael Peyton, Paul Yenerall, Kimberley Avila, Kenneth Huffman, Tomohiro Haruki, Mahboubeh Papari-Zareei, Victor Stastny, Luc Girard, Elizabeth McMillan, Michael Roth, John MacMillan, Bruce Posner, Michael White, Gazdar Adi, John Minna. Identification of two small molecules with small cell lung cancer growth inhibition response profiles different from etoposide/topotecan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2664.

Purpose: Lung cancer is a disease of great oncogenotype complexity (oncogenes and tumor suppressor gene alterations). These alterations can appear in different combinations even within histologically defined lung cancer subtypes. The success of targeted therapy has led to a search for oncogenotype-specific therapies. But, no one therapy fits all oncogenotypes. Here, we investigate whether characterization of oncogene-specific alterations in cellular signaling at single cell level indicate heterogeneity even within cells from the same patient with defined oncogenotype, and whether they can suggest new targets to deal with this heterogeneity. Methods: We compared signaling alterations in single cells for β-CATENIN, SMAD2/3, phospho-STAT3, P65, FOXO1 and phospho-ERK1/2 among a collection of Human Bronchial Epithelial Cells (HBECs) that have been oncogenically transformed with combinations of TP53, K-RAS, and MYC, commonly found alterations in non-small cell lung cancer (NSCLC). We studied ~3000 cells/signaling marker/HBEC oncogenotype variant using immunofluorescence assays and single-cell image analysis (>1M data points). For downstream target identification and validation we utilized gene expression, Western blot and siRNA mediated knockdown assays. We utilized inhibitors to STAT3 and BCL6 in MTT drug sensitivity and colony formation assay in a panel of NSCLC lines. We used xenografted subcutaneous tumors for the in vivo validation of our results. Results: When all three oncogenic changes were present and the HBECs were tumorigenic, we observed STAT3 upregulation and SMAD2/3 downregulation. Interestingly, these STAT3 and SMAD2/3 signaling changes were found to be mutually exclusive in single cells within the transformed HBEC strain. We targeted the STAT3 upregulated subpopulation with the STAT3 inhibitor Stattic. But, Stattic treatment failed to eliminate the SMAD2/3 downregulated subpopulation. To target the SMAD2/3 down-regulated subpopulation, we identified BCL6, a downstream gene of SMAD2/3, as a novel target in transformed HBECs. Next, to test the generality of BCL6 as a target, we studied 5 NSCLC cell lines with various level of BCL6 expression: H1693, H1819, H1993, HCC827 and H2009. Our data suggests that BCL6 can also be a therapeutic target in a subset of NSCLC lines. Then we tested the response of these NSCLC lines to a combination of BBI608 (potent STAT3 inhibitor) and FX1 (BCL6 inhibitor). The combination treatment eliminated more cancer cells than the single treatments alone. Finally, we confirmed the benefit of the combination therapy in H1993 xenografted tumors. Conclusions: We conclude that BCL6 is a new therapeutic target in NSCLC and combination therapy that targets multiple vulnerabilities (Phospho-STAT3 and BCL6) downstream of common oncogenes and tumor suppressors (TP53, K-RAS, and MYC) may provide a potent way to defeat intra-tumor heterogeneity. Citation Format: Dhruba Deb, Satwik Rajaram, Jill E. Larsen, Patrick P. Dospoy, Rossella Marullo, Longshan Li, Kimberley Avila, Leandro Cerchietti, John D. Minna, Lani F. Wu, Steven J. Altschuler. A novel combination therapy targeting BCL6 and phospho-STAT3 defeats intratumor heterogeneity in a subset of non-small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3950. doi:10.1158/1538-7445.AM2017-3950