Fernández-Zapico, Martín E.

Abstract The sparse vascularity of pancreatic ductal adenocarcinoma (PDAC) presents a mystery: What prevents this aggressive malignancy from undergoing neoangiogenesis to counteract hypoxia and better support growth? An incidental finding from prior work on paracrine communication between malignant PDAC cells and fibroblasts revealed that inhibition of the Hedgehog (HH) pathway partially relieved angiosuppression, increasing tumor vascularity through unknown mechanisms. Initial efforts to study this phenotype were hindered by difficulties replicating the complex interactions of multiple cell types in vitro. Here we identify a cascade of paracrine signals between multiple cell types that act sequentially to suppress angiogenesis in PDAC. Malignant epithelial cells promote HH signaling in fibroblasts, leading to inhibition of noncanonical WNT signaling in fibroblasts and epithelial cells, thereby limiting VEGFR2-dependent activation of endothelial hypersprouting. This cascade was elucidated using human and murine PDAC explant models, which effectively retain the complex cellular interactions of native tumor tissues. Significance: We present a key mechanism of tumor angiosuppression, a process that sculpts the physiologic, cellular, and metabolic environment of PDAC. We further present a computational and experimental framework for the dissection of complex signaling cascades that propagate among multiple cell types in the tissue environment. This article is featured in Selected Articles from This Issue, p. 201

Aberrant activation of the Hedgehog (Hh) signaling pathway, through which the GLI family of transcription factors (TF) is stimulated, is commonly observed in cancer cells. One well-established mechanism of this increased activity is through the inactivation of Suppressor of Fused (SUFU), a negative regulator of the Hh pathway. Relief from negative regulation by SUFU facilitates GLI activity and induction of target gene expression. Here, we demonstrate a novel role for SUFU as a promoter of GLI activity in pancreatic ductal adenocarcinoma (PDAC). In non-ciliated PDAC cells unresponsive to Smoothened agonism, SUFU overexpression increases GLI transcriptional activity. Conversely, knockdown (KD) of SUFU reduces the activity of GLI in PDAC cells. Through array PCR analysis of GLI target genes, we identified B-cell lymphoma 2 (BCL2) among the top candidates down-regulated by SUFU KD. We demonstrate that SUFU KD results in reduced PDAC cell viability, and overexpression of BCL2 partially rescues the effect of reduced cell viability by SUFU KD. Further analysis using as a model GLI1, a major TF activator of the GLI family in PDAC cells, shows the interaction of SUFU and GLI1 in the nucleus through previously characterized domains. Chromatin immunoprecipitation (ChIP) assay shows the binding of both SUFU and GLI1 at the promoter of BCL2 in PDAC cells. Finally, we demonstrate that SUFU promotes GLI1 activity without affecting its protein stability. Through our findings, we propose a novel role of SUFU as a positive regulator of GLI1 in PDAC, adding a new mechanism of Hh/GLI signaling pathway regulation in cancer cells.

Abstract Background We sought to determine the safety and efficacy of trifluridine/tipiracil in combination with irinotecan in a phase II trial setting for refractory, advanced unresectable biliary tract carcinoma (BTC). Methods A total of 28 patients (27 were evaluable) with advanced BTCs who progressed on at least one prior systemic therapy were enrolled and were treated with trifluridine/tipiracil 25 mg/m2 (days 1-5 of 14-day cycle) and irinotecan 180 mg/m2 (day 1 of the 14-day cycle). The primary endpoint for the study was 16-week progression-free survival (PFS16) rate. Overall survival (OS), progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), and safety were pre-specified secondary endpoints. Results Of 27 patients, PFS16 rate was 37% (10/27; 95% CI: 19%-58%), thereby meeting the criteria for success for the primary endpoint. The median PFS and OS of the entire cohort were 3.9 months (95% CI: 2.5-7.4) and 9.1 months (95% CI: 8.0-14.3), respectively. In the patients evaluable for tumor response (n = 20), the ORR and DCR were 10% and 50%, respectively. Twenty patients (74.1%) had at least one grade 3 or worse adverse event (AE), and 4 patients (14.8%) had grade 4 AEs. A total of 37% (n = 10/27) and 51.9% (n = 14/27) experienced dose reductions in trifluridine/tipiracil and irinotecan, respectively. Delay in therapy was noted in 56% of the patients while 1 patient discontinued the therapy, primarily due to hematologic AEs. Conclusion The combination of trifluridine/tipiracil plus irinotecan is a potential treatment option for patients with advanced, refractory BTCs with good functional status and no targetable mutations. A larger randomized trial is needed to confirm these results. (ClinicalTrials.gov Identifier: NCT04072445)

Abstract Purpose: Preclinical studies in myeloid neoplasms have demonstrated efficacy of bromodomain and extra-terminal protein inhibitors (BETi). However, BETi demonstrates poor single-agent activity in clinical trials. Several studies suggest that combination with other anticancer inhibitors may enhance the efficacy of BETi. Experimental Design: To nominate BETi combination therapies for myeloid neoplasms, we used a chemical screen with therapies currently in clinical cancer development and validated this screen using a panel of myeloid cell line, heterotopic cell line models, and patient-derived xenograft models of disease. We used standard protein and RNA assays to determine the mechanism responsible for synergy in our disease models. Results: We identified PIM inhibitors (PIMi) as therapeutically synergistic with BETi in myeloid leukemia models. Mechanistically, we show that PIM kinase is increased after BETi treatment, and that PIM kinase upregulation is sufficient to induce persistence to BETi and sensitize cells to PIMi. Furthermore, we demonstrate that miR-33a downregulation is the underlying mechanism driving PIM1 upregulation. We also show that GM-CSF hypersensitivity, a hallmark of chronic myelomonocytic leukemia (CMML), represents a molecular signature for sensitivity to combination therapy. Conclusions: Inhibition of PIM kinases is a potential novel strategy for overcoming BETi persistence in myeloid neoplasms. Our data support further clinical investigation of this combination.

AbstractIt has been challenging to target mutant KRAS (mKRAS) in colorectal cancer (CRC) and other malignancies. Recent efforts have focused on developing inhibitors blocking molecules essential for KRAS activity. In this regard, SOS1 inhibition has arisen as an attractive approach for mKRAS CRC given its essential role as a guanine nucleotide exchange factor for this GTPase. Here, we demonstrated the translational value of SOS1 blockade in mKRAS CRC. We used CRC patient‐derived organoids (PDOs) as preclinical models to evaluate their sensitivity to SOS1 inhibitor BI3406. A combination of in silico analyses and wet lab techniques was utilized to define potential predictive markers for SOS1 sensitivity and potential mechanisms of resistance in CRC. RNA‐seq analysis of CRC PDOs revealed two groups of CRC PDOs with differential sensitivities to SOS1 inhibitor BI3406. The resistant group was enriched in gene sets involving cholesterol homeostasis, epithelial−mesenchymal transition, and TNF‐α/NFκB signaling. Expression analysis identified a significant correlation between SOS1 and SOS2 mRNA levels (Spearman's ρ 0.56, p < 0.001). SOS1/2 protein expression was universally present with heterogeneous patterns in CRC cells but only minimal to none in surrounding nonmalignant cells. Only SOS1 protein expression was associated with worse survival in patients with RAS/RAF mutant CRC (p = 0.04). We also found that SOS1/SOS2 protein expression ratio >1 by immunohistochemistry (p = 0.03) instead of KRAS mutation (p = 1) was a better predictive marker to BI3406 sensitivity of CRC PDOs, concordant with the significant positive correlation between SOS1/SOS2 protein expression ratio and SOS1 dependency. Finally, we showed that GTP‐bound RAS level underwent rebound even in BI3406‐sensitive PDOs with no change of KRAS downstream effector genes, thus suggesting upregulation of guanine nucleotide exchange factor as potential cellular adaptation mechanisms to SOS1 inhibition. Taken together, our results show that high SOS1/SOS2 protein expression ratio predicts sensitivity to SOS1 inhibition and support further clinical development of SOS1‐targeting agents in CRC.

Carcinoma-associated fibroblasts (CAFs) play an important role in the progression of multiple malignancies. Secretion of cytokines and growth factors underlies the pro-tumoral effect of CAFs. Although this paracrine function has been extensively documented, the molecular mechanisms controlling the expression of these factors remain elusive. In this study, we provide evidence of a novel CAF transcriptional axis regulating the expression of SDF1, a major driver of cancer cell migration, involving the transcription factor GLI1 and histone acetyltransferase p300. We demonstrate that conditioned media from CAFs overexpressing GLI1 induce the migration of pancreatic cancer cells, and this effect is impaired by an SDF1-neutralizing antibody. Using a combination of co-immunoprecipitation, proximity ligation assay and chromatin immunoprecipitation assay, we further demonstrate that GLI1 and p300 physically interact in CAFs to co-occupy and drive SDF1 promoter activity. Mapping experiments highlight the requirement of GLI1 N-terminal for the interaction with p300. Importantly, knockdowns of both GLI1 and p300 reduce SDF1 expression. Further analysis shows that knockdown of GLI1 decreases SDF1 promoter activity, p300 recruitment, and levels of its associated histone marks (H4ac, H3K27ac, and H3K14ac). Finally, we show that the integrity of two GLI binding sites in the SDF1 promoter is required for p300 recruitment. Our findings define a new role for the p300–GLI1 complex in the regulation of SDF1, providing new mechanistic insight into the molecular events controlling pancreatic cancer cells migration.

Abstract Pancreatic ductal adenocarcinoma (PDAC) affects many older adults in the United States. This aggressive type of pancreatic cancer is resistant to many chemotherapies, resulting in a poor prognosis and a five-year survival rate of less than ten percent. To study PDAC progression and develop novel therapies, it is important to better understand the tumor microenvironment (TME) and the contributing cell populations and molecular mechanisms. Despite the well-described role of soluble Type I collagen in the regulation of pancreatic cancer TME, the molecular events modulating its expression/activity remain elusive. Here, we describe a novel mechanism controlling the levels of soluble collagen in cancer associated fibroblasts (CAFs), a major producer of Type I collagen in pancreatic cancer TME. Specifically, we provide evidence that the transcription factor GLI2 is required for the expression of COL1A1, a key component of the Type I collagen fiber, stimulated by the transforming growth factor β (TGFβ). Our methodology included expression studies from patient samples, ChIP-qPCR, qPCR, RNA-seq analysis, gene set enrichment analysis, biological assays, and gene ontology analysis. Our results confirm that the TGFβ-GLI2 axis in CAFs is able to activate EGR1-proliferative signaling downstream of collagen signaling in pancreatic cancer cells contributing to our understanding of the molecular underpinnings of pancreatic cancer TME. Further studies that define the complete role of EGR1 in PDAC may lead to the development of novel therapies targeting EGR1 or CAFs to promote enhanced quality of life following a PDAC diagnosis.

BACKGROUND A patient-derived organoid (PDO) platform may serve as a promising tool for translational cancer research. In this study, we evaluated PDO’s ability to predict clinical response to gastrointestinal (GI) cancers. METHODS We generated PDOs from primary and metastatic lesions of patients with GI cancers, including pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and cholangiocarcinoma. We compared PDO response with the observed clinical response for donor patients to the same treatments. RESULTS We report an approximately 80% concordance rate between PDO and donor tumor response. Importantly, we found a profound influence of culture media on PDO phenotype, where we showed a significant difference in response to standard-of-care chemotherapies, distinct morphologies, and transcriptomes between media within the same PDO cultures. CONCLUSION While we demonstrate a high concordance rate between donor tumor and PDO, these studies also showed the important role of culture media when using PDOs to inform treatment selection and predict response across a spectrum of GI cancers. TRIAL REGISTRATION Not applicable. FUNDING The Joan F. & Richard A. Abdoo Family Fund in Colorectal Cancer Research, GI Cancer program of the Mayo Clinic Cancer Center, Mayo Clinic SPORE in Pancreatic Cancer, Center of Individualized Medicine (Mayo Clinic), Department of Laboratory Medicine and Pathology (Mayo Clinic), Incyte Pharmaceuticals and Mayo Clinic Hepatobiliary SPORE, University of Minnesota-Mayo Clinic Partnership, and the Early Therapeutic program (Department of Oncology, Mayo Clinic).