Hedgehog Signaling Research Articles

Soy-derived isoflavones as chemo-preventive agents targeting multiple signalling pathways for cancer prevention and therapy.

The chemopreventive and chemotherapeutic properties of soy and soy-derived compounds, especially isoflavones, have been extensively studied in recent years. However, in contrast to their anticancer effects, such as cell growth inhibition, cell cycle arrest and apoptosis induction, isoflavones have also been found to promote the growth of cancer cells. Therefore, the aim of this comprehensive review article is to present the current state of knowledge regarding the molecular mechanisms by which soy-derived isoflavones target multiple cellular signalling pathways in cancer cells. Our findings indicate that soy-derived isoflavones act as, among other things, potent modulators of HOX transcript antisense RNA (HOTAIR)/SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1), vascular endothelial growth factor (VEGF)/C-X-C motif chemokine ligand 12 (CXCL12)/C-X-C motif chemokine receptor type 4 (CXCR4), 17-β-oestradiol (E2)/oestrogen receptor-α (ERα)/neuroglobin (NGB) and sonic hedgehog signalling pathways, epigenetic modulatory agents (i.a. miR-155, miR-34a and miR-10a-5p) and cancer stem cells and epithelial-to-mesenchymal transition inhibitors. The paper also discusses the latest epidemiological studies and clinical trials and provides an insight into recent extensive research on the chemo-preventive and therapeutic potential of soy-derived isoflavones.

A role for TGFβ signaling in Gli1+ tendon and enthesis cells.

The development of musculoskeletal tissues such as tendon, enthesis, and bone relies on proliferation and differentiation of mesenchymal progenitor cells. Gli1+ cells have been described as putative stem cells in several tissues and are presumed to play critical roles in tissue formation and maintenance. For example, the enthesis, a fibrocartilage tissue that connects tendon to bone, is mineralized postnatally by a pool of Gli1+ progenitor cells. These cells are regulated by hedgehog signaling, but it is unclear if TGFβ signaling, necessary for tenogenesis, also plays a role in their behavior. To examine the role of TGFβ signaling in Gli1+ cell function, the receptor for TGFβ, TbR2, was deleted in Gli1-lineage cells in mice at P5. Decreased TGFβ signaling in these cells led to defects in tendon enthesis formation by P56, including defective bone morphometry underlying the enthesis and decreased mechanical properties. Immunohistochemical staining of these Gli1+ cells showed that loss of TGFβ signaling reduced proliferation and increased apoptosis. Invitro experiments using Gli1+ cells isolated from mouse tail tendons demonstrated that TGFβ controls cell proliferation and differentiation through canonical and non-canonical pathways and that TGFβ directly controls the tendon transcription factor scleraxis by binding to its distant enhancer. These results have implications in the development of treatments for tendon and enthesis pathologies.

Abstract 2139: Identifying molecular mechanisms in triple negative breast cancer disparities: Unveiling the role of Hedgehog signaling in non-Hispanic Black women

Abstract Triple negative breast cancer (TNBC) represents an aggressive and therapeutically challenging breast cancer subtype with higher incidence and mortality rates in non-Hispanic Black (NHB) women. This disparity persists even after adjusting for socioeconomic factors, tumor biology, and quality of care. The study aims to unravel specific molecular mechanisms underlying this disparity, with a focus on Hedgehog signaling pathways in premenopausal NHB women. Understanding these mechanisms is vital for developing targeted therapies and personalized medicine approaches to address these health disparities. Our research utilized a unique cohort of NHB and non-Hispanic White (NHW) women who underwent breast reduction surgery at the Mayo Clinic and donated breast tissue samples for our study. We isolated RNA from whole tissue sections from a cohort of 12 NHB and 40 NHW women who were parous and premenopausal at time of surgery. We performed gene expression profiling using NanoString BC360 and IO360 assays. This allowed us to identify a distinct gene expression signature characteristic of basal-like/TNBC and indicative of heightened activity in the Hedgehog signaling pathway. We also employed advanced cell culture techniques to generate tissue organoids and subculture human mammary epithelial cells (HMECs) that preserve the intrinsic characteristics of the original tissue samples. We found that significantly differential expression of IL20RA and SOX10, principal factors in the gene expression signature, were preserved in both the derived organoids and the HMECs. These results suggest a potential intrinsic difference in the activation of the Hedgehog pathway in the breast epithelial cells of NHB women. This activation may contribute to increased proliferation and transformation of basal stem cells within the breast, possibly fueling the development and progression of TNBC. The implication of activated Hedgehog signaling in NHB women opens new avenues for understanding TNBC disparities. IL20RA and SOX10 represent potential biomarkers for TNBC risk that may serve as a stratification tool for NHB women. The results of this study have the potential to improve assessment and management of TNBC risk among NHB women, thereby contributing significantly to reducing health disparities in breast cancer outcomes. Citation Format: Jennifer Vanessa Cabezas, Savanna Touré, Matthew Wilson, Melody L. Stallings Mann, Laura M. Pacheco-Spann, Joshua W. Ogony, Mark E. Sherman, Derek C. Radisky. Identifying molecular mechanisms in triple negative breast cancer disparities: Unveiling the role of Hedgehog signaling in non-Hispanic Black women [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 2139.

Abstract 1250: Activating PIK3CA mutations and hedgehog signaling may confer resistance to KRAS inhibition in colorectal cancer

Abstract Background: Treatment with KRASG12C inhibitors results in an objective response in only small fraction of patients with KRASG12C mutant colorectal cancer (CRC) (7.1% and 22% objective response rates (ORR) in two prospective trials), in contrast to lung cancer where the ORR exceeds 35%. We hypothesize that co-mutation of other oncogenes and/or transcriptomic factors may contribute to the resistance of KRASG12C CRC to KRAS inhibitors. Methods: Mutation profiles of CRC patients were obtained from the AACR Project-GENIE v14 CRC cohort (N = 9441). The impact of KRAS knockout in 59 CRC cell lines was queried from the DepMap database. KRASG12C mutant CRC cell lines SW837 and SW1463 were treated with Sotorasib (KRASG12C inhibitor), Vismodegib and Taladegib (Hedgehog inhibitors), and cell viability was measured up to 96 hours after treatment. Results: In the AACR Project-GENIE CRC (N = 9441) cohort, KRASG12C tumors (2.6%, 245/9441) were significantly co-mutated with PIK3CAmut (19.2%, 47/245; Odds ratio = 1.7 (95% CI: 1.2 - 2.4), Chi-Square test, p-value = 0.002). Dependence on KRAS in CRISPR knockout viability assays was higher in KRASmut vs. wildtype CRC cell lines (median CERES score -1.3 vs -0.5, p-value < 0.001). Interestingly, KRASmut cell lines with PIK3CA co-mutation (n=10) were more resistant to KRAS knockout than PIK3CAwt cells (n=22) (median -1.0 vs -1.4, p-value = 0.03). The SW837 and SW1463 cell lines initially displayed high sensitivity to Sotorasib (IC50: 0.92 µM and 0.90 µM). However, overexpression of PIK3CAE545K mutant induced resistance to the same treatment (IC50: 5.18 µM and 6.45 µM). Furthermore, gene set enrichment analysis (GSEA) showed that Hedgehog signaling was significantly enriched in KRAS knockout resistant KRASmut CRC cell lines. SW837 cell line treated with Sotorasib (S) and a fixed dosage of 25 µM of Vismodegib (V) or Taladegib (T) showed higher sensitivities in combination treatment assays (IC50 decreased: ~11 folds in S+V vs S; and ~15 folds in S+T vs S) as compared to Sotorasib (S) treatment alone. Similar results were seen for the SW1463 cell line (IC50 decreased: ~18 folds in S+V vs S; and ~15 folds in S+T vs S). Conclusion: Resistance in CRC to KRASG12C inhibitors may be attributed to activated PIK3CA mutations and the Hedgehog signaling pathway. The future study will focus on detailed investigations involving comprehensive transcriptome and exome profiles in tumors and PDX models treated with anti-KRAS drugs in monotherapy and combination therapy. Citation Format: Saikat Chowdhury, Jibran Ahmed, Valsala Haridas, David S. Hong, Scott Kopetz, John Paul Shen. Activating PIK3CA mutations and hedgehog signaling may confer resistance to KRAS inhibition in colorectal cancer [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 1250.

Abstract 3640: RAB26 accelerates endometrial cancer cell progression by regulating the Hedgehog signaling pathway

Abstract As one of the most common gynecological malignant tumors, endometrial cancer (EC) is continuously being studied since its etiology is still not clear. RAB26, a member of the RAB family, has been shown to influence cell proliferation, metastasis and other malignant phenotypes in several cancers. However, whether and how RAB26 plays roles in EC remain unknown. Our previous study showed that RAB26 was related to the cell cycle-associated protein IK, which has been shown to contribute to carcinogenesis. Herein, we further explored the clinical significance and biological function of RAB26 in EC. Immunohistochemical staining of 615 endometrial tissue samples was conducted to explore whether RAB26 expression was related to the clinicopathological characteristics of EC. In vitro and in vivo experiments were used to evaluate the role of RAB26 in EC and investigate the underlying mechanism. RAB26 expression was higher in EC than in normal endometrium (NE), hyperplastic endometrium (HE) and atypical hyperplastic endometrium (AHE). High RAB26 expression was positively correlated with tumor histological grade (P=0.010), FIGO stage (P=0.042) and tumor size (P=0.003) in EC. RAB26 attenuation inhibited cell proliferation and metastasis but promoted cell apoptosis and caused G0/G1 arrest. After RAB26 attenuation, RNA sequencing data showed that the Hedgehog signaling (SHH) pathway was enriched. Further western blot results confirmed that the levels of SHH pathway proteins (SHH, PTCH1, smo and Gli1) decreased as RAB26 expression was attenuated. Moreover, an agonist of the SHH pathway, purmorphamine, blocked the inhibitory effect of RAB26 attenuation on EC progression. The results of an in vivo study also showed that RAB26 attenuation exerted a significant antitumor effect. Accordingly, RAB26 may act as an oncogenic protein to promote EC progression through the SHH pathway. RAB26 may be a potential target for the treatment of EC patients. Citation Format: Chao Gao, Yanyan Liu, Yanyan Han, Fengxia Xue, Yingmei Wang. RAB26 accelerates endometrial cancer cell progression by regulating the Hedgehog signaling pathway [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 3640.

Abstract 4754: Targeted inhibition of the HNF1A/SHH axis by triptolide overcomes paclitaxel resistance in non-small cell lung cancer

Abstract Paclitaxel resistance is commonly associated with a poor prognosis in non-small cell lung cancer (NSCLC) patients, and there are no promising pharmacological treatments for paclitaxel resistance. Here, we constructed human NSCLC-derived paclitaxel-resistant cell lines as an effective tool to explore the mechanisms of chemoresistance. We discovered that triptolide, a monomer compound extracted from Tripterygium wilfordii Hook F, was able to reverse paclitaxel resistance by reducing ABCB1 expression in vivo and in vitro. Through high-throughput sequencing, we found that the SHH-initiated Hedgehog signaling pathway played an important role in this process. More importantly, triptolide could directly target HNF1A, a transcription factor of SHH, and inhibited HNF1A/SHH expression, ensuing in attenuation of Hedgehog signaling. Clinically, we found a positive correlation between HNF1A and SHH expression in both NSCLC tumor tissue microarrays and cancer network databases. These findings illuminated a novel molecular mechanism through which triptolide effectively targets and inhibits HNF1A, thereby impeding the activation of the Hedgehog signaling pathway and reducing the expression of ABCB1. This finding indicated the potential clinical application of triptolide and provided promising prospects in targeting the HNF1A/SHH pathway as a therapeutic strategy for NSCLC patients with paclitaxel resistance. Citation Format: Peichao Li, ngbing Li, Lingxiao Yang, Lei Liu, Xiaogang Zhao. Targeted inhibition of the HNF1A/SHH axis by triptolide overcomes paclitaxel resistance in non-small cell lung cancer [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 4754.

Abstract 7363: Clinical, genomic, transcriptomic and immunologic profile of BRAF mutant colorectal tumors

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. BRAF, a protein kinase of the Mitogen-Activated Protein Kinase (MAPK) pathway, is mutated in 12% of colorectal cancers and is a poor prognostic factor for patients. BRAF mutations at the V600 codon are most studied but non-V600 BRAF mutations also exist and behave differently. BRAF V600 CRC treated with BRAF+MEK inhibitors show 12% response rates while we observe 65% response rates in BRAF V600 Melanoma. Additionally, no approved targeted therapies exist for non-V600 BRAF mutant cancers and retrospective studies show poorer PFS with BRAF+MEK inhibitors vs BRAF V600 cancers. We interrogated publicly available data of BRAF mutant CRC to uncover differences in the tumor microenvironment and signalling pathways of BRAF V600 and non-V600 tumors in hopes of uncovering better treatments. Methods: We analyzed the clinical and genomic profile of BRAF mutant CRC tumors from the GENIE v12 data. Gene Set Enrichment Analysis was performed on RNAseq from the TCGA data (discovery cohort) and the combined data from CPTAC and Sidra-LUMC data (validation cohort). We investigated the composition of tumor infiltrating immune cells using CIBERSORTx (deconvolution algorithm for bulk RNAseq). Results: Non-V600 mutations (n=219/1061) were most represented in patients of Asian or Black race compared to White patients (p<0.0001) and importantly, were found in younger patients (p<0.0001). We identified n=67 and n=74 BRAF mutant RNAseq samples in the discovery and validation cohorts, respectively. In the discovery cohort: BRAF V600 CRC were enriched for Interferon Gamma Signaling, Complement pathway, and IL6 JAK STAT3 Signaling (p<0.05) while the non-V600 BRAF mutant CRC showed an enrichment for Wnt-beta Catenin, Notch, and Hedgehog Signaling pathways (p<0.05). In the validation cohorts these same pathways were enriched in V600 and non-V600 BRAF mutant tumors. Interestingly, enrichment of genes co-mutated (GENIE data) in non-V600 BRAF mutant CRC also revealed the Wnt pathway. Non-V600 CRC tumors have a higher proportion of M0 macrophages (p=0.0035) and CD4 memory resting T cells (p<0.0001) while patients with V600 CRC have higher proportions of CD8 T cells (p=0.006) and activated mast cells (p=0.0341). Conclusion: No optimal therapeutic strategies have been defined for these potentially actionable non-V600 BRAF mutant CRCs. By exploring the multi-omic landscape of these tumors we have identified unique characteristics of non-V600 BRAF mutant CRC that could be therapeutically exploited. Citation Format: Emmanuelle Rousselle, Suzanne Kazandjian, April Rose. Clinical, genomic, transcriptomic and immunologic profile of BRAF mutant colorectal tumors [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 7363.

Abstract 1578: RAS-GTP inhibition modulates Hedgehog signaling, suppressing myCAFs and promoting iCAFs in pancreatic ductal adenocarcinoma

Abstract More than 90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS. RMC-7977 is a potent inhibitor of GTP-bound RAS proteins (RAS(ON), including both wild type and mutant variants of KRAS, NRAS, and HRAS. The related investigational agent, RMC-6236, is a first-in-class, potent, orally bioavailable, RASMULTI(ON) inhibitor currently in Phase 1/1b clinical trials (NCT05379985). We performed preclinical studies in a range of models of PDAC, including the highly chemoresistant K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) genetically engineered mouse. RMC-7977 exhibited broad anti-tumor activity at tolerable doses, provoking radiographic responses in KPC pancreatic tumors and extending overall survival by 3-fold, the largest response observed yet in this model. RMC-7977 dosing produced a metronomic effect on RAS signaling in tumor and normal tissues. This yielded tumor-selective effects on apoptosis and proliferation, consistent with RAS oncogene addition in PDAC. PDAC tumors are characterized by an expansive stroma that harbors multiple subtypes of cancer associated fibroblasts (CAFs) that manufacture a fibrotic, inflammatory extracellular matrix. CAF proliferation is driven by signals emanating from the malignant epithelial cells, such as Sonic Hedgehog (SHH), a secreted ligand that drives downstream Hedgehog pathway signaling in myofibroblastic CAFs (myCAFs). To understand the impact of RAS-GTP inhibition on paracrine signaling to CAFs in PDAC, To understand the dynamic responses of malignant epithelial cells following GTP-RAS inhibition, we performed single cell RNA sequencing (scRNAseq) on KPC pancreatic tumors treated with RMC-7977 or vehicle at multiple timepoints. Using the ARACNe and VIPER algorithms, we performed single cell regulatory network analysis on the expression profiles of >210,000 cells to calculate the activities of thousands of transcriptional regulatory protein in each cell. We found that the RAS-GTP inhibition blocked SHH expression in malignant epithelial cells, resulting in loss of downstream Hh pathway activity in CAFs within 24 hours of treatment. By one week of treatment, myCAFs were significantly depleted whereas inflammatory CAFs (iCAFs) were significantly accumulated. Strikingly, CAF heterogeneity was restored in KPC pancreatic tumors that acquired resistance to RMC-7977, despite continued suppression of Hedgehog pathway signaling. Consistent with prior studies of Smoothened inhibitors, the depletion of myCAFs from RAS-GTP inhibition was associated with the rapid opening and hypersprouting of intra-tumoral blood vessels. Ongoing studies using the mutant-selective KRASG12D inhibitor RM-044 should discriminate between direct effects of wild type RAS inhibition in CAFs versus the paracrine consequences of mutant RAS inhibition in malignant epithelial cells. Citation Format: Marie Hasselluhn, Lorenzo Tomassoni, Urszula Wasko, Alvaro Curiel-Garcia, Tanner Dalton, Steve A. Sastra, Carmine Palermo, Andrea Califano, Kenneth P. Olive. RAS-GTP inhibition modulates Hedgehog signaling, suppressing myCAFs and promoting iCAFs in pancreatic ductal adenocarcinoma [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 1578.

Abstract 69: Versican proteolysis signature as a potential immune infiltration biomarker for colorectal cancer

Abstract Background: A subset of patients with mismatch repair proficient colorectal cancers (CRCs) benefit from immunotherapies, though currently no markers predict which cancers are likely to respond. Our group demonstrated that versican (VCAN), an immunoregulatory tumor matrix proteoglycan, when proteolyzed to an immunostimulatory fragment called versikine (Vkine) results in enhanced CD8+ T cell infiltration across multiple cancers. Here we evaluate if a RNA expression signature of VCAN proteolysis can identify CRCs with an immune cell infiltrated phenotype. Methods: The TCGA PanCancer Atlas colorectal adenocarcinoma dataset (594 patients (pts)) was divided into cohorts based on RNA expression Z-scores for VCAN, TIMP3, and ADAMTS4. The VCAN proteolytic predominant (VPP) cohort (Z-score VCAN >0, TIMP3 <0.5, ADAMST4 >0) was compared to the VCAN proteolytic weak (VPW) cohort (Z-score VCAN >0, TIMP3 >0.5, ADAMST4 >0). Differences in clinical characteristics, mutation profile, differential gene expression, and gene set enrichment analysis (GSEA) were evaluated. Results: The VPP group consisted of 42 pts (20:22 female: male, median age: 68.5 years old). The VPW group consisted of 29 pts (12:17 female: male, median age: 64 years old). The two groups had no significant difference between stages of disease. There was a significant increase in the mutation count in the VPP cohort (VPP median=115.5, VPW median=91, p=0.045). The MSI MANTIS scores were not different between the cohorts (VPP median=0.35, VPW median=0.37, p=0.325). The VPP cohort was enriched for gene sets associated with immune cell infiltration (interferon gamma response (FDR q<0.001), inflammatory response (FDR q<0.001), complement (FDR q<0.001), TNFA signaling via NKFB (FDR q<0.001), IL2 STAT5 signaling (FDR q<0.001), and IL6 JAK STAT3 signaling (FDR q<0.001). The VPW cohort was enriched for gene sets involving cell proliferation (WNT beta catenin signaling (FDR q<0.214), hedgehog signaling (FDR q<0.190), and KRAS signaling DN (q<0.159). Specifically, the VPP group demonstrated enhanced expression of CD274 (PD-L1; log2FC=1.41, p<0.001), LAG3(log2FC=1.41, p<0.001), PDF1(log2FC=1.34, p<0.001), CD8A(log2FC=1.25, p<0.001), CXCL9(log2FC=1.19, p=0.001), CXCL10(log2FC=1.44, p=0.001), IL6(log2FC=2.23, p<0.001), and CD19(log2FC=1.26, p<0.001). Conclusion: VPP expression signature correlates with immune infiltration based on RNA expression and deserves further investigation to predict response to immunotherapies in CRC as well as other cancer types. Citation Format: Derek Buckalew, Sean Kraus, Katie Johnson, Cheri A. Pasch, Dustin A. Deming. Versican proteolysis signature as a potential immune infiltration biomarker for colorectal cancer [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 69.

Abstract 5587: Regulator of chromosome condensation 1 (RCC1) as a novel therapeutic target in pancreatic ductal adenocarcinoma

Abstract Background and Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with limited treatment options. There is an urgent need for the identification of novel therapeutic targets for PDAC. The transport of molecular cargo between the nucleus and cytoplasm, facilitated by Ran-GTPase, is a critical process that dividing cells rely on to maintain their growth. Elevated nuclear Ran-GTP levels are produced by the activity of the Ran guanine nucleotide exchange factor (GEF) known as Regulator of Chromosome Condensation 1 (RCC1), which is found in the nucleus bound to chromatin. This creates a Ran-GTP gradient across the nuclear membrane, which maintains proper directional transport of proteins and RNA. To sustain proliferative signaling, cancer cells become reliant on high rates of nucleocytoplasmic transport through dysregulation of transport machinery. Methods: We examined the role of RCC1 in the biology of PDAC. The impact of RCC1 modulation on PDAC growth was evaluated using RNA interference and CRISPR-Cas9 in vitro and in vivo using PDAC cell lines and LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) tumor-derived cells. The broader impact of RCC1 silencing on PDAC-sustaining signaling was evaluated through RNA-sequencing and proteomics. Results: Transcriptomic sequencing of PDAC tissue (n=5,071) as well as analysis of publicly available data from the TCGA, CPTAC and GTEx revealed that RCC1 expression is higher in PDAC tissues compared to normal pancreas. Moreover, PDAC patients with higher RCC1 expression were more likely to have poorer outcomes. RCC1 silencing by RNAi and CRISPR-Cas9 resulted in reduced proliferation in 2D and 3D cultures and attenuated tumor growth in vivo. RCC1 KD decreased migration and colony formation, enhanced apoptosis, and altered the cell cycle in human and KPC mouse PDAC cells. Subcutaneous RCC1 KO cell line-derived xenografts show arrested growth. Subcellular Ran distribution was disrupted upon RCC1 KO, suggesting that nuclear Ran concentration is important for PDAC proliferation. Nuclear and cytosolic proteomic analysis revealed altered subcellular proteome in RCC1 KD KPC tumor-derived cells. Altered cytoplasmic protein pathways include several metabolic pathways, PI3K-AKT, and Hedgehog signaling. Nuclear enriched pathways include cell cycle, mitosis, metabolic processes, and RNA processing. RNA-seq of RCC1 KO cells showed widespread transcriptional alterations. Upstream of RCC1, c-MYC activates the RCC1-RAN axis, and RCC1 KO cells show differential sensitivity to c-MYC inhibitors. Finally, RCC1 KD resulted in the sensitization of PDAC cells to Gemcitabine. Molecular characterization of conditional Rcc1fl/fl KO transgenic model to study the role of RCC1 in PDAC in vivo is ongoing. Conclusions: Overall, our results show that RCC1 is involved in the regulation of PDAC growth and is a potential target for therapy. Citation Format: Sahar F. Bannoura, Husain Y. Khan, Amro Aboukameel, Md Hafiz Uddin, Rund Nimri, Eliza Beal, Seongho Kim, Mohammed Najeeb Al Hallak, Bassel El-Rayes, Philip A. Philip, Boris Pasche, Ramzi Mohammad, Asfar S. Azmi. Regulator of chromosome condensation 1 (RCC1) as a novel therapeutic target in pancreatic ductal adenocarcinoma [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 5587.

Abstract 275: Deciphering biphasic responses to Sonic Hedgehog pathway inhibition in breast cancer: Insights from a tumor-fibroblast spheroid model and PDXs

Abstract A significant subset (30-40%) of triple-negative breast cancer (TNBC) cases exhibit hyperactivation of the sonic hedgehog (SHH) pathway, correlating with poor clinical outcomes. However, the therapeutic efficacy of SHH inhibitors in TNBC has yielded inconsistent results, showcasing biphasic tumor responses going from complete remission in some patients to faster disease progression in others. Such tumor response discrepancies underscore the need for more relevant culture models to elucidate how stromal cells respond to SHH signals and impact tumor cell reprogramming.We have developed a tumor-fibroblast spheroid model of robust paracrine SHH signaling to examine alterations in aggressive tumor cell phenotypes resulting from altered SHH pathway activity levels to understand better how SHH therapy leads to biphasic effects in tumor tissues. Specifically, tumor stemness alterations were examined as a function of SHH signal strength altered by tumor-fibroblast proximity, and findings were confirmed in two PDX models of TNBC of divergent sensitivity to pharmacological SHH pathway inhibition. In vitro, results show that tumor stemness potency is enriched by tumor-fibroblast proximity and inversely correlates with SHH pathway activity levels. Tumor stemness potency was determined via a mammosphere forming assay and resistance to chemotherapy using docetaxel as treatment. CD44+ sorted tumor cells derived from adjacent fibroblast cultures showed higher mammosphere forming efficiency and docetaxel sensitivity than those from mixed cultures. These effects correlated with differences in SHH pathway activity strengths and were reverted by pharmacological inhibition of SHH confirming pathway activity involvement. In two PDX models, both exhibiting high SHH pathway activity and similar tumor vascularization, we observed variable response to pharmacological SHH inhibition. PDX2 displayed favorable responses with minimal alpha-smooth muscle actin-positive (SMA+) fibroblast infiltration, while PDX1 exhibited no benefit from SHH therapy, accompanied by a high level of SMA+ fibroblasts. Spatial transcriptomic analysis confirmed stemness enrichments in the PDX1 model treated with SHH inhibitor- a scenario our mixed co-culture model resembled. Stemness-enriched tumor regions were positive for the Ki67 proliferation marker and confined to areas of high SMA+ cell abundance. Plotting Ki67 vs SMA+ cell levels revealed a positive correlation, suggesting that SMA levels may predict tumor response to SHH pathway inhibition.In summary, our studies demonstrate that pharmacological SHH inhibition can induce altered tumor plasticity by favoring stemness phenotypes. Strategies targeting tumor stemness may mitigate biphasic effects in response to SHH inhibitors, offering insights for optimizing TNBC therapeutic approaches. Citation Format: Heizel M. Rosado-Galindo, Miosotis Acevedo-Esquillin, Wandaliz Torres-García, Ana M. Reyes-Ramos, Gabriela Ortiz-Soto, Michelle M. Martínez-Montemayor, Maribella Domenech. Deciphering biphasic responses to Sonic Hedgehog pathway inhibition in breast cancer: Insights from a tumor-fibroblast spheroid model and PDXs [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 275.

Abstract 5928: Structure-activity relationships for the pan-quadruplex experimental drug QN-302 and two analogs probed with comparative transcriptome profiling and molecular modeling

Abstract The tetrasubstituted naphthalene diimide compound QN-302 binds to G-quadruplex DNA structures. It inhibits the transcription of cancer-related genes in pancreatic ductal adenocarcinoma (PDAC) cells, shows high potency in PDAC cells in vitro and in PDAC animal models. It is currently in Phase 1 clinical evaluation as an anticancer drug for advanced, metastatic solid tumors. A study of structure-activity relationships of QN-302 and two related analogs (CM03 and SOP1247) is reported here. These have been probed using comparisons of transcriptional profiles from whole-genome RNA-seq analyses, together with qualitative molecular modelling. Compounds CM03 and SOP1247 differ by the presence of a methoxy substituent in the former: these two have closely similar transcriptional profiles, whereas that of QN-302, although showing effects in the same cancer-related pathways, highlights the down regulation of distinct genes, for example in the hedgehog pathway. The distinct pattern of genes affected by QN-302 is hypothesized to contribute to its superior potency compared to CM03 and SOP1247. Its superior ability to stabilize quadruplex structures has been attributed to its benzyl-pyrrolidine substituent fitting into and filling most of the space in a quadruplex groove compared to the hydrogen atom in CM03 or the methoxy group in SOP1247. Citation Format: Ahmed A. Ahmed, Shozeb Haider, Tariq Arshad, Stephen Neidle. Structure-activity relationships for the pan-quadruplex experimental drug QN-302 and two analogs probed with comparative transcriptome profiling and molecular modeling [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 5928.

Abstract 3586: A Hedgehog-dependent signaling axis drives neural plasticity and oncogenic reprogramming in gastroenteropancreatic neuroendocrine tumors

Abstract Background: Neuroendocrine tumors (NETs) represent heterogenous malignancies whose origins and etiology remain poorly understood. Men1-driven reprogramming of neural crest-derived glial cells was recently implicated in NET development. In these studies, aberrant Sonic hedgehog (SHH) pathway activation known to pattern neural cell fate coincided with the neuroendocrine phenotype in mice. Here, we investigated the hypothesis that loss of menin encoded by the MEN1 gene drives SHH-dependent oncogenic reprogramming of NETs by modulating the neural crest cell fate. Methods: Glial cell specific Men1 deletion was accomplished by expressing Cre recombinase downstream of the human glial fibrillary acidic protein promoter (GFAPΔMen1). Hedgehog (HH) activation of Men1-deficient glial cells was blocked by deleting the gene encoding primary ciliary protein Kif3a required for transducing SHH signaling. The resulting GFAPΔMen1 mice were evaluated for NET development and dysregulated hormone profiles. To identify aberrant SHH activation leading to neuroendocrine reprogramming, we performed single cell RNA- and Assay for Transposase Accessible Chromatin sequencing on GFAPΔMen1 NETs and primary glial cultures. Hyperactivation of SHH in human gastroenteropancreatic (GEP)-NETs was confirmed by immunofluorescent staining and western blot. Lastly, primary mouse and human NET organoids were treated with an agonist and inhibitors of HH signaling and evaluated for ERK/AKT activation, proliferation, and transcript fluctuations indicative of neural crest cell reprogramming. Results: GFAPΔMen1 mice developed NETs in the pancreas, pituitary, and small intestine. Astonishingly, impaired SHH activation in GFAP+/Men1−/− cells abolished the development of NETs and restored hormone levels to that of wild type mice. GFAPΔMen1 NETs and glial cultures showed increased SHH signaling and chromatin accessibility of HH pathway genes, whereas additive Kif3a deletion blocked HH activation. Moreover, menin negative glial cells demonstrated a phenotypic shift in neuroglial lineages that suggested SHH-mediated reprogramming of neural crest-derived cells following the loss of menin. Consistent with hyperactive SHH activation in the GFAPΔMen1 mice, human GEP-NETs overexpressed SHH and downstream signaling targets. Functionally, SHH induced ERK/AKT activation, cell proliferation, and neuroendocrine transcript expression in human and mouse NET organoids, whereas pharmacological inhibition of HH signaling reversed these effects. Conclusions: Our observations implicate neural crest-derived glial cells as potential neuroendocrine cell precursors that are susceptible to transformation through increased HH signaling. These studies warrant future investigation into the delivery of Hedgehog inhibitors in the adjuvant setting for NET treatment. Citation Format: Suzann Duan, Ricky A. Sontz, Martin Deymier, Juanita L. Merchant. A Hedgehog-dependent signaling axis drives neural plasticity and oncogenic reprogramming in gastroenteropancreatic neuroendocrine tumors [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 3586.

Abstract 4364: Molecular insight of non-canonical Gsk3β-Gli-AR axis: Therapeutic implications of truncated-Gli3 in castration-resistant prostate cancer

Abstract The aberrant activation of the hedgehog (Hh) signaling pathway causes the progression of therapy-resistant castration-resistant prostate cancer (CRPC). In this study, we aimed to unravel the biological significance of Gli-family proteins and their functional involvement of Gsk3β-mediated regulatory mechanism(s) in CRPC. We have discovered a novel function of the truncated form of Gli (t-Gli3) in advancing CRPC. This was accomplished through in silico analysis, in vitro experiments, PCa patient clinical data, and in vivo PCa MYC overexpression mouse models. Our investigation into the Gli3 regulation, typically influenced by receptor-mediated ciliary localization, uncovered a Smo-independent mechanism. Surprisingly, despite the lack of a transactivation domain in t-Gli3, we found a specific requirement for an androgen receptor variant 7 (AR-V7) for its action, as evidenced by co-immunoprecipitation and chromatin immunoprecipitation assays. Mechanistically, Gsk3β activation leads to the t-Gli3 generation, and inhibition of Gsk3β supported the accumulation of full-length Gli3 through a non-canonical mechanism. Knockdown of Gsk3β (Gsk3β KD) reduces CRPC cell proliferation in 2-D and 3-D culture models, inhibiting key proliferative signaling pathways and inducing apoptosis via mitochondrial fragmentation. These findings were supported using an orthotropic PCa mouse model. RNA-seq analysis also showed upregulated phenotype enrichment of the apoptosis, P53, and glycolysis pathway genes in 22Rv1Gsk3β KD cells. Compared to scramble, a lower OCR/mitochondrial respiratory rate is evident in 22Rv1Gsk3β KD cells. Next, our exploration of potential therapies for PCa showed that individually targeting Smo or Gli1 is insufficient to inhibit cell viability. Interestingly, we uncovered that solely targeting Gli3 was sufficient to inhibit the growth of CRPC cells. In conclusion, our study provides the significance of Gli-family proteins and novel insights into the interplay of Gsk3β, t-Gli3, and AR, offering therapeutic implications in CRPC. Citation Format: Jyoti Bala Kaushal, Pratima Raut, Sarathy Seshacharyulu, Zahraa W. Alsafwani, Gunjan Sharma, Abdullah M. Khan, Sushanta Halder, Satyanarayana Rachagani, Surinder K. Batra, Jawed A. Siddiqui. Molecular insight of non-canonical Gsk3β-Gli-AR axis: Therapeutic implications of truncated-Gli3 in castration-resistant prostate cancer [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 4364.

Characterization and Comparative Analysis of Whole-Transcriptome Sequencing in High- and Low-Fecundity Chongming White Goat Ovaries during the Estrus Phase.

Reproductive performance is one of the most important economic traits in the goat industry. Increasing the number of goats is an effective measure to improve production efficiency and reduce production costs. Ovaries are important reproductive organs in female mammals that directly affect the estrous cycle and reproductive abilities. Understanding the complex transcription network of non-coding RNAs (lncRNAs, circRNAs, and miRNAs) and messenger RNA (mRNA) could lead to significant insights into the ovarian regulation of the reproductive processes of animals. However, the whole-transcriptome analysis of the non-coding RNAs and mRNA of the ovaries in Chongming white goats between high-fecundity (HP) and low-fecundity (LP) groups is limited. In this study, a whole-transcriptome sequencing approach was used to identify lncRNA, circRNA, miRNA, and mRNA expression in the ovaries of Chongming white goats during the estrus phase using RNA-Seq technology. More than 20,000 messenger RNAs (mRNAs), 10,000 long non-coding RNAs (lncRNAs), 3500 circular RNAs (circRNAs), and 1000 micro RNAs (miRNAs) were identified. A total of 1024 differential transcripts (724 mRNAs, 112 lncRNAs, 178 circRNAs, and 10 miRNAs) existing between the HP and the LP groups were revealed through a bioinformatics analysis. They were enriched in the prolactin signaling pathway, the Jak-STAT signaling pathway, and the GnRH signaling pathway, as well as various metabolic pathways. Differentially expressed mRNAs (such as LYPD6, VEGFA, NOS3, TNXB, and EPHA2) and miRNAs (such as miR-10a-5p) play key roles in the regulation of goat ovaries during the estrus phase. The enrichment of pathways related to reproduction, such as the Hippo, Hedgehog, PI3K-AKT, and MAPK signaling pathways, suggests that they might be involved in the prolificacy of goat ovaries. Overall, we identified several gene modules associated with goat fecundity and provided a basis for a molecular mechanism in the ovaries of Chongming white goats.

Abstract 6836: Single-cell RNA sequencing of FUS::DDIT3 myxoid/round cell liposarcoma delineates mechanisms of oncogenic progression and immune escape

Abstract Myxoid/Round Cell Liposarcoma (MRCL) is an understudied soft tissue sarcoma that typically affects young adults and accounts for 30% of all liposarcomas. MRCL is characterized by oncogenic DDIT3 fusions, with FUS::DDIT3 being present in over 90% of cases. However, the cellular origins and oncogenic mechanisms of MRCL remain incompletely defined. Here we report oncogene transcription, and tumor-immune interactions in a cohort of 5 patient tumors with matched normal tissue for which we sequenced the transcriptomes of approximately 45,000 single cells using 10X Genomics, and full-length transcripts in approximately 20,000 single cells via Oxford Nanopore in 2 of these patients. Our results reveal two distinct cancer cell populations: Early Cancer Cells (ECC) and Advanced Cancer Cells (ACC). These cells appear to derive from adipose stem progenitor cells (ASPC) and fall along an ASPC→ECC→ACC pseudo-time axis. Along this axis, a distinct pattern emerges where oncogenes tend to be upregulated and tumor suppressor genes (TSG) tend to be downregulated and these appear to be mutually exclusive. Comparing ECC to ASPC we see increased expression of HIPPO and PI3K pathway oncogenes, FGFR2 and IGF1R RTKs, and downregulation of the TSG CDKN2A and CDKN1A. ACC show upregulation of hedgehog, ErbB, WNT, HIPPO, PI3K, and MAPK signaling pathways compared to ECC. Full-length transcript sequencing at the single-cell level shows a consistent expression of the FUS::DDIT3 fusion transcript in both ECC and ACC populations, confirming that these populations are cancer cells. Furthermore, MRCL displays several immune escape mechanisms, including poor immune infiltration and a marked decline of HLA Class I expression along the pseudo-time axis from ECC to ACC, suggesting a complex interplay between oncogenic signaling and immune evasion. These results suggest that MRCL likely originates from ASPC and expression of FUS:DDIT3 oncoprotein induces early HIPPO, PI3K, FGFR2, and insulin signaling pathway expression in ECC with further hedgehog, ErbB, MAPK, and WNT pathway activation in ACC. This progressive activation in oncogenic signaling is associated with immune evasion. This is the first single-cell RNA seq analysis to detect the presence of the FUS::DDIT3 fusion in ECC and ACC significantly advancing our understanding of the mechanisms of MRCL oncogenesis, immune escape, and identifies potential novel therapeutic targets. Citation Format: Rodrigo Gularte-Mérida, Evan Seffar, George Li, Young-Mi Kim, Narasimhan P. Agaram, Nicholas Socci, Francisco Sanchez-Vega, Nikolaus Schultz, Samuel Singer. Single-cell RNA sequencing of FUS::DDIT3 myxoid/round cell liposarcoma delineates mechanisms of oncogenic progression and immune escape [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 6836.

Abstract 486: Paclitaxel nanoformulation attenuates pancreatic tumor desmoplasia and alter tumor immune responses

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer with a 5-year survival rate of less than 13% due to the lack of effective diagnostic/therapeutic modalities. Paclitaxel (PTX) has been tested in pancreatic cancer (PanCa) therapy with marginally better clinical outcomes, but remains limited by its poor hemocompatibility, biodistribution, and intracellular accumulation in tumor cells. Gemcitabine (GEM) is the most effective therapy for PanCa. However, it shows only a marginal survival benefit of 6 months. The poor therapy response is attributed to high desmoplasia, unfavorable tumor microenvironment (TME), and excessive recruitment of immune suppressive cells. We have recently engineered a multi-layered Pluronic F127 and polyvinyl alcohol stabilized and poly-L-lysine coated paclitaxel loaded poly(lactic-co-glycolic acid) nanoparticle formulation (PPNPs), which effectively inhibits pancreatic tumor. In this study, we demonstrate that PPNPs effectively sensitize tumor cells to GEM via attenuation of tumor desmoplasia. Our findings also show that PPNPs synergize GEM therapy response in cell lines and in vivo mouse models. Mechanistically, PPNPs target the TME via inhibition of the sonic hedgehog (SHH) pathway and other oncogenic signaling axis that inhibits bidirectional tumor-stromal cell interaction as determined by qPCR and Western blot analyses. In addition, PPNPs effectively target tumor-associated macrophages (TAM) by repolarizing M2 into M1 phenotype via inhibiting expression of M2 markers and an increase in M1 markers in mouse macrophage cell line RAW264.7. M2 polarization of RAW264.7 cells were induced by culture with IL-4 (20 ng/mL) in the presence of PPNPs or vehicle control. Furthermore, PPNPs effectively increase phagocytic capacity in murine macrophages as determined by phagocytosis assay (Vybrant Phagocytosis Assay Kit). In conclusion, we observed that our novel PPNP nano formulation effectively targets TME, and facilitates GEM uptake by inhibiting the activation of SHH signaling and reprograming the tumor immune surveillance mechanisms. This study suggests that PPNPs have great potential for future clinical use in management of pancreatic cancer. Citation Format: Vivek K. Kashyap, Godwin P. Darkwah, Neeraj Chauhan, Mohammed Sikander, Eswara N. Ghali, Meghana Kolli, Bilal B. Hafeez, Murali M. Yallapu, Subhash C. Chauhan. Paclitaxel nanoformulation attenuates pancreatic tumor desmoplasia and alter tumor immune responses [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 486.

Abstract 576: The pan-quadruplex drug QN-302 selectively down-regulates key pathway targets in pancreatic cancer cells that are up-regulated in human pancreatic cancer

Abstract We have previously disclosed (Ahmed et al, ACS Med Chem Lett, 2020) a novel small molecule G-quadruplex binding compound, QN-302, that is highly potent in pancreatic ductal adenocarcinoma (PDAC) cells and in several in vivo models for this disease. QN-302 was invented at University College London UK and has been subsequently developed by Qualigen Therapeutics Inc. It was cleared by the FDA in August 2023 for Phase 1a clinical trials in USA and is currently under clinical evaluation. RNA-seq whole-genome transcriptome analysis revealed that QN-302 selectively targets G-quadruplexes in cancer-related genes, notably by forming stable G-quadruplex complexes with appropriate promoter sequences. This results in the transcriptional down regulation of affected genes, which map to for example, the hedgehog, axon guidance, WNT/β-catenin and P38 signal transduction pathways. In many instances individual expression changes are modest, though statistically significant: cumulatively, they affect whole pathways. We have revisited the RNA-seq data for QN-302 in PDAC MIA-PACA2 cells (GSE151741 at https://www.ncbi.nlm.nih.gov/geo/) in detail and now identify a small group of eleven down-regulated genes, all with >3-fold expression changes. Interestingly all these genes and their genes products have been previously identified as playing a role in human PDAC (they also have varying roles in other cancer types). Some have been validated as anticancer targets. 10/11 of these genes (https://www.proteinatlas.org/) have been previously reported as being over-expressed in significant fractions of large (>150) groups of human cancer patients from The Cancer Genome Atlas (TCGA) project. Cellular transcriptome data on these eleven genes is presented here, which together with earlier reports on them, supports the designation of QN-302 as a pan-G-quadruplex agent targeting a group of major genes involved in PDAC maintenance and metastasis. Data from a xenograft model for pancreatic cancer is also available for four of these genes, which concurs with the cellular data. We suggest that the identification of these 11 genes may also provide a basis for biomarker selection and possible patient stratification based on transcriptome data. Citation Format: Stephen Neidle, Ahmed A. Ahmed, Tariq Arshad. The pan-quadruplex drug QN-302 selectively down-regulates key pathway targets in pancreatic cancer cells that are up-regulated in human pancreatic cancer [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 576.

Primary Cilium in Neural Crest Cells Crucial for Anterior Segment Development and Corneal Avascularity.

Intraflagellar transport 46 (IFT46) is an integral subunit of the IFT-B complex, playing a key role in the assembly and maintenance of primary cilia responsible for transducing signaling pathways. Despite its predominant expression in the basal body of cilia, the precise role of Ift46 in ocular development remains undetermined. This study aimed to elucidate the impact of neural crest (NC)-specific deletion of Ift46 on ocular development. NC-specific conditional knockout mice for Ift46 (NC-Ift46F/F) were generated by crossing Ift46F mice with Wnt1-Cre2 mice, enabling the specific deletion of Ift46 in NC-derived cells (NCCs). Sonic Hedgehog (Shh) and Notch signaling activities in NC-Ift46F/F mice were evaluated using Gli1lacZ and CBF:H2B-Venus reporter mice, respectively. Cell fate mapping was conducted using ROSAmTmG reporter mice. The deletion of Ift46 in NCCs resulted in a spectrum of ocular abnormalities, including thickened corneal stroma, hypoplasia of the anterior chamber, irregular iris morphology, and corneal neovascularization. Notably, this deletion led to reduced Shh signal activity in the periocular mesenchyme, sustained expression of key transcription factors Foxc1, Foxc2 and Pitx2, along with persistent cell proliferation. Additionally, it induced increased Notch signaling activity and the development of ectopic neovascularization within the corneal stroma. The absence of primary cilia due to Ift46 deficiency in NCCs is associated with anterior segment dysgenesis (ASD) and corneal neovascularization, suggesting a potential link to Axenfeld-Rieger syndrome, a disorder characterized by ASD. This underscores the pivotal role of primary cilia in ensuring proper anterior segment development and maintaining an avascular cornea.

Abstract 7568: Molecular effects of neo-adjuvant hormone therapy for locally advanced prostate cancer: An ancillary study of the ARNEO phase 2 trial

Abstract Intensified hormone therapy in combination with radiotherapy improves outcome for very high-risk locally advanced prostate cancer. The impact of hormone therapy with surgery, however, is uncertain. In order to understand this better, we ran an ancillary translational study on ARNEO (NCT03080116), a Phase 2 clinical trial, which randomized 89 locally-advanced prostate cancer patients to 12 weeks LHRHa +/- apalutamide (APA) followed by prostatectomy. We performed clinical-grade transcriptome-wide expression array analysis (Veracyte, Inc. San Diego, CA) of FFPE diagnostic core biopsies (N=89) and tumors removed at prostatectomy (N=32 LHRHa, N=23 LHRHa + APA). We also performed RNASeq on fresh frozen biopsies of prostatectomy tumors (N=42 LHRHa, N=45 LHRHa + apalutamide) and LHRHa-naive control cases (N=61) matched for clinical and pathological risk factors. Pre-surgery serum PSA decreased by a median of 98% (IQR:90.2-99.3) after hormone treatment. This decrease was better associated with tumor AR signaling (r2=0.11) versus pre-treatment serum PSA (r2=0.0005), or between pre-treatment serum PSA with diagnostic biopsies (r2=0.008). We confirmed APA more potently suppressed AR signaling than LHRHa alone, with no detectable difference in direction of effect for differentially regulated genes/pathways. LHRHa + APA had less residual tumor (median Residual Cancer Burden, RCB=0.48cm3) than in LHRHa (median RCB=1.7cm3) but in cases with sufficient tumor, we observed residual active AR signaling pathways at prostatectomy in both treatment arms. To orthogonally confirm this, we selected 28 ERG positive cases (by IHC pre-treatment) and found retained ERG expression in 12/13 on LHRHa and 10/15 on LHRHa + APA. Using pathway analysis in the RNAseq data, we identified LHRHa +/- APA induced down-regulation of AR, metabolic pathways, as well as DNA repair, and up-regulation of hallmark NOTCH, Hedgehog, TGF-beta, WNT and immune signaling (adjusted p-value cutoff=0.001). Comparing differentially expressed genes on RNASeq between treatment-naïve versus LHRHa +/- APA treated tumors, we observed STING1 as significantly higher in LHRHa +/- APA vs control (Wilcoxon rank sum test p-value <0.001). We found that expression of STING1 was positively correlated with hallmark interferon alpha response (r=0.7, rs=0.6) that was correlated with apoptosis (r=0.5, rs=0.5). Whilst we show that adding APA to LHRHa increases suppression of AR activity and ERG expression, we find that 12 weeks is insufficient to abrogate AR signaling in most tumors, despite ~98% decrease in serum PSA. We identify novel processes modulated in humans by potent suppression of androgen activity, including upregulation of STING1-type 1 interferon response as a potential key modulator of treatment effect. Citation Format: Mazlina Ismail, Wout Devlies, Marina Parry, Gaetan Devos, Stefanie Friedrich, Charles Parker, Larissa Mendes, Elai Davicioni, Mark Linch, Frank Claessens, Steven Joniau, Gerhardt Attard. Molecular effects of neo-adjuvant hormone therapy for locally advanced prostate cancer: An ancillary study of the ARNEO phase 2 trial [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 7568.