We have previously reported on a novel monoclonal antibody (mAb) we designated F5, which was raised against a glycopeptide derived from the tandem repeat (TR) region of Mucin-4 (MUC4), a heavily O-glycosylated protein that is overexpressed in many pancreatic cancer cells. This mAb was highly specific for the MUC4 glycopeptide antigen in glycan microarrays, ELISA and SPR assays, selectively stained tissue derived from advanced-stage tumors, and bound MUC4+ tumor cells in flow cytometry assays. The mAb was also unique in that it did not cross-react with other commercial anti-MUC4 mAbs that were raised in a similar but non-glycosylated TR sequence. Here we describe the selective conjugation of a novel near-infrared dye to this mAb and in vivo biodistribution of this labeled mAb to various MUC4-expressing tumors in mice. The labeled mAb were selectively distributed to both cell-derived xenograft (CDX) flank tumors and patient-derived xenograft (PDX) tumors that expressed MUC4 compared to those that were MUC4-negative. Organ distribution analysis showed high uptake in MUC4+ relative to MUC4− tumors. These results suggest that mAb F5 may be used to develop MUC4-targeted, passive antibody-based immunotherapies against Pancreatic Ductal Adenocarcinomas (PDACs) which are notorious for being refractory to many chemo- and radiotherapies

Preclinical mouse models for ovarian cancer that faithfully recapitulate human disease on both histopathological and molecular levels are crucial for advancing novel interventions into the clinic. We developed three genetically engineered mouse (GEM) models for fallopian tube-originating ovarian cancer with the loss of Brca1, Trp53, and Rb expression driven by Pax8 or Ovgp1 promoters or virally induced directly in the oviductal epithelium. We profiled the tumors by histology and gene expression and compared them to a previously described ovarian cancer model derived from ovarian surface epithelium. Expression profiles from the oviductal and ovarian epithelium tumors fall within the four subtypes of human high-grade serous carcinoma (HGSC) and represent different patient subpopulations. Allograft tumor models derived from the GEMs are amenable to preclinical intervention studies and respond to standard of care therapies. These well-defined, tractable models present a valuable resource for assessing novel drugs and immunotherapies for patients with HGSC.

Abstract In pancreatic ductal adenocarcinoma (PDAC) the tumor microenvironment (TME) plays a crucial role in therapy resistance, metastasis, and recurrence. ProAgio is a novel therapeutic cytotoxin rationally designed to bind outside the RGD ligand pocket of the alphaV-beta3 integrin pair and then kill cells expressing this target. Pre-clinical studies demonstrated the safety and anti-tumor efficacy of ProAgio in animal models, and ProAgio is currently being tested in a Phase I clinical trial. The effect of ProAgio on TME cellular components has not been closely examined. We hypothesized that ProAgio remodels the PDAC TME to decrease stromal density and local immunosuppression by killing stromal cells thought to express the integrin pair, such as cancer associated fibroblasts (CAFs), proliferating endothelial cells, and tumor associated macrophages (TAMs). To identify potential cellular targets of ProAgio in the PDAC TME, we re-analyzed publicly available single cell RNA-seq datasets of human and mouse PDAC samples. We assessed integrin co-expression using flow cytometry, performed immunophenotyping and histopathological analysis to identify TME cellular and structural alterations induced by ProAgio. Integrins αV and β3 were both expressed at the RNA level in TAMs, myeloid cells, lymphocytes (T, B and NK), endothelial cells and CAFs. In the autochthonous KPC mouse model, we confirmed that granulocytes, macrophages, monocytes, CD4+ and CD8+T cells co-expressed integrin αVβ3 at the protein level. Amongst CAFs, inflammatory CAFs (iCAFs) had the highest percentage of cells co-expressing αV and β3 integrins. No anti-tumor effect was observed after 14 days of ProAgio treatment in a syngeneic KPC-derived PDAC orthotopic model, however, in the KPC autochthonous model the same treatment retarded tumor growth within 1 week. ProAgio treated autochthonous tumors displayed no changes in immune components or CAF subsets, but increased collagen area was observed. In addition, ProAgio treatment resulted in higher blood vessel area while integrin β3 protein levels remained stable. In conclusion, short-course ProAgio remodels the TME and delays tumor growth in an autochthonous PDAC model with mature stromal components, but does not deplete CAFs, TAMs or collagen area. Future studies will seek to refine our understanding of the cell subtypes affected by more prolonged ProAgio treatment and to determine the cellular and inflammatory mediators responsible for these changes. Citation Format: Mayrel Palestino Dominguez, Philip Homan, Xianyu Zhang, Sandra Navas Reyes, Theresa Guerin, Laura Bassel, Liu Zhi-ren, Serguei Kozlov, Christine Alewine. Targeting integrin alpha V beta 3 remodels the tumor microenvironment in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B044.

Pancreatic adenocarcinoma (PDAC) is one the most intractable cancers, in part due to its highly inflammatory microenvironment and paucity of infiltrating dendritic cells (DCs). Here, we find that genetic ablation or antibody blockade of tumor necrosis factor receptor 1 (TNFR1) enhanced intratumor T cell activation and slowed PDAC growth. While anti-PD-1 checkpoint inhibition alone had little effect, it further enhanced intratumor T cell activation in combination with anti-TNFR1. The major cellular alteration in the tumor microenvironment in the absence of TNFR1 signaling was a large increase in DC number and immunostimulatory phenotype. This may reflect a direct effect on DCs, because TNF induced TNFR1-dependent apoptosis of bone-marrow-derived DCs. The therapeutic response to anti-TNFR1 alone was superior to the combination of DC-activating agonistic anti-CD40 and Flt3 ligand (Flt3L). These observations suggest that targeting TNFR1, perhaps in concert with other strategies that promote DC generation and mobilization, may have therapeutic benefits.

PDF file - 242K, EGFR-overexpressing tumors respond after 1 week of treatment by H&E staining and IHC for total EGFR, phospho-EGFR, and Ki-67

<p>Role of metabolic pathways imparting gemcitabine resistance in pancreatic cancer</p>

PDF file - 117K, Reduction of p-EGFR and p-S6 in BIBW 2992 and rapamycin-treated lungs at 4, 8, and 24 hours post-treatment

PDF file - 117K, Reduction of p-EGFR and p-S6 in BIBW 2992 and rapamycin-treated lungs at 4, 8, and 24 hours post-treatment

Legends for the three Supplementary Figures.

<p>Effects of glutamine deprivation on cell viability and NRF2 expression</p>

PDF file - 92K, LC-MS/MS validation for selected metabolites

All-trans-retinoic acid (ATRA), the retinoic acid receptors (RARs) agonist, regulates cell growth, differentiation, immunity, and survival. We report that ATRA-treatment repressed cancer growth in syngeneic immunocompetent, but not immunodeficient mice. The tumor microenvironment was implicated: CD8+ T cell depletion antagonized ATRA’s anti-tumorigenic effects in syngeneic mice. ATRA-treatment with checkpoint blockade did not cooperatively inhibit murine lung cancer growth. To augment ATRA’s anti-tumorigenicity without promoting its pro-tumorigenic potential, an RARγ agonist (IRX4647) was used since it regulates T cell biology. Treating with IRX4647 in combination with an immune checkpoint (anti-PD-L1) inhibitor resulted in a statistically significant suppression of syngeneic 344SQ lung cancers in mice—a model known for its resistance to checkpoints and characterized by low basal T cell and PD-L1 expression. This combined treatment notably elevated CD4+ T-cell presence within the tumor microenvironment and increased IL-5 and IL-13 tumor levels, while simultaneously decreasing CD38 in the tumor stroma. IL-5 and/or IL-13 treatments increased CD4+ more than CD8+ T-cells in mice. IRX4647-treatment did not appreciably affect in vitro lung cancer growth, despite RARγ expression. Pharmacokinetic analysis found IRX4647 plasma half-life was 6 h in mice. Yet, RARα antagonist (IRX6696)-treatment with anti-PD-L1 did not repress syngeneic lung cancer growth. Together, these findings provide a rationale for a clinical trial investigating an RARγ agonist to augment check point blockade response in cancers.

<p>Isotype control for NRF2 in IHC and survival measure for NRF2</p>

Cell cycle analysis of PDAC cells upon NRF2 activation

<p>Effects of NRF2 activation on metabolomics</p>

<p>Gemcitabine IC50 Values</p>

All-trans-retinoic acid (ATRA), a pan-agonist for retinoic acid receptors (RARs), regulates diverse cellular functions including growth, differentiation and immune function. We report here that ATRA-treatment represses tumor growth in syngeneic, immunocompetent but not in immunodeficient mice. Tumor immune microenvironment was implicated since depletion of cytotoxic T lymphocytes antagonized these effects in syngeneic mice. Combining ATRA with immune checkpoint blockade did not inhibit lung cancer growth in mice. We sought to augment retinoid anti-tumor effects without affecting its pro-tumorigenicity. We previously reported that CD38 mediated resistance to checkpoint blockade in murine 344SQ lung cancer cells via RARα transcriptional activation of CD38 expression. Yet, combining the RARα antagonist (IRX6696) with anti-PD-L1 did not augment anti-tumorigenicity in transplanted 344SQ cells in syngeneic mice. Prior work implicated RARγ in regulating T cell response. Combining the novel RARγ agonist (IRX4647) with anti-PD-L1 statistically-significantly repressed 344SQ lung cancer cell growth in syngeneic mice. This line is relatively resistant to checkpoint blockade. Immunofluorescent analysis of these treated tumors revealed that combined IRX4647 and anti-PD-L1 treatments reduced CD38 expression in the tumor stroma relative to IRX4647 or anti-PD-L1 treatment alone. Statistically-significantly elevated helper (CD4+) T cells were detected in treated tumors along with increased IL-5 and IL-13 expression observed in plasma and tumors. These cytokines can activate helper T cells, altering lung cancer growth. These microenvironment effects were associated with in vivo anti-tumorigenicity. IRX4647-treatment did not appreciably alter in vitro growth of lung cancer cells although retinoid receptors expression profiles were affected. Pharmacokinetic study of IRX4647 found its plasma half-life was 6 hours. Combining an RARγ agonist with immune checkpoint blockade exerted superior anti-neoplastic efficacy against lung cancer versus an ATRA-based regimen. Given these findings we propose exploring activity of this RARγ agonist with an optimal checkpoint inhibitor in a lung cancer clinical trial. Citation Format: Cheng-Hsin Wei, Lu Huang, Blair Kreh, Xiuxia Liu, Liliya Tyutyunyk-Massey, Zibo Chen, Mi Shi, Vidyasagar Vuligonda, Martin Sanders, Serguei Kozlov, King Chan, Amir Horowitz, Mary Carrington, Patrick Hwu, Weiyi Peng, Ethan Dmitrovsky, Xi Liu. Combining a novel retinoic acid receptor-γ agonist with immune checkpoint blockade represses lung cancer growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1836.