SOX2 Amplification Research Articles

Abstract 3256: Novel SOX2-targeting bioPROTACs for the treatment of cancers

Abstract Targeted protein degradation (TPD) technology has been developed showing the therapeutic potential to modulate historically ‘undruggable’ disease-associated proteins. Currently, proteolysis targeting chimeras (PROTACs) using small-molecule binders represent the TPD field, and PROTAC-induced proximity between target proteins and E3 ligases enables specific degradation of target proteins. However, substantial amounts of clinically relevant cancer targets such as transcription factors lack active sites or allosteric binding pockets for small molecules which challenges the development of classical PROTACs for these targets. SOX2 is one of the pluripotency transcription factors and has been well-known for its crucial role in tumor development through promoting cancer cell survival, metastasis, stemness, and drug resistance. Numerous studies have also reported SOX2 amplification and overexpression in various tumor tissues. Thus, SOX2 has been validated as an attractive anticancer target, but directly targeting SOX2 has been proved to be difficult since SOX2 is an “undruggable” transcription factor. EPD Biotherapeutics develops EPDegTM, a next-generation engineered protein degrader platform in order to overcome the limitations of small molecule-based PROTAC. Here we report a series of SOX2-targeting bioPROTACs, being composed of a nanobody against SOX2 with high affinity and several ubiquitin E3 ligases with deletion of natural substrate binding domain to allow highly selective degradation of SOX2 protein. To evaluate the in vitro efficacy, SOX2 bioPROTACs were generated by mRNA IVT with CleanCap ® and modified uridine. First, robust degradation of SOX2 with a half-maximal degradation concentration (DC50) of the picomolar range was observed with the treatment of SOX2 bioPROTACs in multiple SOX2-overexpressing cancer cell lines. The degradation was ubiquitin- and proteosome-dependent manner, and neither nanobody nor ubiquitin E3 ligase alone affected SOX2 protein level. Then, SOX2 bioPROTACs led to significant cancer cell growth inhibition caused by apoptotic cell death with a half-maximal growth inhibition concentration (GI50) of the picomolar range. Notably, chemo-resistant cancer cell lines expressing SOX2 protein in high levels were sensitized to chemotherapy in combination with our SOX2 bioPROTACs. Furthermore, i.v. injection of one of our lead candidates formulated with LNP significantly inhibited tumor growth in the A549 xenograft model, proving the in vivo anti-tumor efficacy of SOX2 degraders for the first time in the world. Taken together, EPD’s SOX2 bioPROTACs show potent in vitro and in vivo anti-tumor efficacy as first-in-class SOX2 degraders and provide new insights for the therapeutic potential of transcription factor-degrading strategies. Citation Format: Jaehyun Choi, Saeyi Lim, Xuelian Bai, Hyerin Song, Yeonji Oh, Yeryoung Yong, Seunghyun Lee. Novel SOX2-targeting bioPROTACs for the treatment of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3256.

Combined PIK3CA and SOX2 Gene Amplification Predicts Laryngeal Cancer Risk beyond Histopathological Grading.

The PIK3CA and SOX2 genes map at 3q26, a chromosomal region frequently amplified in head and neck cancers, which is associated with poor prognosis. This study explores the clinical significance of PIK3CA and SOX2 gene amplification in early tumorigenesis. Gene copy number was analyzed by real-time PCR in 62 laryngeal precancerous lesions and correlated with histopathological grading and laryngeal cancer risk. Amplification of the SOX2 and PIK3CA genes was frequently detected in 19 (31%) and 32 (52%) laryngeal dysplasias, respectively, and co-amplification in 18 (29%) cases. The PIK3CA and SOX2 amplifications were predominant in high-grade dysplasias and significantly associated with laryngeal cancer risk beyond histological criteria. Multivariable Cox analysis further revealed PIK3CA gene amplification as an independent predictor of laryngeal cancer development. Interestingly, combined PIK3CA and SOX2 amplification allowed us to distinguish three cancer risk subgroups, and PIK3CA and SOX2 co-amplification was found the strongest predictor by ROC analysis. Our data demonstrate the clinical relevance of PIK3CA and SOX2 amplification in early laryngeal tumorigenesis. Remarkably, PIK3CA amplification was found to be an independent cancer predictor. Furthermore, combined PIK3CA and SOX2 amplification is emerging as a valuable and easy-to-implement tool for cancer risk assessment in patients with laryngeal precancerous lesions beyond current WHO histological grading.

Abstract 5765: Genomic characterization of Chinese biliary tract cancers identifies differences between subtypes and therapeutic biomarkers

Abstract Background: Biliary tract carcinoma (BTC) is a relatively rare cancer worldwide and accounts for 3% gastrointestinal cancers in China. BTCs are mainly classified as gallbladder cancer (GBC) and cholangiocarcinomas (CCA) with dismal prognosis. Genetic landscape of BTC can offer a comprehensive understanding of its carcinogenesis and clinical associations. The present study aims to compare the molecular features between BTC subtypes and explore clues for treatment therapies. Methods: We analyzed 258 BTC tissue samples including 70 GBCs and 188 CCAs with targeted sequencing (NGS) using a 425 caner-relevant genes panel. We identified the somatic alterations and MSI status of all samples. TMB was counted by summing all base substitutions and indels in the coding region of targeted genes. TMB-high (TMB-H) was defined as mutation number ≥ 10mut/Mb. Results: The most frequently mutated genes in 258 BTCs were TP53 (56.6%), KRAS (27.9%), CDKN2A (21.3%), TERT (17.8%) and MCL1 (14.3%). Four gene mutations and 2 gene amplifications were significantly enriched in GBC compared to CCA (mutations P<0.05, amplifications P<0.01): TP53 (78.8% vs 47.9%), ARID2 (15.7% vs 3.7%), STK11 (12.9% vs 4.3%), TERT (24.3% vs 11.2%), CCNE1 and SOX2 amplifications (21.4% vs 2.7%, 4.3% vs 0%). However, ARID1A mutation (15.4% vs 5.7%), PBRM1 mutation (10.1% vs 0%), and MCL1 amplification (17.0% vs 5.7%) were more frequently existed in CAA (all P<0.05). Seven actionable alterations, including IDH1/2 (5.4%), FGFR1-4 (4.7%), ERBB2 (6.2%), PIK3CA (5.4%), MET (3.5%), and BRCA1/2 (5.0%), were detected with different occurrence frequencies in two subtypes. ERBB2 was predominant in GBC (15.7% vs 7.4%, p<0.001), while IDH1/2 and KRAS alteration rates were much higher in CCA (8% vs 0%, 31.4% vs 18.6%, both P<0.05). FGFR3 alterations were also tended to be enriched in CCA (7.1% vs 2.7%), although no significant difference was found. In general, there were 25.7% of GBC patients harboring actionable alterations versus 29.3% of CCA patients. In addition, 40 (15.5%, 13 GBCs and 27 CCAs) and 11 (4.3%, 10 GBCs and 1 CCA) patients were TMB-H and MSI-H, respectively, suggesting the possibility of benefit from immunotherapy. Conclusions: There were distinct molecular features in somatic variations and actionable alterations between GBSs and CCAs. In general, 44.3% of GBC and 46.3% CCA may benefit from targeted therapy and immunotherapy. This study could be helpful to establish treatment therapies for BTCs according to genetic information. Citation Format: Xiaofeng Chen, Jiaohui Pang, Cuicui Liu, Sha Wang, Yang Shao. Genomic characterization of Chinese biliary tract cancers identifies differences between subtypes and therapeutic biomarkers [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 5765.

Pathway-Based Analysis Revealed the Role of Keap1-Nrf2 Pathway and PI3K-Akt Pathway in Chinese Esophageal Squamous Cell Carcinoma Patients With Definitive Chemoradiotherapy

Esophageal squamous cell carcinoma (ESCC) is the major type of EC in China. Chemoradiotherapy is a standard definitive treatment for early-stage EC and significantly improves local control and overall survival for late-stage patients. However, chemoradiotherapy resistance, which limits therapeutic efficacy and treatment-induced toxicity, is still a leading problem for treatment break. To optimize the selection of ESCC patients for chemoradiotherapy, we retrospectively analyzed the clinical features and genome landscape of a Chinese ESCC cohort of 58 patients. TP53 was the most frequent mutation gene, followed by NOTCH1. Frequently, copy number variants were found in MCL1 (24/58, 41.4%), FGF19 (23/58, 39.7%), CCND1 (22/58, 37.9%), and MYC (20/58, 34.5%). YAP1 and SOX2 amplifications were mutually exclusive in this cohort. Using univariate and multivariate analyses, the YAP1 variant and BRIP1 mutant were identified as adverse factors for OS. Patients with PI3K-Akt pathway alterations displayed longer PFS and OS than patients with an intact PI3K-Akt pathway. On the contrary, two patients with Keap1-Nrf2 pathway alterations displayed significantly shortened PFS and OS, which may be associated with dCRT resistance. Our data highlighted the prognostic value of aberrant cancer pathways in ESCC patients, which may provide guidance for better chemoradiotherapy management.

STEM-08. ST6Gal1-MEDIATED SILALYLATION IS CRITICAL FOR GLIOBLASTOMA GROWTH

Abstract Although altered cell surface glycosylation was one of the earliest modifications observed in neoplastic progression, this facet of cancer cell biology has received meager attention, particularly in brain tumors. Among the various glycosyltransferases present in human cells, golgi sialyltransferase ST6Gal1 [beta-galactoside alpha-2,6-sialyltransferase 1] adds sialic acid residues in α2-6 linkage to membrane-bound and secreted N-glycans. ST6Gal1 is known to be pro-tumorigenic in epithelial cancers where it can promote epithelial to mesenchymal transformation, tumor-initiating cell (TIC) phenotypes, and survival of cells exposed to stressors such as chemo- and radiotherapy, hypoxia, or serum starvation. However, roles for this potent TIC regulator have not been well explored in GBM as experiments in standard cell lines suggested ST6Gal1 was epigenetically silenced. To explore our hypothesis that ST6Gal1-mediated α2,6 sialylation is elevated in Brain Tumor Initiating Cells (BTICs) and promotes GBM growth, we utilized GBM patient-derived xenografts (PDXs). ST6Gal1 is expressed in GBM PDX tissue sections and elevated in stem-like BTICs in comparison to differentiated GBM cells or astrocytes. Knockdown of ST6Gal1 in BTICs decreased growth and neurosphere formation capacity in vitro, suggesting that ST6Gal1 regulates BTIC maintenance. Similarly, cells isolated directly from PDXs that were sorted for high and low expression of α2,6 sialylation showed that α2,6 sialylationhigh GBM PDX have elevated neurosphere formation capacity and growth. Further, immunocompromised mice injected with sorted α2,6 sialylationhigh PDX cells had significantly lower survival compared to mice injected with α2,6 sialylationlow cells. Using proteomic analysis of ST6Gal1 KD vs NT PDX, we identified novel regulators of cancer stem cell biology directly modulated by ST6Gal1. As we identified a small subset of IDHwt GBMs with ST6Gal1 and SOX2 amplification, we are generating a novel gliomagenesis model with conditional ST6Gal1 overexpression. Together, our data strongly implicates ST6Gal1 as a regulator of GBM BTIC maintenance and GBM growth.

Comprehensive Profiling of Genomic and Transcriptomic Differences between Risk Groups of Lung Adenocarcinoma and Lung Squamous Cell Carcinoma.

Lung cancer is the second most frequently diagnosed cancer type and responsible for the highest number of cancer deaths worldwide. Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) are subtypes of non-small-cell lung cancer which has the highest frequency of lung cancer cases. We aimed to analyze genomic and transcriptomic variations including simple nucleotide variations (SNVs), copy number variations (CNVs) and differential expressed genes (DEGs) in order to find key genes and pathways for diagnostic and prognostic prediction for lung adenocarcinoma and lung squamous cell carcinoma. We performed a univariate Cox model and then lasso-regularized Cox model with leave-one-out cross-validation using The Cancer Genome Atlas (TCGA) gene expression data in tumor samples. We generated 35- and 33-gene signatures for prognostic risk prediction based on the overall survival time of the patients with LUAD and LUSC, respectively. When we clustered patients into high- and low-risk groups, the survival analysis showed highly significant results with high prediction power for both training and test datasets. Then, we characterized the differences including significant SNVs, CNVs, DEGs, active subnetworks, and the pathways. We described the results for the risk groups and cancer subtypes separately to identify specific genomic alterations between both high-risk groups and cancer subtypes. Both LUAD and LUSC high-risk groups have more downregulated immune pathways and upregulated metabolic pathways. On the other hand, low-risk groups have both up- and downregulated genes on cancer-related pathways. Both LUAD and LUSC have important gene alterations such as CDKN2A and CDKN2B deletions with different frequencies. SOX2 amplification occurs in LUSC and PSMD4 amplification in LUAD. EGFR and KRAS mutations are mutually exclusive in LUAD samples. EGFR, MGA, SMARCA4, ATM, RBM10, and KDM5C genes are mutated only in LUAD but not in LUSC. CDKN2A, PTEN, and HRAS genes are mutated only in LUSC samples. The low-risk groups of both LUAD and LUSC tend to have a higher number of SNVs, CNVs, and DEGs. The signature genes and altered genes have the potential to be used as diagnostic and prognostic biomarkers for personalized oncology.

Deciphering the clonal relationship between glandular and squamous components in adenosquamous carcinoma of the lung using whole exome sequencing

Adenosquamous carcinoma of the lung (ASC) is a rare subtype of non-small cell lung cancer, consisting of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) components. ASC shows morphological characteristics of classic LUAD and LUSC but behaves more aggressively. Although ASC can serve as a model of lung cancer heterogeneity and transdifferentiation, its genomic background remains poorly understood. In this study, we sought to explore the genomic landscape of macrodissected LUAD and LUSC components of three ASC using whole exome sequencing (WES). Identified truncal mutations included the pan-cancer tumor-suppressor gene TP53 but also EGFR, BRAF, and MET, which are characteristic for LUAD but uncommon in LUSC. No truncal mutation of classical LUSC driver mutations were found. Both components showed unique driver mutations that did not overlap between the three ASC. Mutational signatures of truncal mutations differed from those of the branch mutations in their descendants LUAD and LUSC. Most common signatures were related to aging (1, 5) and smoking (4). Truncal chromosomal copy number aberrations shared by all three ASC included losses of 3p, 15q and 19p, and an amplified region in 5p. Furthermore, we detected loss of STK11 and SOX2 amplification in ASC, which has previously been shown to drive transdifferentiation from LUAD to LUSC in preclinical mouse models. Conclusively, this is the first study using WES to elucidate the clonal evolution of ASC. It provides strong evidence that the LUAD and LUSC components of ASC share a common origin and that the LUAD component appears to transform to LUSC.

Abstract PR08: Systematic pan-cancer analyses of Hippo pathway deregulation in cancer

Abstract Hippo signaling is a highly conserved, pleiotropic pathway that is recurrently deregulated across many cancers. With large-scale cancer genomics data, several efforts have been undertaken to quantify Hippo pathway activity in an unbiased manner. However, identifying a comprehensive catalog of Hippo pathway genes can be difficult due to the heterogenous nature of the disease and the limitations of any given study. Here we show the high concordance between different methods of quantifying Hippo pathway activity in cancers. Furthermore, we establish a first-principled approach by experimentally deriving a lineage-independent quantification by leveraging the transcriptomic profiles of nearly 900 cell lines from 28 tissue types. We applied these signature(s) to the genomic profiles of over 11,000 primary human cancer patients to identify several statistically significant biomarkers associated with Hippo pathway dysregulation, including NF2 mutations and deletions, SOX2 amplifications, and CDH1 mutations. To further assess therapeutic implications of pathway inhibition, we applied machine learning approaches to predict a tumor’s sensitivity to Hippo pathway inhibition based on its genomic profile, revealing that Hippo pathway inhibition may be a tractable approach in a wide spectrum of cancers. This abstract is also being presented as Poster B38. Citation Format: Matthew T. Chang, Thijs J. Hagenbeek, Jennifer Lacap, Zora Modrusan, Christiaan Klijn, Anwesha Dey. Systematic pan-cancer analyses of Hippo pathway deregulation in cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr PR08.

Genomic Profile and BRCA-1 Promoter Methylation Status in BRCA Mutated Ovarian Cancer: New Insights in Predictive Biomarkers of Olaparib Response.

Objective: We assessed the genomic profile of four representative BRCA-mutated ovarian cancer (OC) patients treated with olaparib to investigate the relationship between intratumor heterogeneity and response to olaparib treatment. The main aim is to identify possible predictive biomarkers of response to olaparib through the analysis of HRD or not HRD genes and the definition of BRCA1 promoter methylation status.Methods: DNA, isolated from formalin-fixed, paraffin-embedded (FFPE) diagnostic OC tissues, was analyzed by FoundationOneCDx™. This assay detects alterations in a total panel of 324 genes, using the Illumina® HiSeq 4000 platform. Methylation analysis of the BRCA gene promoter was carried out by pyrosequencing with PyroMark Q24 platform (Qiagen), an in vitro nucleic acid sequence-based detection test based on pyrosequencing technology for quantitative measurements of methylation status.Results: Case #1 and #2 were defined Long-term responders since they received olaparib for 27 and 36 months, respectively. These remarkable results could be explained, at least in part, by the presence of somatic IDH1 mutation in case #1 and PI3K and SOX2 amplification in the case #2. In case #3, the somatic NF1 mutation appeared to be related to the short duration of response. In the case #4, in which the patients is on olaparib from 1 year achieving a stable disease, a somatic mutation of BRCA1 was recorded. Moreover, in all cases, levels of BRCA1 promoter were strictly related to olaparib response.Conclusions: Based on our experience, genomic analysis of tumor tissue at diagnosis might help to determine the future response to olaparib in advanced OC setting, revealing predictive biomarkers beyond BRCA 1-2 and HRD status.

Sox2: A Regulatory Factor in Tumorigenesis and Metastasis.

The transcription factor Sox2 plays an important role in various phases of embryonic development, including cell fate and differentiation. These key regulatory functions are facilitated by binding to specific DNA sequences in combination with partner proteins to exert their effects. Recently, overexpression and gene amplification of Sox2 has been associated with tumor aggression and metastasis in various cancer types, including breast, prostate, lung, ovarian and colon cancer. All the different roles for Sox2 involve complicated regulatory networks consisting of protein-protein and protein-nucleic acid interactions. Their involvement in the EMT modulation is possibly enabled by Wnt/ β-catenin and other signaling pathways. There are number of in vivo models which show Sox2 association with increased cancer aggressiveness, resistance to chemo-radiation therapy and decreased survival rate suggesting Sox2 as a therapeutic target. This review will focus on the different roles for Sox2 in metastasis and tumorigenesis. We will also review the mechanism of action underlying the cooperative Sox2- DNA/partner factors binding where Sox2 can be potentially explored for a therapeutic opportunity to treat cancers.

CDK7 inhibition as a promising therapeutic strategy for lung squamous cell carcinomas with a SOX2 amplification.

Despite the development of molecular targeted therapies, few advances have been made in the treatment of lung squamous cell carcinoma (SCC). SOX2 amplification is one of the most common genetic alterations in SCC. Here, we investigated the effects of THZ1, a potent cyclin-dependent kinase 7 (CDK7) inhibitor that plays a key role in gene transcription, in SCC. Lung SCC-derived cell viabilities were assessed using a CCK-8 assay. SOX2 expression and RNAPII-CTD phosphorylation levels after THZ1 treatment were determined by Western blotting. The effect of SOX2 suppression using shRNA was assessed by flow cytometry. Gene expression patterns after THZ1 treatment of lung SCC-derived cells were identified using microarray-based mRNA profiling. We found that THZ1 treatment led to suppression of cell growth and apoptotic cell death in SOX2-amplified SCC-derived cells only, whereas the modest growth-inhibitory effect of cisplatin did not differ according to SOX2 amplification status. We also found that THZ1 decreased the phosphorylation of the carboxyl-terminal domain of RNA polymerase II and the expression of several genes. Specifically, we found that the expression of transcription-associated genes, including SOX2, was down-regulated by THZ1 in SOX2-amplified SCC cells. This inhibition of SOX2 expression resulted in suppression of the growth of these cells. From our data, we conclude that THZ1 may effectively control the proliferation and survival of SOX2-amplified SCC cells through a decrease in global transcriptional activity, suggesting that CDK7 inhibition leading to transcription suppression may be a promising therapeutic option for lung SCC with a SOX2 amplification.

Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

Diffuse gliomas progress by invading neighboring brain tissue to promote postoperative relapse. Transcription factor SOX2 is highly expressed in invasive gliomas and maps to chromosome region 3q26 together with the genes for PI3K/AKT signaling activator PIK3CA and effector molecules of mitochondria fusion and cell invasion, MFN1 and OPA1. Gene copy number analysis at 3q26 from 129 glioma patient biopsies revealed mutually exclusive SOX2 amplifications (26%) and OPA1 losses (19%). Both forced SOX2 expression and OPA1 inactivation increased LN319 glioma cell invasion in vitro and promoted cell dispersion in vivo in xenotransplanted D. rerio embryos. While PI3 kinase activity sustained SOX2 expression, pharmacological PI3K/AKT pathway inhibition decreased invasion and resulted in SOX2 nucleus-to-cytoplasm translocation in an mTORC1-independent manner. Chromatin immunoprecipitation and luciferase reporter gene assays together demonstrated that SOX2 trans-activates PIK3CA and OPA1. Thus, SOX2 activates PI3K/AKT signaling in a positive feedback loop, while OPA1 deletion is interpreted to counteract OPA1 trans-activation. Remarkably, neuroimaging of human gliomas with high SOX2 or low OPA1 genomic imbalances revealed significantly larger necrotic tumor zone volumes, corresponding to higher invasive capacities of tumors, while autologous necrotic cells are capable of inducing higher invasion in SOX2 overexpressing or OPA1 knocked-down relative to parental LN319. We thus propose necrosis volume as a surrogate marker for the assessment of glioma invasive potential. Whereas glioma invasion is activated by a PI3K/AKT-SOX2 loop, it is reduced by a cryptic invasion suppressor SOX2-OPA1 pathway. Thus, PI3K/AKT-SOX2 and mitochondria fission represent connected signaling networks regulating glioma invasion.

1348PD - Genomic landscape and its correlation with TMB, CD8 TILs and PD-L1 expression in Chinese lung squamous cell carcinoma

Background: To comprehensively depict the genomic landscape of Chinese lung squamous cell carcinoma (LSCC) and investigate its correlation with tumor mutation burden (TMB), CD8 tumor infiltrating lymphocytes (TILs) density and PD-L1 expression. Methods: Whole-exome sequencing were performed on tumor tissue collected from 189 patients with surgically resected LSCC. TMB was defined as total number of nonsynonymous single nucleotide and indel variants. High TMB was defined as greater than 75th percentile. CD8+ TILs and PD-L1 expression were assessed by immunohistochemistry. The cut-off point was 5% for high/low CD8+ TIL or PD-L1 +/- expression. Results: We found recurrent mutations (>5%) in 8 genes, including TP53, KMT2C, NFE2L2, KEAP1, CDKN2A, PTEN and FBXW7. FGFR1 and PIK3CA amplifications were found in 19% and 11% of samples. 24.9% of patients had high TMB. Baseline clinical variables were similar except for smoking status. Interestingly, FGFR1, PIK3CA or SOX2 amplification was independently associated with higher TMB (P = 0.020, P = 0.017, P = 0.029; respectively). Patients with copy number variations had significantly higher TMB than those without (P = 0.009). Positive PD-L1 and CD8+ TILs expression were identified in 24.3% and 78.8% of all cases. NFE2L2 mutation and PIK3CA amplification were associated with significantly higher PD-L1 expression (P = 0.003, P = 0.014; respectively). TP53 mutations were associated with higher CD8+ TILs expression (P = 0.008), but FGFR1 amplification was correlated with lower CD8+ TILs expression (P = 0.042). Of note, there is no association between TMB and PD-L1 expression (r = 0.052, P = 0.476), or CD8+ TILs expression (r = 0.026, P = 0.718). None of TMB, PD-L1 and CD8+ TIL expression could individually predict overall survival (OS). However, combination of TMB and PD-L1 could stratify total populations into two groups with distinct prognosis. Patients with negative PD-L1 expression and high TMB had the worst prognosis (P = 0.008). Conclusions: This was the most large-scale study to comprehensively portray genomic landscape of Chinese LSCC and provides several meaningful and referential findings for the future design of clinical trials in LSCC, especially immunotherapy. Legal entity responsible for the study: Caicun Zhou. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

PS02.047: TARGETED SILENCING OF SOX2 BY AN ARTIFICIAL TRANSCRIPTION FACTOR SUPPRESSED THE GROWTH OF ESOPHAGEAL CANCER CELLS

Abstract Background SOX2 is a transcription factor that is fundamental for early development and for the maintenance of stem cells in multiple adult tissues and also plays an important role in squamous cell differentiation. Amplification of chromosome 3q26 is the most common of the genetic alterations found in squamous cell carcinoma (SCC). SOX2 is a candidate oncogene present in this locus and amplification of SOX2 has been reported in lung and esophageal squamous cell SCC. In this study, we have developed a zinc finger-based artificial transcription factor (ATF) to selectively suppress SOX2 expression in cancer cells and termed the system ATF/SOX2. Methods We engineered the ATF using six zinc finger arrays designed to target a 19 bp site in the SOX2 distal promoter and a KOX transcriptional repressor domain. A recombinant adenoviral vector Ad-ATF/SOX2 that expresses ATF/SOX2 suppressed SOX2 at the mRNA and protein levels in esophageal SCC cells(TE1 and TE4) expressing SOX2. Results Ad-ATF/SOX2 decreased esophageal SCC cells proliferation and colony formation more effectively than the recombinant adenoviral vector Ad-shSOX2, which expresses SOX2 short hairpin RNA (shSOX2). Ad-ATF/SOX2 induced the cell cycle inhibitor CDKN1A more strongly than Ad-shSOX2. Moreover, Ad-ATF/SOX2 effectively inhibited tumor growth in a SCC xenograft mouse model. Conclusion In this study, we have shown that the targeted down-regulation of SOX2 using ATF based technologies can be used as an effective tool for the treatment of SCC in esophageal cancers that express SOX2. Our results indicate that ATF/SOX2 would lead to the development of an effective molecular-targeted therapy for esophageal SCC. Disclosure All authors have declared no conflicts of interest.

Abstract 4998: Targeting DHPS to abrogate TGFβ-induced metastasis in breast cancer

Abstract Progression of solid tumors to a metastatic stage accounts for over 90% of cancer mortality. Thus, it is critical to identify therapeutic strategies that target both primary and metastatic tumors. Epithelial-mesenchymal transition (EMT) negatively correlates with therapy response, contributing to intratumoral heterogeneity and systemic dissemination in breast cancer. We previously reported that pseudopodium-enriched atypical kinase one (PEAK1) promotes breast cancer cell EMT and metastasis by potentiating fibronectin-transforming growth factor beta (TGFβ) signaling cross-talk. Since eukaryotic initiation factor five A (eIF5A), a unique translation factor that is activated by deoxyhypusine synthase (DHPS)-dependent post-translational hypusination, is required for PEAK1 expression, we hypothesized that TGFβ may directly regulate eIF5A activity to promote EMT, and that targeted inhibition of this pathway may provide a novel means to inhibit or reverse metastatic progression. In this regard, we provide evidence of an active eIF5A-EMT program in undifferentiated breast cancer tissue. Notably, blockade of DHPS activity and eIF5A hypusination reduces PEAK1 translation, cell viability and TGFβ-induced EMT in vitro and metastasis in vivo. Conversely, we demonstrate that TGFβ induces post-translational hypusination of eIF5A in metastatic breast cancer cells. TGFβ is known to activate histone deacetylase six (HDAC6) and HDAC6 was independently reported to promote eIF5A deacetylation and nuclear export to support its translation functions. When delivered in combination, HDAC6 and DHPS inhibitors synergize to sequester eIF5A to the nucleus, suppress eIF5A-dependent translation and potently kill metastatic breast cancer cells. To identify candidate pathways downstream of the eIF5A/PEAK1 axis during EMT, we generated a Cytoscape interactome using eIF5A signaling and PEAK1-induced EMT genes as search terms. All interactome component genes were then analyzed across two breast cancer patient studies available on the Cancer BioPortal. Interestingly, SOX2, PIK3CA and EIF4A2 were the interactome nodes that exhibited copy number amplifications among patients harboring genomic alterations in the initial interactome search genes, and SOX2 amplification significantly and independently associated with decreased patient survival (p = 0.0476). Taken together, our results establish a novel node/axis by which TGFβ signaling stimulates HDAC6 and/or DHPS function to activate cytoplasmic eIF5A and promote EMT and survival of breast cancer cells within the metastatic niche, identifying new targeted therapy strategies that may improve cancer patient survival. Citation Format: Robert Guth, Lindsay Kutscher, Yvess Adamian, Cameron Geller, Kishan Bahkta, Kayla Meade, Jonathan A. Kelber. Targeting DHPS to abrogate TGFβ-induced metastasis in breast 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 4998.

Abstract PR05: Lineage specifiers SOX2 and NKX2-1 inversely regulate lung tumor immune microenvironment

Abstract The tumor microenvironment is a critical effector of therapeutic response to diverse stimuli including immunotherapies. Lineage-specific drivers of cancer are known to dictate tumor differentiation and response to therapy, but how they influence the tumor immune microenvironment is largely unknown. SOX2 and NKX2-1 are critical regulators of lung development and cancer, with SOX2 amplifications associated with squamous lung cancer and NKX2-1 amplifications associated with lung adenocarcinoma. Here we employ and develop new genetically engineered mouse models (GEMMs) of lung cancer to interrogate the impact of SOX2 and NKX2-1 on innate immune infiltration. Squamous and adenocarcinoma GEMMs accurately recapitulate features of their human counterparts, including an enrichment of tumor-associated neutrophils (TANs) specifically in squamous lung cancer. We show that SOX2 recruits TANs, whereas NKX2-1 suppresses TANs, in the absence of histopathologic changes. SOX2 and NKX2-1 inversely regulate the neutrophil chemoattractant, Cxcl5, which is also upregulated in human squamous lung tumors. Using a novel GEMM of squamous lung cancer with a rapid latency of 3-4 months, preliminary data suggest that neutrophils can regulate adeno- to squamous transdifferentiation in vivo. Together these data reveal how lineage specifiers influence the tumor immune microenvironment, which in turn may regulate cancer cell fate. This abstract is also being presented as Poster B19. Citation Format: Gurkan Mollaoglu, Alex Jones, Anandaroop Mukhopadhyay, Jason Gertz, Kevin Jones, Eric Snyder, Trudy G. Oliver. Lineage specifiers SOX2 and NKX2-1 inversely regulate lung tumor immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr PR05.

LSCC SNP variant regulates SOX2 modulation of VDAC3.

Lung squamous cell carcinoma (LSCC) is a genomically complex malignancy with no effective treatments. Recent studies have found a large number of DNA alterations such as SOX2 amplification in LSCC patients. As a stem cell transcription factor, SOX2 is important for the maintenance of pluripotent cells and may play a role in cancer. To study the downstream mechanisms of SOX2, we employed expression quantitative trait loci (eQTLs) technology to investigate how the presence of SOX2 affects the expression of target genes. We discovered unique eQTLs, such as rs798827-VDAC3 (FDR p-value = 0.0034), that are only found in SOX2-active patients but not in SOX2-inactive patients. SNP rs798827 is within strong linkage disequilibrium (r2 = 1) to rs58163073, where rs58163073 [T] allele increases the binding affinity of SOX2 and allele [TA] decreases it. In our analysis, SOX2 silencing downregulates VDAC3 in two LSCC cell lines. Chromatin conformation capturing data indicates that this SNP is located within the same Topologically Associating Domain (TAD) of VDAC3, further suggesting SOX2's role in the regulation of VDAC3 through the binding of rs58163073. By first subgrouping patients based on SOX2 activity, we made more relevant eQTL discoveries and our analysis can be applied to other diseases.

The management of localized esophageal squamous cell carcinoma: Western approach.

Esophageal cancer remains a significant cause of cancer-related mortality worldwide and carries a poor prognosis both in the locally advanced and metastatic setting. Esophageal squamous cell carcinoma (ESCC) accounts for the majority of esophageal cancers globally, while esophageal adenocarcinoma (EAC) now predominates in the Western world. Historically, clinical trials have not separated the two histologic subtypes, with similar regimens being used for both. This, however, is changing with the recent publication by the Cancer Genome Atlas Research Group confirming that comprehensive analysis reveals ESCC demonstrates frequent genomic amplifications of CCND1 and SOX2 and/or TP63 and biologically are more like head and neck squamous cell carcinomas, whereas EACs strongly resemble the chromosomally unstable variant of gastric cancer. With a lack of viable targets in squamous cell carcinoma, immunotherapeutic approaches are now being investigated in both early stage and metastatic ESCC with promising results being reported in the preliminary data published to date.

35P - CDK7 inhibition as a promising therapeutic strategy in lung squamous cell carcinoma with SOX2 amplification

35P - CDK7 inhibition as a promising therapeutic strategy in lung squamous cell carcinoma with SOX2 amplification

Integrated genomic characterization of oesophageal carcinoma

Oesophageal cancers are prominent worldwide; however, there are few targeted therapies and survival rates for these cancers remain dismal. Here we performed a comprehensive molecular analysis of 164 carcinomas of the oesophagus derived from Western and Eastern populations. Beyond known histopathological and epidemiologic distinctions, molecular features differentiated oesophageal squamous cell carcinomas from oesophageal adenocarcinomas. Oesophageal squamous cell carcinomas resembled squamous carcinomas of other organs more than they did oesophageal adenocarcinomas. Our analyses identified three molecular subclasses of oesophageal squamous cell carcinomas, but none showed evidence for an aetiological role of human papillomavirus. Squamous cell carcinomas showed frequent genomic amplifications of CCND1 and SOX2 and/or TP63, whereas ERBB2, VEGFA and GATA4 and GATA6 were more commonly amplified in adenocarcinomas. Oesophageal adenocarcinomas strongly resembled the chromosomally unstable variant of gastric adenocarcinoma, suggesting that these cancers could be considered a single disease entity. However, some molecular features, including DNA hypermethylation, occurred disproportionally in oesophageal adenocarcinomas. These data provide a framework to facilitate more rational categorization of these tumours and a foundation for new therapies.