Analisa DiFeo

Identification of a TNIK-CDK9 Axis as a Targetable Strategy for Platinum-Resistant Ovarian Cancer

Abstract Up to 90% of patients with high-grade serous ovarian cancer (HGSC) will develop resistance to platinum-based chemotherapy, posing substantial therapeutic challenges due to a lack of universally druggable targets. Leveraging BenevolentAI’s artificial intelligence (AI)–driven approach to target discovery, we screened potential AI-predicted therapeutic targets mapped to unapproved tool compounds in patient-derived 3D models. This identified TNIK, which is modulated by NCB-0846, as a novel target for platinum-resistant HGSC. Targeting by this compound demonstrated efficacy across both in vitro and ex vivo organoid platinum-resistant models. Additionally, NCB-0846 treatment effectively decreased Wnt activity, a known driver of platinum resistance; however, we found that these effects were not solely mediated by TNIK inhibition. Comprehensive AI, in silico, and in vitro analyses revealed CDK9 as another key target driving NCB-0846’s efficacy. Interestingly, TNIK and CDK9 co-expression positively correlated, and chromosomal gains in both served as prognostic markers for poor patient outcomes. Combined knockdown of TNIK and CDK9 markedly diminished downstream Wnt targets and reduced chemotherapy-resistant cell viability. Furthermore, we identified CDK9 as a novel mediator of canonical Wnt activity, providing mechanistic insights into the combinatorial effects of TNIK and CDK9 inhibition and offering a new understanding of NCB-0846 and CDK9 inhibitor function. Our findings identified the TNIK-CDK9 axis as druggable targets mediating platinum resistance and cell viability in HGSC. With AI at the forefront of drug discovery, this work highlights how to ensure that AI findings are biologically relevant by combining compound screens with physiologically relevant models, thus supporting the identification and validation of potential drug targets.

Mutant PP2A Induces IGFBP2 Secretion to Promote Development of High-Grade Uterine Cancer

Abstract Uterine serous carcinoma (USC) and uterine carcinosarcoma (UCS) tumors are uniquely aggressive, suggesting that the primary tumor is intrinsically equipped to disseminate and metastasize. Previous work identified mutational hotspots within PPP2R1A, which encodes the Aα scaffolding subunit of protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine phosphatase. Two recurrent heterozygous PPP2R1A mutations, P179R and S256F, occur exclusively within high-grade subtypes of uterine cancer and can drive tumorigenesis and metastasis. Elucidation of the mechanisms by which PP2A Aα mutants promote tumor development and progression could help identify therapeutic opportunities. Here, we showed that expression of these mutants in USC/UCS cell lines enhanced tumor-initiating capacity, drove a hybrid epithelial-to-mesenchymal plasticity phenotype, and elevated secretion of the tumorigenic cytokine insulin growth factor (IGF) binding protein 2 (IGFBP2). Therapeutic targeting of the IGFBP2/IGF receptor 1 signaling axis using small molecules and genetic approaches resulted in marked tumor growth inhibition. Mechanistically, PP2A regulated IGFBP2 expression through the transcription factor, NF-κB, which harbors a B56 recognition motif. Collectively, these results identify a role for PP2A in regulating paracrine cancer cell signaling that can be targeted to block the initiation and metastasis of high-grade uterine cancer. Significance: Elevated IGFBP2 secretion by uterine cancer cells with heterozygous PPP2R1A mutations supports tumor progression and confers a vulnerability to IGFBP2/IGF1R inhibition as a therapeutic approach for this highly aggressive cancer subtype.

Small-Molecule–Mediated Stabilization of PP2A Modulates the Homologous Recombination Pathway and Potentiates DNA Damage-Induced Cell Death

Abstract High-grade serous carcinoma (HGSC) is the most common and lethal ovarian cancer subtype. PARP inhibitors (PARPi) have become the mainstay of HGSC-targeted therapy, given that these tumors are driven by a high degree of genomic instability (GI) and homologous recombination (HR) defects. Nonetheless, approximately 30% of patients initially respond to treatment, ultimately relapsing with resistant disease. Thus, despite recent advances in drug development and an increased understanding of genetic alterations driving HGSC progression, mortality has not declined, highlighting the need for novel therapies. Using a small-molecule activator of protein phosphatase 2A (PP2A; SMAP-061), we investigated the mechanism by which PP2A stabilization induces apoptosis in patient-derived HGSC cells and xenograft (PDX) models alone or in combination with PARPi. We uncovered that PP2A genes essential for cellular transformation (B56α, B56γ, and PR72) and basal phosphatase activity (PP2A-A and -C) are heterozygously lost in the majority of HGSC. Moreover, loss of these PP2A genes correlates with worse overall patient survival. We show that SMAP-061–induced stabilization of PP2A inhibits the HR output by targeting RAD51, leading to chronic accumulation of DNA damage and ultimately apoptosis. Furthermore, combination of SMAP-061 and PARPi leads to enhanced apoptosis in both HR-proficient and HR-deficient HGSC cells and PDX models. Our studies identify PP2A as a novel regulator of HR and indicate PP2A modulators as a therapeutic therapy for HGSC. In summary, our findings further emphasize the potential of PP2A modulators to overcome PARPi insensitivity, given that targeting RAD51 presents benefits in overcoming PARPi resistance driven by BRCA1/2 mutation reversions. Watch the interview with Analisa DiFeo, PhD, recipient of the 2025 Molecular Cancer Therapeutics Award for Outstanding Journal Article: https://vimeo.com/1100470225

miR-181a initiates and perpetuates oncogenic transformation through the regulation of innate immune signaling

AbstractGenomic instability (GI) predisposes cells to malignant transformation, however the molecular mechanisms that allow for the propagation of cells with a high degree of genomic instability remain unclear. Here we report that miR-181a is able to transform fallopian tube secretory epithelial cells through the inhibition of RB1 and stimulator-of-interferon-genes (STING) to propagate cells with a high degree of GI. MiR-181a targeting of RB1 leads to profound nuclear defects and GI generating aberrant cytoplasmic DNA, however simultaneous miR-181a mediated inhibition of STING allows cells to bypass interferon mediated cell death. We also found that high miR-181a is associated with decreased IFNγ response and lymphocyte infiltration in patient tumors. DNA oncoviruses are the only known inhibitors of STING that allow for cellular transformation, thus, our findings are the first to identify a miRNA that can downregulate STING expression to suppress activation of intrinsic interferon signaling. This study introduces miR-181a as a putative biomarker and identifies the miR-181a-STING axis as a promising target for therapeutic exploitation.

STING pathway expression in low‐grade serous carcinoma of the ovary: an unexpected therapeutic opportunity?

AbstractOvarian carcinoma histotypes are distinct diseases with variable clinical outcomes and response to treatment. There is a need for new subtype‐specific treatment modalities, especially for women with widespread and chemo‐resistant disease. Stimulator of interferon genes (STING) is a part of the cGAS–STING pathway that mediates innate immune defence against infectious DNA‐containing pathogens and also detects tumour‐derived DNA and generates intrinsic antitumour immunity. The STING signalling pathway is suppressed by several mechanisms in a variety of malignant diseases and, in some cancers that may be a requirement for cellular transformation. The aim of this study was to use immunohistochemistry to evaluate STING protein expression across normal tissue, paratubal and ovarian cysts, and ovarian tumour histotypes including ovarian carcinomas. Herein, we show that the fallopian tube ciliated cells express STING protein, whereas the secretory cells are negative. STING expression differs among ovarian cancer histotypes; low‐grade serous ovarian carcinomas and serous borderline tumours have uniform high STING expression, while high‐grade serous and endometrioid carcinomas have heterogeneous expression, and clear cell and mucinous carcinomas show low expression. As low‐grade serous carcinomas are known to be genomically stable and typically lack a prominent host immune response, the consistently high STING expression is unexpected. High STING expression may reflect pathway activation or histogenesis and the mechanisms may be different in different ovarian carcinoma histotypes. Further studies are needed to determine whether the STING signalling pathway is active and whether these tumours would be candidates for therapeutic interventions that trigger innate immunity activation.

The miR–181a–SFRP4 Axis Regulates Wnt Activation to Drive Stemness and Platinum Resistance in Ovarian Cancer

Abstract Wnt signaling is a major driver of stemness and chemoresistance in ovarian cancer, yet the genetic drivers that stimulate its expression remain largely unknown. Unlike other cancers, mutations in the Wnt pathway are not reported in high-grade serous ovarian cancer (HGSOC). Hence, a key challenge that must be addressed to develop effective targeted therapies is to identify nonmutational drivers of Wnt activation. Using an miRNA sensor-based approach, we have identified miR-181a as a novel driver of Wnt/β-catenin signaling. miR-181ahigh primary HGSOC cells exhibited increased Wnt/β-catenin signaling, which was associated with increased stem-cell frequency and platinum resistance. Consistent with these findings, inhibition of β-catenin decreased stem-like properties in miR-181ahigh cell populations and downregulated miR-181a. The Wnt inhibitor SFRP4 was identified as a novel target of miR-181a. Overall, our results demonstrate that miR-181a is a nonmutational activator of Wnt signaling that drives stemness and chemoresistance in HGSOC, suggesting that the miR–181a–SFRP4 axis can be evaluated as a novel biomarker for β-catenin–targeted therapy in this disease. Significance: These results demonstrate that miR-181a is an activator of Wnt signaling that drives stemness and chemoresistance in HGSOC and may be targeted therapeutically in recurrent disease.