Kannan, Nagarajan

Abstract Global incidence and mortality rates for endometrial and cervical cancers have been steadily rising, while these rates for ovarian cancer have decreased. Although these cancers represent unique diseases and challenges, overlapping biology and responses to targeted and immunotherapies create opportunities to advance research through shared knowledge and resources. In 2025, investigators funded by National Cancer Institute (NCI) Specialized Programs of Research Excellence (SPORE) and other NCI-supported grants convened a hybrid meeting to identify challenges and knowledge gaps in translational research, highlight advances, define shareable resources and technologies, and foster collaboration among the Gynecologic Cancer SPORE sites, patient advocates, and NCI staff. Special sessions were arranged for presentations by Patient Advocates, Early-stage Investigators, Investigators Transitioning to Gynecologic Cancer Research. Meeting-wide workshops on Sharing Resources and Reducing Time to Activation of Clinical Trials and networking events were included. Collectively, the presentations converged on three overarching themes: (i) interception and early disease biology, (ii) development of innovative targeted and immune-based therapies, (iii) critical role of shared resources and collaborative infrastructure in accelerating translational progress. The unifying theme across disease sites was molecular advances to improve early detection/prevention and to drive therapeutic advancements. Novel findings discussed included: candidate precursor cells and mechanisms of high grade serous ovarian cancer development; therapeutic vaccines and their delivery methods for cervical dysplasia; identification of immune-targetable ovarian cancer antigens; various novel molecularly targeted drugs and immune-based agents for endometrial and ovarian cancers; and approaches to bypass drug resistance. Workshops delineated actionable strategies to further advance gynecologic cancer translational research.

Context Women with obesity or polycystic ovary syndrome (PCOS) often require repeated ovulation induction (OI) due to ovulatory dysfunction and infertility. However, the cumulative effects of repeated stimulation on ovarian function and embryo development remain poorly understood. Aims The present study aimed to understand the consequences of repeated OI and/or repeated superovulation (SO) on preimplantation embryonic development in obese (high-fat diet-induced) and PCOS (high-fat diet and dehydroepiandrosterone-induced) mouse models. Methods Control (without any metabolic disorder), obese, and PCOS mice were divided into three experimental groups. The mice in one round of superovulation (SO1) were injected with 5 IU intraperitoneal (i.p.) injection of pregnant mare serum gonadotropin, followed by 10 IU of human chorionic gonadotropin 48 h later. In the repeated superovulation (SO3) group, mice were subjected to three rounds of superovulation with a gap of 9 days between each superovulation. Mice in the OI3 + SO3 group were administered with three doses of CC (50 mg/kg body weight, i.p., gap of 9 days between each CC injection), followed by three rounds of superovulation. Key results Both single and three rounds of superovulation resulted in decrease in primordial follicles and a moderate increase in atretic follicles in obese and PCOS mice. Reduction in the number of ovulated oocytes, increased rate of abnormal fertilization, decrease in blastocyst rate and elevated DNA damage in blastocysts were observed in in vitro fertilization-derived embryos. Conclusions These findings demonstrate compromised embryonic development following repeated ovarian stimulation. Implications The study highlights need for mild ovarian stimulation strategies in metabolically compromised patients undergoing infertility treatments.

Abstract High-grade serous ovarian cancer (HGSOC), the most prevalent and lethal ovarian cancer subtype, is typically diagnosed at an advanced stage due to the absence of effective early detection strategies. Mounting evidence indicates that HGSOC originates from the fallopian tube (FT), particularly from secretory epithelial cells in the fimbria. However, incomplete understanding of the cellular and molecular origins of HGSOC continues to hinder early interception efforts. To address this, we established a clinically and genetically annotated living FT organoid biobank at Mayo Clinic, representing over 200 patient donors. Using optimized enrichment protocols, we isolated FT epithelial stem/progenitor cells from tissue and Tao brushings, generating organoids independent of anatomical site or laterality that recapitulate native FT epithelial architecture and lineage differentiation. We applied integrated multi-omic analyses, including single-cell RNA sequencing, single-nucleus RNA/ATAC sequencing, and bulk RNA-seq, across fresh cells and organoids. Integration with public datasets produced the largest single-cell atlas of normal and high-risk FTs to date. This analysis defined high-specificity gene signatures for secretory and multiciliated lineages that outperform canonical lineage markers (e.g. PAX8, FOXJ1), identified lineage-defining transcription factors and regulatory networks, and showed concordant protein expression in the human FT epithelium. Notably, subcluster-level analysis revealed a rare hybrid epithelial-mesenchymal population transcriptionally and proteomically aligned with the mesenchymal-like subtype of HGSOC. Spatial immunoprofiling demonstrated that these cells express multiple defining markers and reside above the basement membrane, intercalated between epithelial cells and lack luminal contact, reminiscent of the histologically described peg cell population. Comparative analyses with fetal mesonephric tissues uncovered developmental parallels, and this population was stably maintained in 3D organoid cultures, providing a robust ex-vivo model to study early transformation. Together, our findings identify a rare, multipotent epithelial population with mesenchymal features as a candidate origin for mesenchymal-like HGSOC and establish the FT organoid biobank as a powerful platform for mechanistic and translational studies in ovarian carcinogenesis. Citation Format: Megan L. Ritting, Wenmei Yang, Syed Mohammed Musheer Aalam, Hui Zhao, Liang Feng, Jianning Song, Mihai G. Dumbrava, Wazim M. Ismail, Kenneth Schaufelberger, S. John Weroha, Scott H. Kaufmann, Alexandre Gaspar-Maia, Mark E. Sherman, Jamie N. Bakkum-Gamez, Nagarajan Kannan. A rare multipotent peg-like epithelial cell is a candidate cell-of-origin for high-grade serous ovarian cancer [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 7636.

Abstract Therapeutic resistance to chemotherapy is frequently the proximate cause of increased tumor burden, cancer treatment failure, and ultimately, patient mortality. The most successful therapy strategy derived from evolutionary practices to date, Adaptive Therapy (AT), leverages the fitness cost of developing therapeutic resistance. AT avoids treating cancer cells with the Maximum Tolerated Dose (MTD) of therapeutic drugs and instead incorporates a series of adaptive dosing schedules. Treatment-sensitive cells are bolstered by modulating or pausing treatment to out-compete and suppress treatment-resistant cell growth. We are evaluating AT for the first time in colorectal cancer, using a syngeneic orthotopic xenograft model. This work represents several key advances: it is the first to longitudinally and non-invasively track sensitive and resistant clones in vivo, to evaluate adaptive therapy in an immunocompetent mouse model, to incorporate multi-drug treatment regimens, and to integrate a targeted therapeutic agent. The dual-bioluminescent sensitive and resistant CT26 cell lines developed for this study allow real-time visualization and quantification of each population throughout tumor progression and treatment. Together, these advances establish a robust and clinically relevant platform for characterizing resistance dynamics and refining adaptive therapy strategies aimed at improving treatment outcomes in colorectal cancer. Citation Format: Harley I. Richker, Bailey Sierra Kane, Gissel Marquez Alcaraz, Ellie Pahl, Ryan Carr, Nagarajan Kannan, Carlo C. Maley. Harnessing clonal dynamics to overcome resistance: An adaptive therapy framework in colorectal cancer [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 702.

ABSTRACT The normal adult male breast has not been characterized at single-cell resolution, leaving the cellular basis of male breast cancer (MBC) biology undefined. Here we present an integrated single-cell RNA sequencing atlas of the adult human breast comprising 174,471 cells from 17 donors (3 male, 14 female), including 18,117 male-derived cells. This revealed that the male breast retains all three epithelial populations, basal (BC), luminal progenitor (LP), and luminal committed cells (LC), but with an increase in LC at the expense of BC and LP across all three male donors. Male LC were distinguished from female by elevated ESR1 and PGR mRNA, enrichment of RNA processing and ribosome biogenesis programs, reduced inflammatory cytokine and growth factor signaling, elevated estradiol gene set enrichment scores, and higher inferred activity of developmental patterning transcription factors. This pattern was observed across differential expression, gene ontology, ligand profiling, and regulon-based analyses, and was not restricted to sex chromosome-linked gene expression. This is consistent with the near-universal estrogen receptor (ER) positivity that characterizes MBC clinically. This atlas provides the first cellular and transcriptional reference for the normal male breast and a resource for investigating sex differences in mammary biology, germline susceptibility variant interpretation, and modeling breast malignancies.

The fallopian tubes are critical segments of the female reproductive tract and are essential for transporting gametes and embryos. It creates a conducive environment necessary for successful fertilization, early embryo development, and embryo transport. The cellular composition and function of the fallopian tube are tightly regulated by the sex hormones estradiol and progesterone. Therefore, any pathological/ metabolic condition or exposure to exogenous agents with the potential to alter endocrine levels can have a significant impact on fallopian tube function and health. This review summarizes the effects of medications, infections, pathological conditions, lifestyle choices, and environmental factors that can significantly impact the morphology, histology, cellularity, and functionality of the fallopian tube.

Abstract Cancer can recur when a subset of tumor cells, termed persister cells, survive therapy and re-enter the cell cycle. The cellular lineages that give rise to persister cells and the mechanisms that confer the persister state remain poorly understood. Through single-cell multiomic profiling (snRNA-seq and snATAC-seq) on a cohort of (1) non-malignant fallopian tube, (2) treatment-naïve, and (3) neoadjuvant chemotherapy (NACT)-treated high-grade serous ovarian cancer (HGSOC) patient samples, we identified an epigenetic signature that defines the chemotherapy-tolerant persister state. The changes in chromatin accessibility characterizing the signature were identified in residual NACT tumors but are also present in treatment-naive samples from patients who later developed resistance. Furthermore, this epigenetic signature independently predicted chemotherapy response in patient-derived xenograft models of HGSOC and in a separate patient cohort. Cells enriched in the persister state arose from multiple lineages and displayed activation of oncogenic pathways, including altered stress responses, epithelial to mesenchymal transition, and changes to the cell cycle promoting quiescence. Finally, we identified a subset of genes that are epigenetically primed for expression before treatment and are upregulated after treatment. These findings suggest that an intrinsic epigenetic program primes tumor cells towards chemotherapy tolerance and reveal new vulnerabilities that could be exploited to delay or prevent cancer recurrence. Citation Format: Mihai G. Dumbrava, Wazim M. Ismail, Leticia Sandoval, Amelia Mazzone, Syed Mohammed Musheer Aalam, Megan L. Ritting, Xiaonan Hou, Yiwen Xie, Shariska Harrington, Scott H. Kaufmann, Nagarajan Kannan, S. John Weroha, Alexandre Gaspar-Maia. Single cell multiomic analysis of high-grade serous ovarian carcinoma reveals an intrinsic epigenetic program that primes chemotherapy tolerance in persister cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr PR008.

Abstract High-grade serous ovarian cancer (HGSOC), the most prevalent and lethal ovarian cancer subtype, is often diagnosed at an advanced stage due to the lack of effective early detection methods. Current evidence suggests that HGSOC originates from the fallopian tube (FT), specifically from secretory epithelial cells in the fimbriated region, based on the detection of early serous lesions and clonal genetic relationships linking them to HGSOC tumors. However, efforts to improve early diagnosis and interception are hindered by an incomplete understanding of HGSOC’s cellular and molecular origin. To address this, we established a diverse, clinically and genetically annotated living FT organoid biobank at the Mayo Clinic, encompassing specimens from over 200 patient donors. Using robust, multi-step protocols, we enriched FT epithelial stem/progenitor cells from tissue samples and Tao brushings, generating bi-lineage differentiated organoids independent of anatomical site (ampulla, fimbria) or laterality (right, left). To gain deeper insight into FT epithelial lineage dynamics, we applied multi-omic modalities—including single-cell RNA sequencing (scRNA-seq), single-nucleus RNA and ATAC sequencing (snMultiome), and bulk RNA-seq—to fresh cells and pooled organoids, integrating findings with public FT datasets to generate the largest known integrated single-cell atlas of normal and high-risk FTs. This enabled the generation and validation of gene expression-derived signatures for secretory and multiciliated lineages, identification of key lineage-defining transcription factors and their regulatory networks, and validation using immunohistochemistry (IHC) data from the Human Protein Atlas. Subcluster-level analysis of epithelial cells revealed transcriptionally distinct populations with unique biological functions, deepening insight into epithelial heterogeneity and FT lineage differentiation. Further, analysis of FT epithelial subclusters identified a rare population with transcriptomic and proteomic association with the mesenchymal-like HGSOC subtype. These cells retained epithelial identity while expressing mesenchymal and ECM-related genes, forming a hybrid phenotype. Spatial profiling via IHC localized them to a rare, basal, peg-like position within the FT epithelium. Developmental analysis showed shared transcriptional features with fetal mesonephric cells, suggesting both developmental plasticity and potential vulnerability to transformation. Pooled organoid scRNA-seq confirmed that they are preserved in culture, indicating they are stably maintained ex-vivo. Together, these findings suggest that this cell cluster represents a rare, multipotent epithelial population with mesenchymal features, offering a candidate cellular origin for the mesenchyme-like subtype of HGSOC. In conclusion, this living FT organoid biobank serves as a powerful, clinically annotated platform for dissecting epithelial lineage hierarchies, identifying rare cell populations, and uncovering mechanisms that may underlie subtype-specific ovarian carcinogenesis. Citation Format: Megan Ritting, Syed Mohammed Musheer Aalam, Mihai Dumbrava, Wazim Mohammed Ismail, Alexandre Gaspar-Maia, Mark Sherman, Jamie Bakkum-Gamez, Nagarajan Kannan. Tubal organoid biobanking and multi-omics framework uncovers rare basal peg cells as a source of epithelial stemness and a candidate for high-grade serous ovarian cancer origin [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr A078.

Abstract Introduction: Despite treatment advances, ovarian cancer (OC) remains the deadliest gynecological cancer, with a ∼70% mortality rate. OCs initially respond to platinum chemotherapy but often develop resistance. While PARP inhibitors improve relapse-free survival in responsive cases, they do not extend overall survival. This underscores the need for effective combination therapies beyond BRCA status. The lysosomal protein cathepsin L (CTSL) can traffic to the nucleus, where it can regulate DNA repair and serve as a chromatin modifier. We found that nucleoside analog clofarabine (CLF) induces CTSL nuclear trafficking in OC cells. Here we tested the hypothesis that accumulation nuclear CTSL (nCTSL) is a biomarker of sensitivity to DNA damaging therapy in OCs for improved therapeutic options. Methods: To determine the role of CLF induced nuclear CTSL in DNA damage and its synergy with PARP and CRM1 inhibitors in PARP inhibitor–sensitive and –resistant ovarian cancer (OC) cell lines, ex vivo cultures of ascites-derived patient OC cells (OVA-3D spheroids), primary ovarian tumors, and patient-derived xenograft (PDX) models and validated by colony formation assays. Nuclear CTSL localization was determined in subcellular fractionated lysates by western blots and immunofluorescence cytometry (IFC). Tumor regression was determined in vivo using ex vivo cultures of PDX derived cells. Results: In ovarian cancer (OC) cell lines, CLF monotherapy induced nuclear CTSL (nCTSL) in a subset of models classified as CLF-responsive (CLF-r), sensitizing them to the PARP inhibitors olaparib and rucaparib. In CLF-non-responsive (CLF-nr) models, nCTSL induction and therapeutic synergy were observed only with the CLF+olaparib combination. Synergy was detected in 47% of CLF-r and 24% of CLF-nr patient-derived ascites samples (OVA). Mechanistically, CLF induced nuclear import of CTSL via KPNB1 in CLF-r cells, while this import was triggered by the CLF+olaparib combination in CLF-nr cells. CLF also downregulated the nuclear export protein CRM1 (XPO1) in both groups. Knockdown of CTSL in either CLF-r or CLF-nr cells abrogated sensitivity to the combination. Notably, in 29% of OVA samples classified as CLF-resistant (CLF-Res), CLF failed to suppress CRM1 expression. In these cases, CRM1 inhibition with KPT8602 restored CLF+PARP inhibitor synergy. In vivo, CLF-r and CLF-nr PDX models treated with the combination exhibited increased DNA damage, reduced tumor burden, and extended survival. Conclusion: These findings identify nuclear CTSL (nCTSL) induction as a potential biomarker of response to DNA-damaging therapies. The combination of CLF and olaparib represents a promising strategy to overcome drug resistance in ovarian cancer by promoting CTSL nuclear localization and enhancing DNA damage. Citation Format: Prabhu Thirusangu, Ling Jin, Upasana Ray, Anya Zhao, Julie Staub, Xinyan Wu, Xiaonan Hou, Jamison L. VanBlaricom, Syed Mohamed Musheer Aalam, Sun-Hee Lee, Ann Oberg, Nagarajan Kannan, John Weroha, Jeremy Chien, Scott H. Kaufmann, Jamie N. Bakkum-Gamez, Viji Shridhar. Role of drug induced nuclear CTSL (nCTSL) in DNA damage response in cancer- therapeutic implications [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr A050.