SCUBE3 regulates FOXR2 to activate pro-growth signaling

Approaches targeting factors that simultaneously promote tumor growth and progression, induce therapy resistance, and inhibit anti-tumor immunity offer clear benefits over therapies targeting only one of these tumor-promoting processes. Through comprehensive loss-of-function genomic screening, we identified SCUBE3 as a pivotal factor that supports survival and therapy resistance and also orchestrates an immunosuppressive tumor microenvironment. Secretory SCUBE3 supported oncogenic activity through interactions with key oncogenic cell surface receptor proteins, including EGFR, mutant CALR, and TGFβRI/II. These interactions activated the transcription factors FOXR2 and c-Myc, promoting cancer cell proliferation and therapy resistance by enhancing DNA damage repair. Additionally, the SCUBE3-FOXR2 axis created an immunosuppressive tumor microenvironment by facilitating recruitment of the DNMT1 epigenetic repressor complex to the transcription regulator IRF1, thereby inhibiting the expression of MHC-I and MHC-II genes. A first-in-class neutralizing antibody targeting SCUBE3, which was developed using a sophisticated antibody discovery platform and engineered with specific mutations in the heavy chain for enhanced specificity and efficacy, demonstrated profound therapeutic potential across various cancer types in preclinical models, including breast and ovarian cancer patient-derived xenografts. This discovery marks an advancement toward developing a targeted therapy for cancers characterized by hyperactive SCUBE3-associated signaling pathways.

Abstract Background: Medulloblastoma (MB) is the most common malignant pediatric brain tumor and a major cause of childhood cancer mortality. It comprises four molecular subgroups: WNT, SHH, Group 3, and Group 4. Group 3 MB, often marked by c-MYC amplification, has the worst prognosis. Despite surgery, radiation, and chemotherapy, high-risk patients face low 5-year survival and frequent relapse. Current treatments cause severe side effects. Cancer stem cells (CSCs) drive MB aggressiveness, therapy resistance, and recurrence. This project aims to uncover mechanisms sustaining CSCs and MB progression. Methods: MB cell lines (HD-MB03, D556, D425, DAOY) with ALKBH5 overexpression, knockdown (siRNA), or knockout (CRISPR), along with controls, were analyzed using viability, migration, invasion, colony formation, cell cycle, and apoptosis assays. RNA sequencing identified ALKBH5-regulated genes, validated by RT-qPCR, western blot, and RNA immunoprecipitation. An orthotopic intracranial xenograft model assessed ALKBH5’s tumor-promoting role in vivo. Results: To explore the role of m6A RNA methylation in medulloblastoma (MB), we silenced key regulators (writers: METTL3, METTL14; erasers: ALKBH5, FTO) in MB cell lines. ALKBH5 depletion caused the greatest reduction in proliferation, suggesting its therapeutic potential. Analysis of pediatric cancer datasets and tissue microarrays confirmed ALKBH5 overexpression and amplification in MB. Knockdown of ALKBH5 increased global m6A levels and reduced MB cell viability, migration, invasion, and stemness (medullosphere formation and NANOG, OCT4, SOX9 expression). Apoptosis assays showed elevated Annexin V-positive cells, and in vivo studies demonstrated suppressed tumor growth in orthotopic xenografts. ALKBH5 loss also increased DNA damage markers (γH2AX, 53BP1). RNA-seq and IPA revealed downregulation of genes involved in glycolysis, lipogenesis, and c-MYC targets, including ChREBP. Western blot confirmed decreased protein levels in these pathways. Conclusion: ALKBH5 is a critical regulator of MB tumorigenesis and CSC maintenance via m6A RNA demethylation. Targeting ALKBH5 may offer a promising therapeutic strategy to inhibit MB progression, overcome therapy resistance, and improve outcomes for high-risk MB patients. Citation Format: Panneerdoss Subbarayalu, Daisy Medina, Prabhakar Pitta Venkata, Shahad Abdulsahib, Saif Nirzhor, Santosh Timilsina, Deepika Singh, Phat Do, Desiree Denman, Krishna Priya Evani, Dhiya Billa, Meera Nair, Esha Reddy, Yogesh Gupta, Peter Houghton, Yidong Chen, Suryavathi Viswanadhapalli, Gangadhara R. Sareddy, Andrew Brenner, Ratna K. Vadlamudi, Manjeet Rao. Targeting ALKBH5 mediated ChREBP signaling impairs cancer stem cell metabolism and tumor growth in medulloblastoma [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 3492.

SCUBE3 regulated tumor cell growth

SCUBE3 interacts with cell surface receptors

Medulloblastoma (MB) is pediatric brain cancer originate from the cerebellum, and accounts for large number of all childhood cancer related deaths. MB grows vigorously, and spreads to central nervous system (CNS) through cerebrospinal fluid, leading to leptomeningeal metastases in 66% brain cancer patients. MB are commonly treated with a combination of surgery, radiation, and chemotherapy drugs like vincristine. Unfortunately, at the young age, radiation exposure and chemotherapy treatment lead to high toxicity which ultimately compromise the quality of life for those kids. Therefore, it is critical to understand the molecular mechanism that regulates MB tumor growth, metastasis and relapse so that safe and novel therapeutics can be developed to treat pediatric MB. In this study, we investigated the role of RNA demethylase ALKBH5, in regulating MB growth and progression. Medulloblastoma cell lines including HD-MB03, D556, D425 and DAOY were purchased from the ATCC. MB cells were transfected either with ALKBH5 overexpression (OE) plasmid or siRNA (KD). These OE/KD MB cells were analyzed for cell viability, migration, invasion, colony formation, cell cycle, apoptosis assays, RNA sequencing, RT-qPCR, western blotting, RNA immunoprecipitation, and in vivo tumor xenograft study. Our results showed that depletion of ALKBH5 significantly reduced short term cell viability, migration, invasion and colony forming ability of MB cells. ALKBH5 knockdown MB cells showed increased apoptosis. Further, our results show that ALKBH5 silencing suppressed the self-renewal/proliferation of MB cancer stem cells (MBCSC), including medullosphere formation. In orthotopic xenograft study, ALKBH5 knockdown cells intracranially injected into mouse showed reduced tumor growth compared to the scrambled siRNA transfected MB cells. To understand the molecular mechanism of ALKBH5, we performed transcriptome analysis of ALKBH5 depleted MB cells. Silencing of ALKBH5 showed alterations in several metabolism related pathways. Further, ALKBH5-depletion led to significantly decreased levels of cancer stem cell marker proteins including Nanog, OCT4 and SOX9. Together, our data suggests that m6A RNA demethylase ALKBH5 may play a critical role in MB growth and tumor progression by regulating MB tumor initiating stem cells and ALKBH5 serves as a potent therapeutic target to treat MB. Panneerdoss Subbarayalu, Daisy Medina, Pitta V. Prabhakar, Shahad Abdulsahib, Saif R. Nirzhor, Desiree Denman, Santosh Timilsina, Deepika Singh, Phat Do, Krishna Evani, Dhiya Billa, Esha Reddy, Meera Nair, Peter Houghton, Yogesh Gupta, Yidong Chen, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi, Gangadhara R. Sareddy, Andrew Brenner, Manjeet K. Rao. m6A demethylase ALKBH5 regulates medulloblastoma growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2620.

Introduction: Cancer cells continue to replicate their DNA and survive mainly due to their unique ability to repair damaged DNA using alternate DNA repair pathways. For example, cancer cells with a deficiency in homologous recombination (HR) proteins (such as BRCA1) can repair their DNA by either relying on other highly expressed HR-related proteins (such as RAD51 or PARP1) or by using backup DNA repair mechanisms such as ALT-NHEJ. Alterations in DNA repair pathways commonly occur during breast cancer (BC) progression. For example, TNBCs have dysfunctional BRCA1/2 but express high levels of RAD51. Further, ER+BC employs ALT- NHEJ, HR, or BER to repair their DNA. The objective of this study was to identify FDA-approved non-cancer drug/s capable of inhibiting DNA repair in BC cells, thereby inhibiting their growth, with potential clinical benefits for BC patients. Methods: BC cells were treated with vehicle and FDA-approved drugs for 72 hours and were subjected to cell-titer Glo assay. The anti-tumor effect of imipramine alone and in combination with Olaparib and tamoxifen was validated using orthotopic xenograft mouse models. The effect of imipramine on DNA repair was determined by immunofluorescence using antibody against 53BP1, and functional DNA repair assays. Based on these results, a window of opportunity clinical trial was conducted to test the efficacy of imipramine in early-stage breast cancer patients. After having a breast core needle biopsy, 15 eligible patients with stage I-III breast cancer were enrolled in the trial and were treated with imipramine at a target dose of 200 mg PO daily for an average of 28 days. Patients were evaluated on day 7, day 14, day 21, and at the end of treatment for toxicity. The primary endpoint for the trial was the absolute change in the Ki67. IHC using an antibody against Ki67 was performed on core biopsy (pre-treatment) and tumor tissue after imipramine treatment. The secondary objectives of this trial were to further define the toxicity profile of imipramine. Results: Imipramine treatment significantly reduced the viability of TNBC and ER+ BC cells. Further, imipramine treatment inhibited the migration and invasion of BC cells. Systemic delivery of imipramine suppressed the growth of BC. Importantly, imipramine blocked the DNA repair capacity of BC cells by inhibiting the expression of DNA repair proteins including FOXM1 and RAD51. Notably, imipramine treatment improved the efficacy of Olapraib in TNBC and sensitized the tamoxifen response in endocrine-resistant ER+ BC cells. The clinical trial on 15 patients treated with imipramine showed a marked reduction in Ki67-positive cells in post-surgical tumor tissues compared to core needle biopsy tissues. Toxicity was mild with only grade 1 and 2 toxicities that included some instances of dizziness and nausea. There was no dose reduction, interruption, or treatment discontinuation. Discussion: Our preclinical and clinical studies showed that imipramine can block DNA repair in both TNBC and HR+ breast cancer. Short-term treatment with imipramine can effectively decrease Ki67 in breast cancer patients. Future clinical trials will involve combining imipramine with other regimens such as Olaparib for TNBC patients and elacestrant/CDK4/6 inhibitor for therapy-resistant ER+ breast cancer patients. Citation Format: Manjeet Rao, Arhan Rao, Santosh Timilsina, Subapriya Rajamanickam, Panneerdoss Subbarayalu, Ismail Jatoi, Yidong Chen, Kate Lathrop, Ratna Vadlamudi, Virginia Kaklamani. Imipramine: A Promising Therapeutic Regimen for Breast Cancer Patients [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-08-26.

The interplay between tumor cells and the microenvironment significantly influences cancer progression. Here, we report a significant role of the transcription factor FOXM1 in shaping the tumor immune landscape. Single-cell sequencing reveals that tumor-intrinsic FOXM1 creates an immune-suppressive tumor microenvironment by inhibiting expression of stress ligands (including ULBP1) on cancer cells, thereby blocking NKG2D-NKG2DL interactions critical for priming natural killer- and T cell-mediated cytotoxicity of cancer cells. FOXM1 suppresses ULBP1 expression by epigenetically silencing the DNA-sensing protein STING using a DNMT1-UHRF1 complex, which in turn inhibits the unfolded protein response protein CHOP from activating ULBP1. Importantly, cancer patients with higher levels of FOXM1 and DNMT1, and lower levels of STING and ULBP1, have worse survival and are less responsive to immunotherapy. Collectively, our findings provide key insight into how a tumor-intrinsic transcription factor epigenetically shapes the tumor immune microenvironment, with strong implications for refining existing and designing new cancer immunotherapies.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy, with a 5-year survival rate of 12%. The dense extracellular matrix (ECM) and cancer-associated fibroblast (CAF)-driven microenvironment contribute to tumor progression and resistance to current therapies, necessitating the development of novel treatments. This study introduces CIDD-8633, a selective small-molecule inhibitor targeting the Discoidin Domain Receptor (DDR) complex, which plays a crucial role in ECM-mediated signaling and PDAC progression. High-throughput screening identified CIDD-8633 as a potent inhibitor of DDR kinase activity. Functional assays demonstrated that CIDD-8633 significantly reduced PDAC cell proliferation, migration, and colony formation while inducing apoptosis and G1/S cell cycle arrest. RNA sequencing revealed that CIDD-8633 treatment downregulated pathways associated with DNA replication and repair, while upregulating apoptosis-related signaling pathways. In vivo, CIDD-8633 effectively suppressed tumor growth in PDAC xenograft models, reducing tumor volume and proliferation markers. Histopathological analysis showed decreased phosphorylated DDR and downstream RTKs expression, accompanied by enhanced apoptotic signaling. Furthermore, combination therapy with gemcitabine demonstrated synergistic effects, enhancing anti-tumor efficacy by reducing PDAC cell viability and metastatic potential more effectively than either agent alone. These findings position CIDD-8633 as a promising therapeutic candidate for PDAC, capable of targeting the DDR complex to disrupt tumor progression. This study highlights the potential of DDR inhibition as a novel therapeutic approach for overcoming the challenges of this aggressive cancer. Chrid TP Do, Prabhakar P. Venkata, Jack Y. Prochnau, Deepika Singh, Santosh Timilsina, Panneerdoss Subbarayalu, Daisy Medina, Shahad Abdulsahib, Sajeed Khan, Manjeet K. Rao. A novel inhibitor targeting DDR complex to suppress pancreatic cancer growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2702.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor, and although multimodal therapies have improved outcomes for standard-risk patients, children diagnosed with high-risk or recurrent MB, particularly those presenting with leptomeningeal dissemination (LMD), continue to experience poor prognoses, frequent relapses, and debilitating long-term treatment-related toxicities. To identify novel therapeutic vulnerabilities, we performed an unbiased genome-wide CRISPR knockout screen in MB cells alongside a meta-analysis of the Cancer Dependency Map (DepMap), which converged on THOC7 as a top-ranked dependency. THOC7, a highly conserved component of the TREX complex responsible for mRNA processing and nuclear export, was found to be significantly overexpressed in MB patient tumor samples compared to normal cerebellum, with elevated expression correlating with reduced overall survival, indicating potential clinical relevance. Functional depletion of THOC7 using stable shRNA-mediated knockdown in multiple MB cell lines resulted in a profound reduction in cell viability, proliferation, and anchorage-independent growth. Transwell migration assays revealed impaired invasive capacity, and apoptosis was significantly induced, as evidenced by Annexin V/PI staining and cleaved PARP accumulation. Mechanistically, THOC7 suppression led to the accumulation of DNA double-strand breaks, marked by persistent γH2AX foci and S/G2 cell cycle arrest, pointing to activation of DNA damage response pathways. In parallel, we observed reduced expression of stemness-associated transcription factors such as SOX2 and NANOG, suggesting that THOC7 supports MB maintenance by preserving tumor-initiating cell features. To further dissect the molecular consequences of THOC7 loss, we will employ RNA immunoprecipitation sequencing (RIP-seq) to identify transcripts directly bound and regulated by THOC7, potentially uncovering downstream oncogenic pathways dependent on its RNA-binding activity. Altogether, our findings define THOC7 as a multifaceted contributor to MB pathogenesis through regulation of genome integrity and stem-like identity, offering a promising therapeutic target for high-risk MB and informing future precision medicine strategies in pediatric neuro-oncology.

This study investigates the role of transcription factor FOXM1 in creating an immune-suppressive tumor microenvironment (TME) by regulating stress ligands and the STING pathway in triple-negative breast cancer (TNBC). We performed in vitro and in vivo assays using CRISPR/Cas9-mediated knockout of FOXM1 in TNBC cell lines and syngeneic mouse models. Using single-cell RNA sequencing, we analyzed the impact of tumor-intrinsic FOXM1 on stress ligand expression and its downstream effects on immune cell interactions. FOXM1’s epigenetic regulation on STING pathway was further validated through chromatin immunoprecipitation. We found tumor-intrinsic FOXM1 establishes an immune-suppressive TME by downregulating stress ligands like ULBP1 on cancer cells. This inhibition disrupts NKG2D-NKG2DL signaling, a key pathway for initiating natural killer and T cell cytotoxicity against cancer cells. FOXM1 achieves this by epigenetically silencing the DNA-sensor protein STING via a DNMT1-UHRF1 complex. Notably, patients with elevated FOXM1 and DNMT1 levels and reduced STING and ULBP1 levels show poorer survival rates and reduced responsiveness to immunotherapy. Our findings identify FOXM1 as a key regulator of immune evasion in TNBC, revealing a new pathway by which it shapes the TME through epigenetic repression. Targeting FOXM1 or its downstream effectors could enhance immunotherapy efficacy, offering new therapeutic strategies for patients with TNBC. Supported by NIH NIGMS/T32GM113896; T32GM145432; T32CA279363; R01CA179120; R01CA239227; P30CA054174; CPRIT RP200110. Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with limited effective treatments, partly because of its complex tumor microenvironment. In this study, we report discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase, as a critical protein that promotes PDAC growth and survival. Our results reveal that DDR2 is highly expressed and its expression correlates with the worst survival outcome in patients with PDAC. Using an unbiased high-throughput screen of small-molecule inhibitor libraries, we identified CIDD-8633, a novel inhibitor targeting DDR2. Our study suggests that CIDD-8633 interacts with DDR2 and inhibits DDR2-associated signaling. Importantly, in vivo studies demonstrate that CIDD-8633 effectively blocks PDAC tumor growth in preclinical mouse models. Additionally, combining CIDD-8633 with gemcitabine enhanced its efficacy synergistically. Mechanistically, CIDD-8633 treatment induces pro-apoptotic genes in PDAC cells. These findings position DDR2 as a promising therapeutic target and CIDD-8633 as a potential DDR2 inhibitor, offering new avenues for the treatment of PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with limited effective treatments, partly because of its complex tumor microenvironment. In this study, we report discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase, as a critical protein that promotes PDAC growth and survival. Our results reveal that DDR2 is highly expressed and its expression correlates with the worst survival outcome in patients with PDAC. Using an unbiased high-throughput screen of small-molecule inhibitor libraries, we identified CIDD-8633, a novel inhibitor targeting DDR2. Our study suggests that CIDD-8633 interacts with DDR2 and inhibits DDR2-associated signaling. Importantly, in vivo studies demonstrate that CIDD-8633 effectively blocks PDAC tumor growth in preclinical mouse models. Additionally, combining CIDD-8633 with gemcitabine enhanced its efficacy synergistically. Mechanistically, CIDD-8633 treatment induces pro-apoptotic genes in PDAC cells. These findings position DDR2 as a promising therapeutic target and CIDD-8633 as a potential DDR2 inhibitor, offering new avenues for the treatment of PDAC.

Background: Breast cancer (BC) stands as the most prevalent cancer and the leading cause of cancer-related deaths among women globally, with an incidence rate of 1 in 8 women developing invasive breast cancer during their lifetime. A significant majority (80%) of these cases are Estrogen Receptor positive (ER+). The standard first-line treatment for locally advanced or metastatic ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer includes endocrine therapy with aromatase inhibitors (AI) or Fulvestrant combined with a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. However, disease progression often leads to endocrine resistance, primarily due to acquired mutations in the estrogen receptor 1 (ESR1). These ESR1 mutations cause estrogen-independent ER activation, resulting in resistance to AIs but not to selective ER degraders (SERD) or selective ER modulators (SERM). Current treatment guidelines recommend continuing sequential endocrine therapy until a visceral crisis occurs or all options are exhausted, revealing a significant unmet clinical need. Repurposing existing or investigational drugs has emerged as a promising strategy for cancer treatment. Consequently, we explored the potential of repurposing the antidepressant Amitriptyline for treating ER+ breast cancer. Methods: We employed both short-term and long-term cell viability assays, as well as apoptosis assay, to evaluate the anticancer activity of Amitriptyline across various BC cell lines. These included wild-type ERα cell lines (MCF7 and ZR75), cell lines with acquired resistance (MCF7-Tam and MCF7-LTLT), and mutant ERα cell lines (MCF7-ERα-D538G, MCF7-ERα-Y537S, ZR75-ERα-D538G, and ZR75-ERα-Y537S). To validate our in vitro findings, we employed patient-derived explants (PDEX), cell-derived xenograft, and patient-derived xenograft (PDX) models to test Amitriptyline's efficacy. Biotin/Streptavidin pull down assay followed by mass spec analysis and RNA sequencing (RNA-seq) analysis were conducted on vehicle-treated and Amitriptyline-treated breast BC cells to elucidate the target protein and mechanism of action respectively. Additionally, we investigated whether Amitriptyline could sensitize ER+ breast cancer cells to Elacestrant, an FDA approved SERD. Results: Amitriptyline treatment significantly reduced both short-term and long-term cell viability of BC cells in a dose-dependent manner. Additionally, Amitriptyline markedly increased apoptosis in BC cells. These in vitro findings were corroborated in vivo, as Amitriptyline treatment inhibited the growth of ER+ BC in a preclinical orthotopic syngeneic model. Furthermore, Amitriptyline significantly lowered Ki67 levels in ESR1 mutant patient-derived explants (PDEX). Notably, Amitriptyline enhanced the sensitivity of ER+ BC to Elacestrant, demonstrating highly synergistic effects. The combination of Amitriptyline and Elacestrant significantly improved cell viability, survival, and apoptosis outcomes compared to Elacestrant alone. Conclusion: Our study highlights the potential of Amitriptyline as a safe and effective treatment option for patients with ER+ breast cancer. Citation Format: Prabhakar Pitta Venkata, Arhan Rao, Timilsina Santosh, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi, Virginia G. Kaklamani, Manjeet K. Rao. Amitriptyline potentiates Elacestrant in treating ER positive ESR1 mutant Breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-01-26.