Aalam, Syed Mohammed Musheer

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 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.

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.

Abstract Cancer can recur when a subset of tumor cells, denoted here as persister cells, are able to survive therapy and re-enter the cell cycle. The precise mechanisms that confer the persister state and whether it is characteristic of a subgroup of cells or arises from multiple cellular lineages remain poorly understood. We hypothesize that an epigenetic signature underlies the drug-tolerant persister state, characterized by transcriptional and chromatin accessibility changes that promote survival of residual cancer following chemotherapy. To identify clinically relevant features of persister cells in untreated tumors and residual disease, we performed single-cell multiomic profiling (snRNA+snATAC) on a cohort of non-malignant fallopian tube, treatment-naïve, and neoadjuvant chemotherapy (NACT)-treated high-grade serous ovarian cancer (HGSOC) samples. We identified differences in gene expression and open chromatin between naïve and residual patient tumors following chemotherapy. Although only a small proportion of the differentially expressed genes enriched in residual HGSOC overlapped with established gene sets for chemo-response and patient prognosis, the epigenomic analysis revealed activity of several DNA-binding factors that are both enriched upon chemotherapy and also high in resistant tumors prior to treatment. From this analysis, we identified an epigenetic signature that precedes expression and defines the persister state. This epigenetic signature also correlated with chemotherapy sensitivity and resistance using patient-derived xenograft models of HGSOC. Gene regulatory networks driven by the persister signature are involved in the activation of oncogenic pathways, including changes to the cell cycle promoting quiescence and stress response. Further study of the persister cells identified by this epigenetic signature may increase understanding of the mechanisms underlying persister cell survival and reveal new vulnerabilities that could be exploited to delay or prevent cancer recurrence.

ABSTRACT In our efforts to enhance sensitivity to PARP inhibitors, we identified clofarabine (CLF) as a potential therapy for drug-resistant ovarian cancer and nuclear trafficking of Cathepsin L (CTSL) as a treatment- responsive biomarker. Using PARP inhibitor-sensitive and -resistant OC cell lines, ex vivo cultures of patient-derived ovarian ascites (OVA), primary ovarian tumors, and xenografts (PDX), we found that CLF monotherapy induces nuclear CTSL (nCTSL) in CLF-responsive cells (CLF-r) and sensitizes them to PARP inhibitors olaparib and rucaparib. In CLF non-responsive cells (CLF-nr), a combination of CLF with olaparib is necessary for nCTSL trafficking and synergy. CLF+olaparib synergy was observed in 47% of CLF-r and 24% of CLF-nr OVA samples. Drug-induced nCTSL is crucial for DNA damage response, including cell cycle arrest and apoptosis. Knockdown of CTSL in both CLF-r and CLF-nr cells conferred resistance to the CLF+olaparib combination, emphasizing nCTSL’s role in the DNA damage response pathway (DDR). Mechanistically, CLF facilitates CTSL nuclear import via KPNB1 in CLF-r cells. In CLF-nr cells, both olaparib and CLF are needed to facilitate CTSL nuclear import. Additionally, CLF downregulates the nuclear export protein CRM1 (XPO1) in both cohorts. Interestingly, CLF does not downregulate CRM1 in a subset of OVAs (29%), and they were classified as CLF-resistant (CLF- Res). In these samples, inhibiting CRM1 with KPT8602 restored synergy between CLF and PARP inhibitors. In vivo, CLF-r and CLF-nr PDX models exhibited enhanced DDR, reduced tumor burden, and prolonged survival with the CLF+olaparib combination. These findings suggest the CLF+olaparib combination is a promising therapeutic strategy for drug-resistant OC by inducing DDR through CTSL nuclear localization.

Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy. Despite therapeutic advances that have vastly improved survival, all patients are expected to relapse. Current standard of care includes autologous stem cell transplant (ASCT) with high-dose melphalan (HDM) administered as a conditioning agent. Cytotoxic chemotherapies such as melphalan have been shown to drive therapy-induced senescence (TIS) in several tissues. We hypothesize that HDM activates TIS pathways in surviving MM cells and that this may correlate with patient outcomes post-ASCT. We developed an in vitro HDM model using 5TGM1 mouse MM cells treated with vehicle (Veh) or HDM (10µM, IC90) for 6h, followed by co-culture with primary mouse bone marrow stromal cells. Image analysis was performed to quantify proliferation by cumulative population doublings (CPDs) over 10 days, after which 5TGM1 cells were evaluated for cell size, telomere-associated DNA damage foci (TAFs), sensitivity to senolysis, and senescence gene expression (scRNA-seq and RT-qPCR). The effect of HDM on CPDs and cell size was also evaluated in human MM cell lines RPMI-8226 and H929. scRNA-seq was used to analyze CD138+ PCs from MM patients at various timepoints post-ASCT. Longitudinal MM patient iliac crest biopsies (diagnosis, post-ASCT; N=20) were stained for CD138+ PCs; PC burden was quantified by AI-assisted histology. Lastly, we established an in vivo model of induction therapy (bortezomib) followed by HDM/SCT in Vk*MYC mouse MM engrafted C57BL/6 mice. Tumor burden was tracked by serum protein electrophoresis for M-spike using submandibular bleeds. Bone marrow was then flushed from long bones and analyzed by flow cytometry and immunofluorescence. HDM-5TGM1 showed decreased CPDs (HDM 0.32±0.31, Veh 6.72±0.69, p<0.0001, N=3), increased size (median pixel size: HDM 413.2±99.98, Veh 207±8.88, p<0.05, N=3), and increased TAF+ cells (Percent TAF+ cells: HDM 34.77%±10.74%, Veh 11.41%±1.58%, p<0.05, N=3) consistent with known TIS features. Preliminary assessment (N=1) of human MM cell lines showed similar growth arrest (RPMI-8226 CPDs: HDM 0.058±0.101, Veh 4.185±0.358; H929 CPDs: HDM 0±0, Veh 4.61±0.243) and size increase (RPMI-8226 mean pixel size: HDM 804.1±6.048, Veh 526.9±2.115; H929 mean pixel size: HDM 371.2±26.95, Veh 130.3±41.43), suggesting a translatable mechanism. HDM-5TGM1 cells were more sensitive to senolysis by dasatinib+quercetin (viability: HDM 54.9%±0.09%, Veh 76.0%±0.05%, N=2). scRNA-seq analysis showed that HDM-5TGM1 were enriched for gene sets related to senescence pathways, including the published set SenMayo (FDR<0.25). HDM-5TGM1 cells also exhibited myeloid gene expression consistent with previous publications on MM dormancy. RT-qPCR confirmed significant increases in HDM-5TGM1 (p<0.001, N=3) in senescence markers (Cdkn1a, Cdkn1c, Glb1), senescent cell anti-apoptosis pathway genes (SCAPs, Bcl2l1), senescence associated secretory phenotype (SASP) genes (Ccl5, Icam1, Mmp13), and myeloid markers (Axl, Fcer1g, Mpeg1). scRNA-seq of patient CD138+ PCs confirmed a myeloid-like cluster that gained prominence in relapsed patients with resistant disease. CD138+ PCs were detected in patient bone biopsies post-ASCT. Interestingly, patients who relapsed (≤3 years, N=9) had a greater percent reduction in PC burden from diagnosis to post-ASCT when compared to patients that did not relapse (N=11) in that time (p=0.02). Further, CD138+ PC burden post-ASCT positively correlated with time in remission in relapsed patients (R=0.697, p=0.03). Bortezomib reduced serum M-spike in Vk*MYC-engrafted mice (p=0.0003); M-spike was further reduced by HDM/SCT compared to Veh-treated mice (p=0.0007). Flow cytometry confirmed a decrease in Vk*MYC cells after HDM/SCT (p=0.0043), and the percentage of Vk*MYC cells with greater size and complexity was increased compared to Veh-treated mice (p=0.0043), consistent with our in vitro data. Vk*MYC cells from HDM-treated mice also trended for more TAF+ cells (>3 TAFs) compared to Veh-treated mice (p=0.0714). Our findings suggest that HDM can induce a TIS and myeloid signature in surviving MM cells. Greater MM burden post-ASCT appears to be associated with longer durable response, again suggesting that these cells may be in growth arrest consistent with TIS. Finally, senolytics may be a novel approach to eliminate surviving MM cells and prevent relapse.

Abstract Multiple myeloma (MM) is an incurable plasma cell (PC) cancer in which all patients are expected to relapse. Cytotoxic agents such as high-dose melphalan (HDM), which is used as a myeloablative agent prior to autologous stem cell transplant (ASCT) in MM, drive therapy-induced senescence (TIS) in non-cancerous tissues. We hypothesized that HDM similarly activates TIS pathways in surviving MM cells and this response may correlate with patient outcomes post-ASCT. To test this, we developed an in vitro model in which 5TGM1 mouse MM cells are cultured with 10uM melphalan (HDM) or vehicle (Veh) for 6 hours, followed by co-culture in normal media with primary mouse bone marrow stromal cells. Cultures were maintained for 10 days and imaged to quantify cumulative population doublings (CPDs). 5TGM1 were then isolated by FACS to measure telomere-associated DNA damage foci (TAFs), sensitivity to senolytic drugs, and senescence gene expression (RT-qPCR and scRNA-seq). To translate these studies, scRNA-seq was used to analyze CD138+ PCs from MM patient bone marrow post-ASCT. Additionally, longitudinal bone biopsies from MM patients at diagnosis and post-ASCT were stained for CD138+ PCs; PC burden was quantified using artificial intelligence assisted histology (HALO). HDM-5TGM1 exhibited stable growth arrest (decreased CPDs) and sustained DNA damage (increased TAFs) compared to Veh-5TGM1. HDM-5TGM1 were also significantly more sensitive to senolytic ablation (Dasatinib+Quercetin or Fisetin). HDM-5TGM1 showed increased senescence (Cdkn1a, Cdkn1c, Glb1), anti-apoptosis (Bcl2l1), and senescence associated secretory phenotype (Ccl5, Icam1, Mmp13) genes, as well as myeloid markers found to be increased in MM dormancy (Axl, Fcer1g, Mpeg1). Analysis by scRNA-seq revealed that HDM-5TGM1 were G0/G1 arrested, and gene set enrichment analysis (GSEA) confirmed upregulation of senescence pathways with downregulation of DNA replication and cell cycle pathways. Consistent with RT-qPCR results, these cells exhibited myeloid gene expression. Analysis of patient CD138+ PCs post-ASCT by scRNA-seq also revealed a myeloid-like cluster. Of interest, of the 491 genes upregulated in this cluster, 268 were also upregulated in HDM-5TGM1. CD138+ PCs could also be detected in patient bone biopsies post-ASCT, although PC burden was significantly reduced post-ASCT. Surprisingly, the percent reduction in PC burden post-ASCT was greater in relapsed (≤3 years) vs non-relapsed patients. Further, in relapsed patients, PC burden post-ASCT positively correlated with time in remission. Altogether, these findings demonstrate that HDM can induce senescence features and a myeloid-dormancy signature in surviving MM cells. This phenotype is associated with longer durable response, suggesting that activation of TIS may be a strategy to extend PFS. Further, senolytic therapy may be a novel approach to eliminate dormant MM cells and prevent relapse. Citation Format: Angelo Jose Guilatco, Marta Diaz-del-Castillo, Gabriel Alvares Borges, Neal I. Sannuli, Megan L. Ritting, Syed Mohammed Aalam, Nagarajan Kannan, Tamar Tchkonia, James L. Kirkland, Yi Lin, Taxiarchis Kourelis, Matthew T. Drake, Thomas Levin Andersen, Megan Weivoda. Melphalan-treated multiple myeloma cells exhibit a senescent-like dormancy phenotype [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 2958.