Musheer Aalam, Syed Mohammed

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, 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 A problematic feature of many human cancers is a lack of understanding of mechanisms controlling organ-specific patterns of metastasis, despite recent progress in identifying many mutations and transcriptional programs shown to confer this potential. To address this gap, we developed a methodology that enables different aspects of the metastatic process to be comprehensively characterized at a clonal resolution. Our approach exploits the application of a computational pipeline to analyze and visualize clonal data obtained from transplant experiments in which a cellular DNA barcoding strategy is used to distinguish the separate clonal contributions of two or more competing cell populations. To illustrate the power of this methodology, we demonstrate its ability to discriminate the metastatic behavior in immunodeficient mice of a well-established human metastatic cancer cell line and its co-transplanted LRRC15 knockdown derivative. We also show how the use of machine learning to quantify clone-initiating cell (CIC) numbers and their subsequent metastatic progeny generated in different sites can reveal previously unknown relationships between different cellular genotypes and their initial sites of implantation with their subsequent respective dissemination patterns. These findings underscore the potential of such combined genomic and computational methodologies to identify new clonally-relevant drivers of site-specific patterns of metastasis.

e18055 Background: Entrectinib, a TRK kinase inhibitor, has been approved for the treatment of tissue agnostic rare tumors positive for TRK fusions. A very low frequency molecular subset of TRK fusion tumors dubbed as secretory carcinoma (SC) are characterized by organ-agnostic epithelial origin, ETV6-NTRK3 (EN) fusion and distinguishable secretory-type tumor cells. These tumors have been frequently miscategorized as acinic cell carcinoma or adenocarcinoma. A previous mouse study identified EGF dependent epithelial progenitors as putative cell-of-origin for SC. Methods: To test the role of EGFR signaling in EN mediated transformation and therapy resistance, we expressed EN, kinase-dead EN-K380M, and drug-resistant EN-G623R in human epithelial MCF10A cells with EGF-dependent primitive function and investigated their ability to grow in the presence and absence of EGF and/or entrectinib. To understand the significance of findings based on our model, we analyzed a total of 22 ‘rare’ patients from Mayo Clinic Tissue Registry and analyzed TCGA PanCan datasets. Results: We report herein that EGF signaling is essential for normal growth but dispensable during EN driven transformation. Our findings suggest that levels equivalent to circulating EGF (0.5-1ng/ml) is sufficient to drive 100% resistance to entrectinib in vitro. Three different strategies to blockade EGF/EGFR axis including depletion of EGF in culture system, genetic depletion of EGFR using shRNA as well as cetuximab antibody-based EGFR neutralization potentiated oncogene-addiction and hypersensitivity to entrectinib in our models. As predicted, models with G623R mutation in EN was refractory to entrectinib under all experimental conditions. Further omics analysis of TCGA PanCan suggested that EN and EGFR mutations are mutually exclusive and entrectinib-resistant G623R mutation were uncommon. Interestingly, nearly all EN tumors from Mayo Clinic Tissue Registry immunostained weakly or strongly for EGFR and showed perfect concordance with pEGFR suggesting pathway activation. Conclusions: Together, these findings raise an important question whether blockade of ‘wildtype’ EGFR signaling could improve medical intervention in SC patients presenting with wildtype EGFR and no drug-resistant mutation in entrectinib, by improving oncogene-addiction and attendant hypersensitization of transformed cells to entrectinib.