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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.

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.

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.

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.

The current study was assumed to identify the novel activities from different plant species for the control of nematodes. To resolve the egg hatchability and Nematode mortality rates of root knot Nematodes. Root knot Nematodes are most abundant and destructive Nematode around all the tropics and sub tropic regions of the world. These root knot Nematodes isolated from the Musa acuminata were treated with extracts of five plant leaves namely Azadirachta indica (neem), Arthemisia pallens (marikolunthu), Ocimum tenuiflorum (tulasi), Hibiscus rosasinensis (Sembaruthi), Ficus hispida (fig) with methanol were evaluated against root knot Nematodes Meloidogyna incognita under laboratory conditions. The Nematicidal activity of Methnolic and leaves of five plant species extract, decreases the Nematodes viability as the concentration of the extracts increases. Hence they examine the Nematicidal capability of the plant species against Meloidogyna incognita. This article reports a reduction in egg hatching and increased Nematicidal activity.

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.

KRAS is the most frequently mutated oncogene in human cancer, particularly in cancers with a high mortality rate such as pancreatic, colorectal, and non-small cell lung cancer (NSCLC) (Ryan and Corcoran, 2018). While effective therapies directly targeting KRAS-mutant tumors have yet to be fully validated, recent clinical trials show positive progress for patients with the KRAS(G12C) mutation (Canon et al. 2019). Moreover, sequencing data has allowed for better understanding of how secondary mutations synergize with oncogenic KRAS to drive tumor progression. For example, activating mutations in KRAS frequently occur with loss-of-function mutations in the gene STK11, which encodes the tumor suppressor liver kinase B1 (LKB1), resulting in decreased patient survival, de novo resistance to targeted treatments and immunotherapies, and increased likelihood of tumor recurrence (Cancer Genome Atlas Research Network 2014, Skoulidis et al. 2018, Caiola et al. 2018). Additionally, previous work from genetically engineered mouse models (GEMMs) suggests loss of Lkb1 is sufficient to promote the progression and metastasis of nascent Kras-driven lung adenocarcinoma (Ji et al. 2007). Therefore, we sought to determine whether knockdown of Lkb1 by RNAi could cooperate with activating mutations in Ras to drive tissue overgrowth in wing imaginal discs of the genetically tractable model organism Drosophila melanogaster. To address this question, we obtained transgenic Drosophila expressing oncogenic RasV12, which on its own causes hyperplastic growth balanced by non-autonomous cell death in imaginal tissues (Karim and Rubin 1998). To knockdown Lkb1 we obtained an RNAi fly stock (Lkb1RNAi) developed by the Transgenic RNAi Project (TRiP) (Dietzl et al. 2007) and validated through the Harvard Medical School RNAi Stock Validation and Phenotypes (RSVP) resource (Perkins et al. 2015). Of note, the Lkb1RNAi stock was determined to have approximately 68% knockdown efficiency when used with the MTD-Gal4 driver (Sopko et al. 2014). Additional validation using the Updated Targets of RNAi Reagents (UP-TORR) Fly resource confirmed no off-target effects with this RNAi sequence (Hu et al. 2013). We generated a combined RasV12/Lkb1RNAi fly line, and crossed our double mutant (along with single transgenes as controls) to the MS1096-Gal4, UAS-GFP wing pouch driver. In order to precisely measure effects on overall organ size, we used confocal microscopy to acquire z-sections through the entire wing disc, followed by 3D reconstruction and volume measurements using IMARIS software. We determined that total wing disc volume was significantly larger in MS1096-Gal4; RasV12/Lkb1RNAi tissues compared to control genotypes. (A-B). Previous investigations have shown that Lkb1 can exert a non-autonomous role in tumor suppression (Katajisto et al. 2008; Tanwar et al. 2012; Ollila et al. 2018). Therefore, we investigated whether the increase in organ size was due to autonomous vs. non-autonomous effects on growth. To do this we measured individual volumes of GFP-positive and GFP-negative tissue across genotypes. Expression of RasV12/Lkb1RNAi led to significant autonomous overgrowth in the GFP-positive MS1096 expression domain, while the GFP-negative (non-autonomous) tissue compartment remained unchanged (C-D). Changes in organ size control can result from any number of combinations of cell growth, proliferation, and cell death phenotypes. To investigate the compartmental effects on cell proliferation and cell death in RasV12/Lkb1RNAi tissues, we used the MS1096-Gal4 driver to express Lkb1RNAi, RasV12, or RasV12/Lkb1RNAi in developing wing pouches. Tissues were either labeled with BrdU or an anti-Death Caspase-1 (DCP-1) antibody (E-F, G-H). Knockdown of Lkb1 alone resulted in no change in the absolute levels of BrdU or DCP-1 relative to control discs (F, H). Expression of RasV12 alone resulted in a mild increase in the amount of autonomous BrdU and non-autonomous DCP-1 (F, H). Alternatively, co-expression of RasV12/Lkb1RNAi led to a dramatic shift in cellular phenotypes with a large increase in autonomous and non- autonomous BrdU – and a rescue of the non-autonomous cell death observed in cells expressing RasV12 alone. Therefore, knockdown of Lkb1 in the context of oncogenic Ras in the Drosophila wing pouch can exert both non-autonomous and autonomous effects that override organ size control. Future studies will focus on the signaling pathways responsible for both phenotypes which could represent novel, targetable pathways for the thousands of cancer patients in the U.S. with LKB1 mutations.

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.

Introduction After a failed transplant, management of a non-functional graft with pain or recurrent infections can be challenging. Transplant nephrectomy (TN) can be a morbid procedure with the potential for significant blood loss. Embolization of the renal artery alone has been proposed as a method of reducing complications from an in vivo failed kidney transplant. While this does yield less morbidity, it may not address an infected graft or refractory hematuria or rejection. We elected to begin preoperative embolization to assess if this would help decrease the blood loss and transfusion rate associated with TN. Materials and Methods We performed a retrospective analysis of all patients who underwent non-emergent TN at our institution. Patients who had functioning grafts that later failed were included in analysis. TN was performed for recurrent infections, pain or hematuria. We evaluated for blood loss (EBL) during TN, transfusion rate and length of hospital stay. Results A total of 16 patients were identified. Nine had preoperative embolization or no blood flow to the graft prior to TN. The remaining 7 did not have preoperative embolization. The shortest time from transplant to TN was 8 months and the longest 18 years with an average of 6.3 years. Average EBL for the embolized patients (ETN) was 143.9cc compared to 621.4cc in the non-embolized (NETN) group (p=0.041). Average number of units of blood transfused was 0.44 in the ETN with only 3/9 patients requiring transfusion. The NETN patients had average of 1.29 units transfused with 5/7 requiring transfusion. The length of stay was longer for the ETN (5.4 days) compared to 3.9 in the NETN. No intraoperative complications were seen in either group and only one patient had a postoperative ileus in the NETN. Conclusion Embolization prior to TN significantly decreases the EBL but does not significantly decrease transfusion rate. However, patients do require a significantly longer hospitalization with embolization due to the time needed for embolization. Larger studies are needed to determine if embolization before transplant nephrectomy reduces the transfusion rates and overall complications.

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.