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Purpose: Patients with advanced non–small cell lung cancer (NSCLC) harboring activating EGFR mutations are initially responsive to tyrosine kinase inhibitors (TKI). However, therapeutic resistance eventually emerges, often via secondary EGFR mutations or EGFR-independent mechanisms such as epithelial-to-mesenchymal transition. Treatment options after EGFR-TKI resistance are limited as anti-PD-1/PD-L1 inhibitors typically display minimal benefit. Given that IL6 is associated with worse outcomes in patients with NSCLC, we investigate whether IL6 in part contributes to this immunosuppressed phenotype. Experimental Design: We utilized a syngeneic genetically engineered mouse model (GEMM) of EGFR-mutant NSCLC to investigate the effects of IL6 on the tumor microenvironment and the combined efficacy of IL6 inhibition and anti-PD-1 therapy. Corresponding in vitro studies used EGFR-mutant human cell lines and clinical specimens. Results: We identified that EGFR-mutant tumors which have oncogene-independent acquired resistance to EGFR-TKIs were more mesenchymal and had markedly enhanced IL6 secretion. In EGFR-mutant GEMMs, IL6 depletion enhanced activation of infiltrating natural killer (NK)- and T-cell subpopulations and decreased immunosuppressive regulatory T and Th17 cell populations. Inhibition of IL6 increased NK- and T cell–mediated killing of human osimertinib-resistant EGFR-mutant NSCLC tumor cells in cell culture. IL6 blockade sensitized EGFR-mutant GEMM tumors to PD-1 inhibitors through an increase in tumor-infiltrating IFNγ+ CD8+ T cells. Conclusions: These data indicate that IL6 is upregulated in EGFR-mutant NSCLC tumors with acquired EGFR-TKI resistance and suppressed T- and NK-cell function. IL6 blockade enhanced antitumor immunity and efficacy of anti-PD-1 therapy warranting future clinical combinatorial investigations.

IL-6 modulates expression of NK receptor and their ligands in EGFR mutant tumors.

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Supplementary tables S1-S6

Purpose:Radiotherapy with or without chemotherapy is a mainstay of treatment for locally advanced non–small cell lung cancer (NSCLC), but no predictive markers are currently available to select patients who will benefit from these therapies. In this study, we investigated the association between alterations in STK11/LKB1, the second most common tumor suppressor in NSCLC, and response to radiotherapy as well as potential therapeutic approaches to improve outcomes.Experimental Design:We conducted a retrospective analysis of 194 patients with stage I–III NSCLC, including 164 stage III patients bearing mutant or wild-type STK11/LKB1 treated with radiotherapy, and assessed locoregional recurrence (LRR), distant metastasis rates, disease-free survival (DFS), and overall survival (OS), and we investigated the causal role of LKB1 in mediating radiotherapy resistance using isogenic pairs of NSCLC cell lines with LKB1 loss or gain.Results:In stage III patients, with 4 years median follow-up, STK11/LKB1 mutations were associated with higher LRR (P = 0.0108), and shorter DFS (HR 2.530, P = 0.0029) and OS (HR 2.198, P = 0.0263). LKB1 loss promoted relative resistance to radiotherapy, which was dependent on the KEAP1/NRF2 pathway for redox homeostasis. Suppression of the KEAP1/NRF2 pathway via KEAP1 expression, or pharmacologic blockade of glutaminase (GLS) 1 sensitized LKB1-deficient tumors to radiotherapy.Conclusions:These data provide evidence that LKB1 loss is associated with LRR and poor clinical outcomes in patients with NSCLC treated with radiotherapy and that targeting the KEAP1/NRF2 pathway or GLS inhibition are potential approaches to radiosensitize LKB1-deficient tumors.

Epidermal growth factor receptor (EGFR) mutations typically occur in exons 18–21 and are established driver mutations in non-small cell lung cancer (NSCLC)1–3. Targeted therapies are approved for patients with ‘classical’ mutations and a small number of other mutations4–6. However, effective therapies have not been identified for additional EGFR mutations. Furthermore, the frequency and effects of atypical EGFR mutations on drug sensitivity are unknown1,3,7–10. Here we characterize the mutational landscape in 16,715 patients with EGFR-mutant NSCLC, and establish the structure–function relationship of EGFR mutations on drug sensitivity. We found that EGFR mutations can be separated into four distinct subgroups on the basis of sensitivity and structural changes that retrospectively predict patient outcomes following treatment with EGFR inhibitors better than traditional exon-based groups. Together, these data delineate a structure-based approach for defining functional groups of EGFR mutations that can effectively guide treatment and clinical trial choices for patients with EGFR-mutant NSCLC and suggest that a structure–function-based approach may improve the prediction of drug sensitivity to targeted therapies in oncogenes with diverse mutations.

Purpose: Radiotherapy with or without chemotherapy is a mainstay of treatment for locally advanced non–small cell lung cancer (NSCLC), but no predictive markers are currently available to select patients who will benefit from these therapies. In this study, we investigated the association between alterations in STK11/LKB1, the second most common tumor suppressor in NSCLC, and response to radiotherapy as well as potential therapeutic approaches to improve outcomes. Experimental Design: We conducted a retrospective analysis of 194 patients with stage I–III NSCLC, including 164 stage III patients bearing mutant or wild-type STK11/LKB1 treated with radiotherapy, and assessed locoregional recurrence (LRR), distant metastasis rates, disease-free survival (DFS), and overall survival (OS), and we investigated the causal role of LKB1 in mediating radiotherapy resistance using isogenic pairs of NSCLC cell lines with LKB1 loss or gain. Results: In stage III patients, with 4 years median follow-up, STK11/LKB1 mutations were associated with higher LRR (P = 0.0108), and shorter DFS (HR 2.530, P = 0.0029) and OS (HR 2.198, P = 0.0263). LKB1 loss promoted relative resistance to radiotherapy, which was dependent on the KEAP1/NRF2 pathway for redox homeostasis. Suppression of the KEAP1/NRF2 pathway via KEAP1 expression, or pharmacologic blockade of glutaminase (GLS) 1 sensitized LKB1-deficient tumors to radiotherapy. Conclusions: These data provide evidence that LKB1 loss is associated with LRR and poor clinical outcomes in patients with NSCLC treated with radiotherapy and that targeting the KEAP1/NRF2 pathway or GLS inhibition are potential approaches to radiosensitize LKB1-deficient tumors.

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.

Epidermal growth factor receptor (EGFR) mutations typically occur in exons 18-21 and are established driver mutations in non-small cell lung cancer (NSCLC)

Approximately 15% of all patients with non-small cell lung cancer (NSCLC) and nearly 35% of Asian patients with NSCLC harbor activating mutations within the epidermal growth factor receptor (EGFR). Although these patients are initially highly sensitive to first or second generation EGFR tyrosine kinase inhibitors (TKIs) including erlotinib or third-generation inhibitors including osimertinib, EGFR TKI-refractory disease inevitably emerges. While therapeutic strategies to target resistant disease that emerges though secondary EGFR mutations or MET amplification have been developed, there remains a void of therapeutic options for patients where resistance occurs through EGFR-independent mechanism such as epithelial to mesenchymal transition (EMT) or transformation to small cell lung cancer (SCLC). To identify cell surface proteins that could be targeted by antibody-based or adoptive cell therapy approaches we interrogated RNAseq data from EGFR mutant NSCLC cell lines (HCC827 and HCC4006) and their associated EGFR TKI resistant variants previously shown to have developed resistance through EMT and filtered gene expression data to include only genes which transcribed proteins localized to the cell surface. We identified CD70 as a being highly upregulated in EGFR TKI resistant cells (p = 7.2e-42). Given that CD70 expression is highly restricted and only transiently expressed on immune cells, CD70 was selected as a top candidate cell surface protein for targeting studies. Western blotting and flow cytometry analysis confirmed CD70 protein levels to be highly upregulated in EGFR TKI resistant cells that had undergone EMT but not in cells harboring secondary EGFR mutations or MET amplifications. We also observed CD70 upregulation in osimertinib-treated drug tolerant persister cells, indicating that CD70 upregulation is an early event in the evolution of TKI resistance. Moreover, patient-derived models of acquired EGFR TKI resistance also exhibited CD70 positivity. Our data also indicated that in EGFR mutant NSCLC cells, CD70 could be upregulated through decreased CD70 promoter methylation as well as by the EMT regulators, transforming growth factor-β (TGF-β) and ZEB1, both of which were upregulated in TKI resistant cells. In EGFR TKI resistant cells, CD70 knockdown impaired cell viability and invasiveness, and stimulation of CD70 using the exogenous binding partner CD27 resulted in activation of AKT and MAPK, pathways known to be re-activated with acquired TKI resistance. CD70-targeting approaches including anti-CD70 antibody drug conjugates (ADCs) and CD70-targeting CAR T cell and CAR NK cells showed promising in vitro and in vivo activity against CD70 positive tumor cells and in osimertinib drug-tolerant persister cells. These results identify CD70 as a novel therapeutic target for EGFR mutant tumors with acquired EGFR TKI resistance that merits further investigation in the clinic. Citation Format: Monique B. Nilsson, Yan Yang, Sonia Patel, Simon Heeke, Xiuning Le, Thiru Aruguman, Jacqulyne Robichaux, Xiaoxing Yu, Alissa Poteete, Xiaoyang Ren, Lixia Diao, Li Shen, Qi Wang, Fahao Zhang, Leticia Campos Clemente, Luisa Solis Soto, Chunhua Shi, Hai Tran, Jason Bock, Jing Wang, Ignacio I. Wistuba, John D. Minna, John V. Heymach. CD70 is a novel therapeutic target for EGFR mutant NSCLC with acquired, EMT-associated EGFR TKI resistance [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 1827.

Genomic alterations that result in loss of function of the tumor suppressor serine/threonine kinase STK11/LKB1 occur in 20-30% of lung adenocarcinomas. We previously observed that STK11/LKB1 mutations are genomic drivers of primary resistance in lung adenocarcinoma. Moreover, LKB1-mutant NSCLCs exhibit higher hypoxia and glycolysis rates, resulting in enhanced production and secretion of lactate. Accordingly, we hypothesize that high production of lactate of LKB1 mutant tumor may contribute to its immunologically cold phenotype and that blockade of the lactate pathway may potentiate the efficacy of immune checkpoint blockade (ICB) therapies. We characterized the immune landscape of LKB1 mutant clinical samples and performed scRNAseq analysis in KRAS mutant (K) and KRAS mutant LKB1 knockout (KL) syngeneic murine models. To evaluate inhibition of lactate metabolism as a therapeutic strategy, we knocked out the lactate transporter SLC16A3/MCT4 and characterized the impact on the tumor microenvironment (TME), and response to ICB. Clinical analysis of LKB1 mutant NSCLC patients from the MD Anderson’s ICON and PROSPECT cohorts suggested that LKB1 mutant tumors showed reduced immune cell infiltration, restricted T cell function, and enhanced M2-like macrophages phenotypes. Moreover, in preclinical models, LKB1 mutant tumors showed enhanced glycolysis and upregulation of MCT4 expression in a variety of human and murine cell lines. Deletion of MCT4 dramatically reduced glycolysis, energy production, and cell proliferation. By scRNAseq, we identified distinct immune subclusters modulated by LKB1 mutation. Hypofunctional T cells and M2-like macrophages were abundant in LKB1 mutant tumors, while these populations were significantly reduced in KL tumors with MCT4 KO. The conditioned medium from KL cells impaired T cell activation and decreased T cell killing, IFNγ production and glycolysis rate. Moreover, conditioned medium from KL cells induced M2-associated genes expression, as well as CD206+ expression in both peritoneal macrophages and Raw264.7 cells. These effects were at least in part MCT-dependent, as medium from MCT4 KO cells induced the opposite effects on T cells and macrophages, and the effects could be reversed by introducing exogenous lactate, suggesting that blockade of lactate transport reactivated T cells and reversed M2 polarization. Importantly, MCT4 KO in LKB1-mutant tumors sensitized tumors to anti-PD1 immunotherapy in syngeneic murine tumors and promoted long-term anti-tumor immunity. Collectively, our data indicate that LKB1 mutant tumors enhanced lactate secretion into the TME and this results in decreased T cell cytotoxic potential as well as higher pro-tumor M2 polarization, leading to resistance to immunotherapy. These data suggest that therapeutic inhibition of MCT4 is a promising strategy to overcome immunotherapy resistance in NSCLC patients harboring LKB1 mutant tumors. Citation Format: Yu Qian, Irene Guijarro, Ana Galan-Cobo, Minghao Dang, Alissa Poteete, Fahao Zhang, Qi Wang, Jing Wang, Edwin Parra, Ferdinandos Skoulidis, Ignacio Wistuba, Svena Verma, Taha Merghoub, Linghua Wang, Jedd Wolchok, Alexandre Reuben, John Heymach. MCT4 blockade reverses lactate-mediated immunosuppression in LKB1-deficient NSCLC [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 2160.

Around 10-15% of non-small cell lung cancer (NSCLC) cases in the United States harbor an activating mutation in epidermal growth factor receptor (EGFR) which are initially highly sensitive to treatment EGFR tyrosine kinase inhibitors (TKI) before eventually developing resistance to these agents. Therefore, there is a current unmet clinical need to identify novel treatment options for this patent population. While anti-PD-1/PD-L1 immunotherapy has been effective for many NSCLC patients, EGFR-mutant tumors have response rates of less than 10%. The mechanisms driving resistance to immunotherapy in EGFR-mutant NSCLC are not well understood. We previously reported that IL-6 is highly upregulated in NSCLC cells with acquired resistance to EGFR-TKIs. Because IL-6 is a pleiotropic cytokine known to impact immune populations within the tumor microenvironment, we hypothesized that IL-6 may drive immunosuppression in EGFR mutant NSCLC. Using genetically engineered mouse models (GEMMs) of EGFR-mutant NSCLC with and without IL-6 knockout, we evaluated the effects of IL-6 on the tumor microenvironment. EGFR-mutant mice with knockout of IL-6 had a significantly increased overall survival compared to those with intact-IL-6. Knockout of IL-6 increased total immune infiltration in these tumors as assessed by flow cytometry. Infiltrating T cells from IL-6 knockout mice displayed a smaller population of T helper 17 cells compared to IL-6-expressing tumors. Knockout of IL-6 also increased the population of infiltrating activated CD8 T cells and a reduced T-regulatory cell population. Due to these changes in T cell activity, we wanted to determine any potential synergistic effects of IL-6 blockade used during anti-PD-1 therapy. We observed that combination treatment extended survival in EGFR-mutant NSCLC mice. Corresponding in vitro T cell cytotoxicity assays confirmed that EGFR-TKI resistant cells treated with IL-6 blocking antibody were more sensitive to T cell-mediated killing. Furthermore, infiltration of NK cells was increased in tumors with knockout of IL-6. Infiltrating NK cells found in these IL-6 knockout mice displayed a more activated phenotype as demonstrated by increased expression of NKG2D. Next, we evaluated the effects of IL-6 from conditioned media collected from human EGFR-mutant and TKI-resistant cells lines on human T and NK cells cultured ex vivo. e determined that IL-6 suppressed expression of activation markers including granzyme B and IFN-y on the surface of both T and NK cells. Blockade of IL-6 increased NK-mediated cytotoxic killing of EGFR-TKI resistant cells in vitro. In conclusion, our data indicates that in EGFR-TKI resistance, upregulated IL-6 suppresses T and NK cell cytotoxic potential and drives immunosuppression. Citation Format: Sonia Patel, Monique Nilsson, Yan Yang, Xiaoxing Yu, Fahao Zhang, Alissa Poteete, Xiaoyang Ren, Li Shen, Jing Wang, Xiuning Le, John Heymach. IL-6 contributes to the suppression of T and NK cell anti-tumor activity in EGFR-mutant NSCLC [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 3125.

Background: KEAP1, which regulates the degradation of the antioxidant transcription factor NRF2, is the third most commonly mutated tumor suppressor in lung adenocarcinoma (LUAD). Recent reports have provided clinical evidence that mutations in STK11/LKB1 and KEAP1 are strongly associated with immune checkpoint blockade resistance in LUAD, particularly those harboring KRAS mutations. Nevertheless, the specific mechanisms by which loss of KEAP1 impacts anti-tumor immunity in KRAS mutant tumors remains to be determined. Methods: KRAS-mutant (K) and LKB1 (KL), and/or KEAP1-deficient (KK and KLK) murine tumor models were profiled using single-cell RNA sequencing (scRNA-seq) and multiplex staining, and response to anti-PD1 treatment was assessed. Clinical samples from the MD Anderson ICON study (a cohort of 148 resected tumors from early-stage lung cancer patients) and TCGA lung cohorts were used to validate pre-clinical findings. Results: While K tumors were sensitive to anti-PD1 treatment, KEAP1-deficient isogenic tumors (KK; KLK) were refractory. KEAP1-deficient tumors were found to exhibit low immune cell infiltration and an enrichment of cancer associated fibroblasts (CAFs) and endothelial cells. scRNA-seq analysis indicated that KEAP1-deficient tumors had reduced T cell infiltration, in particular, CD8 and NK T cells, decreased B cell populations, and a marked change in M2 macrophage polarization as compared to KEAP1-proficient tumors. Multiplex analysis of CD3 and F4/80 markers confirmed these findings. In the TCGA lung cancer cohort, CD8B expression was dramatically decreased while MIF (macrophage migration inhibitory factor) was upregulated in KK tumors as compared to K LUAD tumors, and expression of KEAP1 inversely correlated with CD163, ARG2 and IL10, which are mainly secreted by macrophages. KEAP1-deficient pre-clinical tumor models showed a significant upregulation of MIF expression and secretion. CRISPR-Cas9 deletion of MIF dramatically impaired in vivo tumor growth, and enhanced T cell cytotoxic effects, anti-tumor immune response and anti-PD1 treatment in KK and KLK tumor models. Conclusions: These findings indicate that loss of KEAP1, alone or in combination with STK11/LKB1 alterations, contributes to an immunosuppressed tumor immune microenvironment. These changes appear to be mediated at least in part through MIF upregulation, providing a potential therapeutic strategy for overcoming KEAP1-dependent resistance to immunotherapy. Citation Format: Ana Galan-Cobo, Yu Qian, Fuduan Peng, Daniel James McGrail, Sonia Patel, Fahao Zhang, Xiang Zhang, Ferdinandos Skoulidis, Edwin Parra, Minghao Dang, Saxon Rodriguez, Alexandre Reuben, Ignacio Wistuba, Linghua Wang, John Victor Heymach. Suppression of macrophage migration inhibitory factor (MIF) impairs tumor growth and overcomes immunotherapy resistance in KEAP1-deficient NSCLC tumors [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 2113.

HPV-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) tumors typically have p53 loss due to the activity of the human papillomavirus (HPV)-encoded E6 protein and the E6-associated protein (HPVE6-AP) which mediate the degradation of wild-type (WT) p53 (p53α). The loss of p53 is thought to be a major contributor to the pathogenesis of HPV+ HNSCC, which comprise approximately 35% of all HNSCC. Currently, standard care for HPV+HNSCC includes radiation and chemotherapy. However long-term toxicity related to these treatments is a concern, and there is a need for newer therapeutic strategies. Previously, we reported that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising of exons 1 to 9β or 9γ, respectively) are degraded by nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. Here, using HPV+HNSCC cell line models, we show that NMD inhibition rescues p53β/γ isoforms and activates p53 pathway. Furthermore, we show that p53β/γ isoforms are more stable compared to p53α in these cells, with reduced vulnerabililty to HPVE6-AP- mediated degradation, and that p53β/γ isoforms contribute to increased expression of p53 transcriptional targets p21 and PUMA following NMD inhibition. Consistent with p53 pathway activation, NMD inhibition enhanced radiosensitivity of HNSCC cells. NMD inhibition attenuated colony forming ability and disrupted cell cycle progression. To evaluate the therapeutic implications of NMD inhibition, we assessed the in vivo growth of HPV+ UMSCC47 tumors. Nude mice were injected with UMSCC47 cells either subcutaneously or orthotopically in the tongue and randomized to receive vehicle or with an NMD inhibitor. In both tumor models, we observed a significant reduction in tumor volume with NMD inhibition as compared to the vehicle-treated animals. To investigate whether NMD inhibition induced the expression of p53β/γ isoforms and activated the p53 pathway in vivo, we collected tumor tissues from animals and evaluated expression of p53 isoforms and transcriptional targets by RT-PCR. We observed increased expression of p53γ, p21, GADD45A and PUMA mRNAs in NMD inhibitor treated UMSCC47 tumors, compared to their respective vehicle treated controls. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in major subgroups of p53-deficient HPV+ HNSCC tumors. Citation Format: Jayanthi Gudikote, Tina Cascone, Alissa Poteete, Piyada Sitthideatphaiboon, Sonia Patel, Yan Yang, Fahao Zhang, Lerong Li, Li Shen, Monique Nilsson, Phillip Jones, Jing Wang, Jean-Christophe Bourdon, Faye M. Johnson, John V. Heymach. Targeting nonsense-mediated decay restores p53 function in HPV-associated head and neck cancers [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 5733.

The leading cause of cancer-related deaths in the US is non-small cell lung cancer (NSCLC), and about 10-15% of NSCLC cases harbor EGFR activating mutations. While these patients are initially responsive to EGFR tyrosine kinase inhibitors (TKIs), they eventually experience progression and develop acquired TKI resistance. Currently, there are no approved therapies for NSCLC patients with acquired resistance to the third-generation EGFR TKI osimertinib (OSI). Moreover, EGFR-TKI resistant NSCLCs are refractory to immune checkpoint inhibitors. Therefore, novel treatment strategies are urgently needed. Chimeric antigen receptors (CARs) have been used to enhance the anti-tumor activity of immune effector cells. Here, we developed CAR-based therapies targeting EGFR as a novel strategy to overcome immune suppression and target EGFR TKI-resistant NSCLC. We designed multi-generational EGFR CAR constructs and transduced them into T and natural killer (NK) cells to generate CAR-T and CAR-NK cells, respectively. We then evaluated their activity against NSCLC cell lines in vitro using firefly luciferase assays. EGFR CAR-T and CAR-NK cells showed potent cytotoxicity against NSCLC cells expressing EGFR including EGFR wild-type and mutant NSCLCs, as well as NSCLC cells with acquired resistance to OSI. However, EGFR CAR-T cells showed decreased killing activity against OSI-resistant NSCLC cells compared to their parental cells. In contrast, EGFR CAR-NK cells showed stronger cytotoxicity against NSCLC with acquired OSI resistance as compared to parental cells. Transcriptomic analysis revealed that NSCLC cells with acquired resistance to TKI had undergone epithelial-mesenchymal transition and had downregulated expression of genes related to antigen presentation and upregulated expression of B7-H6 (NCR3LG1), which have been reported to be associated with increased responsiveness to NK cells. Moreover, treatment of TKI resistant cells with OSI resulted in upregulation of cell surface EGFR and potentiated CAR-NK cells-mediated killing. TGF-β is a critical immune-suppressive cytokine. We found that EGFR-TKI resistant NSCLC cells elaborated expression of TGF-β as compared to parental cells. Blockade of the TGF-β pathway using galunisertib significantly enhanced the activity of EGFR CAR-NK cells against OSI-resistant NSCLC cells. Next, we engineered the dominant-negative TGF-β receptor II (DNTGFBRII) into our EGFR CAR constructs. The expression of DNTGFBRII inhibited the phosphorylation of SMAD2 and the downregulation of granzyme B and perforin induced by TGF-β. DNTGFBRII-CAR-NK cells showed higher killing activity against OSI-resistant NSCLC cells and were resistant to TGF-β-mediated immunosuppression. In conclusion, EGFR CAR-NK cells show significant anti-tumor activity to EGFR TKI-resistant NSCLC cells, and the cytotoxicity is enhanced in combination with OSI treatment and blockade of the TGF-β pathway. Citation Format: Yan Yang, Monique Nilsson, Sonia Patel, Jacqulyne Robichaux, Alissa Poteete, Xiaoxing Yu, Fahao Zhang, Simon Heeke, Yu Qian, Junqin He, John Heymach. EGFR CAR-NK cells demonstrate potent activity against EGFR TKI resistant NSCLC [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 548.

Introduction The treatment of patients with EGFR-mutant NSCLC with vascular endothelial growth factor (VEGF) inhibitors in combination with EGFR inhibitors provides a greater benefit than EGFR inhibition alone, suggesting that EGFR mutation status may define a patient subgroup with greater benefit from VEGF blockade. The mechanisms driving this potentially enhanced VEGF dependence are unknown. Methods We analyzed the effect of EGFR inhibition on VEGF and HIF-1α in NSCLC models in vitro and in vivo. We determined the efficacy of VEGF inhibition in xenografts and analyzed the impact of acquired EGFR inhibitor resistance on VEGF and HIF-1α. Results NSCLC cells with EGFR-activating mutations exhibited altered regulation of VEGF compared with EGFR wild-type cells. In EGFR-mutant cells, EGFR, not hypoxia, was the dominant regulator of HIF-1α and VEGF. NSCLC tumor models bearing classical or exon 20 EGFR mutations were more sensitive to VEGF inhibition than EGFR wild-type tumors, and a combination of VEGF and EGFR inhibition delayed tumor progression. In models of acquired EGFR inhibitor resistance, whereas VEGF remained overexpressed, the hypoxia-independent expression of HIF-1α was delinked from EGFR signaling, and EGFR inhibition no longer diminished HIF-1α or VEGF expression. Conclusions In EGFR-mutant NSCLC, EGFR signaling is the dominant regulator of HIF-1α and VEGF in a hypoxia-independent manner, hijacking an important cellular response regulating tumor aggressiveness. Cells with acquired EGFR inhibitor resistance retained elevated expression of HIF-1α and VEGF, and the pathways were no longer EGFR-regulated. This supports VEGF targeting in EGFR-mutant tumors in the EGFR inhibitor–naive and refractory settings.

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.

Sotorasib (AMG 510) is the first KRAS inhibitor to be FDA-approved for the treatment of KRASG12C mutant lung adenocarcinomas which comprise ~42% of KRAS mutations in lung adenocarcinomas. Preclinical studies have shown that within hours of KRASG12C inhibition, synthesis of new KRASG12C protein and increased KRAS signaling was observed, leading to enhanced tumor cell growth and survival. This appeared to be due, at least in part, to a feeback loop leading to activation of EGFR signaling but the role of other ErbB family members including ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4) has not been fully evaluated. We hypothesize that short-term adaptation to KRAS inhibition is driven by multiple ErbB family members, and that the combination of pan-ErbB inhibitors with KRASG12C inhibitors could enhance KRASG12C inhibitor activity and prolong survival in preclinical models harboring KRASG12C mutations to a greater extent than more specific EGFR inhibitors. To investigate the role of different ErbB family members in this feedback signaling, we evaluated the impact of an EGFR specific inhibitor, erlotinib, an EGFR/HER2 inhibitor, afatinib, or pan-ErbB inhibitor (EGFR/HER2/3/4), poziotinib on anti-tumor activity of KRASG12C inhibitors in KRASG12C mutant NSCLC cell lines. We initially tested the different inhibitors in combination with sotorasib and adagrasib (MRTX849) and calculated the BLISS index (BI) for each drug pair matrix, using SynergyFinder. We found that when combined with either sotorasib or adagrasib, poziotinib had a synergistic effect (BI>10) in H23 (BI: 17, p=0.01), HCC44 (BI: 11, p=0.04), and H1792 (BI: 13, p=0.01) and an additive effect (BI= -10–10) in H2122 (BI: 2.8). Afatinib and erlotinib were additive in H23 (BI: 9.2 & 9.4), HCC44 (BI:8.8 & 7.9), H2122 (BI: -4.5 & -3.3) and H1792 (BI: 6.6 & 3.3). Poziotinib yielded a higher BI across all four cell lines compared to afatinib (p Citation Format: Jacqulyne P. Robichaux, Ana Galan-Cobo, Ried T. Powell, Kelly A. Gale, Jun He, Fahao Zhang, Monique B. Nilsson, Xiang Zhang, Mary M. Sobieski, Nghi Nguyen, Stephan C. Clifford, John V. Heymach. Pan-ErbB inhibition enhances activity of KRASG12C inhibitors in preclinical models of KRASG12C mutant cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P256.

BackgroundIn LUAD, KEAP1 is the third most common tumor suppressor and loss-of-function mutations in KEAP1 commonly co-occur with STK11/LKB1 and KRAS mutations. KEAP1 protein that regulates the degradation of the antioxidant transcription factor NRF2. The role of STK11/LKB1 mutations in immunotherapy resistance has been characterized, however the mechanistic understanding of KEAP1 deficiency in shaping LUAD phenotype and therapy response is still very limited. Recent clinical data has been reported suggesting that mutations in STK11/LKB1 and KEAP1 are strongly associated with immune checkpoint blockade resistance in LUAD, particularly those with KRAS mutations. Nevertheless, the biology of KEAP1-deficient tumors and the immune suppression mechanisms are to be characterized.MethodsWe have first validated response to anti-PD1 treatment in vivo using subcutaneous murine models, and performed a deep profiling and characterization of tumor microenvironment (TME) heterogeneity of KRAS-mutant (K) and LKB1 (KL), and/or KEAP1 deficient (KK and KLK) tumors using single-cell RNA sequencing (scRNA-seq) and multiplex staining. Data from pre-clinical models has been used to survey the immune genomic data available from the MD Anderson ICON study (a cohort of early stage lung cancer untreated 148 resected tumors) and TCGA lung cohorts to further validate our findings.ResultsWhile K tumors showed significant response to anti-PD1 treatment, KEAP1 loss completely impaired therapeutic response to this immunotherapy. KEAP1-deficient tumors were characterized by low immune infiltration while displayed an enrichment of cancer associated fibroblasts (CAFs) and endothelial cells. scRNA-seq data indicated a significant reduction of T cell infiltration, in particularly, CD8 and NK T cells, pronounced decreased of B cell population and a marked M2 macrophages polarization. Likewise, IHC and multiplex analysis of CD3 and F4/80 markers confirmed these previous findings. In TCGA lung cancer cohort, CD8B expression was dramatically decreased while MIF (macrophage migration inhibitory factor) was upregulated in KK compared to K LUADs tumors, and expression of KEAP1 inversely correlated with CD163, ARG2 and IL10, which are mainly secreted by macrophages. Concordantly, KEAP1-deficient pre-clinical tumors showed a significant upregulation of MIF expression and secretion, and CRISPR-Cas9 deletion of MIF dramatically impaired in vivo tumor growth in KK and KLK but not in K or KL models.ConclusionsThese findings indicate that loss of KEAP1, alone or in combination with STK11/LKB1 alterations, unfavorably reprograms TME. These changes appear to be mediated at least in part through MIF upregulation, providing a potential therapeutic strategy for overcoming KEAP1-dependent resistance to immunotherapy.

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

We characterized the landscape and drug sensitivity of ERBB2 (HER2) mutations in cancers. In 11 datasets (n = 211,726), ERBB2 mutational hotspots varied across 25 tumor types. Common HER2 mutants yielded differential sensitivities to eleven EGFR/HER2 tyrosine kinase inhibitors (TKIs) in vitro, and molecular dynamics simulations revealed that mutants with a reduced drug-binding pocket volume were associated with decreased affinity for larger TKIs. Overall, poziotinib was the most potent HER2 mutant-selective TKI tested. Phase II clinical testing in ERBB2 exon 20-mutant non-small cell lung cancer resulted in a confirmed objective response rate of 42% in the first 12 evaluable patients. In pre-clinical models, poziotinib upregulated HER2 cell-surface expression and potentiated the activity of T-DM1, resulting in complete tumor regression with combination treatment.

Clinical studies of HER2 targeting agents have shown mutational variant & cancer-specific differences in patient outcomes. Furthermore, the drug sensitivity profiles of HER2 mutational variants have not been fully characterized. To this end, we expressed the 16 most frequently detected HER2 mutations across exons 19-21 into Ba/F3 cells & determined activating potential & drug sensitivity to 13 HER1/2 targeting agents including afatinib, neratinib, dacomitinib, tarloxotinib-TKI, poziotinib, pyrotinib, TDM-1, & trastuzumab. Third generation EGFR TKIs, osimertinib, ibrutinib, & nazartinib were not effective at inhibiting cell viability in cells expressing exon 20 mutations; however, 3rd generation TKIs demonstrated activity against cells expressing D769 exon 19 variants & exon 21 variants. By comparison, covalent, quinazoline-based TKIs, afatinib, neratinib, dacomitinib, tarloxotinib-TKI, & poziotinib, differentially inhibited HER2 mutants across all three exons. Across all HER2 mutation variants & TKIs tested, poziotinib had the lowest average IC50 (1.73nM) & was significantly more effective in reducing cell viability than neratinib & tarloxotinib-TKI (p Citation Format: Jacqulyne P. Robichaux, Monique B. Nilsson, Fahao Zhang, Limei Hu, Junqin He, Marlese Pisegna, Alissa Poteete, John V. Heymach. Identification of poziotinib alone or in combination with TDM1 as a pan-HER2 inhibitor [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 347.

The gold standard for determining the tumorigenic potential of human cancer cells is a xenotransplantation into immunodeficient mice. Higher tumorigenicity of cells is associated with earlier tumor onset. Here, we used xenotransplantation to assess the tumorigenic potential of human breast cancer cells following RNA interference-mediated inhibition of over 5000 genes. We identify 16 candidate tumor suppressors, one of which is the zinc-finger transcription factor SALL1. Analyzing this particular molecule in more detail, we show that inhibition of SALL1 correlates with reduced levels of CDH1, an important contributor to epithelial-to-mesenchymal transition. Furthermore, SALL1 expression led to an increased migration and more than twice as many cells expressing a cancer stem cell signature. Also, SALL1 expression correlates with the survival of breast cancer patients. These findings cast new light on a gene that has previously been described to be relevant during embryogenesis, but not carcinogenesis.

The Y-box binding protein-1 (YB-1) transcription factor is associated with unfavorable clinical outcomes. However, the mechanisms underlying this association remain to be fully elucidated. We demonstrate that YB-1 phosphorylation, indicative of YB-1 activation, is a powerful marker of outcomes for ovarian cancer patients. In ovarian cancer, YB-1 phosphorylation is induced by activation of the lysophosphatidic acid (LPA) receptor (LPAR) via SRC-dependent transactivation of the epidermal growth factor receptor (EGFR) that is coupled to MAPK/p90 ribosomal S6 kinase (p90RSK), but not phosphatidylinositol 3-kinase (PI3K)/AKT signaling. Activation of the LPAR/SRC/EGFR/MAPK/p90RSK/YB-1 axis leads to production of the EGFR ligand amphiregulin (AREG). AREG induces ongoing YB-1 phosphorylation as well as YB-1-dependent AREG expression, thus constituting an AREG/YB-1 self-reinforcing loop. Disruption of transactivation of the EGFR and the downstream self-reinforcing loop decreases invasiveness of ovarian cancer cells in vitro and limits ovarian cancer growth in xenograft models. These findings established the regulation and significance of YB-1 phosphorylation, therefore further exploration of this signaling axis as a therapeutic avenue in ovarian cancer is warranted.

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.

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.

To identify the roles of various circulating cells (eg, endothelial and/or stem and progenitor cells) in angiogenesis, we parabiosed a wild-type syngeneic mouse with a transgenic syngeneic green fluorescent protein mouse. Following the establishment of a common circulation between these parabionts, we investigated acute (7 to 10 days), subacute (2 to 3 weeks), and chronic (4 to 6 weeks) phases of angiogenesis in wild-type mice using wound healing, implanted gel foam fragments, and subcutaneous tumor assays, respectively. We found that under in vitro conditions, circulating murine monocytes expressed F4/80, CD31, and vascular endothelial growth factor receptor 2, but neither CD133 nor von Willebrand factor, whereas murine endothelial cells expressed CD31, vascular endothelial growth factor receptor 2, and von Willebrand factor, but neither CD133 nor F4/80. Immunofluorescence analysis revealed that green fluorescent protein-positive cells in the walls of new vessels in wounds, gel foam blocks, and tumors expressed both F4/80 and CD31, that is, macrophages. Pericytes, cells that express both CD31 and desmin, were found both in the walls of tumor-associated vessels and within tumors. Collectively, these data demonstrate that monocytes (ie, cells that express both CD31 and F4/80) may be recruited to the site of tissue injury and directly contribute to angiogenesis, reaffirming the close relationships between various cell types within the reticuloendothelial system and suggesting possible targets for anticancer treatments.

Acute myeloid leukemia (AML) 1 is often disrupted by chromosomal translocations generating oncogenic fusions in human leukemias. However, its role in epithelial cancers has not been extensively investigated. Herein, we show a marked accumulation of AML1 transcripts including a high frequency of a novel alternatively spliced AML1b transcript lacking exon 6 (AML1b(Del179-242)) in ovarian cancer patients. The increases in RNA transcripts for total wild-type AML1 and AML1b(Del179-242) are associated with poor patient outcomes. We have shown that although both wild-type AML1b and AML1b(Del179-242) are localized to nuclear speckles, AML1b(Del179-242) was observed to have dramatically reduced transactivation potential with the plasminogen activator inhibitor-1 promoters and behaved as a weak dominant negative of wild-type AML1b. Wild-type AML1b was found to inhibit the growth of immortalized ovarian epithelial cells (T29) decreasing colony-forming ability. Moreover, we have identified a novel function of AML1b where it inhibits ovarian cell migration. In contrast, AML1b(Del179-242) has lost the ability to inhibit both ovarian cell proliferation and migration indicating that the functional effects observed with wild-type AML1b are dependent on amino acids 179-242. Collectively, these studies suggest that deregulated alternative splicing of AML1b transcripts may potentially contribute to the pathophysiology of ovarian cancers.

We have previously reported that adenoviral vector-mediated interferon (IFN)-beta gene therapy inhibits orthotopic growth of human prostate cancer cells in nude mice. The purpose of this study was to determine efficacy and mechanisms of this therapy in immune-competent mice. TRAMP-C2Re3 mouse prostate cancer cells infected with 100 multiplicity of infection (MOI) of adenoviral vector encoding for mouse IFN-beta (AdmIFN-beta), but not AdE/1 (a control adenoviral vector), produced approximately 60 ng/10(5) cells/24 h of IFN-beta. The tumorigenicity of AdmIFN-beta-transduced cells was dramatically reduced in the prostates of C57BL/6 mice. A single intratumoral injection of 2 x 10(9) PFU (plaque-forming unit) of AdmIFN-beta inhibited tumor growth by 70% and prolonged survival of tumor-bearing mice. Intriguingly, this AdmIFN-beta therapy did not alter the growth of tumors in inducible nitric oxide synthase (iNOS)-null C57BL/6 mice. Immunohistochemical analysis revealed that treatment of tumors with AdmIFN-beta in wild-type C57BL/6 mice led to increased iNOS expression, decreased microvessel density, decreased cell proliferation, and increased apoptosis. Furthermore, quantitative reverse-transcriptional PCR analysis showed that AdmIFN-beta therapy, in C57BL/6 but not the iNOS-null counterparts, reduced levels of the mRNAs for angiopoietin, basic fibroblast growth factor, matrix metalloproteinase-9, transforming growth factor-beta1, vascular endothelial growth factor (VEGF)-A, and VEGF-B, as well as the antiapoptotic molecule endothelin-1. These data indicated that IFN-beta gene therapy could be effective alternative for the treatment of locally advanced prostate cancer and suggest an obligatory role of NO in IFN-beta antitumoral effects in vivo.

We investigated the role of transforming growth factor-β (TGF-β) signaling in the growth and metastasis of PC-3MM2 human prostate cancer cells. Highly metastatic PC-3MM2 human prostate cancer cells were engineered to constitutively overexpress a dominant-negative type II TGF-β receptor (DNR). Transfection of DNR had minimal direct effects on cell growth and attenuated TGF-β-induced cell growth inhibition and TGF-β1 production. There were no discernable differences in tumorigenicity (tumor incidence) among PC-3MM2 variants when the cells were implanted into the prostates of nude mice. Growth rate and metastatic incidence of DNR-engineered PC-3MM2 cells, however, were significantly reduced. Most cells in the control tumors were positively stained by an antibody to proliferation cell nuclear antigen and very few cells were stained by terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL). In sharp contrast, tumors formed by PC-3MM2-DNR cells contained fewer proliferation cell nuclear antigen–positive cells and many more TUNEL-positive cells. Staining with antibody against CD31 showed that control tumors contained more blood vessels than PC-3MM2-DNR tumors. Expression of interleukin-8 (IL-8) in tumors formed by PC-3MM2 cells was significantly reduced as revealed by both Northern blotting and ELISA. Finally, transfection of antisense IL-8 cDNA significantly reduced IL-8 production by PC-3MM2 cells and antisense IL-8-transfected PC-3MM2 cells grew slower in comparison with parental and control vector-transfected cells. Taken together, our data suggest that TGF-β signaling, by regulating IL-8 expression in tumor cells and hence tumor angiogenesis, is critical for progressive growth of PC-3MM2 cells in the prostate of nude mice.

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

The purpose of this study was to determine the effects of interferon-beta (IFN-beta) gene transfer on the growth of PC3MM2 human prostate cancer cells in nude mice. Intralesional delivery of an adenoviral vector encoding murine IFN-beta (AdIFN-beta), but not a vector encoding bacterial beta-galactosidase (AdLacZ), suppressed PC3MM2 tumors in a dose-dependent manner. At the highest dose (2x10(9) plaque-forming units, PFU), a single injection of AdIFN-beta (but not AdLacZ) suppressed orthotopic PC3MM2 tumors and development of metastasis by 80%, and eradicated the tumors in 20% of mice. Immunohistochemical staining showed that AdIFN-beta-treated tumors contained fewer microvessels, fewer proliferating cells, and more apoptotic cells than did the control tumors. Compared with controls, tumors injected with AdIFN-beta expressed higher levels of IFN-beta and inducible nitric oxide synthase (iNOS) and lower levels of basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1). In vitro analysis indicated that expression of bFGF and TGF-beta1 in PC3MM2 cells could be suppressed by the nitric oxide donor sodium nitroprusside. These data suggest that intratumoral delivery of the IFN-beta gene with adenoviral vectors could be an effective therapy for prostate cancer and that tumor suppression by AdIFN-beta correlated with up-regulation of iNOS and down-regulation of angiogenesis.

Introduction Tumor cells growing in the brain are resistant to chemotherapy. We have recently demonstrated that the activation of the endothelin receptors A and B axis is involved in the induction of resistance to chemotherapy by primary brain tumor cells and by brain metastases. Treatment with a dual endothelin receptor antagonist in combination with chemotherapy produced significant therapy of orthotopically growing glioblastoma cells as well as breast cancer and lung cancer brain metastases. Since the milieu of tumors in the brain is hypoxic (0.5% - 1% O2), we investigated whether the chemo-resistance of the tumor cells is linked to hypoxia mediated activation of the endothelin receptor axis. Materials and Methods Human glioma (LN229), breast cancer (MDA231), and lung adenocarcinoma (PC14) cell lines were cultured in DMEM supplemented with 5% FBS. The endothelin receptor A (ETAR) and/or B (ETBR) of cancer cells were knocked down by shRNA. ETAR and ETBR were activated by incubating the tumor cells with exogenous endothelin-1 for 15 minutes or blocked by treating the parental or engineered cells with ETAR-specific antagonist, BQ123 (100nM), and/or ETBR-specific antagonist, BQ788 (100nM), for 2 hours prior to the addition of paclitaxel (5ng/ml), temozolomide (100μg/ml) or cisplatinum (5μg/ml). The cultures were placed into incubators with an atmosphere of 20%, 6% or 1% oxygen for 48 hours (MDA231 cells) or 72 hours (LN229 and PC14 cells). Tumor cells were then plated into 96-well plates to determine chemosensitivity by the MTT assay or into 6-well plates for western blots, or into chamber slides for immunohistochemical analyses. Expression of survival-related proteins such as pETAR, pETBR, pAkt, pMAPK, pNFκB, GSTA5, TWIST1 or Bcl2L1, were then determined. Results Stimulation of parental MDA-231, PC-14, or LN229 cells with exogenous ET-1 induced significant resistance against all tested chemotherapeutic agents. The resistance was abolished only when both the ETAR and ETBR were antagonized by a combination of BQ123 and BQ788. Parental cells cultured under hypoxia were also significantly resistant to chemotherapy and treatment of these cells with BQ123 and BQ788 reversed this resistance. The effects of exogenous ET-1 on the induction of chemo-resistance or BQ123 and BQ788 on restoration of chemo-sensitivity were not found in any cancer cells devoid of ETAR and ETBR. In all cases, the chemo-resistance of tumor cells was associated with increased level of expression of proteins related to cell survival. Conclusion These data suggest that the endothelin receptor axis plays a role in hypoxia induced resistance of cancer cells to chemotherapy. Additional studies are warranted to determine the functional changes of the endothelin receptor axis in hypoxia. Citation Format: Hyun Kyung Yu, Ho Jeong Lee, Fahao Zhang, Qiuyu Wu, Isaiah J. Fidler, Sun Jin Kim. Hypoxia induced resistance to chemotherapy is regulated by the endothelin receptor axis. [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 2943.

PRMT5 (protein arginine methyltransferase 5) is an enzyme that catalyses transfer of methyl groups from S-adenosyl methionine to the arginine residues of histones or non-histone proteins and is involved in a variety of cellular processes. Although it is highly expressed in some tumours, its direct role in cancer growth has not been fully investigated. In the present study, in human lung tissue samples we found that PRMT5 was highly expressed in lung cancer cells, whereas its expression was not detectable in benign lung tissues. Silencing PRMT5 expression strongly inhibited proliferation of lung adenocarcinoma A549 cells in tissue culture, and silencing PRMT5 expression in A549 cells also abolished growth of lung A549 xenografts in mice. In vitro and in vivo studies showed that the cell growth arrest induced by loss of PRMT5 expression was partially attributable to down-regulation of fibroblast growth factor receptor signalling. These results suggest that PRMT5 and its methyltransferase activity is essential for proliferation of lung cancer cells and may serve as a novel target for the treatment of lung cancer.

During lung development, cells proliferate for a defined length of time before they begin to differentiate. Factors that control this proliferative process and how this growth process is related to lung cancer are currently unknown. Here, we found that the WD40-containing protein (p44/wdr77) was expressed in growing epithelial cells at the early stages of lung development. In contrast, p44/wdr77 expression was diminished in fully differentiated epithelial cells in the adult lung. Loss of p44/wdr77 gene expression led to cell growth arrest and differentiation. Re-expression of p44/wdr77 caused terminally differentiated cells to re-enter the cell cycle. Our findings suggest that p44/wdr77 is essential and sufficient for proliferation of lung epithelial cells. P44/Wdr77 was re-expressed in lung cancer, and silencing p44/wdr77 expression strongly inhibited growth of lung adenocarcinoma cells in tissue culture and abolished growth of lung adenocarcinoma tumor xenografts in mice. The growth arrest induced by loss of p44/wdr77 expression was partially through the p21-Rb signaling. Our results suggest that p44/wdr77 controls cellular proliferation during lung development, and this growth process is reactivated during lung tumorigenesis.

Potential treatments for ovarian cancers that have become resistant to standard chemotherapies include modulators of tumor cell survival, such as endothelin receptor (ETR) antagonist. We investigated the therapeutic efficacy of the dual ETR antagonist, macitentan, on human ovarian cancer cells, SKOV3ip1 and IGROV1, growing orthotopically in nude mice. Mice with established disease were treated with vehicle (control), paclitaxel (weekly, intraperitoneal injections), macitentan (daily oral administrations), or a combination of paclitaxel and macitentan. Treatment with paclitaxel decreased tumor weight and volume of ascites. Combination therapy with macitentan and paclitaxel reduced tumor incidence and further reduced tumor weight and volume of ascites when compared with paclitaxel alone. Macitentan alone occasionally reduced tumor weight but alone had no effect on tumor incidence or ascites. Immunohistochemical analyses revealed that treatment with macitentan and macitentan plus paclitaxel inhibited the phosphorylation of ETRs and suppressed the survival pathways of tumor cells by decreasing the levels of pVEGFR2, pAkt, and pMAPK. The dose of macitentan necessary for inhibition of phosphorylation correlated with the dose required to increase antitumor efficacy of paclitaxel. Treatment with macitentan enhanced the cytotoxicity mediated by paclitaxel as measured by the degree of apoptosis in tumor cells and tumor-associated endothelial cells. Collectively, these results show that administration of macitentan in combination with paclitaxel prevents the progression of ovarian cancer in the peritoneal cavity of nude mice in part by inhibiting survival pathways of both tumor cells and tumor-associated endothelial cells.

Murine melanomas produce site-specific experimental brain metastases that reflect clinical reality. When injected into the internal carotid artery of mice, K-1735 melanoma cells produce metastatic lesions only in the brain parenchyma, whereas B16 melanoma cells and the somatic hybrid cells of B16 × K-1735 melanoma cells produce metastatic lesions only in the leptomeninges and ventricles. In the present study, we identified transforming growth factor-β2 (TGF-β2), an isoform of the TGF-β family, as a molecular determinant of melanoma cell growth in the brain parenchyma. We found that the TGF-β2 mRNA was highly expressed by the K-1735 cells, whereas the B16 cells or any B16 × K-1735 somatic cell-cell fusion hybrids have low expression. Transfection of the TGF-β2 gene into B16 cells resulted in the production of microscopic metastatic lesions in the brain parenchyma, without a decrease in metastasis to the leptomeninges or ventricles. TGF-β2 knockdown in the K-1735 melanoma cells significantly reduced metastasis to the brain parenchyma but did not induce metastasis to the leptomeninges or ventricles. These data show that TGF-β2 expression by murine melanoma cells is necessary for the establishment and growth of metastases in the brain parenchyma. [Cancer Res 2009;69(3):828–35]

GN963 is a tyrosine kinase inhibitor with activity against platelet-derived growth factor receptor (PDGFR) and Src kinases. We determined whether oral administration of GN963, alone or in combination with gemcitabine produces therapy against L3.6pl human pancreatic cancer cells growing orthotopically in nude mice. The optimal biological dosage of oral GN963 was determined to be 100 mg/kg every 48 h. Seven days after injection of L3.6pl cells into the pancreas of nude mice, mice (n=10) were treated with vehicle (control), thrice-weekly oral GN963 (100 mg/kg), twice-weekly intraperitoneal gemcitabine (100 mg/kg), or GN963 plus gemcitabine. Treatment with gemcitabine did not significantly differ from control. In contrast, treatment with GN963 (100 mg/kg) or GN963 plus gemcitabine produced a 52% and 81% decrease in tumor volume, respectively. GN963 plus gemcitabine completely inhibited the incidence of liver metastasis. Administration of GN963 inhibited PDGFR phosphorylation in both tumor and tumor-associated endothelial cells, decreased Src and Akt kinase activity in tumor cells, decreased microvessel density, and decreased tumor cell proliferation, while increasing apoptosis of tumor and tumor-associated endothelial cells. Collectively, these data indicate that targeting PDGFR, Src, and Akt on tumor and tumor-associated endothelial cells may be an effective therapy for human pancreatic carcinoma.

GN963 is a tyrosine kinase inhibitor with activity against platelet-derived growth factor receptor (PDGFR) and Src kinases. We determined whether oral administration of GN963, alone or in combination with gemcitabine produces therapy against L3.6pl human pancreatic cancer cells growing orthotopically in nude mice. The optimal biological dosage of oral GN963 was determined to be 100 mg/kg every 48 h. Seven days after injection of L3.6pl cells into the pancreas of nude mice, mice (n=10) were treated with vehicle (control), thrice-weekly oral GN963 (100 mg/kg), twice-weekly intraperitoneal gemcitabine (100 mg/kg), or GN963 plus gemcitabine. Treatment with gemcitabine did not significantly differ from control. In contrast, treatment with GN963 (100 mg/kg) or GN963 plus gemcitabine produced a 52% and 81% decrease in tumor volume, respectively. GN963 plus gemcitabine completely inhibited the incidence of liver metastasis. Administration of GN963 inhibited PDGFR phosphorylation in both tumor and tumor-associated endothelial cells, decreased Src and Akt kinase activity in tumor cells, decreased microvessel density, and decreased tumor cell proliferation, while increasing apoptosis of tumor and tumor-associated endothelial cells. Collectively, these data indicate that targeting PDGFR, Src, and Akt on tumor and tumor-associated endothelial cells may be an effective therapy for human pancreatic carcinoma.

Androgen receptor (AR) is a ligand-activated transcription factor that mediates the action of androgens and is essential for the growth, function, and cell differentiation of the prostate gland. Here, we demonstrated that the prostate apoptosis response factor-4 (par-4) functions as a novel AR coactivator. Par-4 physically interacted with the DNA-binding domain of AR, enhanced AR interaction with DNA, and increased AR-dependent transcription. Par-4 enhanced the c-FLIP promoter activity and was recruited on to the c-FLIP gene in the presence of androgens, and the dominant-negative par-4 decreased c-FLIP expression. These results suggest that, in addition to its proapoptotic function, par-4 acts as a novel transcription cofactor for AR to target c-FLIP gene expression. In addition, we demonstrated that loss of c-FLIP expression was essential for castration-induced apoptosis in the prostate gland and that enhanced c-FLIP expression was associated with prostate cancer progression to the androgen-resistant stage. Our data shed light on a transcription-mediated mechanism for the effects of the AR pathway on cell survival and apoptosis.

The purpose of this study was to investigate the therapeutic potential of interferon-beta (IFN-beta) against tumors that resist its antiproliferative effects. Mouse fibrosarcoma cells (UV-2237m-P) and their counterparts, transfected with either IFN-beta cDNA (UV-2237m-IFN-beta) or its control vector (UV-2237m-neo), were used in the study. UV-2237m-IFN-beta cells, still expressing functional IFN receptors, were resistant to the antiproliferative effects of IFN-beta. UV-2237m-P and UV-2237m-neo cells produced progressive tumors in both nude and IFN receptor-null nude (IFNAR-/-nude) mice. In contrast, growth of UV-2237m-IFN-beta cells was significantly delayed in nude mice. UV-2237m-IFN-beta tumors from nude mice contained fewer microvessels, fewer proliferating cells, and more apoptotic cells than did UV-2237m-P and UV-2237m-neo tumors. They expressed high levels of inducible nitric oxide synthase (iNOS) and were densely infiltrated by macrophages. Incubation with macrophages from nude mice, but not those from IFNAR-/- nude mice or iNOS-null/nude mice, led to more significant killing of UV-2237m-IFN-beta cells than that of control cells, which was blocked by iNOS inhibitor N-methylarginine. Similarly, more UV-2237m-IFN-beta cells were killed when they were incubated with spleen lymphocytes from nude mice. These data indicate that IFN-beta can inhibit growth of IFN-beta-resistant tumors by T cell-independent host-mediated mechanisms, including the role of macrophages, natural killer (NK) cells, and iNOS activity.

Interleukin (IL)-8 and transforming growth factor (TGF)-beta1 are proangiogenic factors overexpressed in advanced human melanoma. We investigated the effects of TGF-beta1 on IL-8 expression in the well-characterized A375 human melanoma system. We demonstrated by enzyme-linked immunoassay and Northern blot analysis that TGF-beta1 selectively induced IL-8 expression, at both protein and mRNA levels, in highly metastatic A375SM cells but not cells of their poorly metastatic parental line A375P. Transient transfection with luciferase reporter gene constructs revealed that TGF-beta1 activated IL-8 promoter activity in A375SM cells but not A375P cells. Studies with progressive 5' deletion constructs and site-specific mutations demonstrated that a construct containing -133 to +44 of the 5'-flanking sequence was necessary and sufficient for maximal TGF-beta1-induced transcription response and that TGF-beta1-induced activation of IL-8 promoter depended on AP-1 (-126 to -120 bp), NF-kappaB (-94 to -71 bp), and C/EBP-like factor NF-IL6 (-94 to -81 bp) in this region. Interestingly, both A375P and A375SM cells expressed type I and type II TGF-beta receptors and TGF-beta1 induced the nuclear translocation of Smad3 protein in both A375P and A375SM cells. Moreover, both A375P and A375SM cells were susceptible to TGF-beta1-induced growth inhibition. Our data thus demonstrated that TGF-beta1 selectively induced IL-8 expression in highly metastatic A375SM melanoma cells. This TGF-beta1-induced IL-8 expression could be an amplification cascade responsible for overexpression of IL-8 in human melanoma and one of potential mechanisms by which TGF-beta1 promotes angiogenesis, growth, and metastasis of human melanoma.