Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and treatment refractory disease with a 5-year overall survival of approximately 10%. Molecular subtyping of PDAC remains in its nascent stages and does not currently inform clinical management or therapeutic development. Previously identified bulk expression subtypes in the untreated setting were influenced by contaminating stroma whereas single-cell RNA-seq (scRNA-seq) of fresh tumors under-represented key cell types (Guo, Hoffman, Clin Cancer Res 2021). Nevertheless, two consensus subtypes have arisen from these prior efforts: (1) classical, encompassing a spectrum of pancreatic lineage precursors, and (2) basal-like/squamous/quasi-mesenchymal, characterized by loss of endodermal identity and aberrations in chromatin modifiers. Basal-like tumors were associated with poorer responses to chemotherapy and worse survival in the metastatic setting but attempts to refine this binary classification have failed to further stratify patient survival. Recent clinical trials have supported the increasing adoption of neoadjuvant chemotherapy and/or radiotherapy (CRT) to aggressively address the risk of micro-metastatic spread and to circumvent concerns of treatment tolerance in the postoperative setting.There is an urgent need to understand how preoperative treatment impacts residual tumor cells to identify additional therapeutic vulnerabilities that can be exploited in combination with neoadjuvant CRT.
Towards this end, we developed and optimized a single-nucleus RNA-seq method that is compatible with frozen biobank samples and pretreated specimens, with better recovery of malignant and stromal cells. We applied snRNA-seq to primary PDAC specimens from 43 patients that either received neoadjuvant therapy or were treatment naive, yielding over 220,000 high-quality single-nucleus profiles (Hwang, Jagadeesh, Guo, Hoffman, Nat Genetics 2022). We uncovered recurrent expression programs across malignant cells and fibroblasts, including a novel neural-like progenitor (NRP) malignant cell program featuring pathways and genes involved in neuronal development/migration/adhesion, stem-like state, and endoderm organ development. Moreover, the NRP program is significantly enriched in ‘brain tissue enhanced’ genes in the Human Protein Atlas and we validated the program in situ by multiplexed immunofluorescence, showing that a subset of malignant cells/glands co-express cytokeratins and NRXN3, a program gene typically expressed in neuronal and glial cells of the cerebral cortex and caudate (Hwang, Jagadeesh, Guo, Hoffman, Nat Genetics 2022). Notably, the NRP program was found to be enriched after CRT in tumors and matched organoids and associated with poor prognosis in independent cohorts, suggesting this malignant cell state may play an important role in resistance to cytotoxic therapy.
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Identifying the key regulators, context dependence, and therapeutic vulnerabilities of resistant cell states
While several transcription factor drivers of the classical and basal-like/quasi-mesenchymal subtypes have been described, the key regulators of the NRP state are unknown. Through in silico approaches, we identified candidate transcription factors of the NRP state. To enable systematic study of the classical, basal-like/quasi-mesenchymal and NRP phenotypes, we engineered isogenic KP (Kras G12D/+; Trp 53FL/FL) murine organoids with a germline dCas9-VPR system to enable facile overexpression of state-specific transcription factors through CRISPR activation. We are performing drug sensitivity assays with a panel of targeted therapies in murine and patient-derived organoids and cell lines to build a framework for defining cell state-specific vulnerabilities that will aid in stratifying and treating pancreatic cancer patients more effectively.
Elucidating the role of cell state plasticity in therapeutic resistance and the underlying genetic and epigenetic mechanisms
The enrichment of the NRP phenotype after CRT can be mediated by selection and/or plasticity but the contribution of each mechanism is unknown. We are investigating this question through single-cell multiomics and barcode-based lineage tracing to identify the genetic and epigenetic mechanisms that mediate transcriptional modulation in individual cancer cells to effect therapeutic resistance.
Investigating mechanisms of tumorigenesis using single-cell multiomics to enable chemoprevention and early detection
One of the major reasons for the poor prognosis of PDAC is the fact that most cases are detected at advanced stages. Hence, detecting and inhibiting progression of early oncogenic lesions would be of great value. While PDAC is formally a neoplasm of epithelial ductal tissue, observations made primarily in animal models have suggested that acinar cells may give rise to neoplastic precursors and eventually invasive cancer through an initial process of trans-differentiation known as acinar-to-ductal metaplasia (ADM), but the putative role of ADM lacks direct evidence in humans. In our snRNA-seq data, we identified two subsets of epithelial nuclei with low inferred copy number aberrations (CNAs; non-malignant) that either (1) co-expressed markers of ductal and acinar lineages, which may reflect ADM, or (2) expressed high levels of both ductal and malignant markers, which we termed atypical ductal cells (ADC). Partition-based graph abstraction inferred a dominant pseudotemporal trajectory from acinar to ADM to ductal to atypical ductal to malignant cells, paralleling a monotonic increase in theHALLMARK_KRAS_SIGNALING_UP signature, supporting ADM and ADCs as relevant intermediate states in PDAC tumorigenesis. To further elucidate the genetic and epigenetic mechanisms of tumorigenesis, we will perform single-cell multiomics on resected patient specimens featuring precursor lesions with or without early invasive disease. Moreover, we are interested in identifying blood-based biomarkers of ADM and ADC to better enable early detection of precursor lesions and chemoprevention efforts, especially those that are not radiographically apparent (e.g., pancreatic intraepithelial neoplasia/PanIN).
Studying developmental lineages and mechanisms of metastasis in pancreatic neuroendocrine tumors using paired single-nucleus RNA-seq and whole exome sequencing
Pancreatic neuroendocrine tumors (PNETs) are diverse and derive from cells in the pancreatic islets. Some PNETs secrete excess hormones (functional) whereas others do not (non-functional). The clinical behavior of PNETs varies widely but approximately half of cases progress to metastases and cancer-related death after surgery but the molecular drivers of metastasis are not well understood. While roughly two-thirds of PNETs harbor mutations in MEN1, ATRX or DAXX and 15% exhibit activation of mammalian target of rapamycin signaling, molecular classification to date is not sufficiently informative to guide therapeutic management. We are adapting our snRNA-seq approach to PNETs and performing matched snRNA-seq and whole exome sequencing on primary and metastatic PNETs to better understand the genetic and transcriptomic mediators of metastasis as well as refine the molecular classification of PNETs to guide improved therapeutic strategies.