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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.

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.

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.

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.

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.

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.

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.