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CRISPR/Cas9 gene editing technology is an indispensable and powerful tool in the field of cancer biology. To conduct successful CRISPR-based experiments, it is crucial that sgRNAs generate their designed alterations. Here, we describe a simple and efficient sgRNA screening method for validating sgRNAs that generate oncogenic gene rearrangements. We used IL3-independence in Ba/F3 cells as an assay to identify sgRNA pairs that generate fusion oncogenes involving theRetandNtrk1tyrosine kinases. We confirmed these rearrangements with PCR or RT-PCR as well as sequencing. Ba/F3 cells harboringRetorNtrk1rearrangements acquired sensitivity to RET and TRK inhibitors, respectively. Adenoviruses encoding Cas9 and sgRNAs that catalyze theKif5b-RetandTrim24-Retrearrangements were intratracheally instilled into mice and yielded lung adenocarcinomas. A cell line (TR.1) was established from aTrim24-Retpositive tumor that exhibited highin vitrosensitivity to RET-specific TKIs. Moreover, orthotopic transplantation of TR.1 cells into the left lung yielded well-defined tumors that shrank in response to LOXO-292 treatment. The method offers an efficient means to validate sgRNAs that successfully target their intended loci for the generation of novel murine oncogene-driven tumor models.

Flow cytometry gating strategy for identifying CD4+ and CD8+ T cells

Flow gating strategy for identification of PD-L1(high) cells in the TME

Flow cytometry gating strategy for recovery of lung cancer cells from tumor-bearing mice

Heatmap of cytokines that were expressed >5-fold higher in CMT167 cells in vivo compared to LLC cells in vivo

S3. HIF activity is sufficient to promote AS of HIF target genes.

Silencing of PD-L1 in CMT167 cells using shRNA

Figure S1. Fluorescence in situ hybridization (FISH) analysis of FGFR1 gene copy number status in mesothelioma cell lines. Figure S2. Bioluminescence imaging of orthotopically implanted H226 tumors treated with or without ponatinib. Supplementary Tables S1 through S3.

Description for supplemental files and supplemental figures

Supplemental File 2: genes in each pathway

S4. Activation of endogenous HIF target genes promotes AS of PDK1 minigene.

Supplemental File 1: genes that are hypoxia induced or exhibit alternative splicing under hypoxia

Flow cytometry gating strategy for identifying Foxp3 expression in CD4+ T cells and PD-1, CD44, and CD69 on CD8+ T cells

E-cadherin expression and Jak mutational status of LLC cells

Immune checkpoint inhibitors targeting the interaction between programmed cell death-1 (PD-1) and its ligand PD-L1 induce tumor regression in a subset of non–small cell lung cancer patients. However, clinical response rates are less than 25%. Evaluation of combinations of immunotherapy with existing therapies requires appropriate preclinical animal models. In this study, murine lung cancer cells (CMT167 and LLC) were implanted either orthotopically in the lung or subcutaneously in syngeneic mice, and response to anti–PD-1/PD-L1 therapy was determined. Anti–PD-1/PD-L1 therapy inhibited CMT167 orthotopic lung tumors by 95%. The same treatments inhibited CMT167 subcutaneous tumors by only 30% and LLC orthotopic lung tumors by 35%. CMT167 subcutaneous tumors had more Foxp3+ CD4+ T cells and fewer PD-1+ CD4+ T cells compared with CMT167 orthotopic tumors. Flow cytometric analysis also demonstrated increased abundance of PD-L1high cells in the tumor microenvironment in CMT167 tumor–bearing lungs compared with CMT167 subcutaneous tumors or LLC tumor–bearing lungs. Silencing PD-L1 expression in CMT167 cells resulted in smaller orthotopic tumors that remained sensitive to anti–PD-L1 therapy, whereas implantation of CMT167 cells into PD-L1− mice blocked orthotopic tumor growth, indicating a role for PD-L1 in both the cancer cell and the microenvironment. These findings indicate that the response of cancer cells to immunotherapy will be determined by both intrinsic properties of the cancer cells and specific interactions with the microenvironment. Experimental models that accurately recapitulate the lung tumor microenvironment are useful for evaluation of immunotherapeutic agents. Cancer Immunol Res; 5(9); 767–77. ©2017 AACR.

Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and 2α (HIF2α/EPAS1) to activate gene transcription, which promotes tumor cell survival. The majority of human genes are alternatively spliced, producing RNA isoforms that code for functionally distinct proteins. Thus, an effective hypoxia response requires increased HIF target gene expression as well as proper RNA splicing of these HIF-dependent transcripts. However, it is unclear if and how hypoxia regulates RNA splicing of HIF targets. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in hepatocellular carcinoma cells and characterized the role of HIF in regulating AS of HIF-induced genes. The results indicate that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia-reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF targets, including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirm that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of a PDK1 minigene by other transcription factors in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing.Implications: This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. Mol Cancer Res; 12(9); 1233–43. ©2014 AACR.

Direct DNA sequencing chromatograms of Kras in CMT167, LLC, and LL/2 cells

Purpose: FGFR1 copy-number gain (CNG) occurs in head and neck squamous cell cancers (HNSCC) and is used for patient selection in FGFR-specific inhibitor clinical trials. This study explores FGFR1 mRNA and protein levels in HNSCC cell lines, primary tumors, and patient-derived xenografts (PDX) as predictors of sensitivity to the FGFR inhibitor, NVP-BGJ398.Experimental Design: FGFR1 status, expression levels, and BGJ398 sensitive growth were measured in 12 HNSCC cell lines. Primary HNSCCs (n = 353) were assessed for FGFR1 CNG and mRNA levels, and HNSCC TCGA data were interrogated as an independent sample set. HNSCC PDXs (n = 39) were submitted to FGFR1 copy-number detection and mRNA assays to identify putative FGFR1-dependent tumors.Results: Cell line sensitivity to BGJ398 is associated with FGFR1 mRNA and protein levels, not FGFR1 CNG. Thirty-one percent of primary HNSCC tumors expressed FGFR1 mRNA, 18% exhibited FGFR1 CNG, 35% of amplified tumors were also positive for FGFR1 mRNA. This relationship was confirmed with the TCGA dataset. Using high FGFR1 mRNA for selection, 2 HNSCC PDXs were identified, one of which also exhibited FGFR1 CNG. The nonamplified tumor with high mRNA levels exhibited in vivo sensitivity to BGJ398.Conclusions: FGFR1 expression associates with BGJ398 sensitivity in HNSCC cell lines and predicts tyrosine kinase inhibitor sensitivity in PDXs. Our results support FGFR1 mRNA or protein expression, rather than FGFR1 CNG as a predictive biomarker for the response to FGFR inhibitors in a subset of patients suffering from HNSCC. Clin Cancer Res; 21(19); 4356–64. ©2015 AACR.

Figure S6. Correlation of HPV-expression and FGFR1 mRNA expression.

XLSX file - 66KB, Table S1. Compiled and tabulated ponatinib IC50 values, FGF and FGFR expression, FGFR1 gene copy number and characteristics of the 58 lung cancer cell lines.

Table 1: Cell lines, fusions, tyrosine kinase inhibitors and targets. Table 2: IC50 values for Figure 1C and relative fold change.

Table S1. The panel of HNSCC PDXs was submitted to FGFR1 gene copy number (GCN) by CISH and mRNA levels by in situ hybridization (ISH) assay.

Purpose: We previously reported that a fibroblast growth factor (FGF) receptor (FGFR) signaling pathway drives growth of lung cancer cell lines of squamous and large cell histologies. Herein, we explored FGFR dependency in cell lines derived from the tobacco-related malignancy, head and neck squamous cell carcinoma (HNSCC).Experimental Design: FGF and FGFR mRNA and protein expression was assessed in nine HNSCC cell lines. Dependence on secreted FGF2 for cell growth was tested with FP-1039, an FGFR1-Fc fusion protein. FGFR and epidermal growth factor receptor (EGFR) dependence was defined by sensitivity to multiple inhibitors selective for FGFRs or EGFR.Results: FGF2 was expressed in eight of the nine HNSCC cell lines examined. Also, FGFR2 and FGFR3 were frequently expressed, whereas only two lines expressed FGFR1. FP-1039 inhibited growth of HNSCC cell lines expressing FGF2, identifying FGF2 as an autocrine growth factor. FGFR inhibitors selectively reduced in vitro growth and extracellular signal-regulated kinase signaling in three HNSCC cell lines, whereas three distinct lines exhibited responsiveness to both EGFR and FGFR inhibitors. Combinations of these drugs yielded additive growth inhibition. Finally, three cell lines were highly sensitive to EGFR tyrosine kinase inhibitors (TKI) with no contribution from FGFR pathways.Conclusions: FGFR signaling was dominant or codominant with EGFR in six HNSCC lines, whereas three lines exhibited little or no role for FGFRs and were highly EGFR dependent. Thus, the HNSCC cell lines can be divided into subsets defined by sensitivity to EGFR and FGFR-specific TKIs. FGFR inhibitors may represent novel therapeutics to deploy alone or in combination with EGFR inhibitors in HNSCC. Clin Cancer Res; 17(15); 5016–25. ©2011 AACR.

Oncogenic kinase fusions of ALK, ROS1, RET, and NTRK1 act as drivers in human lung and other cancers. Residual tumor burden following treatment of ALK or ROS1+ lung cancer patients with oncogene-targeted therapy ultimately enables the emergence of drug-resistant clones, limiting the long-term effectiveness of these therapies. To determine the signaling mechanisms underlying incomplete tumor cell killing in oncogene-addicted cancer cells, we investigated the role of EGFR signaling in drug-naïve cancer cells harboring these oncogene fusions. We defined three distinct roles for EGFR in the response to oncogene-specific therapies. First, EGF-mediated activation of EGFR blunted fusion kinase inhibitor binding and restored fusion kinase signaling complexes. Second, fusion kinase inhibition shifted adaptor protein binding from the fusion oncoprotein to EGFR. Third, EGFR enabled bypass signaling to critical downstream pathways such as MAPK. While evidence of EGFR-mediated bypass signaling has been reported after ALK and ROS1 blockade, our results extended this effect to RET and NTRK1 blockade and uncovered the other additional mechanisms in gene fusion–positive lung cancer cells, mouse models, and human clinical specimens before the onset of acquired drug resistance. Collectively, our findings show how EGFR signaling can provide a critical adaptive survival mechanism that allows cancer cells to evade oncogene-specific inhibitors, providing a rationale to cotarget EGFR to reduce the risks of developing drug resistance. Cancer Res; 77(13); 3551–63. ©2017 AACR.

PDF file - 786KB, Figure S1. FGFR1 immunoblots of extracts from lung cancer cell lines representative of the 58 cell lines employed in the studies. Figure S2. Images and quantification of clonogenic growth assays in selected lung cancer cell lines following shRNA-mediated FGFR1 silencing. Figure S3. Dose response curve for inhibition of Colo699 cell growth by the FGF ligand trap, FP-1039. Figure S4. Venn diagram of the overlap of FGFR1, FGF2 and FGF9 expression in ponatinib-sensitive lung cancer cell lines and splicing status of FGFR2 in H1581 and H1048 cells. Figure S5. Graph of ROC analysis of FGFR1 GCN and mRNA expression in a TMA comprised of 21 lung cancer cell lines. Figure S6. Kaplan-Meier analysis of overall survival of lung cancer patients with increased FGFR1 GCN or high mRNA. Figure S7. Venn diagram showing overlap of FGF2 and FGF9 expression with FGFR1 mRNA positivity in primary lung squamous cell carcinomas and adenocarcinomas analyzed by the Cancer Genome Atlas.

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XLSX file - 106KB, Table S5. Tabulated FGFR1, FGF2 and FGF9 mRNA expression values, FGFR1 CNV values and mutation status of TCGA lung adenocarcinomas and squamous cell carcinomas.

Fig. S1: EGFR inhibition or stimulation affects normal and catalytically inactive fusion oncogene signaling. Fig. S2: EGFR knockdown reduces fusion kinase cancer cell proliferation. Fig. S3: Conserved re-phosphorylated activation loop tyrosines enabled signaling PLA design and representative image analyses of fusion kinase and EGFR signaling PLAs. Fig. S4: Optimization of the EGFR-GRB2 PLA and fusion kinase PLA antibody controls. Fig. S5: The GRB2 adaptor does not switch from ROS1 to MET with FKI treatment, but MET can signal through GRB2. FIg. S6: GRB2 and SHC1 signaling rewire in a RET+ cell line treated with a fusion kinase inhibitor (FKI). Fig. S7: TRK, RET, ROS1 and ALK fusion kinases interact specifically with EGFR. Fig. S8: EGFR is highly expressed, phosphorylated, and signaling in vivo in an NTRK1+ patient-derived xenograft (PDX) model. Fig. S9: Fusion kinase, and EGFR signaling complexes are present in RET, and ALK, fusion resistant patient samples.

Purpose: Patients with anaplastic lymphoma kinase (ALK) gene rearrangements often manifest dramatic responses to crizotinib, a small-molecule ALK inhibitor. Unfortunately, not every patient responds and acquired drug resistance inevitably develops in those who do respond. This study aimed to define molecular mechanisms of resistance to crizotinib in patients with ALK+ non–small cell lung cancer (NSCLC).Experimental Design: We analyzed tissue obtained from 14 patients with ALK+ NSCLC showing evidence of radiologic progression while on crizotinib to define mechanisms of intrinsic and acquired resistance to crizotinib.Results: Eleven patients had material evaluable for molecular analysis. Four patients (36%) developed secondary mutations in the tyrosine kinase domain of ALK. A novel mutation in the ALK domain, encoding a G1269A amino acid substitution that confers resistance to crizotinib in vitro, was identified in two of these cases. Two patients, one with a resistance mutation, exhibited new onset ALK copy number gain (CNG). One patient showed outgrowth of epidermal growth factor receptor (EGFR) mutant NSCLC without evidence of a persistent ALK gene rearrangement. Two patients exhibited a KRAS mutation, one of which occurred without evidence of a persisting ALK gene rearrangement. One patient showed the emergence of an ALK gene fusion–negative tumor compared with the baseline sample but with no identifiable alternate driver. Two patients retained ALK positivity with no identifiable resistance mechanism.Conclusions: Crizotinib resistance in ALK+ NSCLC occurs through somatic kinase domain mutations, ALK gene fusion CNG, and emergence of separate oncogenic drivers. Clin Cancer Res; 18(5); 1472–82. ©2012 AACR.

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PDF file - 90KB, Table S2. Tabulated expression values for selected FGFs in lung cancer cell lines from the Cancer Cell Line Encyclopedia. Table S3. Tabulated FGFR1 gene copy number values determined by SISH and associated patient data for the surgical lung cancer cohort. Table S4. Tabulated FGFR1 mRNA expression values determined by ISH and associated patient data for the surgical lung cancer cohort.

Supplementary Figure 1 from Protein Kinase C δ Is a Downstream Effector of Oncogenic K-ras in Lung Tumors

The FGFR1 is a therapeutic target under investigation in multiple solid tumors and clinical trials of selective tyrosine kinase inhibitors (TKI) are underway. Treatment with a single TKI represents a logical step toward personalized cancer therapy, but intrinsic and acquired resistance mechanisms limit their long-term benefit. In this study, we deployed RNAi-based functional genomic screens to identify protein kinases controlling the intrinsic sensitivity of FGFR1-dependent lung cancer and head and neck squamous cell cancer (HNSCC) cells to ponatinib, a multikinase FGFR-active inhibitor. We identified and validated a synthetic lethal interaction between MTOR and ponatinib in non–small cell lung carcinoma cells. In addition, treatment with MTOR-targeting shRNAs and pharmacologic inhibitors revealed that MTOR is an essential protein kinase in other FGFR1-expressing cancer cells. The combination of FGFR inhibitors and MTOR or AKT inhibitors resulted in synergistic growth suppression in vitro. Notably, tumor xenografts generated from FGFR1-dependent lung cancer cells exhibited only modest sensitivity to monotherapy with the FGFR-specific TKI, AZD4547, but when combined with the MTOR inhibitor, AZD2014, significantly attenuated tumor growth and prolonged survival. Our findings support the existence of a signaling network wherein FGFR1-driven ERK and activated MTOR/AKT represent distinct arms required to induce full transformation. Furthermore, they suggest that clinical efficacy of treatments for FGFR1-driven lung cancers and HNSCC may be achieved by combining MTOR inhibitors and FGFR-specific TKIs. Cancer Res; 75(20); 4398–406. ©2015 AACR.

Supplementary Figure 2 from Protein Kinase C δ Is a Downstream Effector of Oncogenic K-ras in Lung Tumors

Supplementary Methods, Figures S1-S12, Tables S1-S2. Figure S1. Overview of workflow for the synthetic lethal and essential gene screens. Figure S2. Procedure for isolating and barcoding PCR-amplified shRNA sequences. Figure S3. Growth inhibition of H157 cells by ponatinib in combination with MTOR inhibitor, AZD8055. Figure S4. Relative sensitivity of cancer cell lines to MTOR inhibitor AZD8055 using a cell proliferation assay. Figure S5. Dose response curves for growth inhibition of cancer cell lines by rapamycin. Figure S6. Growth inhibition by FGFR inhibitors in combination with MTOR inhibitor, AZD8055. Figure S7. Synergistic growth inhibition of CCL30 HNSCC cells by AZD4547 and AZD8055. Figure S8. Growth inhibition of Colo699 and H1581 cells by AZD4547 and rapamycin. Figure S9. Signal pathway inhibition by FGFR TKIs and rapamycin alone, and in combination. Figure S10. Growth inhibition by AKT inhibitors alone and combined with ponatinib on Colo699 cells. Figure S11. Combination ponatinib and AKT inhibitor GSK690693 yields synergistic in vitro growth inhibition. Figure S12. Combination AZD4547 and AZD2014 provides superior growth inhibition of Colo699 xenografts. Table S1. Primers used for sample preparation for Illumina sequencing. Table S2. Ranking of Synthetic Lethal Hits with ponatinib Treatment.

Supplementary Figure Legends 1-2 from Protein Kinase C δ Is a Downstream Effector of Oncogenic K-ras in Lung Tumors

Lung cancers bearing oncogenically-mutated EGFR represent a significant fraction of lung adenocarcinomas (LUADs) for which EGFR-targeting tyrosine kinase inhibitors (TKIs) provide a highly effective therapeutic approach. However, these lung cancers eventually acquire resistance and undergo progression within a characteristically broad treatment duration range. Our previous study of EGFR mutant lung cancer biopsies highlighted the positive association of a TKI-induced interferon γ transcriptional response with increased time to treatment progression. To test the hypothesis that host immunity contributes to the TKI response, we developed novel genetically-engineered mouse models of EGFR mutant lung cancer bearing exon 19 deletions (del19) or the L860R missense mutation. Both oncogenic EGFR mouse models developed multifocal LUADs from which transplantable cancer cell lines sensitive to the EGFR-specific TKIs, gefitinib and osimertinib, were derived. When propagated orthotopically in the left lungs of syngeneic C57BL/6 mice, deep and durable shrinkage of the cell line-derived tumors was observed in response to daily treatment with osimertinib. By contrast, orthotopic tumors propagated in immune deficientnu/numice exhibited modest tumor shrinkage followed by rapid progression on continuous osimertinib treatment. Importantly, osimertinib treatment significantly increased intratumoral CD3+ T cell content relative to diluent treatment. The findings provide strong evidence supporting the requirement for adaptive immunity in the durable therapeutic control of EGFR mutant lung cancer.

The KRAS(G12C) mutation is the most common genetic mutation in North American lung adenocarcinoma patients. Recently, direct inhibitors of the KRASG12C protein have been developed and demonstrate clinical response rates of 37-43%. Importantly, these agents fail to generate durable therapeutic responses with median progression-free survival of ~6.5 months. To provide models for further preclinical improvement of these inhibitors, we generated three novel murine KRASG12C-driven lung cancer cell lines. The co-occurring NRASQ61L mutation in KRASG12C-positive LLC cells was deleted and the KRASG12V allele in CMT167 cells was edited to KRASG12C with CRISPR/Cas9 methods. Also, a novel murine KRASG12C line, mKRC.1, was established from a tumor generated in a genetically-engineered mouse model. The three lines exhibit similar in vitro sensitivities to KRASG12C inhibitors (MRTX-1257, AMG-510), but distinct in vivo responses to MRTX-849 ranging from progressive growth with orthotopic LLC-NRAS KO tumors to marked shrinkage with mKRC.1 tumors. All three cell lines exhibited synergistic in vitro growth inhibition with MRTX-1257 and the SHP2 inhibitor, RMC-4550 and the MRTX-849/RMC-4550 combination yielded tumor shrinkage in orthotopic LLC-NRAS KO tumors propagated in syngeneic mice. Notably, this synergistic combination response was lost in athymic nu/nu mice, supporting a growing literature demonstrating a role for adaptive immunity in the response to this class of drugs. These new models of murine KRASG12C mutant lung cancer should prove valuable for identifying improved therapeutic combination strategies with KRASG12C inhibitors.Contribution to the Field StatementThe development of KRASG12C inhibitors has not impacted treatment of lung cancers bearing the KRASG12C mutation to the degree that tyrosine kinase inhibitors have changed the treatment outcomes for patients bearing oncogenic mutations in receptor tyrosine kinases. Thus, the field is now exploring combination strategies with KRASG12C inhibitors that may enhance their clinical benefit. Moreover, published findings indicate that host immunity contributes to efficacy of oncogene-directed inhibitors including KRASG12C inhibitors. Thus, these novel murine KRASG12C-driven lung cancer cell lines will provide valuable models for preclinical evaluation of novel drug combinations in immune competent hosts.

PurposeLung cancers bearing oncogenic EML4-ALK fusions respond to targeted tyrosine kinase inhibitors (TKIs; e.g. alectinib), with variation in the degree of shrinkage and duration of treatment (DOT). We previously demonstrated a positive association of a TKI-induced interferon gamma (IFNγ) transcriptional response with DOT in EGFR-mutant lung cancers. Herein, we used murine models of EML4-ALK lung cancer to test a role for host immunity in the therapeutic response to alectinib.Experimental DesignThree murine EML4-ALK cell lines (EA1, EA2, EA3) were implanted orthotopically into the lungs of immunocompetent and immunodeficient mice and treated with alectinib. Tumor volumes were serially measured by μCT. Immune cell content was measured by flow cytometry, multispectral immunofluorescence and CyTOF. Transcriptional responses to alectinib were assessed by RNAseq and secreted chemokines were measured by ELISA.ResultsAll cell lines were sensitive to alectinib in vitro. EA1 and EA3 tumors retained residual disease that rapidly progressed upon termination of treatment while EA2 tumors were eliminated by TKI treatment. Alectinib induced inflammatory transcriptional programs and multiple chemokines in all cell lines while untreated tumors exhibited distinct baseline chemokine expression patterns and content of CD8+ T cells and myeloid subsets. When propagated in immune-deficient mice, all three cell line-derived lung tumor models exhibited significant shrinkage followed by prompt progression despite continuous alectinib treatment.ConclusionsThe findings support an hypothesis that host and TKI-stimulated production of chemokines by tumor cells promotes functional engagement of adaptive immune cells within the tumor microenvironment that enhances the durability and depth of TKI response.Statement of Translational RelevancePatients with metastatic lung cancer harboring ALK fusions are treated with targeted tyrosine kinase inhibitors (TKI) in the first line setting. Despite bearing the same driver oncogene, patients experience a range of tumor burden reduction and variable amounts of residual disease. Residual disease burden associates with patient survival and contributes to the emergence of drug resistance yielding treatment failure. The factors mediating this differential response to TKI and residual disease are incompletely understood. Our group has developed a panel of murine ALK driven lung cancer cell lines that reproducibly show differences in the depth and duration of response when implanted into immunocompetent mice. Data using this model indicate that the presence of CD8+ T cells is a major contributor to the depth and duration of response. These models will be critical in developing rational combination therapies to augment the immune microenvironment engagement along with TKIs to improve outcomes for these patients.