Margaret Soucheray

Activation of the PD-1 Pathway Contributes to Immune Escape in EGFR-Driven Lung Tumors

UNLABELLED The success in lung cancer therapy with programmed death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between EGF receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, CTL antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased CTLs and increased markers of T-cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T-cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape and mechanistically link treatment response to PD-1 inhibition. SIGNIFICANCE We show that autochthonous EGFR-driven lung tumors inhibit antitumor immunity by activating the PD-1/PD-L1 pathway to suppress T-cell function and increase levels of proinflammatory cytokines. These findings indicate that EGFR functions as an oncogene through non-cell-autonomous mechanisms and raise the possibility that other oncogenes may drive immune escape.

Efficacy of BET Bromodomain Inhibition in Kras-Mutant Non–Small Cell Lung Cancer

Purpose: Amplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion, and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as the dependency of mutant Kras tumors on MYC function. Unfortunately, drug-like small-molecule inhibitors of KRAS and MYC have yet to be realized. The recent discovery, in hematologic malignancies, that bromodomain and extra-terminal (BET) bromodomain inhibition impairs MYC expression and MYC transcriptional function established the rationale of targeting KRAS-driven non–small cell lung cancer (NSCLC) with BET inhibition. Experimental Design: We performed functional assays to evaluate the effects of JQ1 in genetically defined NSCLC cell lines harboring KRAS and/or LKB1 mutations. Furthermore, we evaluated JQ1 in transgenic mouse lung cancer models expressing mutant kras or concurrent mutant kras and lkb1. Effects of bromodomain inhibition on transcriptional pathways were explored and validated by expression analysis. Results: Although JQ1 is broadly active in NSCLC cells, activity of JQ1 in mutant KRAS NSCLC is abrogated by concurrent alteration or genetic knockdown of LKB1. In sensitive NSCLC models, JQ1 treatment results in the coordinate downregulation of the MYC-dependent transcriptional program. We found that JQ1 treatment produces significant tumor regression in mutant kras mice. As predicted, tumors from mutant kras and lkb1 mice did not respond to JQ1. Conclusion: Bromodomain inhibition comprises a promising therapeutic strategy for KRAS-mutant NSCLC with wild-type LKB1, via inhibition of MYC function. Clinical studies of BET bromodomain inhibitors in aggressive NSCLC will be actively pursued. Clin Cancer Res; 19(22); 6183–92. ©2013 AACR.

Suppression of heat shock protein 27 induces long-term dormancy in human breast cancer

The mechanisms underlying tumor dormancy have been elusive and not well characterized. We recently published an experimental model for the study of human tumor dormancy and the role of angiogenesis, and reported that the angiogenic switch was preceded by a local increase in VEGF-A and basic fibroblast growth factor. In this breast cancer xenograft model (MDA-MB-436 cells), analysis of differentially expressed genes revealed that heat shock protein 27 (HSP27) was significantly up-regulated in angiogenic cells compared with nonangiogenic cells. The effect of HSP27 down-regulation was further evaluated in cell lines, mouse models, and clinical datasets of human patients with breast cancer and melanoma. Stable down-regulation of HSP27 in angiogenic tumor cells was followed by long-term tumor dormancy in vivo. Strikingly, only 4 of 30 HSP27 knockdown xenograft tumors initiated rapid growth after day 70, in correlation with a regain of HSP27 protein expression. Significantly, no tumors escaped from dormancy without HSP27 expression. Down-regulation of HSP27 was associated with reduced endothelial cell proliferation and decreased secretion of VEGF-A, VEGF-C, and basic fibroblast growth factor. Conversely, overexpression of HSP27 in nonangiogenic cells resulted in expansive tumor growth in vivo. By clinical validation, strong HSP27 protein expression was associated with markers of aggressive tumors and decreased survival in patients with breast cancer and melanoma. An HSP27-associated gene expression signature was related to molecular subgroups and survival in breast cancer. Our findings suggest a role for HSP27 in the balance between tumor dormancy and tumor progression, mediated by tumor–vascular interactions. Targeting HSP27 might offer a useful strategy in cancer treatment.

STK11/LKB1 Deficiency Promotes Neutrophil Recruitment and Proinflammatory Cytokine Production to Suppress T-cell Activity in the Lung Tumor Microenvironment

STK11/LKB1 is among the most commonly inactivated tumor suppressors in non-small cell lung cancer (NSCLC), especially in tumors harboring KRAS mutations. Many oncogenes promote immune escape, undermining the effectiveness of immunotherapies, but it is unclear whether the inactivation of tumor suppressor genes, such as STK11/LKB1, exerts similar effects. In this study, we investigated the consequences of STK11/LKB1 loss on the immune microenvironment in a mouse model of KRAS-driven NSCLC. Genetic ablation of STK11/LKB1 resulted in accumulation of neutrophils with T-cell-suppressive effects, along with a corresponding increase in the expression of T-cell exhaustion markers and tumor-promoting cytokines. The number of tumor-infiltrating lymphocytes was also reduced in LKB1-deficient mouse and human tumors. Furthermore, STK11/LKB1-inactivating mutations were associated with reduced expression of PD-1 ligand PD-L1 in mouse and patient tumors as well as in tumor-derived cell lines. Consistent with these results, PD-1-targeting antibodies were ineffective against Lkb1-deficient tumors. In contrast, treating Lkb1-deficient mice with an IL6-neutralizing antibody or a neutrophil-depleting antibody yielded therapeutic benefits associated with reduced neutrophil accumulation and proinflammatory cytokine expression. Our findings illustrate how tumor suppressor mutations can modulate the immune milieu of the tumor microenvironment, and they offer specific implications for addressing STK11/LKB1-mutated tumors with PD-1-targeting antibody therapies.

Intratumoral Heterogeneity in EGFR-Mutant NSCLC Results in Divergent Resistance Mechanisms in Response to EGFR Tyrosine Kinase Inhibition

Non-small cell lung cancers (NSCLC) that have developed resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI), including gefitinib and erlotinib, are clinically linked to an epithelial-to-mesenchymal transition (EMT) phenotype. Here, we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGFβ secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFβ was sufficient to induce EMT and resistance to EGFR TKI treatment. Furthermore, NSCLC HCC4006 cells with acquired resistance to gefitinib were characterized by a mesenchymal phenotype and displayed a higher prevalence of the EGFR T790M mutated allele. Notably, combined inhibition of EGFR and the TGFβ receptor in HCC4006 cells prevented EMT but was not sufficient to prevent acquired gefitinib resistance because of an increased emergence of the EGFR T790M allele compared with cells treated with gefitinib alone. Conversely, another independent NSCLC cell line, PC9, reproducibly developed EGFR T790M mutations as the primary mechanism underlying EGFR TKI resistance, even though the prevalence of the mutant allele was lower than that in HCC4006 cells. Thus, our findings underscore heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations that give rise to divergent resistance mechanisms in response to treatment and anticipate the complexity of EMT suppression as a therapeutic strategy.

Sympathetic inputs regulate adaptive thermogenesis in brown adipose tissue through cAMP-Salt inducible kinase axis

Various physiological stimuli, such as cold environment, diet, and hormones, trigger brown adipose tissue (BAT) to produce heat through sympathetic nervous system (SNS)- and β-adrenergic receptors (βARs). The βAR stimulation increases intracellular cAMP levels through heterotrimeric G proteins and adenylate cyclases, but the processes by which cAMP modulates brown adipocyte function are not fully understood. Here we described that specific ablation of cAMP production in brown adipocytes led to reduced lipolysis, mitochondrial biogenesis, uncoupling protein 1 (Ucp1) expression, and consequently defective adaptive thermogenesis. Elevated cAMP signaling by sympathetic activation inhibited Salt-inducible kinase 2 (Sik2) through protein kinase A (PKA)-mediated phosphorylation in brown adipose tissue. Inhibition of SIKs enhanced Ucp1 expression in differentiated brown adipocytes and Sik2 knockout mice exhibited enhanced adaptive thermogenesis at thermoneutrality in an Ucp1-dependent manner. Taken together, our data indicate that suppressing Sik2 by PKA-mediated phosphorylation is a requisite for SNS-induced Ucp1 expression and adaptive thermogenesis in BAT, and targeting Sik2 may present a novel therapeutic strategy to ramp up BAT thermogenic activity in humans.

Cross-talk Signaling between HER3 and HPV16 E6 and E7 Mediates Resistance to PI3K Inhibitors in Head and Neck Cancer

Human papillomavirus (HPV) type 16 is implicated in approximately 75% of head and neck squamous cell carcinomas (HNSCC) that arise in the oropharynx, where viral expression of the E6 and E7 oncoproteins promote cellular transformation, tumor growth, and maintenance. An important oncogenic signaling pathway activated by E6 and E7 is the PI3K pathway, a key driver of carcinogenesis. The PI3K pathway is also activated by mutation or amplification of PIK3CA in over half of HPV(+) HNSCC. In this study, we investigated the efficacy of PI3K-targeted therapies in HPV(+) HNSCC preclinical models and report that HPV(+) cell line- and patient-derived xenografts are resistant to PI3K inhibitors due to feedback signaling emanating from E6 and E7. Receptor tyrosine kinase profiling indicated that PI3K inhibition led to elevated expression of the HER3 receptor, which in turn increased the abundance of E6 and E7 to promote PI3K inhibitor resistance. Targeting HER3 with siRNA or the mAb CDX-3379 reduced E6 and E7 abundance and enhanced the efficacy of PI3K-targeted therapies. Together, these findings suggest that cross-talk between HER3 and HPV oncoproteins promotes resistance to PI3K inhibitors and that cotargeting HER3 and PI3K may be an effective therapeutic strategy in HPV(+) tumors.Significance: These findings suggest a new therapeutic combination that may improve outcomes in HPV(+) head and neck cancer patients. Cancer Res; 78(9); 2383-95. ©2018 AACR.

Abstract PR04: HER3 crosstalk with HPV16-E6E7 is a feedback resistance mechanism to PI3K-targeted therapies in head and neck cancer

Human papillomavirus (HPV) 16 plays an etiologic role in a growing subset of head and neck squamous cell carcinoma (HNSCC), where viral expression of the E6 and E7 oncoproteins is necessary for tumor growth and maintenance. Although patients with HPV(+) tumors have a more favorable prognosis, there are currently no HPV-selective therapies. Accumulating evidence indicates that HPV oncoproteins can activate the Phosphoinositol-3-Kinase (PI3K) pathway, which contributes to transformation. Furthermore, PI3K is genomically activated by PIK3CA mutation or amplification in a disproportionately high number of HPV(+) tumors as compared to HPV(-) tumors. Based on this knowledge, we investigated the efficacy of PI3K-targeted therapies in preclinical models of HPV(+) HNSCC. Our results indicate that HPV(+) preclinical models were less sensitive to the PI3K inhibitors BYL719, BKM120, and BEZ235 as compared with HPV(-) models. Sensitivity of HPV(+) cell lines to PI3K inhibitors was increased upon knockdown of the E6 and E7 oncoproteins. Reciprocally, overexpression of E6 and E7 in HPV(-) cells rendered them resistant to PI3K-targeted therapies. Proteomic analyses indicated that treatment of HPV(+) cell lines and patient-derived xenografts (PDXs) with the PI3Kα inhibitor BYL719 induced expression of the receptor tyrosine kinase HER3, as well as E6 and E7. HER3 was found to regulate the abundance of E6 and E7 in the HPV(+) models. Targeting HER3 with siRNAs or the monoclonal antibody, KTN3379, blocked the increase in E6 and E7 protein levels following BYL719 treatment, and enhanced the efficacy of PI3K inhibitors in HPV(+) cell line and PDX models. Taken together, these results suggest that crosstalk between HER3 and HPV16-E6E7 can limit the efficacy of PI3K inhibitors, and that co-targeting HER3 and PI3K may be an effective therapeutic strategy in HPV(+) tumors. This abstract is also being presented as Poster 67. Citation Format: Toni M. Brand, Stefan Hartmann, Neil E. Bhola, Hua Li, Yan Zeng, Rachel O9Keefe, Max V. Ranall, Sourav Bandyopadhyay, Margaret Soucheray, Danielle L. Swaney, Nevan Krogan, Carolyn Kemp, Umamaheswar Duvvuri, Daniel E. Johnson, Michelle A. Ozbun, Julie E. Bauman, Jennifer R. Grandis. HER3 crosstalk with HPV16-E6E7 is a feedback resistance mechanism to PI3K-targeted therapies in head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr PR04.

Abstract C75: Overcoming KRAS/LKB1 mutant NSCLC resistance to BET bromodomain inhibitors with gemcitabine or Mcl-1 inhibition

The purpose of our study was to define a method and mechanism for overcoming the resistance of clinically relevant KRAS-mutant/LKB1-deficient NSCLC cells to the BET-bromodomain inhibitor JQ1. LKB1 (Serine/threonine kinase 11) is mutated with loss of function in conjunction with mutated KRAS in 7-10% of NSCLC. Importantly, KRAS-mutant/LKB1-deficiency is associated with tumor aggressiveness and poor survival in human patients as well as in genetically engineered mouse models. Indeed, although the BET bromodomain inhibitor JQ1 dramatically reduces tumor volume in KRAS mutant mice, it has little effect in KRAS-mutant/LKB1-deficient mice. BET bromodomain proteins are chromatin readers that facilitate binding and activity of transcription factors such as the oncogene MYC. As such, JQ1 inhibits the transcriptional program of MYC and decreases MYC in mice. To overcome JQ1 resistance in KRAS-mutant/LKB1-deficient NSCLC, we proposed to test JQ1 combination with chemotherapy, as such a combination would be a necessary arm of a clinical trial testing a BET bromodomain inhibitor. Here, we present data showing that gemcitabine permits and potentiates JQ1-mediated apoptosis of multiple KRAS-mutant/LKB1-deficient human NSCLC cell lines above either monotherapy. Increased apoptosis was observed by AnnexinV and TUNEL flow cytometry, PARP cleavage, and Comet assays. Mechanistically, the drug combination had minimal effects on DNA damage and repair molecules in the KRAS-mutant/LKB1 deficient NSCLC cell line A549. Interestingly, however, JQ1 and combination with gemcitabine had profound effects on apoptotic molecules. JQ1 alone showed a robust increase in pro-apoptotic BimEL, BimL, and BimS that was balanced by a large increase in anti-apoptotic Mcl-1 24 hrs post-treatment. Similarly, MYC knockdown also increased Mcl-1, suggesting that JQ1 upregulation of Mcl-1 was at least partly dependent on JQ1 suppression of MYC. On the other hand, JQ1 combination with gemcitabine showed an increase in Bim that was not associated with increased Mcl-1. Given these findings, we reasoned that KRAS-mutant/LKB1-deficient cells were resistant to JQ1-induced apoptosis executed by Bim because of protection provided by increased Mcl-1. In support of this hypothesis, Bim knockdown prevented JQ1-induced PARP cleavage in JQ1-sensitive H441 cells. Furthermore, Mcl-1 knockdown cells showed increased Bim, decreased Bcl-2, and increased PARP cleavage 48 hrs after JQ1 treatment in otherwise JQ1-resistant A549 cells. Collectively, KRAS-mutant/LKB1-deficient cells are resistant to JQ1-induced apoptosis because of a compensatory increase in Mcl-1. This resistance can be overcome by combinatorial treatment with gemcitabine or direct Mcl-1 suppression, restoring apoptosis. Therefore, combinatorial treatment of a BET bromodomain inhibitor with gemcitabine or a Mcl-1 inhibitor may represent a potential novel strategy for treating the clinically relevant KRAS-mutant/LKB1-deficient subtype of NSCLC. Citation Format: Michael Kahle, Margaret Soucheray, Jeffrey Becker, Eiki Kikuchi, Ines Pulido, Esra Akbay, Camilla Christensen, Wei Qiu, Fatima Al-Shahrour, Neil Johnson, Julian Carretero, Kwok-Kin Wong, Takeshi Shimamura. Overcoming KRAS/LKB1 mutant NSCLC resistance to BET bromodomain inhibitors with gemcitabine or Mcl-1 inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C75.

Abstract LB-C21: CXCR7 expression is necessary for the maintenance of mesenchymal phenotype in acquired EGFR TKI resistance in NSCLC

Activating EGFR mutations in non-small lung cancer (NSCLC) confer sensitivity to reversible EGFR tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib. Despite promising initial response, acquired resistance develops mediated by the emergence of the secondary T790M mutation or by focal amplification of MET. An epithelial-to-mesenchymal transition (EMT) is clinically linked to NSCLCs with acquired EGFR TKI resistance. The exact mechanisms of EGFR TKI resistance with EMT phenotype remain elusive; therefore, we have engineered EGFR-mutated NSCLC cell lines with mesenchymal phenotype by stably depleting E-Cadherin or by overexpressing Snail or chronically exposing the cells to TGFβ1. The resulting mesenchymal cells are resistant to EGFR TKIs. We employed genomic analyses to identify that C-X-C chemokine receptor type 7 (CXCR7) is commonly overexpressed in the engineered cells with mesenchymal phenotype. We also discovered that CXCR7 is overexpressed in EGFR-mutated HCC4006 NSCLC cells grown resistant to gefitinib that developed a mesenchymal phenotype (HCC4006 Ge-R). To extend our findings to in vivo, we assessed if CXCR7 is overexpressed in mouse lung cancers driven by human EGFR exon19 deletion/T790M (TD) that initially respond to the EGFR mutant-specific irreversible TKI WZ4002 to promote tumor regression but later develops tumors with acquired resistance. We discovered that the murine tumors with acquired resistance to WZ4002 present mesenchymal phenotype and overexpress CXCR7. Stable depletion of CXCR7 in HCC4006Ge-R mesenchymal gefitinib resistant cells promoted gradual mesenchymal to epithelial transition. Sustained depletion of CXCR7 in HCC4006Ge-R cells resulted in inactivation of PI3K and MAPK pathways upon gefitinib treatment and greatly restored the sensitivity to gefitinib. Furthermore, the depletion of CXCR7 in HCC4006GeR cells resulted in the downregulation of mesenchymal transcription factors essential including TWIST, ZEB1 and ZEB2, suggesting but not proving that CXCR7 maintains mesenchymal phenotype. To determine if the resistance mechanisms to gefitinib with mesenchymal cells expressing CXCR7 evolve under the selective pressure of gefitinib or pre-exist prior to treatment, we sorted EGFR-mutated NSCLC cells with CXCR7 and mesenchymal markers to find less than 5% of the cells express CXCR7 with mesenchymal phenotype. Interestingly, ectopic expression of CXCR7 in EGFR-mutated cells was not sufficient to confer resistance to EGFR TKIs or to promote EMT. Taken together, we discovered that CXCR7 is necessary for the maintenance of EGFR TKI resistance with mesenchymal phenotype and the population of mesenchymal cells that overexpress CXCR7 pre-exists and is selected upon chronic EGFR TKI treatment. Citation Format: Jeffrey H. Becker, Yandi Gao, Ines Pulido, Eiki Kikuchi, Margaret Soucheray, Rutu Gandhi, Camilla L. Christensen, Fatima Al-shahrour, Kwok-Kin Wong, Julian Carretero, Takeshi Shimamura. CXCR7 expression is necessary for the maintenance of mesenchymal phenotype in acquired EGFR TKI resistance in NSCLC. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C21.