Pandya, Prita

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal human malignancies due to its silent clinical onset, rapid progression, and limited responsiveness to current treatment options. These features underscore the urgent need for early detection strategies and for new molecular targets that can be exploited therapeutically. Epithelial Cell Transforming Sequence 2 (ECT2) is a Rho family guanine nucleotide exchange factor that was originally identified as an oncoprotein and later shown to regulate cytokinesis. In this study, we examined the clinical relevance of ECT2 expression in PDAC and determined its functional contribution to transformed growth and tumorigenicity. Our analyses revealed that ECT2 expression increases at the earliest stages of PDAC development, remains persistently elevated during disease progression, and associates with reduced overall patient survival. Furthermore, a substantial fraction of ECT2 is aberrantly localized to the cytoplasm of PDAC cells. Functional studies demonstrated that depletion of ECT2 suppresses three-dimensional growth, invasive behavior, and tumor formation in vivo, while having little impact on PDAC cell cytokinesis. Mechanistically, we found that ECT2 is required for activation of Rac1 and RhoA and downstream MEK/ERK and ROCK signaling, respectively. Consistent with these results, analyses of PDAC patient datasets revealed a strong positive association between ECT2 expression and Rho GTPase as well as MEK/ERK and ROCK pathway signatures. Together, our data identify ECT2 as an early driver of PDAC transformation and highlight it as a promising therapeutic target. Citation Format: Dania Al-Qasrawi, Nayya N. Murray, Ryan A. Argo, Alicia K. Fleming Martinez, Prita Pandya, Anaya Y. Clarke, Kayla C. Winter, Murli Krishna, Peter Storz, Nicole R. Murray, Verline Justilien, . The Rho-GEF ECT2 links Rho GTPases to MEK/ERK pathway activation in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5993.

Abstract Lung cancer remains the leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of all cases. Despite advances in targeted and immuno-therapies, overall survival for NSCLC remains below 25%, due to late-stage detection and therapeutic resistance. Cancer stem cells (CSCs), a highly tumorigenic subpopulation within NSCLC, drive relapse, metastasis, and treatment failure. Understanding the mechanisms by which CSCs promote the aggressiveness of NSCLC may reveal new diagnostic and therapeutic opportunities. CSCs establish an intercellular communication network through the secretion of extracellular vesicles (EVs), membrane-bound nanoparticles that transport proteins, lipids, and nucleic acids that can influence recipient cell behavior. Our previous work demonstrated that CSCs secrete significantly more EVs compared to bulk cancer cells (BCCs) and induce an enhanced tumorigenic phenotype in BCCs. However, the EV protein cargo that mediates these CSC-driven phenotypes and the signaling they promote remain poorly understood. This study aimed to identify and characterize CSC-derived EV protein cargo that contributes to NSCLC growth and metastasis. CSC- and BCC-derived EVs were isolated using tangential flow filtration and characterized according to MISEV guidelines. Mass spectrometry and pathway analysis were performed to compare the proteome of CSC- and BCC-derived EVs, followed by functional characterization in vitro and in vivo. Our results showed that EVs exhibited expected morphological, biochemical, and physical properties. Pathway analyses showed that CSC-derived EVs were enriched in proteins that localize to exosomes and associate with metabolism and cell adhesion. Comparison of BCC and CSC-derived EVs revealed Desmoglein-2 (DSG2) as one of the proteins exclusively detected in CSC-derived EVs. shRNA-mediated knockdown of DSG2 significantly reduced invasion, clonal expansion, and transformed growth in vitro and decreased tumor growth and metastasis in vivo. Furthermore, DSG2-competent EVs partially rescued the effects of DSG2 knockdown on CSC tumor growth in vivo. Together, these findings identify DSG2 as a key CSC-specific EV protein cargo that promotes an enhanced tumorigenic phenotype and NSCLC tumor growth. This work highlights the role of CSC-derived EV proteins as mediators of NSCLC aggressiveness and as candidates for biomarker and therapeutic development. Citation Format: Prita Pandya, Aamena Ahmed, Nayya Murray, Dania S. Al-Qasrawi, Anaya Y. Clarke, Reagan Carris, Verline Justilien. Desmoglein-2 carrying cancer stem cell-derived extracellular vesicles promote lung cancer aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 833.

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide, and despite advances in treatment, immune suppression remains an obstacle to effective therapy. Effector CD4+ T cells (CD4+ Teff) are critical for antitumor immunity, but their function is often inhibited by regulatory T cells (Treg), which accumulate in lung tumors and mediate suppressive functions through multiple mechanisms. This suppression leads to tumor progression and poor patient outcomes. However, the mechanisms underlying Treg-mediated suppression are not fully understood. Herein, we identify the extracellular adenosine 5-triphosphate receptor P2RX7 as a key regulator of Treg function in lung tumors. In a murine lung cancer model induced by Lewis lung carcinoma cells, we found that P2RX7 enhanced the suppressive capacity of tumor-infiltrating Tregs, promoting tumor growth. In T cell–specific P2RX7-knockout (P2RX7-KO) mice, reduced Treg infiltration was accompanied by increased CD4+ Teff accumulation and improved tumor control. Treg-specific P2RX7-KO mice exhibited reduced tumor growth, confirming a Treg-intrinsic role of P2RX7. Suppression assays revealed that tumor-infiltrating wild-type Tregs had greater suppressive activity compared with P2RX7-KO Tregs, which failed to inhibit type 1 and follicular helper T–like responses. This was associated with increased tumor-specific IgG production by lung B cells in P2RX7-KO mice. We also observed that wild-type Tregs expressed higher levels of the immunosuppressive molecule CTLA-4 when compared with P2RX7-KO Tregs. Thus, we conclude that P2RX7 expression on Tregs is essential for their suppressive function in lung cancer and targeting P2RX7 may constitute a strategy to improve lung cancer treatment by alleviating Treg-mediated immune suppression.

Abstract Lung cancer is the leading cause of cancer deaths in the US. Non-small cell lung cancer (NSCLC) accounts for ~80% of lung cancer cases and is further classified into adenocarcinoma (LUAD), squamous cell carcinoma (LUSC), and large cell carcinoma. New approaches targeting pathways in NSCLC have emerged, resulting in encouraging new treatments that benefit LUAD patients, but very few advances have been made in the treatment options for LUSC which accounts for ~30% of lung cancer cases. NSCLCs, including LUSCs, contain a sub-population of cells that exhibit properties of cancer stem cells (CSCs) and are responsible for tumor initiation, maintenance, therapeutic resistance, and relapse. Therefore, CSCs must be effectively targeted to elicit long lasting therapeutic results in LUSC patients. Our studies have shown that: 1) PKCι is an oncogene and prognostic marker in LUSC; 2) copy number gains (CNGs) of the PKCι gene (PRKCI) drives overexpression of PKCι in 90% of LUSCs; 3) PKCι phosphorylates SOX2, and controls SOX2-mediated transcriptional activation of Hedgehog Acyl Transferase, resulting in Hedgehog (Hh) pathway activation that drives a CSC phenotype in LUSC cells; and 4) the FDA approved compound Auranofin (ANF) is a potent inhibitor of oncogenic PKCι. In this study, we assessed the effect of ANF in combination with the Hh pathway inhibitor, LDE225, on LUSC CSCs and tumor growth. Cell viability, sphere formation and soft agar assays were used to assess the effects of ANF and LDE225 alone or in combination on the growth of LUSC CSCs with PRKCI CNGs or LUAD and LUSC CSCs without PRKCI CNG to determine if PRKCI CNG can be used to predict response to ANF and LDE225 and test the selectivity of response for LUSC. The effects of ANF and LDE225 on the levels of PKCι-Hh signaling components was assessed by qPCR and western blotting. Finally, the efficacy of ANF and LDE225 in combination was evaluated in human primary LUSC patient-derived xenograft (PDX) models that harbor PRKCI CNGs and the ability of PKCι-Hh signaling intermediates to serve as biomarkers of PKCι-Hh pathway inhibition was validated in vivo. We observed a dose-dependent decrease in cell proliferation, clonal expansion and transformed growth in CSCs treated with ANF and LDE225 alone. ANF and LDE225 combined treatment of LUSC CSCs with PRKCI CNGs (but not LUAD or LUSC CSCs without PRKCI CNG) synergistically inhibited CSC phenotypes based on results of combination index analysis. Furthermore, treatment of LUSC PDX models with ANF and LDE225 in combination had a synergistic effect on tumor growth inhibition. Lastly, ANF and LDE225 treatment led to inhibition of PKCι-Hh signaling intermediates demonstrating the on-target effects of treatment. Our data indicate that a PKCι-Hh vertical blockade strategy of combined ANF and LDE225 effectively targets LUSC CSCs and blocks tumor growth in vivo and provides compelling rationale to develop this novel combination strategy further in clinical trials. Citation Format: Dania Alqasrawi, Ryan Argo, Christine Ratliff Rang, Prita Pandya, Skyeler Klinge, Jennifer Lindemann, Nyla Searl, Alan Fields, Verline Justilien. A novel combination therapy for lung squamous cell carcinoma [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 5434.

Cancer stem cells (CSC) within non–small cell lung carcinoma (NSCLC) tumors drive NSCLC progression, metastasis, relapse, and intrinsic chemoresistance. Understanding the mechanisms that support the malignant phenotypes of NSCLC CSCs may provide insights for improved NSCLC therapeutic interventions. Here, we report that expression of RAB27B, a small GTPase, is significantly upregulated in NSCLC CSCs when compared with bulk cancer cells (BCC). Short hairpin RNA–mediated knockdown of RAB27B leads to a loss of stem cell marker gene expression and reduced NSCLC spheroid growth, clonal expansion, transformed growth, invasion, and tumorigenicity. We find that NSCLC CSCs secrete significantly more extracellular vesicles (EV) than BCCs, and that this is RAB27B-dependent. Furthermore, CSC-derived EVs, but not BCC-derived EVs, induce spheroid growth, clonal expansion, and invasion in BCCs. Finally, RAB27B is required for CSC-derived EV-induced stemness in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating, invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Our findings further suggest that inhibition of RAB27B-dependent EV secretion may be a potential therapeutic strategy for NSCLC. Significance: Expression of RAB27B in CSCs leads to elevated levels of EVs that mediate communication between CSCs and BCCs that maintains a stem-like phenotype in NSCLC cells.

Abstract Non-small cell lung cancer (NSCLC), a major subtype of lung cancer, accounts for ~85% of all lung cancer diagnoses. A sub-population of cells within human NSCLC tumors called cancer stem cells (CSCs) have been found to exhibit distinct properties that contribute to factors associated with poor outcomes in NSCLC patients including metastasis, relapse, and therapeutic resistance. We previously performed RNAseq analysis on bulk cancer cells (BCCs) and CSCs from a panel of primary NSCLC tumors and cell lines to identify signaling mechanisms involved in the tumorigenic behavior of NSCLC CSCs. Our analysis revealed that expression of RAB27B, a small GTPase that plays a critical role in extracellular vesicle (EV) formation and secretion, was significantly upregulated in CSCs when compared to BCCs. Interestingly, RAB27B is overexpressed and predicts patient survival in NSCLC. The goal of this study was to assess the role of RAB27B in the tumorigenic behavior of NSCLC CSCs and determine the mechanism by which RAB27B promotes an aggressive stem-cell like phenotype in NSCLC cells an aspect of RAB27B function that has not been investigated in NSCLC. QPCR and immunoblot analysis was used to compare RAB27B mRNA and protein expression in a panel of NSCLC BCCs and CSCs. Two independent shRNA constructs were used to knockdown (KD) RAB27B in NSCLC CSCs. The effects of RAB27B KD on CSC expansion, transformed growth and invasion were analyzed by clonal expansion, soft agar colony formation and Boyden chamber assays, respectively. Xenograft and tail vein mouse models were used to assess the effects of RAB27B KD on CSC tumor growth and lung colonization. EVs were isolated from the conditioned media of BCCs, CSCs and RAB27B KD CSCs using differential ultracentrifugation, and characterized by nanoparticle tracking analysis, transmission electron microscopy and immunoblotting according to MISEV guidelines. Our analysis revealed that RAB27B is significantly upregulated in NSCLC CSCs compared to BCCs. RAB27B KD cells exhibited a significant decrease in clonal expansion, transformed growth and invasion when compared to non-target shRNA control CSCs. In vivo studies demonstrated that RAB27B is important for the tumorigenicity of CSCs and promotes tumor cell proliferation, and angiogenesis. EVs were positive for EV-specific markers and negative for an intracellular vesicle marker. CSCs secrete significantly more EVs when compared to BCCs and KD of RAB27B in CSCs led to a decrease in the levels of EVs secreted. Furthermore, delivery of CSC-derived, but not BCC-derived EVs to BCCs induced spheroid growth, clonal expansion, and invasion in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating and invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Citation Format: Kayleah M. Meneses, Jennifer A. Lindemann, Prita Pandya, Dania S. Al-Qasrawi, Ryan Argo, Celeste Weems, Danielle J. Beetler, Irene K. Yan, Joy Wolfram, Tushar C. Patel, Verline Justilien. RAB27B drives a cancer stem cell phenotype in non-small cell lung cancer cells through enhanced extracellular vesicle secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2444.

Abstract Nicotinamide phosphoribosyltransferase (NAMPT) plays a critical role in cellular metabolism and as such has been an active target for cancer drug therapy. NAMPT inhibition, via small molecule NAMPT inhibitors, disrupts cellular energy metabolism as well as other NAD+-dependent processes and has been shown to suppress cancer cell proliferation. The early clinical translation of NAMPT inhibitors has been hindered by toxicity, therefore the successful development of newer classes of NAMPT inhibitors will depend on identifying cancers with unique sensitivity to, and molecular biomarkers of susceptibility to NAMPT inhibition. Through our pan-cancer screens using several different NAMPT inhibitors on molecularly characterized cancer cell line panels, we have identified specific cancer types and subtypes that are consistently sensitive to NAMPT inhibition. In order to determine the efficacy of NAMPT inhibitors in different cancer cells, we performed proliferation assays on a large and fully characterized panel of human cancer cell lines (n=496) for response to several NAMPT inhibitors including FK866, KPT9274 and LSN3154567. Briefly, cells were treated with six dilutions of these inhibitors in duplicate, counted following a 6-day treatment window and IC50 values were determined for each cell line. Reverse-phase protein array (RPPA) analysis, comparative genomic hybridization (CGH), point mutation and RNAseq data for each cell line were then queried for correlation with NAMPT sensitivity/resistance. Proliferation assays indicate a strong correlation between sensitive and resistant cancer cell types to all three compounds despite their different mechanisms of action. Most notably, acute myeloid leukemia (AML), ovarian cancer, Ewing’s sarcoma and small cell lung cancer cells (SCLC) consistently demonstrated sensitivity to treatment with NAMPT inhibitors. Additional analysis revealed several baseline genomic and proteomic markers that were strongly associated with response to NAMPT inhibition. Our screening process provides a comprehensive overview of the cancer types and subtypes that demonstrate sensitivity to NAMPT inhibition. Furthermore, the molecular predictors of response identified in our screen may ultimately be useful in developing diagnostic tools for enrollment in biomarker-enriched clinical trials aimed at determining the potential use of NAMPT inhibitors. These results suggest the inhibitors’ therapeutic potential in patient populations presenting with AML, ovarian cancer, Ewing’s sarcoma and SCLC all of which currently have high unmet needs. Citation Format: Jenny J. Hong, Martina S. McDermott, Jewel K. Ng, Prita Pandya, Dennis J. Slamon. Pan-cancer analysis of NAMPT inhibitors reveals unique sensitivities to multiple NAMPT inhibitors in several cancer types [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 2336.