Search

Liver kinase B1 (LKB1)–inactivated tumors are vulnerable to the disruption of pyrimidine metabolism, and leflunomide emerges as a therapeutic candidate because its active metabolite, A77–1726, inhibits dihydroorotate dehydrogenase, which is essential for de novo pyrimidine biosynthesis. However, it is unclear whether leflunomide inhibits LKB1-inactivated tumors in vivo, and whether its inhibitory effect on the immune system will promote tumor growth. Here, we carried out a comprehensive analysis of leflunomide treatment in various LKB1-inactivated murine xenografts, patient-derived xenografts, and genetically engineered mouse models. We also generated a mouse tumor–derived cancer cell line, WRJ388, that could metastasize to the lung within a month after subcutaneous implantation in all animals. This model was used to assess the ability of leflunomide to control distant metastasis. Leflunomide treatment shrank a HeLa xenograft and attenuated the growth of an H460 xenograft, a patient-derived xenograft, and lung adenocarcinoma in the immune-competent genetically engineered mouse models. Interestingly, leflunomide suppressed tumor growth through at least three different mechanisms. It caused apoptosis in HeLa cells, induced G1 cell-cycle arrest in H460 cells, and promoted S-phase cell-cycle arrest in WRJ388 cells. Finally, leflunomide treatment prevented lung metastasis in 78% of the animals in our novel lung cancer metastasis model. In combination, these results demonstrated that leflunomide utilizes different pathways to suppress the growth of LKB1-inactivated tumors, and it also prevents cancer metastasis at distant sites. Therefore, leflunomide should be evaluated as a therapeutic agent for tumors with LKB1 inactivation.

Supplemental Figures and Tables

Figure S1. Bak BH3 peptide position in Bak protein. Figure S2. Dephosphorylation of Bax by lambdaPPase in vitro. Figure S3. Bax activators CYD-2-11 and SMBA1 failed to induce Bak oligomerization. Figure S4. Analysis of CYD-2-11 toxicity in vivo. Figure S5. CYD-2-11 has no significant effect on total Bax expression in SCLC PDX models. Figure S6. Toxicity analysis of CYD-2-11 in 2 PDX models. Figure S7. Bax phosphorylation is up-regulated in KRAS driven lung cancer. Figure S8. Quantification of tumor burden. Figure S9. H&E staining of normal tissues from various organs after CYD-2-11 treatment of KL mice. Figure S10. The dual PI3K/mTOR inhibitor BEZ235 inhibits ATK activity leading to down-regulation of Bax phosphorylation. Figure S11. CYD-2-11 synergizes with RAD001 to reverse rapalog resistance of lung cancer cells. Figure S12. RAD001 and CYD-2-11 did not affect total Bax in A549 xenografts. Figure S13. Toxicity analysis following treatment with RAD001, CYD-2-11 or in combination in A549 xenografts. Figure S14. CYD-2-11 synergizes with RAD001 against lung cancer in PDX model.

A rationale exists for pharmacologic manipulation of the serine (S)184 phosphorylation site of the proapoptotic Bcl2 family member Bax as an anticancer strategy. Here, we report the refinement of the Bax agonist SMBA1 to generate CYD-2-11, which has characteristics of a suitable clinical lead compound. CYD-2-11 targeted the structural pocket proximal to S184 in the C-terminal region of Bax, directly activating its proapoptotic activity by inducing a conformational change enabling formation of Bax homooligomers in mitochondrial membranes. In murine models of small-cell and non–small cell lung cancers, including patient-derived xenograft and the genetically engineered mutant KRAS-driven lung cancer models, CYD-2-11 suppressed malignant growth without evident significant toxicity to normal tissues. In lung cancer patients treated with mTOR inhibitor RAD001, we observed enhanced S184 Bax phosphorylation in lung cancer cells and tissues that inactivates the propaoptotic function of Bax, contributing to rapalog resistance. Combined treatment of CYD-2-11 and RAD001 in murine lung cancer models displayed strong synergistic activity and overcame rapalog resistance in vitro and in vivo. Taken together, our findings provide preclinical evidence for a pharmacologic combination of Bax activation and mTOR inhibition as a rational strategy to improve lung cancer treatment. Cancer Res; 77(11); 3001–12. ©2017 AACR.

Purpose: Vimentin is an epithelial-to-mesenchymal transition (EMT) biomarker and intermediate filament protein that functions during cell migration to maintain structure and motility. Despite the abundance of clinical data linking vimentin to poor patient outcome, it is unclear if vimentin is required for metastasis or is a correlative biomarker. We developed a novel genetically engineered mouse model (GEMM) to probe vimentin in lung adenocarcinoma metastasis.Experimental Design: We used the LSL-KrasG12D/Lkb1fl/fl/Vim−/− model (KLV−/−), which incorporates a whole-body knockout of vimentin and is derived from the Cre-dependent LSL-KrasG12D/Lkb1fl/fl model (KLV+/+). We compared the metastatic phenotypes of the GEMMs and analyzed primary tumors from the KLV models and lung adenocarcinoma patients to assess vimentin expression and function.Results: Characterization of KLV+/+ and KLV−/− mice shows that although vimentin is not required for primary lung tumor growth, vimentin is required for metastasis, and vimentin loss generates lower grade primary tumors. Interestingly, in the KLV+/+ mice, vimentin was not expressed in tumor cells but in cancer-associated fibroblasts (CAFs) surrounding collective invasion packs (CIPs) of epithelial tumor cells, with significantly less CIPs in KLV−/− mice. CIPs correlate with tumor grade and are vimentin-negative and E-cadherin–positive, indicating a lack of cancer cell EMT. A similar heterotypic staining pattern was observed in human lung adenocarcinoma samples. In vitro studies show that vimentin is required for CAF motility to lead tumor cell invasion, supporting a vimentin-dependent model of collective invasion.Conclusions: These data show that vimentin is required for lung adenocarcinoma metastasis by maintaining heterotypic tumor cell–CAF interactions during collective invasion. Clin Cancer Res; 24(2); 420–32. ©2017 AACR.

Purine nucleoside phosphorylase (PNP) gene transfer represents a promising approach to treatment of head and neck malignancies. We tested recombinant adenovirus already in phase I/II clinical testing and leading-edge patient-derived xenografts (PDX) as a means to optimize this therapeutic strategy.Our experiments investigated purine base cytotoxicity, PNP enzyme activity following treatment of malignant tissue, tumor mass regression, viral receptor studies, and transduction by tropism-modified adenovirus.Replication deficient vector efficiently transduced PDX cells and mediated significant anticancer effect following treatment with fludarabine phosphate in vivo. Either 6-methylpurine or 2-fluoroadenine (toxic molecules generated by the PNP approach) ablated head and neck cancer cell proliferation. High levels of adenovirus-3 specific receptors were detected in human tumor models, and vector was evaluated that utilizes this pathway.Our studies provide the scientific foundation necessary to improve PNP prodrug cleavage and advance a new treatment for head and neck cancer.

KRAS and LKB1 are frequently co-mutated in lung adenocarcinoma and characterize a particularly aggressive, metastatic, and treatment-resistant subtype of this disease. We recently published that the outputs of biological rhythm pathways downstream of the AMPK kinase are altered in KRAS/LKB1-mutant tumors. In a live-cell phenotyping screen we used RNAseq to identify the circadian clock genes PER1 and PER2 as uniquely upregulated in partially transformed and invasive KRAS/LKB1-mutant human bronchial epithelial cells. We have analyzed expression of individual circadian clock genes in KRAS-mutant and KRAS/LKB1-mutant patient tumors and found that expression of the PER genes, particularly PER1, is increased in the KRAS/LKB1 subtype of lung adenocarcinoma. At the protein level, PER1 expression is increased in KRAS/LKB1-mutant HBECs as compared to KRAS-mutant cells. Addback of wild-type LKB1 in A549 cells, which are KRAS/LKB1-mutant, results in a decrease in PER1 protein levels, confirming that LKB1 has a role in controlling PER1 levels. Multiple phosphorylation events lead to PER1 nuclear translocation to control its role as a transcriptional repressor in the core clock. In circadian-synced HBECs, PER1 enters the nucleus in KRAS-mutant cells, but remains cytosolic in KRAS/LKB1-mutant cells. In circadian-synced A549 cells, we also observed a lack of nuclear PER1 localization. Addback of wild-type LKB1 restored nuclear localization of PER1, as did expression of kinase-dead LKB1, suggesting that LKB1’s regulation of PER1 localization is kinase-independent. Our current and future experiments aim to determine how clock-controlled gene expression may be altered in KRAS/LKB1-mutant cells, what the functional impact of altered PER1 expression and localization is on growth of KRAS/LKB1-mutant tumors, and whether pharmacological manipulation of LKB1-related signaling pathways can restore nuclear localization of PER1. Citation Format: Rebecca E. Parker, Junghui Koo, Bhakti Dwivedi, Adam I. Marcus, Melissa Gilbert-Ross. Defining the role of PER1 and circadian rhythm dysregulation in KRAS/LKB1-mutant lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A027.

The genetic and metabolic heterogeneity of RAS-driven cancers has confounded therapeutic strategies in the clinic. To address this, rapid and genetically tractable animal models are needed that recapitulate the heterogeneity of RAS-driven cancers in vivo. Here, we generate a Drosophila melanogaster model of Ras/Lkb1 mutant carcinoma. We show that low-level expression of oncogenic Ras (RasLow) promotes the survival of Lkb1 mutant tissue, but results in autonomous cell cycle arrest and non-autonomous overgrowth of wild-type tissue. In contrast, high-level expression of oncogenic Ras (RasHigh) transforms Lkb1 mutant tissue resulting in lethal malignant tumors. Using simultaneous multiview light-sheet microcopy, we have characterized invasion phenotypes of Ras/Lkb1 tumors in living larvae. Our molecular analysis reveals sustained activation of the AMPK pathway in malignant Ras/Lkb1 tumors, and demonstrate the genetic and pharmacologic dependence of these tumors on CaMK-activated Ampk. We further show that LKB1 mutant human lung adenocarcinoma patients with high levels of oncogenic KRAS exhibit worse overall survival and increased AMPK activation. Our results suggest that high levels of oncogenic KRAS is a driving event in the malignant transformation of LKB1 mutant tissue, and uncovers a vulnerability that may be used to target this aggressive genetic subset of RAS-driven tumors., Rackley, Seong et al use a Drosophila model of Ras/Lkb1 mutant carcinoma to show that high levels of the KRAS oncogene drive the malignant transformation of LKB1 mutant tissue. This pathway could be used as a target for future therapies that aim to treat RAS-driven tumours.

Lung cancer is among the most commonly diagnosed cancers and the leading cause of cancer-related death globally, with approximately 85% of all lung cancers having non-small cell lung cancer (NSCLC). The protein kinase LKB1 is the 3rd most frequently mutated gene in lung adenocarcinoma, and serves as a master regulator of cell metabolism and growth. LKB1-inactivating mutations have a significant impact on NSCLC development. Although KRAS mutation is most frequently co-mutated with the tumor suppressor p53, the strongest cooperation was seen with homozygous inactivation of LKB1. Loss of LKB1 on a KRAS-mutant (KL) background leads to increased tumor burden, shortened survival time and increased metastasis compared to loss of TP53 (KP). Furthermore, inactivation or loss of LKB1 correlated with poor prognosis, where tumors are typically aggressive and chemoresistant. To discover therapeutic vulnerabilities for this aggressive genetic subtype of lung adenocarcinoma, we evaluated the contribution of multiple single- and double-genetic alterations (KRAS G12D (K), LKB1 knockdown (L), and p53 knockdown (P)) to 3D invasion phenotypes using immortalized human bronchial epithelial cells (HBEC3-KT) and conducted RNA transcriptome profiling of the invading HBEC3-KT 3D spheroids. We found that bone morphogenetic protein 6 (BMP-6) is upregulated in invasive KL spheroids only compared to K and KP, suggesting that the loss of both KL drives increased BMP6 expression. Consistent with this finding, the iron regulator hepcidin, which is regulated by BMP6 is also upregulated in KL spheroids. Both BMP-6 and hepcidin expression can be repressed by restoring exogenous LKB1 in KL spheroids suggesting that LKB1 loss is at least in part responsible for the overexpression of the BMP6/hepcidin pathway. In addition, inhibition of BMP-6 signaling via BMP type I receptor ALK2 using small molecule inhibitor LDN214117 suppressed of cell invasion in 3D culture and decreased tumor growth in a KL syngeneic mouse model. Collectively, our findings show that upregulation of hepcidin via BMP6 signaling and followed by iron sequestration in tumor cells is part of the metastatic invasion strategy of LKB1 inactivated NSCLC. Therefore, current clinical strategies targeting BMP6 signaling could improve outcomes for LKB1-mutant lung cancer patients. Citation Format: Junghui Koo, Chang-Soo Seong, Bhakti Dwivedi, Carol Tucker-Burden, Adam I. Marcus, Melissa Gilbert-Ross. Targeting bone morphogenetic protein 6 as a therapeutic target for LKB1-inactivated lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1036.

The genetic and metabolic heterogeneity of RAS-driven cancers has confounded therapeutic strategies in the clinic. To address this, rapid and genetically tractable animal models are needed that recapitulate the heterogeneity of RAS-driven cancers in vivo. Here, we generate aDrosophila melanogastermodel of Ras/Lkb1mutant carcinoma. We show that low-level expression of oncogenic Ras (RasLo) promotes the survival of Lkb1 mutant tissue, but results in autonomous cell cycle arrest and non-autonomous overgrowth of wild-type tissue. In contrast, high-level expression of oncogenic Ras (RasHi) transforms Lkb1 mutant tissue resulting in lethal malignant tumors. Using simultaneous multiview light-sheet microcopy, we have characterized invasion phenotypes ofRas/Lkb1tumors in living larvae. Our molecular analysis reveals sustained activation of the AMPK pathway in malignantRas/Lkb1tumors, and demonstrate the genetic and pharmacologic dependence of these tumors on CaMK-activated Ampk. We further show that LKB1 mutant human lung adenocarcinoma patients with high levels of oncogenic KRAS exhibit worse overall survival and increased AMPK activation. Our results suggest that high levels of oncogenic KRAS is a driving event in the malignant transformation of LKB1 mutant tissue, and uncover a novel vulnerability that may be used to target this aggressive genetic subset of RAS-driven tumors.One Sentence SummaryA multivariable Ras-drivenDrosophilamodel reveals a novel LKB1 mutant lung adenocarcinoma patient subpopulation and targetable effector pathway.

Introduction The clinical and biological significance of the newly described SCLC subtypes, SCLC-A, SCLC-N, SCLC-Y, and SCLC-P, defined by the dominant expression of transcription factors ASCL1, NeuroD1, YAP1, and POU2F3, respectively, remain to be established. Methods We generated new RNA sequencing expression data from a discovery set of 59 archival tumor samples of neuroendocrine tumors and new protein expression data by immunohistochemistry in 99 SCLC cases. We validated the findings from this discovery set in two independent validation sets consisting of RNA sequencing data generated from 51 SCLC cell lines and 81 primary human SCLC samples. Results We successfully classified 71.8% of SCLC and 18.5% of carcinoid cases in our discovery set into one of the four SCLC subtypes. Gene set enrichment analysis for differentially expressed genes between the SCLC survival outliers (top and bottom deciles) matched for clinically relevant prognostic factors revealed substantial up-regulation of interferon-γ response genes in long-term survivors. The SCLC-Y subtype was associated with high expression of interferon-γ response genes, highest weighted score on a validated 18-gene T-cell–inflamed gene expression profile score, and high expression of HLA and T-cell receptor genes. YAP1 protein expression was more prevalent and more intensely expressed in limited-stage versus extensive-stage SCLC (30.6% versus 8.5%; p = 0.0058) indicating good prognosis for the SCLC-Y subtype. We replicated the inflamed phenotype of SCLC-Y in the two independent validation data sets from the SCLC cell lines and tumor samples. Conclusions SCLC subtyping using transcriptional signaling holds clinical relevance with the inflamed phenotype associated with the SCLC-Y subset.

Purpose: Vimentin is an epithelial-to-mesenchymal transition (EMT) biomarker and intermediate filament protein that functions during cell migration to maintain structure and motility. Despite the abundance of clinical data linking vimentin to poor patient outcome, it is unclear if vimentin is required for metastasis or is a correlative biomarker. We developed a novel genetically engineered mouse model (GEMM) to probe vimentin in lung adenocarcinoma metastasis. Experimental Design: We used the LSL-KrasG12D/Lkb1fl/fl/Vim−/− model (KLV−/−), which incorporates a whole-body knockout of vimentin and is derived from the Cre-dependent LSL-KrasG12D/Lkb1fl/fl model (KLV+/+). We compared the metastatic phenotypes of the GEMMs and analyzed primary tumors from the KLV models and lung adenocarcinoma patients to assess vimentin expression and function. Results: Characterization of KLV+/+ and KLV−/− mice shows that although vimentin is not required for primary lung tumor growth, vimentin is required for metastasis, and vimentin loss generates lower grade primary tumors. Interestingly, in the KLV+/+ mice, vimentin was not expressed in tumor cells but in cancer-associated fibroblasts (CAFs) surrounding collective invasion packs (CIPs) of epithelial tumor cells, with significantly less CIPs in KLV−/− mice. CIPs correlate with tumor grade and are vimentin-negative and E-cadherin–positive, indicating a lack of cancer cell EMT. A similar heterotypic staining pattern was observed in human lung adenocarcinoma samples. In vitro studies show that vimentin is required for CAF motility to lead tumor cell invasion, supporting a vimentin-dependent model of collective invasion. Conclusions: These data show that vimentin is required for lung adenocarcinoma metastasis by maintaining heterotypic tumor cell–CAF interactions during collective invasion. Clin Cancer Res; 24(2); 420–32. ©2017 AACR.

Treatment with fludarabine phosphate (9-β-D-arabinofuranosyl-2-F-adenine 5'-phosphate, F-araAMP) leads to regressions and cures of human tumor xenografts that express Escherichia coli purine nucleoside phosphorylase (EcPNP). This occurs despite the fact that fludarabine (F-araA) is a relatively poor substrate for EcPNP, and is cleaved to liberate 2-fluoroadenine at a rate only 0.3% that of the natural E. coli PNP substrate, adenosine. In this study, we investigated a panel of naturally occurring PNPs to identify more efficient enzymes that may be suitable for metabolizing F-araA as part of experimental cancer therapy. We show that Trichomonas vaginalis PNP (TvPNP) cleaves F-araA with a catalytic efficiency 25-fold greater than the prototypic E. coli enzyme. Cellular extracts from human glioma cells (D54) transduced with lentivirus stably expressing TvPNP (D54/TvPNP) were found to cleave F-araA at a rate similar to extracts from D54 cells expressing EcPNP, although much less enzyme was expressed per cell in the TvPNP transduced condition. As a test of safety and efficacy using TvPNP, human head and neck squamous cell carcinoma (FaDu) xenografts expressing TvPNP were studied in nude mice and shown to exhibit robust tumor regressions, albeit with partial weight loss that resolved post-therapy. F-araAMP was also a very effective treatment for mice bearing D54/TvPNP xenografts in which approximately 10% of tumor cells expressed the enzyme, indicating pronounced ability to kill non-transduced tumor cells (high bystander activity). Moreover, F-araAMP demonstrated activity against D54 tumors injected with an E1, E3 deleted adenoviral vector encoding TvPNP. In that setting, despite higher F-araA cleavage activity using TvPNP, tumor responses were similar to those obtained with EcPNP, indicating factors other than F-Ade production may limit regressions of the D54 murine xenograft model. Our results establish that TvPNP is a favorable enzyme for activating F-araA, and support further studies in combination with F-araAMP for difficult-to-treat human cancers.

9019 Background: The dominant expression of transcription factors ASCL1, NeuroD1, YAP1 or POU2F3 characteristically defines four small cell lung cancer (SCLC) subtypes (SCLC-A, SCLC-N, SCLC-Y and SCLC-P). The clinical validation and biological relevance of these emerging SCLC subtypes is currently lacking. Methods: Using the Illumina TruSeq RNA Exome Kit, we generated RNA-Seq data from 61 cases of SCLC and pulmonary carcinoid to interrogate gene expression differences in SCLC subtypes as well as in survival outliers (top and bottom decile) matched for clinically relevant prognostic factors and treatment. We also assessed YAP1 protein expression in a blinded fashion by immunohistochemistry in 130 SCLC cases. Results: We successfully classified 68% of SCLC into one of the four SCLC subtypes whereas 81.5% of carcinoids did not fit into any of these categories. GSEA for differentially expressed genes between outlier subgroups showed significant upregulation of interferon gamma and interferon alpha response genes in late survivors. Moreover, a previously validated 18-gene T-cell inflamed gene expression profile was upregulated in late survivors and in the SCLC-Y subtype. Furthermore, the SCLC-Y subtype and late survivors showed higher expression of HLA gene family and reduced expression of cancer testis antigens. The median (95%CI) OS was 14 (4.3, 28.8), 16.7 (0.9, NA), 8.1 (2, 9.7) and 20.1 (0.6, 39.5) months respectively, for SCLC-A, N, P and Y subtypes. YAP-1 protein expression was positive in 17 of 130 (13%) SCLC cases. The majority of cases with positive YAP1 expression by immunohistochemistry, 12 of 17 cases (70.6%), were limited stage SCLC at the time of original diagnosis. Conclusions: SCLC subtypes have clinical implication as predictive and prognostic biomarker. SCLC-Y subtype is enriched for T-cell inflamed phenotype and long term survival, and may predict for clinical benefit of immunotherapy.

Although activating mutations in the GTPase KRAS are oncogenic and implicated in various malignancies, mutations in KRAS alone are inefficient to transform epithelia. In lung adenocarcinoma, KRAS mutations occur at high frequency and co-segregate with loss of the LKB1 tumor suppressor, a master serine/threonine kinase that phosphorylates and activates multiple AMPK family kinases to control energy homeostasis, cell polarity, and cell:matrix signaling. Despite our current knowledge of LKB1 biology, the complexity of the signaling pathway has precluded investigations into the precise mechanism by which alteration of this kinase contributes to oncogenic transformation and tumor progression in vivo. In the present study, we introduced mutations in Kras and Lkb1 in the genetically tractable organism Drosophila melanogaster. We show that low-level expression of oncogenic Kras (KrasLow) promotes the survival of Lkb1-mutant tissue, eventually leading to a G1 arrest, and concomitant overgrowth of the surrounding tumor microenvironment. Alternatively, high-level expression of oncogenic Kras (KrasHigh) promotes the neoplastic transformation of Lkb1-mutant tissue, leading to autonomous growth and increased cell cycle progression, invasion of mutant tissue into the brain, and the survival and growth of explanted tumor tissue in a wild-type Drosophila host. Interestingly, phosphorylation of the canonical Lkb1 target AMPK is maintained in KrasHigh/Lkb1 neoplastic tissue, but not in KrasLow/Lkb1 tissue. Finally, we analyzed TCGA Pan Lung Cancer data and show that high-level, but not low-level, expression of oncogenic KRAS results in worse overall survival in the context of LKB1 loss. Taken together, these data suggest that increasing levels of oncogenic KRAS, perhaps through mutant KRAS copy gains, are a driving event in the malignant transformation of LKB1-mutant tissue, and that the rewired AMPK signaling axis may offer a potential therapeutic target for patients with this subset of mutations. Citation Format: Briana B. Rackley, Chang-Soo Seong, Melissa Gilbert-Ross. High-level expression of oncogenic KRAS is required to transform LKB1 mutant tissue in vivo [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B04.

A rationale exists for pharmacologic manipulation of the serine (S)184 phosphorylation site of the proapoptotic Bcl2 family member Bax as an anticancer strategy. Here, we report the refinement of the Bax agonist SMBA1 to generate CYD-2-11, which has characteristics of a suitable clinical lead compound. CYD-2-11 targeted the structural pocket proximal to S184 in the C-terminal region of Bax, directly activating its proapoptotic activity by inducing a conformational change enabling formation of Bax homooligomers in mitochondrial membranes. In murine models of small-cell and non–small cell lung cancers, including patient-derived xenograft and the genetically engineered mutant KRAS-driven lung cancer models, CYD-2-11 suppressed malignant growth without evident significant toxicity to normal tissues. In lung cancer patients treated with mTOR inhibitor RAD001, we observed enhanced S184 Bax phosphorylation in lung cancer cells and tissues that inactivates the propaoptotic function of Bax, contributing to rapalog resistance. Combined treatment of CYD-2-11 and RAD001 in murine lung cancer models displayed strong synergistic activity and overcame rapalog resistance in vitro and in vivo. Taken together, our findings provide preclinical evidence for a pharmacologic combination of Bax activation and mTOR inhibition as a rational strategy to improve lung cancer treatment. Cancer Res; 77(11); 3001–12. ©2017 AACR.

Loss of LKB1 activity is prevalent in KRAS mutant lung adenocarcinoma and promotes aggressive and treatment-resistant tumors. Previous studies have shown that LKB1 is a negative regulator of the focal adhesion kinase (FAK), but in vivo studies testing the efficacy of FAK inhibition in LKB1 mutant cancers are lacking. Here, we took a pharmacologic approach to show that FAK inhibition is an effective early-treatment strategy for this high-risk molecular subtype. We established a lenti-Cre–induced Kras and Lkb1 mutant genetically engineered mouse model (KLLenti) that develops 100% lung adenocarcinoma and showed that high spatiotemporal FAK activation occurs in collective invasive cells that are surrounded by high levels of collagen. Modeling invasion in 3D, loss of Lkb1, but not p53, was sufficient to drive collective invasion and collagen alignment that was highly sensitive to FAK inhibition. Treatment of early, stage-matched KLLenti tumors with FAK inhibitor monotherapy resulted in a striking effect on tumor progression, invasion, and tumor-associated collagen. Chronic treatment extended survival and impeded local lymph node spread. Lastly, we identified focally upregulated FAK and collagen-associated collective invasion in KRAS and LKB1 comutated human lung adenocarcinoma patients. Our results suggest that patients with LKB1 mutant tumors should be stratified for early treatment with FAK inhibitors.

Loss of LKB1 activity is prevalent in KRAS mutant lung adenocarcinoma and promotes aggressive and treatment-resistant tumors. Previous studies have shown that LKB1 is a negative regulator of the focal adhesion kinase (FAK), but in vivo studies testing the efficacy of FAK inhibition in LKB1 mutant cancers are lacking. Here, we took a pharmacologic approach to show that FAK inhibition is an effective early-treatment strategy for this high-risk molecular subtype. We established a lenti-Cre-induced Kras and Lkb1 mutant genetically engineered mouse model (KLLenti) that develops 100% lung adenocarcinoma and showed that high spatiotemporal FAK activation occurs in collective invasive cells that are surrounded by high levels of collagen. Modeling invasion in 3D, loss of Lkb1, but not p53, was sufficient to drive collective invasion and collagen alignment that was highly sensitive to FAK inhibition. Treatment of early, stage-matched KLLenti tumors with FAK inhibitor monotherapy resulted in a striking effect on tumor progression, invasion, and tumor-associated collagen. Chronic treatment extended survival and impeded local lymph node spread. Lastly, we identified focally upregulated FAK and collagen-associated collective invasion in KRAS and LKB1 comutated human lung adenocarcinoma patients. Our results suggest that patients with LKB1 mutant tumors should be stratified for early treatment with FAK inhibitors.

The major cause of cancer-associated mortality is tumor metastasis, but our understanding of this process is far from complete. During successful dissemination, tumor cells invade the surrounding tissue of the primary tumor, intravasate into blood and lymphatic vessels, translocate to distant tissues, extravasate, adapt to the new microenvironment, and eventually seed, proliferate, and colonize to form metastases. Because dissemination mostly occurs through the blood, circulating tumor cells (CTCs) that have been shed into the vasculature and may be on their way to potential metastatic sites are of obvious interest (1). KRAS mutations are the most frequent oncogenic drivers of non-small cell lung cancer and when associated with co-mutations lead to a decrease in overall survival. We have previously established a lenti-Cre-induced Kras and Lkb1 mutant and Kras and p53 mutant genetically engineered mouse modes (KLLenti) and (KPLenti) that develop 100% lung adenocarcinoma to conduct the first study to isolate and maintain primary, CTC and metastatic cells that are KLLenti or KPLenti. We are able to isolate TTF-1+ and pan-cytokeratin+ CTCs from the KLlenti and KPlenti mice and validated their mutational status by genotyping, western blot, and immunofluorescence. Moreover, lkb1-mutant or p53-mutant primary tumor cells have different 3-D invasive properties and patterns, as well as the respective CTCs and metastatic cells when compared across different genetic sub-types. To further study gene expression patterns between primary, CTC, and metastatic sites RNAseq will be employed to identify pathways that drive metastasis within the genetic sub-types. Citation Format: Liza J. Burton, Junghui Koo, Carol Tucker-Burden, Wei Zhou, Melissa Gilbert-Ross, Chunzi Huang, Gabriel Sica, Adam Marcus. Isolation of circulating tumors cells from genetically engineered mouse models of lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 865.

Introduction: LKB1 is the third mostly frequently mutated gene in lung adenocarcinoma, and is mutually exclusive with EGFR mutations. Furthermore, a recent clinical trial indicated that lung cancers with LKB1 mutations are unlikely to respond to immune checkpoint therapy. Therefore, there is an urgent need for novel therapies for LKB1-inactivated lung cancer. We previously demonstrated that leflunomide preferentially induces apoptosis in LKB1-null lung cancer cell lines in vitro. Here, we evaluated the effect of leflunomide in both a xenograft model and immune-competent genetically engineered mouse model (GEMM). Methods: LKB1-null H460 xenograft mice were treated with leflunomide (35mg/kg/day) for 23 days through oral gavage and tumor volume was measured every the other day. KrasG12D/Lkb1-/-Luciferase GEMM mice were treated with leflunomide (30mg/kg/day) for 44 days and tumor burden was measured by bioluminescence imaging (BLI) score every week. At the end of experiments, tumors were dissected, weighed, and analyzed by immunohistochemistry. Results: Although leflunomide is an FDA-approved agent to treat rheumatoid arthritis, its immune suppression function did not facilitate but inhibited the growth of KrasG12DLkb1-/- lung adenocarcinoma. This suppression was also observed in H460 xenografts, and leflunomide treatment did not alter animal weight in either model. Furthermore, distant cancer metastasis was not observed in leflunomide-treated GEMM. In residual tumors from the treated group, cleaved caspase-3 was not detectable but there was a significant decrease in Ki67 staining. This is consistent with our in vitro findings that leflunomide primarily inhibits cell proliferation through the promotion of G1 cell cycle arrest. Conclusion: Our findings indicate that leflunomide may be a viable reagent for the treatment of LKB1-mutated lung adenocarcinoma. Citation Format: Rui Jin, Xiuju Liu, Wei Peng, Yijian Fan, Boxuan Liu, Shuanying Yang, Chun-gen Xing, Melissa Gilbert-Ross, Adam Marcus, Wei Zhou. Leflunomide inhibits the growth of LKB1-inactivated tumors in both xenograft and immunocompetent genetically engineered mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4838.

Liver kinase β1 (LKB1, also known as STK11) is a serine/threonine kinase that has multiple cellular functions including the regulation of cell polarity and motility. Murine proteomic studies show that LKB1 loss causes aberrant adhesion signaling; however, the mechanistic underpinnings of this relationship are unknown. We show that cells stably depleted of LKB1 or its co-activator STRADα have increased phosphorylation of focal adhesion kinase (FAK) at Tyr397/Tyr861 and enhanced adhesion to fibronectin. LKB1 associates in a complex with FAK and LKB1 accumulation at the cellular leading edge is mutually excluded from regions of activated Tyr397-FAK. LKB1-compromised cells lack directional persistence compared with wild-type cells, but this is restored through subsequent pharmacological FAK inhibition or depletion, showing that cell directionality is mediated through LKB1-FAK signaling. Live cell confocal imaging reveals that LKB1-compromised cells lack normal FAK site maturation and turnover, suggesting that defects in adhesion and directional persistence are caused by aberrant adhesion dynamics. Furthermore, re-expression of full-length wild-type or the LKB1 N-terminal domain repressed FAK activity, whereas the kinase domain or C-terminal domain alone did not, indicating that FAK suppression is potentially regulated through the LKB1 N-terminal domain. Based upon these results, we conclude that LKB1 serves as a FAK repressor to stabilize focal adhesion sites, and when LKB1 function is compromised, aberrant FAK signaling ensues, resulting in rapid FAK site maturation and poor directional persistence. Background: LKB1 is a serine/threonine kinase important for cell polarity and motility. Results: LKB1 loss causes focal adhesion kinase hyperactivation and aberrant cell motility. Conclusion: LKB1 represses focal adhesion kinase to regulate its turnover. Significance: This provides information on how LKB1 regulates the cell adhesion pathway during cell motility.

Pharmacologic manipulation of the serine (S)184 phosphorylation site of Bax protein to functionally regulate its proapoptotic activity is an attractive anticancer strategy. We recently identified three small molecule Bax agonists. SMBA1 was selected as the lead compound based on its chemical structure and its drug-like properties, from which a more effective analog, CYD-2-11, was generated. CYD-2-11 targets the structural pocket around S184 in the C-terminal tail of Bax, directly activating its proapoptotic activity via 6A7 conformational change and formation of Bax homo-oligomers in mitochondrial membranes. CYD-2-11 suppresses tumor growth in SCLC, NSCLC and patient-derived lung cancer xenografts as well as the genetically engineered mutant KRAS-driven lung cancer model with no significant normal tissue toxicity. Inhibition of mTOR by RAD001 enhances S184 Bax phosphorylation in lung cancer cell lines and tumor tissues from lung cancer patients treated with RAD001, which inactivates the propaoptotic function of Bax, contributing to rapalog-resistance. Combined CYD-2-11 and RAD001 treatment not only displays strong synergistic activity against lung cancer but also overcomes rapalog-resistance in vitro and in vivo. Therefore, a mechanism-driven combination of Bax agonist and mTOR inhibitor represents a highly attractive therapeutic strategy to improve lung cancer patient outcomes. Citation Format: Rui Li, Chunyong Ding, Jun Zhang, Maohua Xie, Dongkyoo Park, Ye Ding, Guo Chen, Guojing Zhang, Melissa Gilbert-Ross, Wei Zhou, Adam Marcus, Shi-Yong Sun, Zhuo Chen, Gabriel Sica, Suresh Ramalingam, Andrew Magis, Haian Fu, Fadlo Khuri, Walter Curran, Taofeek Owonikoko, Dong Shin, Jia Zhou, Xingming Deng. Modulation of Bax and mTOR for cancer therapeutics [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 2333. doi:10.1158/1538-7445.AM2017-2333

Lung cancer continues to be fatal, in part due to the inability to prevent and treat metastases. Highly metastatic cancers exhibit enhanced glucose uptake to sustain proliferation and importantly, tumor invasion. Among the SLC2A family of facilitative glucose transporters, GLUT1 is largely attributed to be responsible for increased glucose uptake of cancer cells. GLUT1 is however responsible for glucose transport across the blood-brain barrier, expressed in many normal cell types and therefore a less desirable therapeutic target. We previously reported that multiple myeloma cells rely on overexpression and constitutive plasma membrane localization of insulin-responsive glucose transporter, GLUT4. In this study we investigated a role for GLUT4 in lung cancer. To interrogate contributions of GLUT1 and GLUT4 in proliferation, invasion and migration we generated H1299 and A549 GLUT 1 or GLUT4 knockdowns. Knockdown (KD) of GLUT4 did not inhibit proliferation but suppressed migration and invasion assessed through scratch and Boyden chamber assays, respectively. On the contrary, knockdown of GLUT1 reduced proliferation of these lines. Treatment of H1299 and A549 with our newly developed GLUT4-selective inhibitors also reduced invasion, phenocopying the effects detected with GLUT4 KD. GLUT4 inhibition also reduced H1299 invasion in a spheroid invasion model. We utilized H1299 cells to isolate highly invasive less proliferative “leader cells” and less invasive but highly proliferative “follower cells”. Interestingly, examination of these two cell types exhibited a differential expression pattern of GLUT1/GLUT4. Leader cells have elevated expression of GLUT4 and decreased GLUT1. On the contrary, follower cells have high GLUT1 and low GLUT4 expression. Leader cells are more sensitive to GLUT4 inhibitors indicating they are more dependent on GLUT4 than follower cells. In addition, leader cells are more sensitive to mitochondrial complex I inhibitors compared to follower cells, suggesting they rely more on oxidative phosphorylation. A differential reliance on glycolysis/OXPHOS was further supported by evaluation of glucose uptake/oxygen consumption. Isotope tracer and bioenergetics analyses further support altered nutrient dependencies of leader and follower cells. Lastly, we found that GLUT4 is expressed in patient lung adenocarcinoma specimens including more aggressive micropapillary lung adenocarcinoma. Examination of collective invasion packs in human adenocarcinoma demonstrated patchy GLUT1 expression suggestive of a subset of more proliferative “follower” cells. These results suggest that in a lung cancer population a subset of more invasive cells are reliant on GLUT4 with reduced GLUT1 expression while more proliferative cells rely on high GLUT1 expression, making GLUT4 a promising candidate for targeting metastasis in lung cancer. Citation Format: Changyong Wei, Abhinav Achreja, Jessica Konen, Gabriel Sica, Melissa Gilbert-Ross, Deepak Nagrath, Adam Marcus, Mala Shanmugam. GLUT4 exhibits a non-canonical role of regulating lung cancer metastasis [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 4904. doi:10.1158/1538-7445.AM2017-4904

Ras homolog gene family, member A (RhoA) is a small GTPase that plays critical roles in several essential cell functions, such as migration, adhesion, proliferation, and gene expression. 1 RhoA switches between a GTP-bound active form and a GDP-bound inactive form. The activated RhoA directly interacts with its downstream effectors, such as Rho kinase (ROCK) to regulate actomyosin dynamics, or mDia1 to control stress fiber and filopodia formation. The activity of RhoA is primarily regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating protein (GAP), and guanine nucleotide-dissociation inhibitors (GDIs). RhoA was initially postulated as an oncogene in 1989. 2 Even though the amplification of RhoA was capable of transforming mouse fibroblasts, point mutations at codon 14 and 64 were not tumorigenic in the same model. 2 Previous cancer genome sequencing analysis also failed to identify RhoA mutations in most common human cancers, and consequently, it was not thought to be altered by somatic mutation in human cancers. In February of 2014, a recurrent mutation of RhoA (G17V) was reported to be present in 67% of angioimmunoblastic T cell lymphoma (AITL) and 18% of peripheral T cell lymphoma (PTCL), but not otherwise specified (PTCL-NOS) samples. 3 This finding was quickly validated by two other groups.4, 5 In addition, RhoA mutations were found in pediatric Burkitt lymphoma treated according to the NHL-BFM protocols. 6 However, RhoA mutation is not limited to a subset of lymphoma, as three large studies published this year have indicated that RhoA is mutated in 14% of diffuse-type gastric carcinoma samples but not in intestinal type tumors.7, 8, 9 Therefore, RhoA is quickly emerging as a somatic mutational target in these tumor types. The first interesting aspect of this emerging story is that RhoA mutations are limited to these specific tumor types, which suggests that the function of RhoA may be cell type-specific. It is known that the expression of many RhoA regulators is tissue or cell type-specific, and recent mouse model studies have indicated that the regulation of these downstream signaling pathways by RhoA is also cell type-specific. 10 Consequently, the biological significance of RhoA activity will vary among different cell types, and it will be important to determine in the future the biological effect of RhoA depletion in these cell types in mouse models. The type of recurrent RhoA mutations observed in these tumors is another topic of interest. In AITL and PTCL, the dominant mutation observed is G17V, which resides in the GTP/GDP binding site. G17V-mutant RhoA does not interact with its effector molecule rhotekin and suppresses F-actin stress fiber formation. 3 In addition, G17V-mutant RhoA appears to act in a dominant-negative capacity to promote cell proliferation and invasion. 4 The mutational hotspots of RhoA in diffuse-type gastric carcinoma are Y42C, R5Q/W, L57V and G17E. Y42C resides at the C-terminal edge of the core effector binding region of RhoA, and a previous study suggested that this mutation only attenuates the activation of protein kinase N but does not abrogate the activation of mDia or ROCK1. 8 A Rho binding domain assay also suggested that Y42C and L57V mutants have attenuated abilities to associate with GTP. 9 Together, these studies suggest that wild-type RhoA has tumor suppressor functions, while mutated RhoA inhibits wild-type function through a dominant negative mechanism. However, if RhoA is truly a tumor suppressor, one would expect this gene to be frequently inactivated by other gene inactivation mechanisms, such as nonsense or frame-shift mutations in these tumor types. The recurrent nature of RhoA mutations in AITL, PTCL and diffuse-type gastric carcinoma strongly suggests that these hotspot mutations result in a gain-of-function alteration in an unidentified signaling pathway; nevertheless, in the absence of any supporting data, the question still remains whether RhoA is an oncogene or tumor suppressor gene. From the cancer treatment perspective, the recurrent mutational hotspots of this protein represent ideal targets for small molecule inhibitors as therapeutic reagents. If the RhoA mutants act in a dominant negative fashion, such molecules could disrupt their interaction with the wild-type protein to restore RhoA function. On the other hand, if RhoA mutants are oncogenes, the suppression of their activities by these molecules should inhibit tumorigenesis. In either case, the future development of these therapeutic reagents holds promise for cancer patients with RhoA mutations.

Vimentin is an intermediate filament protein that has been used in the clinic as a biomarker for metastatic potential and poor patient prognosis across numerous solid tumor types; however, little is known about how vimentin might contribute to cancer progression and metastasis. Here we created a novel genetically engineered mouse model (GEMM) to study the role of vimentin in lung cancer progression and metastasis. We first re-developed an LSL-KrasG12D,LKB1fl/fl mouse model whereby intranasal administration of lentiviral Cre recombinase causes a KrasG12D mutation and LKB1 knockout in the lungs. In this model Kras/Lkb1 GEMM (termed KLV+/+), approximately half the mice that develop primary lung tumors in 8-10 weeks also develop metastasis to the mediastinal lymph nodes in weeks 12-16. We used this model to test the hypothesis that vimentin loss prevents lung cancer metastasis by crossing this GEMM with a Vim-/- mouse to create a novel LSL-KrasG12D, LKB1fl/fl, Vim-/- (termed here KLV-/-) mouse. Our findings show that in the KLV-/- mouse, primary tumor burden and cell proliferation were not significantly different than wild-type KLV+/+ mice; however, KLV-/- mice exhibit significantly less metastasis to the mediastinal lymph nodes compared to their wild type counterpart indicating that vimentin contributes to the metastatic cascade. Consistent with this finding, histological analysis of the primary tumors show that KLV-/- mice exhibit less focal invasion than KLV+/+ and +/- mice, further confirming that KLV-/- mice have reduced invasiveness. Interestingly, vimentin staining in invasive regions of KLV+/+ mice was not found in the cancer cells but rather in fibroblast-like cells surrounding invasive cell buds that we term collective invasion packs (CIPs). These fibroblast-like, vimentin-positive cells also stain positive for alpha smooth muscle actin, which is consistent with cancer-associated fibroblasts (CAFs). Taken together, these results suggest that in this highly metastatic genetic background, vimentin may play a central role in recruiting CAFs to invading cancer cells but not necessarily in cancer cell epithelial to mesenchymal transition (EMT). Citation Format: Alessandra M. Salgueiro, Melissa Gilbert-Ross, Lauren S. Havel, John Shupe, Allyson E. Koyen, Hans E. Grossniklaus, Gabriel Sica, Adam I. Marcus. Beyond EMT: Vimentin function in lung cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C24.

LKB1 is the 2rd most commonly mutated tumor suppressor gene in human lung adenocarcinoma, is commonly co-mutated with KRAS, and leads to more aggressive, treatment-resistant tumors in mouse models. The identification of druggable signaling molecules that result from specific alterations in LKB1 could result in a personalized clinical strategy to target this high-risk patient population. We have previously published that LKB1 acts to limit focal adhesion kinase (FAK) activity in human lung cancer cells to restrict cell adhesion and migration. Based on our prior published data we hypothesize that FAK pathway inhibition will suppress invasion and metastasis in LKB1-mutant tumors in vivo. To investigate our hypothesis, we have designed the first rolling-enrollment pre-clinical mouse trial to target invasion and metastasis using a small-molecule FAK inhibitor. To enroll mice with early-stage lung adenocarcinoma, we developed a novel lentiviral-Cre induced KrasG12D; Lkb1fl/fl genetically engineered mouse model (GEMM) (KLLLenti) that develops 100% adenocarcinomas, expresses a luciferase reporter gene, and has elevated levels of active FAK in late stage invasive tumors. Importantly, short-term treatment of KLLLenti mice with a pharmacologic FAK inhibitor potently suppresses the invasive progression of primary tumors. Moreover, long-term treatment results in improved progression-free survival, and delays metastatic spread to the lymph nodes. We further pursue mechanistic studies to investigate how LKB1-mutant tumor tissue gains a metastatic advantage in vivo, and using a combination of 3D tumor spheroid assays, and multiphoton microscopy, present results that LKB1-mutant tumors use a unique form of hybrid invasion that relies both on cell:cell and cell-matrix adhesion, and in doing so, are equipped to more efficiently invade into the collagen-dense microenvironment of the lung. We will also present data that similar molecular and cell biologic phenotypes can be found in a subset of KRAS, LKB1-mutant human clinical samples. Our studies suggest that when used early, FAK inhibitors may be a viable clinical strategy to prevent or delay metastasis in the KRAS, LKB1-mutant patient population, and begin to define alternate escape pathways by which this highly invasive cell population may escape first-line therapy. Citation Format: Melissa Gilbert-Ross, Jessica Konen, Junghui Koo, John Shupe, Gabriel L. Sica, Zhengjia Chen, Brian S. Robinson, Madhusmita Behera, Michael R. Rossi, Geoffrey H. Smith, Charles E. Hill, Suresh M. Ramalingam, Haian Fu, Fadlo R. Khuri, Wei Zhou, Adam Marcus. Developing a personalized anti-metastatic therapy to treat KRAS, LKB1-mutant lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-348.

LKB1 is the 2rd most commonly mutated tumor suppressor gene in human lung adenocarcinoma, and a biomarker of aggressive disease in stage IV KRAS mutant lung cancer patients. We have previously published that LKB1 acts to limit focal adhesion kinase (FAK) activity in human lung cancer cells to restrict cell adhesion and migration. Based on our prior published data we hypothesize that FAK inhibition will suppress invasion and metastasis in LKB1-mutant tumors in vivo , and represent a viable clinical strategy to target this high-risk patient population. To investigate our hypothesis, we have developed a novel lentiviral-Cre KrasG12D; Lkb1fl/fl Rosa-luciferase genetically engineered mouse model (GEMM) (KLLLenti) that develops 100% adenocarcinomas, and produces a bioluminescent output that correlates with tumor burden, tumor grade, and degree of metastatic colonization of the lymph node. We show that stage IV KLLLenti primary tumors have elevated levels of active FAK in a subset of invasive cells that maintain collective contact via E-Cadherin. Importantly, treatment of KLLLenti mice with a pharmacologic FAK inhibitor potently suppresses invasive progression of primary tumors. Moreover, KLLLenti tumors treated at later stages progress to the same degree as vehicle-treated mice. This result suggests a ‘therapeutic window’ of opportunity, and argues that FAK inhibitors should be part of first-line therapy to treat LKB1-mutant lung cancer patients. We further pursue mechanistic studies to investigate how Lkb1-mutant tumor tissue gains a metastastic advantage in vivo, and using a combination of 3D tumor spheroid assays and multiphoton microscopy present results that Lkb1-mutant tumors use a unique form of hybrid or transitional invasion that on depends on both FAK-based adhesion and collective migration and in doing so, are equipped to more efficiently invade into the collagen-dense microenvironment of the lung. Our studies suggest that when used early, FAK inhibitors may be a viable clinical strategy to prevent metastasis in the LKB1-mutant patient population, and begin to define alternate escape pathways by which this highly invasive cell population may escape first-line therapy. Citation Format: Melissa Gilbert-Ross, Jessica Konen, John Shupe, Junghui Koo, Gabriel L. Sica, Suresh S. Ramalingam, Haian Fu, Fadlo R. Khuri, Wei Zhou, Adam I. Marcus. An early treatment window to target FAK-dependent collective invasion in Lkb1-mutant lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B45.

A major mechanism of metastasis is the epithelial to mesenchymal transition (EMT) where epithelial cells from the primary site undergo genetic and epigenetic alterations to become motile and mesenchymal. A canonical marker of EMT and a clinical marker of metastasis is the intermediate filament protein, vimentin. Vimentin expression correlates with increased metastatic potential and poor patient prognosis across most solid tumor types including lung, prostate, and breast cancers; however little is known about how vimentin functions to promote the metastatic cascade. Here we use a lung cancer mouse model to study the role of vimentin in lung cancer metastasis in vivo. We have re-developed an LSL-KrasG12D,LKB1fl/fl mouse model whereby intranasal administration of lentiviral Cre recombinase causes a KrasG12D mutation and LKB1 knockout in the lungs. Consequently, about half of the mice that develop primary lung tumors after 10 weeks also develop metastasis to the mediastinal lymph nodes by week 16. We crossed this mouse with a Vim-/- mouse to create a LSL-KrasG12D,LKB1fl/fl, Vim-/- (termed here KLV) mouse to determine if loss of vimentin prevents lung cancer metastasis in this model. Our findings show that the KLV mouse exhibits significantly less metastasis to the mediastinal lymph nodes (12%) as compared with its vimentin wild type counterpart but does not show a decrease in primary tumor formation. These knockout mice also exhibit less focal invasion at the primary tumor site while maintaining a well-differentiated adenocarcinoma histology independent of vimentin genotype. Taken together, our work indicates that vimentin is critical for lung cancer metastasis but not primary tumor formation. Citation Format: Alessandra M. Salgueiro, Melissa Gilbert-Ross, Lauren S. Havel, John Shupe, Adam I. Marcus. More than a biomarker: Studying the role of vimentin in lung cancer metastasis. [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 2290. doi:10.1158/1538-7445.AM2015-2290