Khazaie, Khashayarsha

Abstract Bacteriolytic strategies offer unique advantages to combat a broad range of cancers often refractive to conventional chemotherapies and/or radiotherapies. C. novyi-NT is an attenuated strain of Clostridium novyi, a spore-forming, gram-positive, obligate anaerobe that lacks a lethal alpha- toxin, expressed by the parental strain. When administered intravenously or intratumorally with percutaneous injection, C. novyi-NT colonizes and replicates within the hypoxic region of the tumors, eliciting robust, tumor-confined cell lysis. In this first-in-man phase 1 study, patients with injectable treatment-refractory solid tumors received a single intratumoral injection of C. novyi-NT spores across 6 dose cohorts (spore concentrations of 104, 3x104, 105, 3x105, 106, 3x106) using a 3+3 trial design. The primary endpoints were to assess the safety profile, the dose limiting toxicity, and the maximum tolerated dose of C. novyi-NT. Key secondary endpoints included assessments of the preliminary antitumor activity of the injected tumor, an overall response evaluated by RECIST v. 1.1, and the host immune and inflammatory response to C. novyi-NT. Twenty-four patients were enrolled between November, 2013 and April, 2017. A single intratumoral injection of C. novyi-NT led to germination and resultant tumor lysis of injected tumor masses in 46% of patients across all dosing cohorts. The cohort 5 dose of 106 spores was defined as the maximum tolerated dose. Dose-limiting toxicities were grade 4 sepsis and grade 4 gas gangrene (n=1), all in patients with germination. In the 22 evaluable patients, 21 (95%) had stable disease (SD) as the best response for the injected lesion (tumor shrinkage of > 10% was observed in 21% of patients) and 19 (86%) had overall SD as the best response per RECIST 1.1. C. novyi-NT injection resulted in transient serum cytokine responses consistent with inflammasome activity, activation of innate immunity, tissue remodeling, and angiogenesis. Tumor antigen specific ELISPOT assays pre- and post- treatment documented enhanced secretion of IFNγ and TNFα by circulating T-cells, indicating improved systemic tumor specific T-cell responses. Multiparametric in situ immunostaining of core biopsies suggested improved immune cell infiltration in metastatic lesions. These early signs of improved antitumor activity in patients with advanced solid tumors have encouraged a new trial of C. novyi-NT in combination with immune checkpoint inhibitors. Citation Format: Filip Janku, Mrinal Gounder, Abdul Mohammad Pezeshki, Ravi Murthy, Andrea Wang-Gillam, Dale Shepard, David S. Hong, Sarina A. Piha-Paul, Anjali Raina, Alexey A. Leontovich, Gary DeCrescenzo, Brent L. Kreider, David Tung, Mary Varterasian, Halle H. Zhang, Khashayarsha Khazaie. First-in-man clinical trial of intratumoral injection of Clostridiumnovyi-NT spores in patients with treatment-refractory advanced solid tumors: Safety, activity, and immune responses [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A011.

Abstract In the present study we provide a temporal and mechanistic explanation of how β-catenin signaling skews regulatory T cells (Treg) into a pathogenic, pro-inflammatory phenotype that co-express the Th17 lineage transcription factor RORγt along with Foxp3 during colon cancer (CC) development. We previously described this distinct subset of Tregs in the peripheral blood (PB) of established CC patients that suppresses effector T cell functions but simultaneously maintains inflammation by failing to regulate mast cell degranulation and production of IL-17. Moreover, we showed in mice that RORγt expression correlates with elevated β-catenin levels in T cells and that RORγt is a transcriptional target of β-catenin interaction partners TCF-1 (Tcf7) and HEB (Tcf12). To elucidate how Wnt/β-catenin signaling orchestrates the induction of pathogenic properties in Tregs specifically we analyzed inflammatory bowel disease (IBD) patients' tissue and PB. As IBD can progress to colon cancer, this disease presents a unique platform to study the temporal emergence of RORγt+/Foxp3+ Tregs during the course of the disease. To further mechanistically establish how activated β-catenin signaling facilitates re-programming of Tregs and acquisition of pro-inflammatory properties we over-expressed β-catenin specifically in Tregs and/or knocked out its interaction partners TCF-1 and HEB in a Foxp3-Cre mouse model. Indeed, in a specific sub-population of IBD patients we were able to detect RORγt+/Foxp3+ double positive Tregs in the patients' blood and tissue, expressing high levels of β-catenin and producing IL-17 upon re-stimulation. Mice expressing β-catenin Treg-specifically showed striking X-linked immune pathology. Males died 4 weeks after birth of a severe scurfy-like phenotype, indicating failure of Treg function. β-catenin overexpressing Tregs in mice displayed a reduced suppressive capacity, higher rate of apoptosis and produced effector cytokines IL-17 and IFN-γ. Furthermore, the chromatin accessibility of bona fide Treg genes was drastically reduced, whereas accessibility in T cell activation and IL-17 signature genes increased compared to wild-type Tregs in ATACseq analysis. An additional Treg-specific knock-out of HEB or TCF-1 reduced or delayed auto-immunity, respectively, with increased survival in both cases and partial rescue of systemic Treg numbers. We herewith provide evidence that induction of β-catenin signaling is indeed responsible for the pathogenic, pro-inflammatory conversion of Tregs through activation of RORγt expression. Further transcriptome analysis will reveal how the epigenetic changes in the chromatin translate to gene expression. We anticipate, that this mechanistic insight into β-catenin mediated pathogenic conversion of Tregs will reveal novel targets for cancer immuno-therapy. Citation Format: Jasmin Quandt, Leila Haghi, Akinola O. Emmanuel, Khashayarsha Khazaie, Fotini Gounari. β-catenin signaling mediates pathogenic pro-inflammatory conversion of Foxp3+ Treg cells early during colon cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-281.

Epidemiological studies propose a protective role for dietary fiber in colon cancer (CRC). One possible mechanism of fiber is its fermentation property in the gut and ability to change microbiota composition and function. Here, we investigate the role of a dietary fiber mixture in polyposis and elucidate potential mechanisms using TS4Cre × cAPCl°x468 mice. Stool microbiota profiling was performed, while functional prediction was done using PICRUSt. Stool short-chain fatty acid (SCFA) metabolites were measured. Histone acetylation and expression of SCFA butyrate receptor were assessed. We found that SCFA-producing bacteria were lower in the polyposis mice, suggesting a decline in the fermentation product of dietary fibers with polyposis. Next, a high fiber diet was given to polyposis mice, which significantly increased SCFA-producing bacteria as well as SCFA levels. This was associated with an increase in SCFA butyrate receptor and a significant decrease in polyposis. In conclusion, we found polyposis to be associated with dysbiotic microbiota characterized by a decline in SCFA-producing bacteria, which was targetable by high fiber treatment, leading to an increase in SCFA levels and amelioration of polyposis. The prebiotic activity of fiber, promoting beneficial bacteria, could be the key mechanism for the protective effects of fiber on colon carcinogenesis. SCFA-promoting fermentable fibers are a promising dietary intervention to prevent CRC.

Significance We show that distinct subsets of mast cells (MCs) expand with sequential oncogenic events in small bowel cancer. Mucosal mast cells (MMCs) previously detected early during Trichinella spiralis infection expand in adenomatous polyps in an IL-10–dependent manner. Connective tissue mast cells (CTMCs), earlier shown to expand during the resolution of inflammation following clearance of T. spiralis, are independent of IL-10 and associate with the transition of polyps to adenocarcinoma. IL-33 upregulates the CTMC lineage-specific protease murine mast cell protease 6 (mMCP6). Ablation of mMCP6 attenuates tumor growth. Thus, tissue sentinel cells respond to oncogenic events and cellular transformation in effect to help promote cancer. Delineating the types of MCs present at various stages of disease offers actionable cellular targets for therapeutic intervention in disease progression.

Abstract The interactions between programmed death-1 (PD-1) and its ligands hamper tumor-specific CD8+ T cell (TCD8) responses, and PD-1-based “checkpoint inhibitors” have shown promise in certain cancers, thus revitalizing interest in immunotherapy. PD-1–targeted therapies reverse TCD8 exhaustion/anergy. However, whether they alter the epitope breadth of TCD8 responses remains unclear. This is an important question because subdominant TCD8 are more likely than immunodominant clones to escape tolerance mechanisms and may contribute to protective anticancer immunity. We have addressed this question in an in vivo model of TCD8 responses to well-defined epitopes of a clinically relevant oncoprotein, large T Ag. We found that unlike other coinhibitory molecules (CTLA-4, LAG-3, TIM-3), PD-1 was highly expressed by subdominant TCD8, which correlated with their propensity to favorably respond to PD-1/PD-1 ligand-1 (PD-L1)-blocking Abs. PD-1 blockade increased the size of subdominant TCD8 clones at the peak of their primary response, and it also sustained their presence, thus giving rise to an enlarged memory pool. The expanded population was fully functional as judged by IFN-γ production and MHC class I–restricted cytotoxicity. The selective increase in subdominant TCD8 clonal size was due to their enhanced survival, not proliferation. Further mechanistic studies utilizing peptide-pulsed dendritic cells, recombinant vaccinia viruses encoding full-length T Ag or epitope mingenes, and tumor cells expressing T Ag variants revealed that anti–PD-1 invigorates subdominant TCD8 responses by relieving their lysis-dependent suppression by immunodominant TCD8. To our knowledge, our work constitutes the first report that interfering with PD-1 signaling potentiates epitope spreading in tumor-specific responses, a finding with clear implications for cancer immunotherapy and vaccination.

Background: Colorectal cancer (CRC) is associated with the modern lifestyle. Chronic alcohol consumption—a frequent habit of majority of modern societies—increases the risk of CRC. Our group showed that chronic alcohol consumption increases polyposis in a mouse mode of CRC. Here we assess the effect of circadian disruption—another modern life style habit—in promoting alcohol-associated CRC. Method: TS4Cre × adenomatous polyposis coli (APC)lox468 mice underwent (a) an alcohol-containing diet while maintained on a normal 12 h light:12 h dark cycle; or (b) an alcohol-containing diet in conjunction with circadian disruption by once-weekly 12 h phase reversals of the light:dark (LD) cycle. Mice were sacrificed after eight weeks of full alcohol and/or LD shift to collect intestine samples. Tumor number, size, and histologic grades were compared between animal groups. Mast cell protease 2 (MCP2) and 6 (MCP6) histology score were analyzed and compared. Stool collected at baseline and after four weeks of experimental manipulations was used for microbiota analysis. Results: The combination of alcohol and LD shifting accelerated intestinal polyposis, with a significant increase in polyp size, and caused advanced neoplasia. Consistent with a pathogenic role of stromal tryptase-positive mast cells in colon carcinogenesis, the ratio of mMCP6 (stromal)/mMCP2 (intraepithelial) mast cells increased upon LD shifting. Baseline microbiota was similar between groups, and experimental manipulations resulted in a significant difference in the microbiota composition between groups. Conclusions: Circadian disruption by Light:dark shifting exacerbates alcohol-induced polyposis and CRC. Effect of circadian disruption could, at least partly, be mediated by promoting a pro-tumorigenic inflammatory milieu via changes in microbiota.

Abstract The extent of T cell infiltrates in the primary tumor is one of the most important prognostic factors in colorectal cancer (CRC). Still, a putative causal role of T cell infiltrates for prolonged patient survival remains unclear. To answer this question, we studied the presence and functional activity of tumor infiltrating lymphocytes (TIL) together with their specificity in CRC patients and correlated the obtained results to long term survival. We have utilized intracytoplasmic cytokine staining, conjointly with human leukocyte antigen (HLA)- multimers loaded with peptides derived from tumor antigen (TA), as well as antigen specific cytokine secretion assays, to demonstrate that tumor necrosis factor alpha (TNFα) expression characterizes a population of TA specific, in situ active cytotoxic T cells. TNFα was increased in CRC tumors only when patients develop spontaneous systemic T cell responses, as elucidated by IFN-γ Elispot, and this increase correlated with the number of TNFα expressing tumor infiltrating T cells. Based on these observations we concluded that intratumoral TNFα can be a direct indicator of the in situ functionality of tumor specific effector T cells in CRC. In accordance, intratumoral TNFα concentration was an independent long term prognostic factor in a retrospective multivariate analysis of a cohort of 102 CRC patients involving various other immune parameters, such as CD4+, CD8+ T cell infiltration, presence of regulatory T cells or mast cells. In conclusion, our findings provide evidence for a causal relationship between T cell infiltrates and improved survival in CRC patients as the prognostic impact of T cell infiltrates depends on small subpopulations of in situ active TA specific T cells. Furthermore TNFα, as an indicator for the functionality of TIL, might be better and more accurate prognostic parameter in CRC than mere T cell infiltration. Citation Format: Slava Stamova, Christoph Reissfelder, Christina Gossmann, Marion Braun, Andreas Bonertz, Ute Walliczek, Moritz Koch, Axel Benner, Dirk Jäger, Niels Halama, Khashayarsha Khazaie, Jürgen Weitz, Philipp Beckhove. In situ activity of tumor specific CTL is a prognostic factor in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1283. doi:10.1158/1538-7445.AM2015-1283