Alexander Y. Deneka

In pancreatic ductal adenocarcinoma (PDAC), the fibroblastic stroma constitutes most of the tumor mass and is remarkably devoid of functional blood vessels. This raises an unresolved question of how PDAC cells obtain essential metabolites and water-insoluble lipids. We have found a critical role for cancer-associated fibroblasts (CAFs) in obtaining and transferring lipids from blood-borne particles to PDAC cells via trogocytosis of CAF plasma membranes. We have also determined that CAF-expressed phospholipid scramblase anoctamin 6 (ANO6) is an essential CAF trogocytosis regulator required to promote PDAC cell survival. During trogocytosis, cancer cells and CAFs form synapse-like plasma membranes contacts that induce cytosolic calcium influx in CAFs via Orai channels. This influx activates ANO6 and results in phosphatidylserine exposure on CAF plasma membrane initiating trogocytosis and transfer of membrane lipids, including cholesterol, to PDAC cells. Importantly, ANO6-dependent trogocytosis also supports the immunosuppressive function of pancreatic CAFs towards cytotoxic T cells by promoting transfer of excessive amounts of cholesterol. Further, blockade of ANO6 antagonizes tumor growth via disruption of delivery of exogenous cholesterol to cancer cells and reverses immune suppression suggesting a potential new strategy for PDAC therapy.

Supplementary Data from Association of TP53 and CDKN2A Mutation Profile with Tumor Mutation Burden in Head and Neck Cancer

Purpose:Head and neck squamous cell carcinoma (HNSCC) is a frequently devastating cancer that affects more than a half million people annually worldwide. Although some cases arise from infection with human papillomavirus (HPV), HPV-negative HNSCC is more common, and associated with worse outcome. Advanced HPV-negative HNSCC may be treated with surgery, chemoradiation, targeted therapy, or immune checkpoint inhibition (ICI). There is considerable need for predictive biomarkers for these treatments. Defects in DNA repair capacity and loss of cell-cycle checkpoints sensitize tumors to cytotoxic therapies, and can contribute to phenotypes such as elevated tumor mutation burden (TMB), associated with response to ICI. Mutation of the tumor suppressors and checkpoint mediators TP53 and CDKN2A is common in HPV-negative HNSCC.Experimental Design:To gain insight into the relation of the interaction of TP53 and CDKN2A mutations with TMB in HNSCC, we have analyzed genomic data from 1,669 HPV-negative HNSCC tumors with multiple criteria proposed for assessing the damaging effect of TP53 mutations.Results:Data analysis established the TP53 and CDKN2A mutation profiles in specific anatomic subsites and suggested that specific categories of TP53 mutations are more likely to associate with CDKN2A mutation or high TMB based on tumor subsite. Intriguingly, the pattern of hotspot mutations in TP53 differed depending on the presence or absence of a cooccurring CDKN2A mutation.Conclusions:These data emphasize the role of tumor subsite in evaluation of mutational profiles in HNSCC, and link defects in TP53 and CDKN2A to elevated TMB levels in some tumor subgroups.

Supplementary Data from Association of TP53 and CDKN2A Mutation Profile with Tumor Mutation Burden in Head and Neck Cancer

Purpose:Head and neck squamous cell carcinoma (HNSCC) is a frequently devastating cancer that affects more than a half million people annually worldwide. Although some cases arise from infection with human papillomavirus (HPV), HPV-negative HNSCC is more common, and associated with worse outcome. Advanced HPV-negative HNSCC may be treated with surgery, chemoradiation, targeted therapy, or immune checkpoint inhibition (ICI). There is considerable need for predictive biomarkers for these treatments. Defects in DNA repair capacity and loss of cell-cycle checkpoints sensitize tumors to cytotoxic therapies, and can contribute to phenotypes such as elevated tumor mutation burden (TMB), associated with response to ICI. Mutation of the tumor suppressors and checkpoint mediators TP53 and CDKN2A is common in HPV-negative HNSCC.Experimental Design:To gain insight into the relation of the interaction of TP53 and CDKN2A mutations with TMB in HNSCC, we have analyzed genomic data from 1,669 HPV-negative HNSCC tumors with multiple criteria proposed for assessing the damaging effect of TP53 mutations.Results:Data analysis established the TP53 and CDKN2A mutation profiles in specific anatomic subsites and suggested that specific categories of TP53 mutations are more likely to associate with CDKN2A mutation or high TMB based on tumor subsite. Intriguingly, the pattern of hotspot mutations in TP53 differed depending on the presence or absence of a cooccurring CDKN2A mutation.Conclusions:These data emphasize the role of tumor subsite in evaluation of mutational profiles in HNSCC, and link defects in TP53 and CDKN2A to elevated TMB levels in some tumor subgroups.

The purpose of the study was to assess the prognostic significance of a scaffolding NEDD9 protein in patients with non-small cell lung cancer (NSCLC). NEDD9 (Neural precursor cell expressed, developmentally down-regulated 9) is a scaffolding protein in many intracellular protein cascades, such as PI3K-AKT-mTOR, Ras-ERK, which play key roles in tumor progression. several studies define the regulatory role of NEDD9 in progression of several solid cancers, including non-small cell lung cancer (NSCLC), however its role as a prediction biomarker is minimally assessed. Materials and methods. In the current study quantitative immunohistochemical (IHC) approach was utilized to characterize NEDD9 expression in a cohort of NSCLC primary tumor samples (n=16), and correlative statistical analysis was performed between NEDD9 expression and a set of patients’ clinical and pathological characteristics. Results of the IHC analysis were validated using broader TCGA RNAseq dataset (n=566). Results. The study demonstrated significant difference between the expression of Nedd9 in stage iii tumors versus stages II and IV (pConclusion. This study suggests that the expression of the scaffolding protein NEDD9, both at the protein and RNA levels, positively correlates with a negative prognosis in NSCLC, suggesting that it can potentially be used as a novel biomarker of disease progression. Further basic and clinical research defining the role of NEDD9 in the progression and metastasis of NSCLC are strongly warranted.

Lung cancer holds second place in incidence and first place in mortality among all cancers, both in men and women, with metastasis contributing to the vast majority of deaths. Recently, expression of vascular endothelial growth factor receptor-2 (VEGFR2) on both endothelial and tumor cells was demonstrated as one of the key mechanisms contributing to growth of non-small cell lung cancer (NSCLC), the most common subtype of lung cancer. VEGFR2 dependent signaling is critical both for tumor autocrine functions related to NSCLC migration, and in paracrine control of tumor angiogenesis through action on tumor-proximal endothelial cells, suggesting a potential mechanism for growth control. Several drugs targeting VEGFR2 signaling pathway demonstrated their clinical efficacy in NSCLC setting, including VEGFR2 and VEGF-A inhibitors, VEGF-A/PIGF trap. Recently our group demonstrated that RNA-binding protein Musashi-2 (MSI2) is driving progression and metastasis of NSCLC. MSI2 regulates mRNA translation of multiple targets, its expression is upregulated in the metastasis-competent murine and human lung cancer cell lines and is progressively elevated in lung cancer samples. The goal of this study is to define the role of MSI2 in sustaining VEGFR2 signaling during NSCLC progression. Using reverse RNA immunoprecipitation (RIP) qPCR analysis, we demonstrate that MSI2 directly binds VEGFR2 mRNA. Reverse protein phase array (RPPA) of several NSCLC cell line models with expression or knockdown of MSI2 and subsequent direct validation showed that MSI2 strongly and positively regulates expression of VEGFR2. Subsequent mechanistical analysis revealed that MSI2 knockdown leads to decreased VEGFR2 protein and mRNA levels expression in several NSCLC cell lines in vitro, resulting in increased apoptosis via inhibition of VEGFR2 signaling pathway. To better characterize MSI2 biology, we’ve crossed the B6/129S4-Msi2^tm1.1Cjl/J (M2) mice with B6/129S/Sv-Kras^tm3Tyj/J;Trp53^tm1Brn/J (KP) mice and generated novel B6/129S/Sv-KP;Msi2^-/- (KPM2) mouse model. Induction of tumorigenesis in KP mice using Cre adenovirus leads to development of lung adenocarcinoma. Induction of lung-specific Cre resulted in tumorigenesis in both KP and KPM2 mice. Interestingly, we observed significant decrease in both total lung tumor number and total lung tumor burden in KPM2 compared to KP mice. Next, we established three novel KP and KPM2 derived cell lines from mouse lung tumors and found that KPM2 cell lines demonstrate reduced proliferation capacity and VEGFR2 mRNA level relative to KP cell lines. Additional studies to understand an impact of MSI2 deletion in KP cell lines are ongoing. Taken together, MSI2 is a promising target for NSCLC treatment, as this protein regulates VEGFR2 signaling and apoptosis in cell models and contributes to KP tumorigenesis in vivo. Citation Format: Igor Bychkov, Alexander Deneka, Iuliia Topchu, Petr Makhov, Alexander Kudinov, Anna Nikonova, John Karanicolas, Erica Golemis, Christopher Lengner, Hossein Borghaei, Jyoti Patel, Yanis Boumber. MSI2 regulates VEGFR2 signaling and tumor progression in 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 874.

Colorectal cancer (CRC) is one of the most common cancers, with an annual incidence of ~135,000 in the US, associated with ~ 50,000 deaths. CRC typically arises from mutations that inactivate the tumor suppressor APC, which activates the WNT/CTNNB1 pathway to drive cell transformation and tumor growth. Identifying factors that reduce incidence of CRC is of high interest. Intriguingly, a large population study has found a decreased incidence of CRC in patients with autosomal dominant polycystic kidney disease (ADPKD), an inherited disease affecting approximately 1 in 1,000 individuals. ADPKD typically arises from germline mutations in the PKD1 gene, which encodes a transmembrane signaling protein that is ubiquitously expressed. Puzzlingly, activated WNT/CTNNB1 signaling is also found in ADPKD, which would be expected to enhance rather than reduce likelihood of CRC. To investigate the role of PKD1 in modulating CRC tumorigenesis, we established a mouse model with tamoxifen-inducible loss of Apc with or without parallel loss of Pkd1 in the colon. Pkd1 loss vey significantly reduced tumor incidence (~80%) and rate of growth. In addition, growth of Pkd1-/-;Apc-/- organoids was reduced relative to Apc-/-organoids, indicating a cancer cell-intrinsic activity. Analysis of signaling pathway activation by RNA-seq indicated that Pkd1-deficient CRC organoids had enhanced WNT/CTNNB1 signaling, excluding reduced activation of this core driver pathway as a mechanism of action. Considering alternative mechanisms, a prominent feature of ADPKD is reprogramming barrier function in renal cells to reduce permeability, supporting cyst development; notably, compromised integrity of the epithelial barrier is associated with CRC. We tested the hypothesis that loss of Pkd1 increased colon barrier integrity. As a first approach, we compared susceptibility of wt versus Pkd1-/- mice to dextran-sodium sulfate (DSS), which induces colitis by damaging epithelial barriers. Histopathological assessment confirmed significantly less DSS-induced damage of colon tissue in Pkd1-/- mice. Further, treatment of DSS-treated mice with orally gavaged FITC-dextran led to elevated FITC-dextran in the serum of wt versus Pkd1-/- mice, indicating greater passage through a functionally impaired barrier. In ADPKD, decreased barrier permeability is mediated by upregulation of specific claudins (CLDN4, 7); in CRC, these claudins are typically reduced during and promote tumor formation. We detected strong elevation of CLDN4 and CLDN7 in the colonic epithelium of Pkd1-/- versus wt mice, with the expressed proteins having consistently greater localization to cell junctions. Based on these and other data, we identify PKD1 control of barrier function as an important regulatory mechanism in CRC tumorigenesis, which may suggest novel therapeutic strategies based on targeting PKD1-associated effector pathways. Citation Format: Anna S. Nikonova, Alexander Y. Deneka, Flaviane Silva, Rosella Tricarico, Anna Kiseleva, Sergey Grivennikov, Erica A. Golemis. Loss of Pkd1 limits colorectal cancer by increasing colon barrier function [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 940.

DNA damaging modalities are the backbone of treatments for non-small cell lung cancer (NSCLC). Alterations in DNA damage response (DDR) in tumor cells commonly contribute to emerging resistance to platinating agents, other targeted therapies, and radiation. The goal of this study is to identify the previously unreported role of NEDD9 scaffolding protein in controlling DDR processes and sensitivity to DNA damaging therapies. Using a siRNA-mediated approach to deplete NEDD9 in a group of human and murine KRAS/TP53-mutant NSCLC cell lines, coupled with a set of cell viability and clonogenic assays, flow cytometry analysis, and Western blotting, we evaluated the effects of NEDD9 silencing on cellular proliferation, DDR and epithelial-to-mesenchymal transition (EMT) signaling, cell cycle, and sensitivity to cisplatin and UV irradiation. Using publicly available NSCLC datasets (TCGA) and an independent cohort of primary NSCLC tumors, subsequent in silico and immunohistochemical (IHC) analyses were performed to assess relevant changes in NEDD9 RNA and protein expression across different stages of NSCLC. The results of our study demonstrate that NEDD9 depletion is associated with the increased tumorigenic capacity of NSCLC cells. These phenotypes were accompanied by significantly upregulated ATM-CHK2 signaling, shifting towards a more mesenchymal phenotype in NEDD9 depleted cells and elevated sensitivity to UV-irradiation. IHC analyses revealed an association between reduced NEDD9 protein expression and a decrease in overall (OS) and progression-free survival (PFS) of the NSCLC patients. These data, for the first time, identified NEDD9 as a negative regulator of ATM kinase activity and related DDR signaling in numerous KRAS/TP53 mutated NSCLC, with its effects on the regulation of DDR-dependent EMT signaling, sensitivity to DNA damaging modalities in tumor cells, and the survival of the patients.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is a frequently devastating cancer that affects more than a half million people annually worldwide. Although some cases arise from infection with human papillomavirus (HPV), HPV-negative HNSCC is more common, and associated with worse outcome. Advanced HPV-negative HNSCC may be treated with surgery, chemoradiation, targeted therapy, or immune checkpoint inhibition (ICI). There is considerable need for predictive biomarkers for these treatments. Defects in DNA repair capacity and loss of cell-cycle checkpoints sensitize tumors to cytotoxic therapies, and can contribute to phenotypes such as elevated tumor mutation burden (TMB), associated with response to ICI. Mutation of the tumor suppressors and checkpoint mediators TP53 and CDKN2A is common in HPV-negative HNSCC. Experimental Design: To gain insight into the relation of the interaction of TP53 and CDKN2A mutations with TMB in HNSCC, we have analyzed genomic data from 1,669 HPV-negative HNSCC tumors with multiple criteria proposed for assessing the damaging effect of TP53 mutations. Results: Data analysis established the TP53 and CDKN2A mutation profiles in specific anatomic subsites and suggested that specific categories of TP53 mutations are more likely to associate with CDKN2A mutation or high TMB based on tumor subsite. Intriguingly, the pattern of hotspot mutations in TP53 differed depending on the presence or absence of a cooccurring CDKN2A mutation. Conclusions: These data emphasize the role of tumor subsite in evaluation of mutational profiles in HNSCC, and link defects in TP53 and CDKN2A to elevated TMB levels in some tumor subgroups.