Faculty Bibliography

Non-small cell lung cancers (NSCLCs) harboring KEAP1 mutations are often resistant to immunotherapy. Here, we show that KEAP1 targets EMSY for ubiquitin-mediated degradation to regulate homologous recombination repair (HRR) and anti-tumor immunity. Loss of KEAP1 in NSCLC induces stabilization of EMSY, producing a BRCAness phenotype, i.e., HRR defects and sensitivity to PARP inhibitors. Defective HRR contributes to a high tumor mutational burden that, in turn, is expected to prompt an innate immune response. Notably, EMSY accumulation suppresses the type I interferon response and impairs innate immune signaling, fostering cancer immune evasion. Activation of the type I interferon response in the tumor microenvironment using a STING agonist results in the engagement of innate and adaptive immune signaling and impairs the growth of KEAP1-mutant tumors. Our results suggest that targeting PARP and STING pathways, individually or in combination, represents a therapeutic strategy in NSCLC patients harboring alterations in KEAP1.

Macropinocytosis, an evolutionarily conserved endocytic mechanism that mediates non-specific fluid-phase uptake, is potently upregulated by various oncogenic pathways. It is now well appreciated that high macropinocytic activity is a hallmark of many human tumors, which use this adaptation to scavenge extracellular nutrients for fueling cell growth. In the context of the nutrient-scarce tumor microenvironment, this process provides tumor cells with metabolic flexibility. However, dependence on this scavenging mechanism also illuminates a potential metabolic vulnerability. As such, there is a great deal of interest in understanding the molecular underpinnings of macropinocytosis. In this review, we will discuss the most recent advances in characterizing macropinocytosis: the pathways that regulate it, its contribution to the metabolic fitness of cancer cells, and its therapeutic potential.

Tumor genotype can influence the immune microenvironment, which plays a critical role in cancer development and therapy resistance. However, the immune effects of gain-of-function Trp53 mutations have not been defined in pancreatic cancer. We compare the immune profiles generated by Kras^G12D-mutated mouse pancreatic ductal epithelial cells (PDECs) engineered genetically to express the Trp53^R172H mutation with their p53 wild-type control. Kras^G12D/+;Trp53^R172H/+ tumors have a distinct immune profile characterized by an influx of CD11b⁺Ly6G⁺ neutrophils and concomitant decreases in CD3⁺ T cells, CD8⁺ T cells, and CD4⁺ T helper 1 cells. Knockdown of CXCL2, a neutrophil chemokine, in the tumor epithelial compartment of CRISPR Kras^G12D/+;Trp53^R172H/+ PDEC tumors reverses the neutrophil phenotype. Neutrophil depletion of mice bearing CRISPR Kras^G12D/+;Trp53^R172H/+ tumors augments sensitivity to combined CD40 immunotherapy and chemotherapy. These data link Trp53^R172H to the presence of intratumoral neutrophils in pancreatic cancer and suggest that tumor genotypes could inform selection of affected individuals for immunotherapy.

Immune checkpoint blockade (ICB) has demonstrated efficacy in multiple cancers, offering the potential of long-term disease control not achievable with cytotoxic or targeted therapies. However, the field has not yet achieved the crucial next steps - the expansion of the response rate and achievement of clinical efficacy in so-called "cold tumours". Mechanistic studies of tumour-type specific immunosuppressive pathways can reveal underlying biological hurdles to immunotherapy and offer new therapeutic insights. Our finding that tumour-derived IL-1β mediates immunosuppression in pancreatic cancer has precipitated a new clinical trial.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States. Glycans, such as carbohydrate antigen 19-9, are biomarkers of PDAC and are emerging as important modulators of cancer phenotypes. Herein, we used a systems-based approach integrating glycomic analysis of the well-established KC mouse, which models early events in transformation, and analysis of samples from human pancreatic cancer patients to identify glycans with potential roles in cancer formation. We observed both common and distinct patterns of glycosylation in pancreatic cancer across species. Common alterations included increased levels of α-2,3-sialic acid and α-2,6-sialic acid, bisecting GlcNAc and poly-N-acetyllactosamine. However, core fucose, which was increased in human PDAC, was not seen in the mouse, indicating that not all human glycomic changes are observed in the KC mouse model. In silico analysis of bulk and single-cell sequencing data identified ST6 beta-galactoside alpha-2,6-sialyltransferase 1, which underlies α-2,6-sialic acid, as overexpressed in human PDAC, concordant with histological data showing higher levels of this enzyme at the earliest stages. To test whether ST6 beta-galactoside alpha-2,6-sialyltransferase 1 promotes pancreatic cancer, we created a novel mouse in which a pancreas-specific genetic deletion of this enzyme overlays the KC mouse model. The analysis of our new model showed delayed cancer formation and a significant reduction in fibrosis. Our results highlight the importance of a strategic systems approach to identifying glycans whose functions can be modeled in mouse, a crucial step in the development of therapeutics targeting glycosylation in pancreatic cancer.

Peptides and peptidomimetics represent the middle space between small molecules and large proteins-they retain the relatively small size and synthetic accessibility of small molecules while providing high binding specificity for biomolecular partners typically observed with proteins. During the course of our efforts to target intracellular protein-protein interactions in cancer, we observed that the cellular uptake of peptides is critically determined by the cell line-specifically, we noted that peptides show better uptake in cancer cells with enhanced macropinocytic indices. Here, we describe the results of our analysis of cellular penetration by different classes of conformationally stabilized peptides. We tested the uptake of linear peptides, peptide macrocycles, stabilized helices, β-hairpin peptides, and cross-linked helix dimers in 11 different cell lines. Efficient uptake of these conformationally defined constructs directly correlated with the macropinocytic activity of each cell line: high uptake of compounds was observed in cells with mutations in certain signaling pathways. Significantly, the study shows that constrained peptides follow the same uptake mechanism as proteins in macropinocytic cells, but unlike proteins, peptide mimics can be readily designed to resist denaturation and proteolytic degradation. Our findings expand the current understanding of cellular uptake in cancer cells by designed peptidomimetics and suggest that cancer cells with certain mutations are suitable mediums for the study of biological pathways with peptide leads.

Pancreatic ductal adenocarcinoma (PDAC) evolves a complex microenvironment comprised of multiple cell types, including pancreatic stellate cells (PSCs). Previous studies have demonstrated that stromal supply of alanine, lipids, and nucleotides supports the metabolism, growth, and therapeutic resistance of PDAC. Here we demonstrate that alanine crosstalk between PSCs and PDAC is orchestrated by the utilization of specific transporters. PSCs utilize SLC1A4 and other transporter(s) to rapidly exchange and maintain environmental alanine concentrations. Moreover, PDAC cells upregulate SLC38A2 to supply their increased alanine demand. Cells lacking SLC38A2 fail to concentrate intracellular alanine and undergo a profound metabolic crisis resulting in markedly impaired tumor growth. Our results demonstrate that stromal-cancer metabolic niches can form through differential transporter expression, creating unique therapeutic opportunities to target metabolic demands of cancer.

Protein-protein interactions (PPIs) play a critical role in fundamental biological processes. Competitive inhibition of these interfaces requires compounds that can access discontinuous binding epitopes along a large, shallow binding surface area. Conformationally- defined protein surface mimics present a viable route to target these interactions. However, the development of minimal protein mimics that engage intracellular targets with high affinity remains a major challenge because mimicry of a portion of the binding interface is often associated with the loss of critical binding interactions. Covalent targeting provides an attractive approach to overcome the loss of non-covalent contacts but have the inherent risk of dominating non-covalent contacts and increasing the likelihood of non-selective binding. Here, we report the iterative design of a proteolytically-stable helix mimic that covalently targets oncogenic G12C Ras as a model system. We explored several electrophiles to optimize preferential alkylation with the desired C12 on Ras. The designed lead peptide modulates nucleotide exchange, inhibits activation of the Ras-mediated signalling cascade, and is selectively toxic towards mutant G12C Ras cancer cells. The relatively high frequency of acquired cysteines as missense mutations in cancer and other diseases suggests that covalent peptides may offer an untapped therapeutic approach for targeting aberrant protein interactions.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy typified by a highly stromal and weakly immunogenic tumor microenvironment that promotes tumor evolution and contributes to therapeutic resistance. Here, we demonstrate that PDA tumor cell-derived pro-inflammatory cytokine, interleukin-1β (IL-1β) is essential for the establishment of the pro-tumorigenic PDA microenvironment. Tumor cell-derived IL-1β promoted the activation and secretory phenotype of quiescent pancreatic stellate cells (PSC) and established an immunosuppressive milieu mediated by M2 macrophages, MDSCs, CD1dhiCD5+ regulatory B cells and Th17 cells. Loss of tumor cell-derived IL-1 signaling in tumor stroma enabled intra-tumoral infiltration and activation of CD8+ cytotoxic T cells, attenuated growth of pancreatic neoplasia and conferred survival advantage to PDA bearing mice. Accordingly, antibody-mediated neutralization of IL-1β significantly enhanced the anti-tumor activity of α-PD-1, and was accompanied by increased tumor infiltration of CD8+ T cells. Tumor cell expression of IL-1β in vivo was driven by microbial-dependent activation of toll-like receptor 4 (TLR4) signaling and subsequent engagement of the NLRP3 inflammasome. Collectively, these findings identify a hitherto unappreciated role for tumor cell-derived IL-1β in orchestrating an immune modulatory program that supports pancreatic tumorigenesis.