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We have previously demonstrated that PD-1 blockade decreased the incidence of high-grade dysplasia in a carcinogen-induced murine model of oral squamous cell carcinoma (OSCC). It remains unknown, however, whether there are additional factors involved in escape from immune surveillance that could serve as additional targets for immunoprevention. We performed this study to further characterize the immune landscape of oral premalignant lesions (OPL) and determine the impact of targeting of the PD-1, CTLA-4, CD40, or OX40 pathways on the development of OPLs and oral carcinomas in the 4-nitroquinoline 1-oxide model. The immune pathways were targeted using mAbs or, in the case of the PD-1/PD-L1 pathway, using PD-L1–knockout (PD-L1ko) mice. After intervention, tongues and cervical lymph nodes were harvested and analyzed for malignant progression and modulation of the immune milieu, respectively. Targeting of CD40 with an agonist mAb was the most effective treatment to reduce transition of OPLs to OSCC; PD-1 alone or in combination with CTLA-4 inhibition, or PD-L1ko, also reduced progression of OPLs to OSCC, albeit to a lesser extent. Distinct patterns of immune system modulation were observed for the CD40 agonists compared with blockade of the PD-1/PD-L1 axis with or without CTLA-4 blockade; CD40 agonist generated a lasting expansion of experienced/memory cytotoxic T lymphocytes and M1 macrophages, whereas PD-1/CTLA-4 blockade resulted in a pronounced depletion of regulatory T cells among other changes. These data suggest that distinct approaches may be used for targeting different steps in the development of OSCC, and that CD40 agonists merit investigation as potential immunoprevention agents in this setting.Prevention Relevance:PD-1/PD-L1 pathway blockade, as well as activation of the CD40 pathway, were able to prevent OPL progression into invasive OSCC in a murine model. A distinct pattern of immune modulation was observed when either the CD40 or the PD-1/PD-L1 pathways were targeted.

Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non–small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising exons 1 to 9β or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53β and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.

In KRAS-mutant lung adenocarcinoma, tumors with LKB1 loss (KL) are highly enriched for concurrent KEAP1 mutations, which activate the KEAP1/NRF2 pathway (KLK). Here, we investigated the biological consequences of these cooccurring alterations and explored whether they conferred specific therapeutic vulnerabilities. Compared with KL tumors, KLK tumors exhibited increased expression of genes involved in glutamine metabolism, the tricarboxylic acid cycle, and the redox homeostasis signature. Using isogenic pairs with knockdown or overexpression of LKB1, KEAP1, and NRF2, we found that LKB1 loss results in increased energetic and redox stress marked by increased levels of intracellular reactive oxygen species and decreased levels of ATP, NADPH/NADP+ ratio, and glutathione. Activation of the KEAP1/NRF2 axis in LKB1-deficient cells enhanced cell survival and played a critical role in the maintenance of energetic and redox homeostasis in a glutamine-dependent manner. LKB1 and the KEAP1/NRF2 pathways cooperatively drove metabolic reprogramming and enhanced sensitivity to the glutaminase inhibitor CB-839 in vitro and in vivo. Overall, these findings elucidate the adaptive advantage provided by KEAP1/NRF2 pathway activation in KL tumors and support clinical testing of glutaminase inhibitor in subsets of KRAS-mutant lung adenocarcinoma. Significance: In KRAS-mutant non–small cell lung cancer, LKB1 loss results in enhanced energetic/redox stress, which is tolerated, in part, through cooccurring KEAP1/NRF2–dependent metabolic adaptations, thus enhancing glutamine dependence and vulnerability to glutaminase inhibition.

Common mechanisms for p53 loss in cancer include expression of MDM2 or the human papilloma virus (HPV)-encoded E6 protein which both mediate degradation of wild-type (WT) p53 (p53α). Here, we show that two alternatively-spliced, functional, truncated isoforms of p53 (p53β and p53γ, containing exons 1-9 of the p53 gene) can be markedly upregulated by pharmacologic or genetic inhibition of nonsense mediated decay (NMD), a regulator of aberrant mRNA stability. These isoforms lack the MDM2 binding domain and hence have reduced susceptibility to MDM2-mediated degradation. In MDM2-overexpressing cells bearing wildtypeTP53gene, NMD blockade increased p53β/γ expression and p53 pathway activation, enhanced radiosensitivity, and inhibited tumor growth. A similar pattern was observed in HPV+cancer cells and in cancer cells with p53 mutations downstream of exon 9. These results identify a novel therapeutic strategy for restoration of p53 function in tumors rendered p53 deficient through MDM2 overexpression, HPV infection, or certain p53 mutations.

Although most activating mutations of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancers (NSCLCs) are sensitive to available EGFR tyrosine kinase inhibitors (TKIs), a subset with alterations in exon 20 of EGFR and HER2 are intrinsically resistant and lack an effective therapy. We used in silico, in vitro, and in vivo testing to model structural alterations induced by exon 20 mutations and to identify effective inhibitors. 3D modeling indicated alterations restricted the size of the drug-binding pocket, limiting the binding of large, rigid inhibitors. We found that poziotinib, owing to its small size and flexibility, can circumvent these steric changes and is a potent inhibitor of the most common EGFR and HER2 exon 20 mutants. Poziotinib demonstrated greater activity than approved EGFR TKIs in vitro and in patient-derived xenograft models of EGFR or HER2 exon 20 mutant NSCLC and in genetically engineered mouse models of NSCLC. In a phase 2 trial, the first 11 patients with NSCLC with EGFR exon 20 mutations receiving poziotinib had a confirmed objective response rate of 64%. These data identify poziotinib as a potent, clinically active inhibitor of EGFR and HER2 exon 20 mutations and illuminate the molecular features of TKIs that may circumvent steric changes induced by these mutations.

PD-1 checkpoint inhibitors pembrolizumab and nivolumab are approved for the treatment of recurrent or metastatic oral squamous cell carcinoma (OSCC). Previously we have demonstrated that PD-1/PD-L1 pathway blockade can reduce the progression of oral premalignant lesions (OPL) to carcinoma in the 4-NQO carcinogen-induced murine model of OSCC. Furthermore, pembrolizumab is currently being evaluated for prevention of OSCC in patients with high risk OPLs in the Immune Prevention of Oral Cancer clinical trial (NCT02882282). Building on these earlier studies, here we have conducted pre-clinical evaluation of other possible targets including CD40, OX40, and 4-1BB for the immunoprevention of OSCC, and determined the impact of combined or sequential treatment with PD-1/PD-L1 pathway blockade. We treated C57BL/6 mice with the carcinogen 4-nitroquinoline 1-oxide for 8 weeks in drinking water. Eight weeks after discontinuing 4-NQO, mice were treated with either IgG (n: 20), used as control group, anti-PD-1 monotherapy, concomitant anti-PD-1+OX4, anti-PD-1+CD40, anti-PD-1+4-1BB, or OX40, CD40, and 4-1BB followed by anti-PD-1 therapy (n:10 treatment groups). Mice were sacrificed 8 weeks after treatment. We assessed serial H&E-stained cross sections of tongues harvested at the same time point for total OSCC area. Tumor draining lymph nodes (TDLN) lymphocytes were analyzed by flow cytometry. Anova and Mann-Whitney test were used for statistical comparison. Treatment of mice harboring OPLs with CD40 followed by anti-PD-1 immunotherapy resulted in a decrease in the median total OSCC area when compared with our IgG control group (p= 0.034). Treatment with anti-PD-1 monotherapy showed a trend towards reducing progression of OPL to OSCC. Concomitant therapy with CD40 and anti-PD-1 along with the other combination immunotherapy regimens did not demonstrate any significant reduction in the progression of OPL to OSCC. Treatment with sequential CD40+anti-PD-1 increased both the CD8+PD-1+ lymphocyte population and the MFI values for PD-1 in the CD4+Foxp3- population in TDLN. When the TDLN of both concomitant and sequential 4-1BB+anti-PD-1 treatment groups were analyzed, a decrease in the CD4+PD-L1+ population, and an increase in both CD8+PD-1+ population and CD8/CD4+PD-L1+ ratio was found, although this change in the immune milieu was not associated to a reduction of OPL progression to OSCC. Targeting CD40 and PD-1 pathways sequentially, but not concomitantly, during the OPL stage led to a reduction in the progression of OPL to OSCC. The TDLN analysis of this treatment group revealed an increase in both CD8+PD-1+ lymphocytes and MFI values for PD-1 in CD4+Foxp3- lymphocytes. No other treatment group was able to significantly reduce progression of OPL to OSCC or showed a similar modulation of the lymphocyte population. These results support further investigation on how sequential CD40+anti-PD-1 immunotherapy hinder tumor immune evasion, making it a potential regimen for prevention of OSCC. Citation Format: Jose A. Monteiro de Oliveira Novaes, Alissa Poteete, Marlese Pisegna, William N. William, John V. Heymach. CD40/anti-PD-1 sequential immunotherapy outperforms multiple immunotherapy combinations in an oral cancer prevention mouse model [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO048.

Background: The serine/threonine kinase STK11/LKB1 is the second most commonly altered tumor suppressor in NSCLC. Non-functional mutations or loss of LKB1 expression occur more frequently in NSCLC than other alterations; however, there are currently no effective treatment strategies for this subset of tumors. KRAS-mutant LKB1 deficient NSCLC tumors often also have alterations in KEAP1 or NRF2 gene, which activate the KEAP1/NRF2 pathway known to be involved in antioxidant response. Inhibitors of ATM and ATR, two key proteins in the DNA damage response (DDR) pathway, are currently undergoing clinical testing but there are no validated biomarkers established for identifying which subgroups of patients are more likely to benefit from treatment. Here we have identified that alterations of LKB1, and the KEAP1/NRF2 pathway, are associated with enhanced response to ATM and ATR inhibitors (ATMi and ATRi) as well as other inhibitors of the DDR and may be useful biomarkers for predicting therapeutic response. Methods: To investigate the impact of LKB1 loss and KEAP1/NRF2 pathway activation on DDR and replication stress, we first tested replication fork protection in LKB1 deficient cells (KL). DNA fiber assay showed a defect in fork protection in KL cells compared with LKB1 wild type cells (K). Also, RPPA analysis revealed an activation of ATR/Chk1/Cdk1/CyclinB1 axis as well as Wee1 activation in cells harboring LKB1 and/or KEAP1 loss (KL/KLK/KK). Therefore, to evaluate response to DDR inhibitors (DDRi) we analyzed the in vitro activity of ATM, ATR, Wee1 and PARP inhibitors in NSCLC murine cell lines with or without knock out of LKB1 and/or KEAP1. In these cells, the loss of LKB1 and/or KEAP1 significantly sensitized to AZD0156 (ATMi), AZD6738 (ATRi) and AZD1775 (Wee1i) relative to cells with intact LKB1 and KEAP1. Next, we investigated whether the activity of ATR and ATMi in KL, KK or KLK tumor cells could be enhanced by the addition of a PARP inhibitor (olaparib). Although all NSCLC cells were resistant to the PARP inhibitor olaparib when used as a single agent, treatment of LKB1, KEAP1 or LKB1 plus KEAP1 deficient cells with the combination of olaparib plus ATM or ATR inhibitors significantly enhanced the antitumor cell activity of ATM or ATR inhibitors alone in vitro. We confirmed these data in an additional panel of LKB1 deficient NSCLC human cell lines treated with a broad spectrum of ATR and ATM inhibitors. In all human cell lines re-expression of LKB1 clearly reduced the sensitivity to ATR inhibition. LKB1 loss was also associated with sensitivity to PARP and ATM inhibitors, although these effects seemed to be less significant compared with ATR inhibitors. Interestingly, in vivo experiments performed in K, KL and KLK syngeneic models as well as PDX models showed greater response to ATRi and Wee1i monotherapy only in KLK but not in K or KL tumor models. Conclussions: Tumors with LKB1 deficiency or KEAP/NRF2 mutations are typically resistant to standard chemotherapy drugs and immunotherapy. Our data indicate that LKB1 and KEAP1/NRF2 loss significantly enhance the sensitivity to ATR, ATM and Wee1 inhibitors in vitro while only ATR and Wee1 inhibitor show significant tumor growth impairment in syngeneic and PDX KLK models. Thus, we have identified that NSCLC tumors bearing STK11 and KEAP1/NRF2 mutations are highly sensitive to DDR inhibitors and that genes may serve as biomarkers for selecting appropriate patients for treatment alone or in combination, such as PARPi, chemo or immunotherapy. Citation Format: Ana Galan-Cobo, Marlese Pisegna, Kavya Ramkumar, Alissa Poteete, Sungnam Cho, Fahao Zhang, You-Hong Fan, Qi Wang, Lixia Diao, Katharina Schlacher, Jing Wang, Lauren A Byers, John V. Heymcach. LKB1 deficiency and KEAP1/NRF2 pathway alterations as biomarkers of response for ATR and ATM inhibitors and other inhibitors of DNA damage response (DDR) in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A096. doi:10.1158/1535-7163.TARG-19-A096

Oral premalignant lesion (OPL) expression of PD-L1 is associated with increased cancer risk. These data suggest that immune evasion is already present at the OPL stage and warrant an evaluation of immune modulatory therapies for cancer prevention. We conducted this preclinical study in a carcinogen-induced mouse model of oral cancer to evaluate the efficacy of multiple immunomodulatory strategies, given at a time point when only OPLs are present, to reduce the incidence of oral squamous cell carcinoma (OSCC). We treated C57BL/6 mice with the carcinogen 4-nitroquinoline 1-oxide (4-NQO) for 8 weeks in drinking water. Eight weeks after discontinuing 4-NQO treatment, a group of mice (N=6) was sacrificed for assessment of invasive and non-invasive tongue lesions. A second group of mice was treated with either anti-PD-1 (N=40), anti-CTLA-4 (N=20), anti-OX40 (N=20), anti-PD-1 + anti-CTLA-4 (N=20), anti-PD-1 + anti-OX40 (N=20) antibodies, or IgG (N=40) for a total of 3 doses every 3 days, initiating 8 weeks after the cessation of 4-NQO. Antibodies used in combination were given on the same day . Mice were sacrificed 56 days after the last dose of treatment. We assessed serial H&E-stained cross sections of the tongues harvested at that same time point for development of OPLs and cancer and measured the OSCC area of each tongue using aperio imagescope software. Mann-Whitney and Fisher's exact tests were used for statistical comparisons between groups. Eight weeks after 4NQO cessation, 100% of mice developed tongue hyperplasia or dysplasia and invasive lesions were not identified, indicating this to be an ideal time point to initiate treatment strategies addressing OPLs. Tongues from mice treated with anti-PD-1 antibody displayed a decrease in the mean OSCC area when compared with mice treated with IgG (mean 3.53 mm2 versus 6.62 mm2, respectively; p= 0.018). At this time point, invasive oral cancers had developed in 75% versus 50% of IgG and anti-PD-1 treated mice, respectively (p=0.03). There were also non-significant decreases in the mean OSCC area in tongues from mice treated with anti-CTLA-4 (mean = 5.07 mm2), anti-OX40 (mean = 3.79 mm2) and anti-PD-1 + anti-CTLA-4 (mean = 3.86 mm2) antibodies when compared to mice treated with IgG (mean = 6.62 mm2) control. In the group treated with anti-PD-1 + anti-OX40 combination therapy, there was an increase in the mean OSCC area when compared to anti-PD-1 monotherapy (mean 3.53 mm2 versus 9.03 mm2), respectively (p= 0.01). Short-term anti-PD-1 immune checkpoint inhibitor therapy in the context of OPLs led to a reduction in the incidence of oral cancer. When comparing the mean area of OSCC in each group, anti-PD-1 monotherapy was superior to IgG and all other treatment strategies. Paradoxically, combining anti-OX40 and anti-PD-1 antibodies created an antagonizing effect on the therapy and increased total tumor burden when compared with anti-PD-1 monotherapy. Citation Format: Jose Augusto Monteiro de Oliveira Novaes, Marlese A. Pisegna, Alissa Poteete, Fahao Zhang, John V. Heymach, William N. William. Anti-PD-1 monotherapy outperforms multiple immunotherapy combination strategies in an oral cancer prevention mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1268.

Metastatic Small cell lung cancer (SCLC) is exceptionally lethal, having a median survival of 9-10 months from the time of diagnosis and a 5 year survival rate of less than 2 percent. More effective treatment approaches to SCLC are desperately needed. Poly ADP-ribose polymerase (PARP) inhibitor such as olaparib exploit the concept of synthetic lethality by selectively targeting cancer cells with defective DNA repair pathways. Preclinical data indicates that SCLC cell lines overexpress PARP and are sensitive to PARP inhibition, and PARP inhibitors show promise in the clinical setting of SCLC. The identification of combination regimens that potentiate the efficacy of PARP inhibition would be of great clinical value. Recent findings indicate that olaparib and the vascular endothelial growth factor receptor (VEGFR) inhibitor, cediranib, is an effective therapeutic combination in patients with BRCA wild-type ovarian cancer. Therefore, we evaluated the efficacy of the olaparib and cediranib combinations in SCLC models. Human SCLC cell lines, NCI-H841, H526, COR-L24 and murine SCLC cell line TKOmTmG were sensitive to olaparib or cediranib therapy in vitro as determined by Cell Titer Glo® assay. Moreover, a significantly greater effect on cell viability was observed when olaparib and cediranib were used in combination. Likewise, in H841 xenograft models, single agent treatment of olaparib or cediranib inhibited tumor growth, and combination treatment of olaparib plus cediranib induced tumor regression. Western blot analysis indicated that treatment with cediranib or the combination of olaparib and cediranib induced PD-L1 protein expression both in vivo and in vitro. In TKOmTmG xenograft model in syngeneic mice, programmed death-ligand 1 (PD-L1) inhibitor plus olaparib or olaparib/cediranib combination resulted in superior combined efficacy compared to the respective single agent therapies in terms of tumor growth inhibition. These data indicate that olaparib plus cediranib may be an effective therapeutic strategy for the treatment of SCLC. Further study is warranted to understand the mechanism of action and clinical activity. Citation Format: Naoto Morikawa, Monique B. Nilsson, Irene Guijarro, You-Hong Fan, Alissa Poteete, John V. Heymach. Evaluation of the combination of olaparib and cediranib in small-cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4847.

KRAS is the most commonly mutated oncogenic driver in non-small cell lung cancer (NSCLC) and other solid tumors. Recently we conducted an integrative analysis and found three major subgroups of KRAS-mutated cancer defined by co-occurring genomic events with distinct biology, molecular vulnerabilities, and therapeutic sensitivities. One of these genes, the serine/threonine kinase STK11 (LKB1), represents the second most commonly altered tumor suppressor in NSCLC and there are currently no treatment strategies tailored for LKB1-deficient NSCLC. KRAS-mutant/LKB1-deficient (KL) tumors are characterized by high co-occurrence of KEAP1 mutational inactivation. Inactivation of KEAP1 protects cells against REDOX stress via upregulation of NRF2 target genes, in part by production of glutathione. We evaluated the effects of blocking glutamine metabolism using an isogenic series of NSCLC cell lines harboring mutations in STK11 and KEAP1. Through sequential silencing or overexpression of LKB1, KEAP1, or NRF2 we demonstrated that glutaminase inhibitors (GLSi) can block cell proliferation while increasing energetic and REDOX stress specifically in LKB1 deficient cells with hyperactivation of the KEAP1/NRF2 pathway driven by KEAP1 mutations (KLK subtype). In KLK models, overexpression of LKB1 or KEAP1 partially reduced GLSi sensitivity, while siRNA-mediated down-regulation of NRF2 showed a similar effect. Furthermore, the combination of LKB1 add back coupled with down-regulation of NRF2 conferred even greater resistance to GLSi. To confirm the LKB1/KEAP1-driven response to GLSi, we performed in vivo experiments examining the response of subcutaneous xenografts of an A549 isogenic series; A549 (KLK), A549 LKB1 add back (KK) or A549 KEAP1 add back (KL); to GLS inhibition. These experiments demonstrated that GLSi impaired tumor growth in A549 (KLK) tumors, exhibiting significant statistical differences compared with the vehicle group from 18 days of treatment. Conversely, GLS inhibition did not significantly affect the growth of A549/LKB1 (KK) or A549/KEAP1 (KL) tumors. Collectively, our data indicate that in KLK tumors both pathways, LKB1 and KEAP1/NRF2, cooperatively drive a glutamine-addicted metabolic program, making KLK tumors selectively vulnerable to GLSi treatment. These findings have immediate clinical implications and support the future clinical testing of GLS inhibitors in KLK NSCLC. Citation Format: Ana Galan-Cobo, Piyada Sitthideatphaiboon, Xiao Qu, Jeffrey J. Kovacs, Alissa Poteete, Pan Tong, Sungnam Cho, Varsha V. Gandhi, Ferdinandos Skoulidis, Jing Wang, Timothy P. Heffernan, John V. Heymach. LKB1 and KEAP1/NRF2 pathways cooperatively promote glutamine dependence and vulnerability to glutaminase inhibitors in KRAS-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2408.

The serine/threonine kinase LKB1 is mutated and inactivated in 20-30% of lung adenocarcinoma representing the second most commonly altered tumor suppressor in non-small cell lung cancer (NSCLC), and to date there are no effective targeting strategies for tumors bearing this mutation. Therefore, new therapeutic approaches are urgently needed for LKB1-deficient tumors. LKB1 activates AMPK, the master sensor of cellular energy and because of this, many of the best known functions of LKB1 are attributed to its ability to control metabolic alterations. Our laboratory and others have demonstrated that loss of LKB1 can promote enhanced glucose metabolism and a shift from aerobic to anaerobic respiration. A result of enhanced glycolysis is an elevated lactate production even under normoxic conditions. It has been reported that lactate-mediated extracellular acidification is a key factor mediating tumor cell invasion and metastasis. We found that LKB1-deficient NSCLC tumors significantly upregulate lactate transporters and, because these tumors exhibit an immunologically inert phenotype, we propose that inhibition of lactate transporters could represent a rational strategy to inhibit tumor growth and enhance immune responses in LKB1 mutant NSCLC. To characterize lactate transporter expression, a panel of NSCLC cell lines was stably transduced to overexpress LKB1 and with shRNA targeting LKB1. We analyzed gene expression of lactate transporters in TCGA dataset of lung adenocarcinoma (LUAD). We validated these results by qPCR and western blot analysis of expression levels of MCT1, MCT4 and MCT14 in human LKB1-intact and deficient cells and in LKB1 KO murine NSCLC cell lines that were generated using CRISPR/Cas9 in a KRASG12D mutant background. To study lactate transporter expression in tumors in vivo, we generated syngeneic NSCLC mouse models via s.c. injection of LKB1-intact and KO murine cells in immunocompetent mice and analyzed the protein expression levels of MCT4 in these tumor samples. The analysis of TCGA LUAD dataset revealed a significant upregulation of lactate transporter SLC16A14 (MCT14) gene expression levels in LKB1-deficient tumors compared with LKB1 wild-type (p Citation Format: Irene Guijarro, Alissa Poteete, Pan Tong, Jing Wang, John Heymach. Lactate transporters are a potential therapeutic target for LKB1-deficient lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5147.

Introduction: Blockade of immune checkpoints has improved clinical outcomes for patients with metastatic non-small cell lung cancer (NSCLC), but its role in the perioperative setting for early-stage disease is unclear. We generated preclinical models of resectable NSCLC expressing an antigen that permits quantitative assessment of the immune response and compared survival, tumor recurrence, and immune response after neoadjuvant or adjuvant immunotherapy. Experimental Procedures: We transfected murine 344SQ NSCLC cells with an ovalbumin-expression plasmid to generate OVA+ cells that can be identified with an antibody against the peptide SIINFEKL bound to H-2Kb of MHC-I. We implanted 344SQ-OVA+ cells in the flank of syngeneic mice and then randomized mice with established tumors to either 3 doses of neoadjuvant IgG, anti-PD-1, anti-CTLA-4, anti-PD-1+anti-CTLA-4, or to observation. Primary tumors were resected in all mice 2 days after mice in the neoadjuvant group had received their last dose of therapy. Two days post-surgery, mice in the observation group were treated with 3 doses of adjuvant IgG, anti-PD-1, anti-CTLA-4, anti-PD-1+anti-CTLA-4. Mice were euthanized 4 weeks after injection or when moribund and their survival recorded and lung metastases counted. We determined the composition of CD3+CD8+ tumor-infiltrating lymphocytes (TILs) with flow cytometry using tetramers against SIINFEKL-specific T cell receptors and immunohistochemical staining of tumors. Results: Single agent and combined immunotherapy significantly prolonged survival compared to controls in both neoadjuvant and adjuvant groups (p Conclusions: Neoadjuvant combined immune checkpoint blockade is superior to adjuvant immunotherapy at prolonging survival, reducing distal recurrence and inducing anti-tumor immunity in preclinical models of resectable NSCLC. Citation Format: Tina Cascone, Haifa Hamdi, Fahao Zhang, Alissa Poteete, Lerong Li, Courtney W. Hudgens, Leila J. Williams, Qiuyu Wu, Jayanthi Gudikote, Weiyi Peng, Patrick Hwu, Jing Wang, Michael Tetzlaff, William N. William, John V. Heymach. Superior efficacy of neoadjuvant compared to adjuvant immune checkpoint blockade in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1719.

Introduction: Immunotherapy with single agent anti-PD-1 antibody confers modest overall survival benefits in patients with recurrent/metastatic HNSCCs. Combined immune checkpoint targeting has shown promising results for melanomas and other solid tumors. Thus, evaluation of combination immunotherapy in animal models specific to HNSCCs could inform prioritization and design of human clinical trials for this disease. We conducted this pre-clinical study to test the hypothesis that combining anti-PD-1 with either anti-CTLA-4 or co-stimulatory immune checkpoint agonist antibodies would be more effective than anti-PD-1 monotherapy in a mouse model of oral carcinoma. We also aimed at identifying the combination regimen with highest anti-tumor activity. Methods: C57BL/6 mice were subcutaneously grafted with 2x106 mouse oral cancer 1 (MOC1) cells derived from a carcinogen-induced tumor molecularly similar to human HNSCC. After 25 days, mice were randomized into one of 12 treatment groups (IgG, or antibodies targeting PD-1, CTLA-4, CD40, OX-40, GITR, 4-1BB, PD-1+CTLA-4, PD1+CD40, PD-1+OX-40, PD-1+GITR, or PD-1+4-1BB), N=8-9 mice/group. Antibodies were administered every 4 days for 3 doses. Tumors were measured twice per week. The primary endpoint was overall survival. Mice were sacrificed when tumor diameter was >2 cm or tumor ulceration was >5 mm. Survival curves were computed using the Kaplan-Meier method and compared using the log-rank (Mantel-Cox) test. Results: In analogy to human clinical trials, anti-PD-1 therapy led to a modest improvement in survival compared to IgG [HR=0.41; 95%CI 0.06-0.53; P=0.015], indicating that this model recapitulates, in part, the effects of immunotherapy in HNSCC patients; none of the animals survived long term. Monotherapy with anti-CTLA-4, anti-CD40, anti-OX-40, or anti-4-1BB also improved survival compared to IgG (P=0.0001, 0.003, 0.02, and 0.003, respectively), but were not more effective than anti-PD-1 alone (P=0.09, 0.12, 0.72, and 0.77, respectively). Combination therapy did not prolong survival compared to anti-PD-1 alone, with the exception of anti-PD-1+ anti-CTLA4 [HR=0.36; 95% CI 0.07-0.6; P=0.016], and anti-PD-1+ anti-CD40 [HR=0.125, 95% CI 0.02-0.24; P=0.0003]. Notably, 50% of mice receiving anti-PD-1+ anti-CD40 antibodies had complete tumor eradication and are alive and disease-free 120 days after tumor grafting. Conclusions: Single agent immunotherapy targeting PD-1, CTLA-4, CD40, OX-40 and 4-1BB modestly improved survival in a mouse model of oral cancer. Combination with anti-PD-1+ anti-CTLA4 and anti-PD-1+ anti-CD40 antibodies outperformed anti-PD-1 monotherapy. Complete responses were exclusively seen with PD-1+CD40 dual targeting. This regimen should be prioritized for evaluation in HNSCC clinical trials. Citation Format: Jose Augusto Monteiro de Oliveira Novaes, Alissa R. Poteete, Marlese A. Pisegna, Uma Giri, Fahao Zhang, Patrick Hwu, John V. Heymach, William N. William. Combined immune checkpoint targeting with anti-PD-1 plus anti-CD40 antibodies as the most effective approach to eradicate head and neck squamous cell carcinomas (HNSCCs) in mouse models [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 5648. doi:10.1158/1538-7445.AM2017-5648

e20099Background: Small cell lung cancer (SCLC) is an aggressive form of lung cancer characterized by loss of the tumor suppressor Rb. Chemotherapy and radiation remain the standard of care for SCL...