Research

Our growing collection of samples in a real world setting enables us to address multiple questions about cancer cell and TME biology. In an initial study, we performed scSeq on freshly collected human PDAC samples either before or after chemotherapy. Overall, we found a heterogeneous mixture of basal and classical cancer cell subtypes, along with distinct cancer-associated fibroblast and macrophage subpopulations. Strikingly, classical and basal-like cancer cells exhibited similar transcriptional responses to chemotherapy and did not demonstrate a shift towards a basal-like transcriptional program among treated samples. We observed decreased ligand-receptor interactions in treated samples, particularly TIGIT on CD8+ T cells and its receptor on cancer cells, and identified TIGIT – not PD1 – as the major inhibitory checkpoint molecule of CD8+ T cells. Our results suggest that chemotherapy profoundly impacts the PDAC TME and may actually promote resistance to immunotherapy. 

Our ongoing projects focus on the differences between the tumor microenvironment in primary PDAC and liver metastases, CNV analysis of cancer cells to assesses subclonal growth patterns and interacting neighborhoods within tumors, and using matched pre- and on-treatment samples from the same patient to get a deeper understanding of response to therapy and identify predictive biomarkers for clinical applications. In these studies, we are incorporating multiplex IHC and spatial transcriptomic analysis to define the spatial relationships of our findings.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer characterized by profound desmoplasia and cellular heterogeneity, which cannot be fully resolved using traditional bulk sequencing approaches. To understand the contribution of this heterogeneity to PDAC biology, we analyzed a large cohort of primary human PDAC samples (n = 62), profiling 443,451 single cells and 53,236 spatial transcriptomic spots using a combined single-cell RNA sequencing and spatial transcriptomics approach. Our analysis revealed significant intratumoral heterogeneity, with multiple genetically distinct neoplastic clones co-existing within individual tumors. These clones exhibited diverse transcriptional states and subtype profiles, challenging the traditional binary classification of PDAC into basal and classical subtypes; instead, our findings support a transcriptional continuum influenced by clonal evolution and spatial organization. Additionally, these clones each interacted uniquely with surrounding cell types in the tumor microenvironment. Phylogenetic analysis uncovered a rare but consistent classical-to-basal clonal transition associated with MYC amplification and immune response depletion, which were validated experimentally, suggesting a mechanism driving the emergence of a more aggressive basal clonal phenotype. Spatial analyses further revealed dispersed clones enriched for epithelial-to-mesenchymal transition (EMT) activity and immune suppression, correlating with metastatic potential and colonization of lymph node niches. These dispersed clones tended to transition toward a basal phenotype, contributing to disease progression. Our findings highlight the critical role of clonal diversity, transcriptional plasticity, and TME interactions in shaping human PDAC biology. This work provides new insights into the molecular and spatial heterogeneity of PDAC and offers potential avenues for therapeutic intervention targeting clonal evolution and the mechanisms driving metastasis.

Our scRNAseq and spatial transcriptomics analysis revealed a rare but consistent classical-to-basal clonal transition associated with MYC amplification and immune response depletion in PDAC. We validated this finding experimentally using our signature genetically engineered mouse models (GEMMs), demonstrating a mechanism by which MYC drives the emergence of a more aggressive basal clonal phenotype. Importantly, we showed that knocking out MYC promoted a basal-to-classical transition of tumors, which typically correlates with higher treatment sensitivity and improved prognosis. However, the exact molecular mechanisms and therapeutic implications remain to be elucidated.

We aim to explore further the underlying mechanisms through which MYC influences these phenotypic transitions. Given that MYC is known to regulate transcription and alleviate transcriptional stress via liquid-liquid phase separation (LLPS), understanding these processes may reveal novel therapeutic opportunities for targeting MYC-driven phenotypic plasticity in PDAC. Our research will focus on two complementary aims: 1. Elucidate the role of MYC-mediated LLPS in transcriptional regulation during PDAC phenotypic plasticity. 2. Investigate the interplay between MYC amplification and immune microenvironment changes. 

KRAS is a critical oncogene frequently mutated in PDAC. KRAS G12C inhibitors have demonstrated clinical success with significant impact, but they benefit only a small subset of patients, and treatment resistance commonly emerges. With new KRAS inhibitors targeting different mutants or functioning as pan-RAS inhibitors in development, the mechanistic investigation is essential.

We are developing multiple RAS activity assays that combine pioneering work from Cell Signaling San Diego on RAS activity biosensors with our expertise in pancreatic cancer models. Our models range from patient-derived primary cells to organoids, xenografts, and genetically engineered mouse models. These assays will serve as a comprehensive platform to evaluate new RAS inhibitors, understand their mechanisms and efficacy, and address therapeutic resistance in PDAC.

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal malignancy. Recent genomic studies reveal that 20-25% of human PDA harbor recurrent mutations in genes involved in the DNA damage response, including BRCA1/2, PALB2, and ATM, which participate in homologous recombination (HR) repair. This subgroup of PDAs has emerged as a defined biological entity associated with a more aggressive disease course. Defects in HR in these tumors impart cells with a specific vulnerability to poly-ADP ribose polymerase (PARP) inhibitors and platinum-containing chemotherapy. Still, only a fraction of HR-defective patients responds to PARP inhibition. More so, many patients that initially respond often develop resistance and progress. Therefore, new therapies that can be effective against HR-defective PDA, alone or in combination with PARP inhibitors or other combinatorial regimens, are needed.

We have determined that inactivation of the HR pathway in PDA is associated with overexpression of polymerase theta (PolƟ, also known as POLQ). POLQ is a key enzyme that regulates the alternative non-homologous end-joining (alt-NHEJ) pathway of double-strand break (DSB) repair. In this study, in partnership with our collaborator Dr. Agnel Sfeir ( Skirball Institute, NYU) we will use a large portfolio of HR-defective PDA model systems, both murine and human, to interrogate the impact of POLQ levels on mutational load and genomic stability, tumor growth characteristics and the role of the alt-NHEJ pathway in mediating these effects. With POLQ inhibitors in clinical development, we seek to establish the pre-clinical data set that guide the use of these inhibitors for pancreatic cancer patients into the clinic.

This has indeed been shown to be the case with BRCA1 and BRCA2 mutant PDAC, which display outlier responses to platinum agents and PARP inhibitors, resulting in the first potential subtype of PDAC (beyond the 1% of pancreatic cancers that have mutations in mismatch repair genes and respond more favorably to checkpoint blockade). Mutations commonly associated with DDR are in the genes BRCA1, BRCA2, and ATM.

ATM, a DDR gene that serves as a master regulator gene of the DDR, is upstream of both BRCA1 and BRCA2 in the DDR. ATM is a serine/threonine protein kinase that regulates cell cycle arrest, DNA repair and apoptosis.  ATM germline mutations occur in roughly 4% of PDAC patients, and ATM sporadic mutations have been detected in up to 18% of PDACs (Russell et al., 2015), with the majority been missense mutations potentially impairing its kinase activity (Cremona et al., 2014). ATM germline and sporadic mutations are also present in other tumor types, including breast and gastric cancer, however therapeutic strategies to target ATM overall are not well developed.

The goal of this project is to leverage out large portfolio of ATM mutant human PDAC cell lines and genetically engineered mouse models to develop a tiered strategy to design new, more effective therapeutics to target ATM-mutant pancreatic tumors. Mouse and patient derived-ATM mutant PDAC cell lines will be used to screen standard therapies (e.g. platinums and PARP inhibitors) and perform a large scale, unbiased screen to identify targets/pathways that result in novel synthetic lethality.

 In further studies by our group to evaluate the tumorigenic function of ATDC, we generated ATDC;LSL-KrasG12D;p48-Cre (AKC) mouse in which ATDC is overexpressed in the pancreas in the setting of oncogenic KrRAS mutations. We observed that LSL-KrasG12D;p48-Cre (KC) mice developed PanINs with a long latency, and PDAC was not observed until mice reached an age of 15 months or greater. However, ATDC overexpression combined with KrasG12D accelerated PanIN progression and 85% of AKC mice at 6-8 months of age developed highly invasive and metastatic PDAC, whereas none of the age-matched KC mice displayed those lesions. It suggests, as an invasive trigger, ATDC promotes the biologic aggressiveness of pancreatic cancer. In a follow-up study, Also, by the generation of LSL-KrasG12D; p53-/+; pdx1-Cre; ATDC-/- (KPCA) mice, we demonstrated that deletion of ATDC prevents KRAS-driven acinar– ductal metaplasia (ADM), its progression to pancreatic intraepithelial neoplasia (PanIN), and PDAC development. These novel mouse models provide a platform for an improved understanding of the pathogenesis of PDAC and the development of targeted treatment strategies. Furthermore, we found that ATDC not only causes the cancer cells to grow faster and be more aggressive, but it also makes the cancer cells particularly resistant to chemotherapy and radiation.

By targeting ATDC, we may be able to make cancer cells more sensitive to the therapies we already have in hand. Efforts to develop new therapeutics targeting ATDC are underway. 

ATDC also appears to be critical to the function of other cancer types, including bladder cancer and lung cancer. We are continuing to investigate the biological function of ATDC in those cancers.