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With only a fraction of patients responding to cancer immunotherapy, a better understanding of the entire tumor microenvironment is needed. Using single-cell transcriptomics, we chart the fibroblastic landscape during pancreatic ductal adenocarcinoma (PDAC) progression in animal models. We identify a population of carcinoma-associated fibroblasts (CAF) that are programmed by TGFβ and express the leucine-rich repeat containing 15 (LRRC15) protein. These LRRC15+ CAFs surround tumor islets and are absent from normal pancreatic tissue. The presence of LRRC15+ CAFs in human patients was confirmed in >80,000 single cells from 22 patients with PDAC as well as by using IHC on samples from 70 patients. Furthermore, immunotherapy clinical trials comprising more than 600 patients across six cancer types revealed elevated levels of the LRRC15+ CAF signature correlated with poor response to anti–PD-L1 therapy. This work has important implications for targeting nonimmune elements of the tumor microenvironment to boost responses of patients with cancer to immune checkpoint blockade therapy.Significance:This study describes the single-cell landscape of CAFs in pancreatic cancer during in vivo tumor evolution. A TGFβ-driven, LRRC15+ CAF lineage is associated with poor outcome in immunotherapy trial data comprising multiple solid-tumor entities and represents a target for combinatorial therapy.This article is highlighted in the In This Issue feature, p. 161

Figure S5,6. Effects of Nrf2 knockdown in different growth conditions

Figure S15. Relationship between KEAP1 mutant status and other genes in NSCLC

S1. Sequences of RNAi reagents S2. Data from siRNA screens S3. Genes decreased following Nrf2 KD and increased in KEAP1 mutant lung cancer cells S4. Data from CRISPR screens

Supplementary methods and figure legends

Figure S7,8. Effects of different compounds cell viability and genes included in CRISPR library

Mutations in KEAP1 and NFE2L2 (encoding the protein Nrf2) are prevalent in both adeno and squamous subtypes of non–small cell lung cancer, as well as additional tumor indications. The consequence of these mutations is stabilized Nrf2 and chronic induction of a battery of Nrf2 target genes. We show that knockdown of Nrf2 caused modest growth inhibition of cells growing in two-dimension, which was more pronounced in cell lines expressing mutant KEAP1. In contrast, Nrf2 knockdown caused almost complete regression of established KEAP1-mutant tumors in mice, with little effect on wild-type (WT) KEAP1 tumors. The strong dependency on Nrf2 could be recapitulated in certain anchorage-independent growth environments and was not prevented by excess extracellular glutathione. A CRISPR screen was used to investigate the mechanism(s) underlying this dependence. We identified alternative pathways critical for Nrf2-dependent growth in KEAP1-mutant cell lines, including the redox proteins thioredoxin and peroxiredoxin, as well as the growth factor receptors IGF1R and ERBB3. IGF1R inhibition was effective in KEAP1-mutant cells compared with WT, especially under conditions of anchorage-independent growth. These results point to addiction of KEAP1-mutant tumor cells to Nrf2 and suggest that inhibition of Nrf2 or discrete druggable Nrf2 target genes such as IGF1R could be an effective therapeutic strategy for disabling these tumors.Significance:This study identifies pathways activated by Nrf2 that are important for the proliferation and tumorigenicity of KEAP1-mutant non–small cell lung cancer.

Figure S1. Consequences of KEAP1 mutations on Nrf2 expression and ubiquitination Figure S2-4. Inducible knockdown of Nrf2 in KEAP1 mutant and WT cells and tumors

Figures S9-11. Effects of knocking down genes involved in oxidative stress

With only a fraction of patients responding to cancer immunotherapy, a better understanding of the entire tumor microenvironment is needed. Using single-cell transcriptomics, we chart the fibroblastic landscape during pancreatic ductal adenocarcinoma (PDAC) progression in animal models. We identify a population of carcinoma-associated fibroblasts (CAF) that are programmed by TGFβ and express the leucine-rich repeat containing 15 (LRRC15) protein. These LRRC15+ CAFs surround tumor islets and are absent from normal pancreatic tissue. The presence of LRRC15+ CAFs in human patients was confirmed in >80,000 single cells from 22 patients with PDAC as well as by using IHC on samples from 70 patients. Furthermore, immunotherapy clinical trials comprising more than 600 patients across six cancer types revealed elevated levels of the LRRC15+ CAF signature correlated with poor response to anti–PD-L1 therapy. This work has important implications for targeting nonimmune elements of the tumor microenvironment to boost responses of patients with cancer to immune checkpoint blockade therapy. Significance: This study describes the single-cell landscape of CAFs in pancreatic cancer during in vivo tumor evolution. A TGFβ-driven, LRRC15+ CAF lineage is associated with poor outcome in immunotherapy trial data comprising multiple solid-tumor entities and represents a target for combinatorial therapy. This article is highlighted in the In This Issue feature, p. 161

Mutations in KEAP1 and NFE2L2 (encoding the protein Nrf2) are prevalent in both adeno and squamous subtypes of non–small cell lung cancer, as well as additional tumor indications. The consequence of these mutations is stabilized Nrf2 and chronic induction of a battery of Nrf2 target genes. We show that knockdown of Nrf2 caused modest growth inhibition of cells growing in two-dimension, which was more pronounced in cell lines expressing mutant KEAP1. In contrast, Nrf2 knockdown caused almost complete regression of established KEAP1-mutant tumors in mice, with little effect on wild-type (WT) KEAP1 tumors. The strong dependency on Nrf2 could be recapitulated in certain anchorage-independent growth environments and was not prevented by excess extracellular glutathione. A CRISPR screen was used to investigate the mechanism(s) underlying this dependence. We identified alternative pathways critical for Nrf2-dependent growth in KEAP1-mutant cell lines, including the redox proteins thioredoxin and peroxiredoxin, as well as the growth factor receptors IGF1R and ERBB3. IGF1R inhibition was effective in KEAP1-mutant cells compared with WT, especially under conditions of anchorage-independent growth. These results point to addiction of KEAP1-mutant tumor cells to Nrf2 and suggest that inhibition of Nrf2 or discrete druggable Nrf2 target genes such as IGF1R could be an effective therapeutic strategy for disabling these tumors. Significance: This study identifies pathways activated by Nrf2 that are important for the proliferation and tumorigenicity of KEAP1-mutant non–small cell lung cancer.

Lung adenocarcinoma is often discovered as metastatic disease with very poor prognosis. However, much remains unknown about the mechanisms of lung adenocarcinoma tumor progression. In this study we showed that knockdown of BUB1B, a critical mitotic checkpoint protein, significantly inhibited anchorage-independent growth of lung adenocarcinoma cell lines. In allograft and tail vein mouse model studies, BUB1B suppression inhibited primary tumor growth and reduced metastasis to the lung and lymph nodes, resulting in prolonged survival in both tumor prevention and tumor intervention settings. Mechanistic studies revealed that BUB1B knockdown sensitized cells to anoikis. The N-terminal region of BUB1B was required for its functions in both anchorage-independent growth and anoikis resistance, whereas the kinase domain was less critical. Overexpression of BUB1B is associated with disease progression and poor survival in human lung adenocarcinoma patients. Collectively, these data reveal a novel function for BUB1B in mediating anchorage-independent survival and growth, thereby facilitating lung adenocarcinoma dissemination during metastasis. Thus, targeting BUB1B could provide potential therapeutic benefit in suppressing metastasis and prolonging survival in lung adenocarcinoma patients. Citation Format: Honglin Chen, James Lee, Noelyn M. Kljavin, Benjamin Haley, Anneleen Daemen, Yuxin Liang, Leisa Johnson. Requirement for BUB1B in tumor progression of lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2259. doi:10.1158/1538-7445.AM2015-2259

We have discovered a potential oncogenic role of a gene in non-small cell lung cancer (NSCLC) using RNAi technology. Silencing of the gene significantly inhibits soft agar colony formation of NSCLC cell lines, whereas its inhibition on cell proliferation in liquid culture is modest. This effect was observed in multiple murine and human NSCLC cell lines with varied Kras and p53 status. The inhibition on soft agar colony formation is reversed with ectopic overexpression of a cDNA encoding the full-length gene. Knocking down the expression of the gene also significantly impairs migration and enhances detachment-induced anoikis of NSCLC cells. An investigation of the molecular mechanisms reveals that silencing of the gene cytoskeleton signaling network. In xenograft studies using stable luciferase-expressing inducible shRNA cell lines, silencing of the gene upon doxycycline treatment leads to a modest reduction in tumor volumes while significantly decreases bioluminescent signals at metastatic sites including lung and lymph nodes. Furthermore, gene expression analyses in KrasG12D/p53 deletion-driven mouse tumors and clinical tumor samples show significantly increased expression of the gene in lung cancer as well as multiple other cancer types. Taken together, we have uncovered a novel oncogenic function of a gene in NSCLC, which could provide a potential new therapeutic target for cancer intervention. Citation Format: Honglin Chen, James Lee, Noelyn M. Kljavin, Leisa Johnson, Yuxin Liang. Identification of a novel gene target involved in tumor growth and metastasis of non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3015. doi:10.1158/1538-7445.AM2013-3015 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

The Ras GTPase family controls numerous downstream signaling cascades in response to signals that regulate cellular processes including proliferation and survival. While Ras is one of the most prevalent targets for gain-of-function mutations in human tumors, questions remain regarding how the Ras effector pathway functions in mutant K-ras-driven tumorigenesis. Since an important function of K-ras involves B-Raf activation within the canonical MAPK signaling pathway, we initiated a study to determine B-raf9s role in the context of mutant K-ras-driven tumor promotion and maintenance. We began by delivering adenovirus expressing the Cre recombinase to the lungs of genetically engineered mice possessing a conditional K-ras G12D allele ( K-rasLSL- G12D ) and either 0, 1 or both copies of the B-raf gene flanked by LoxP sites ( B-raf CKO ). This procedure results in expression of mutant K-ras G12D in the presence or absence of one or both B-raf alleles deleted within the mouse lung. Surprisingly, we observe that B-raf deletion significantly enhances lung tumor number and burden and decreases overall survival. When we used a highly specific small-molecule inhibitor that targets B-raf in a murine non-small cell lung carcinoma line harboring the K-ras G12D mutation, we observed an increase in cell proliferation and soft agar colony formation. Further investigation revealed that treating K-ras G12D expressing cells with the B-raf inhibitor enhanced MEK and Erk phosphorylation. Therefore, our data suggests that while B-raf deletion does not inhibit K-ras-driven tumor initiation and disease progression, its presence may play a pivotal role in establishing negative feedback regulation of constitutive mutant K-ras activity. Citation Information: Cancer Res 2009;69(23 Suppl):B31.

The Ras GTPase family controls numerous downstream signaling cascades in response to signals that regulate cellular processes including proliferation and survival. While Ras is one of the most prevalent targets for gain-of-function mutations in human tumors, questions remain regarding how the Ras effector pathway functions in mutant K-ras-driven tumorigenesis. Since an important function of K-ras involves B-Raf activation within the canonical MAPK signaling pathway, we initiated a study to determine B-raf's role in the context of mutant K-ras-driven tumor promotion and maintenance. We began by delivering adenovirus expressing the Cre recombinase to the lungs of genetically engineered mice possessing a conditional K-rasG12D allele (K-rasLSL-G12D) and either 0, 1 or both copies of the B-raf gene flanked by LoxP sites (B-rafCKO). This procedure results in expression of mutant K-rasG12D in the presence or absence of one or both B-raf alleles deleted within the mouse lung. Surprisingly, we observe that B-raf deletion significantly enhances lung tumor number and burden and decreases overall survival. When we used a highly specific small-molecule inhibitor that targets B-raf in a murine non-small cell lung carcinoma line harboring the K-rasG12D mutation, we observed an increase in cell proliferation and soft agar colony formation. Further investigation revealed that treating K-rasG12D expressing cells with the B-raf inhibitor enhanced MEK and Erk phosphorylation. Therefore, our data suggests that while B-raf deletion does not inhibit K-ras-driven tumor initiation and disease progression, its presence may play a pivotal role in establishing negative feedback regulation of constitutive mutant K-ras activity. Citation Information: Cancer Res 2009;69(23 Suppl):B31.