Khazaie, Khashayarsha

Abstract Colorectal cancer (CRC) is one of the most common cancers in the world, yet it remains one of the most difficult to treat. Immunotherapy of CRC using checkpoint inhibitors is only efficacious in approximately 4% of patients with high microsatellite instability (MSI-H). In earlier studies we showed that in patients with inflammatory bowel diseases (IBD) and CRC as well as in the APCΔ468 murine polyposis model, Tregs overexpress β-catenin. Furthermore, we demonstrated that expression of a dominant active β-catenin in Tregs induces RORγt, compromising suppressive activity and promoting TH17-like properties. This results in a scurfy-like phenotype. By contrast loss of TCF-1 upregulated RORγt, but only compromised Treg anti-inflammatory functions. These observations suggested cooperation of β-catenin and TCF-1 in regulating Treg plasticity. We show here that in healthy mice, β-catenin expression is significantly higher in Tregs as compared to conventional CD4+ T-cells. In Tregs that express a dominant active β-catenin, ablation of TCF-1 or HEB provide partial rescue, while ablation of both TCF-1 and HEB recovers Treg numbers and prevents the scurfy-like phenotype. These observations suggest that TCF-1, HEB and β-catenin cooperate to determine Treg functions. In search for additional partners, we show that loss of TCF-1 upregulates SATB-1, a chromatin modifier that is known to determine T-cell development and function. Upregulation of SATB-1 is enhanced with the loss of both TCF-1 and HEB. All three transcription factors upregulate RORγt suggesting that they cooperatively determine Treg function and plasticity. Elucidating this cooperation will enhance our ability to control Treg plasticity in health and disease. NIH/NIAID - R01 AI108682-0A1

Abstract T-cell Acute lymphoblastic leukemia (T-ALL) is an aggressive disease with only a 10% 5-year survival rate for patients over 60, due to genomic instability. We have shown that β-catenin and TCF-1 cooperate in DP thymocytes to promote genomic instability leading to lymphomagenesis. We have also established that TCF-1 and HEB cooperate in the DP stage. Now, we aim to determine the molecular underpinnings of T-ALL and thymic development, by elucidating the interplay of all three factors, β-catenin, TCF-1, and HEB. Aberrant activation of β-catenin promotes the development of Lymphoma/Leukemia equivalent to the human disease T-ALL and blocks thymocyte development in the early DP stage. Ablation of TCF-1 in thymocytes with activated β-catenin rescues from lymphomagenesis, but the developmental block persists. Antithetically, developmental progression is achieved only when HEB is conditionally knocked out. In an effort to explain this phenomenon, we found that genes associated with the transition of DP thymocytes from DPsmall to CD69+ are downregulated by activating β-catenin (e.g. Runx-3, Ccr-7, TOX-2, etc.) and revert upon deletion of HEB. This is associated with changes in the binding of TCF-1 to DNA. Genomic sites that acquire TCF-1 binding upon β-catenin activation forfeit this binding when HEB is deleted, while at the same time novel TCF-1 binding sites emerge. These new binding sites are significantly more enriched for the conserved TCF-1 motif than TCF-1-bound sites in WT thymocytes. This observation promotes the notion that HEB guides TCF-1 towards alternative binding sites, thus altering gene expression. These findings highlight the complex interactive relationship of TCF-1 and HEB in both normal development and disease. NIH/NIAID R01 AI147652-01A1

Abstract Checkpoint blockade has improved patient outcome through reinvigorating T cells against cancer. Despite this success, many patients don’t respond, leaving them with few options. Tumors utilize many immunosuppressive mechanisms, including altering the function of innate immune cells. Cancer cells can promote differentiation of macrophages into tolerogenic “M2-like” macrophages, which support tumor growth. Macrophage differentiation by cancer cells is influenced by many receptors, including Siglec-E. Siglec-E binds to α2,8-linked disialic acids, generated by the enzyme ST8Sia6, on O-linked glycoproteins, and functions to inhibit activation of innate immune cells. In some tumor types, higher expression of ST8Sia6 correlates with poorer survival. Therefore, ST8Sia6 overexpressing tumors may engage Siglec-E, leading to immune suppression and accelerating tumor growth. Mice injected with ST8Sia6 overexpressing MC38 and B16-F10 tumors had increased tumor burden, reduced survival and increased M2 macrophage polarization, which suppresses the immune response. This acceleration of tumor growth was abrogated when injected into Siglec-E knockout mice. Thus, the primary function of ST8Sia6 in tumors is an extrinsic alteration of the immune response, rather than an intrinsic effect on cell growth. Similarly, in a spontaneous colon cancer model, ST8Sia6 overexpression in a murine colon cancer model accelerated the development of tumors, decreasing survival to 2.5 months instead of 6 months. The overexpression of ST8Sia6 leads to increased Arg-1 expression by colonic tumor macrophages. Therefore, ST8Sia6 expression in tumors inhibits the immune response to promote tumor growth in a Siglec-E dependent manner.

Abstract The transcription factor TCF-1 is essential for the development and function of T regulatory (Treg) cells, however its function is poorly understood. Here, we show that TCF-1 primarily suppresses transcription of genes that are co-bound by Foxp3. Single-cell RNA-seq analysis identified effector- and central-memory Treg-cells with differential expression of Klf2 and memory and activation markers. TCF-1 deficiency did not change the core Treg transcriptional signature, but promoted alternative signaling pathways whereby Treg-cells became activated and gained gut-homing and TH17 characteristics. TCF-1-deficient Treg-cells strongly suppressed T-cell proliferation and cytotoxicity, but were compromised in controlling CD4+ T-cell polarization and inflammation. In mice with polyposis, Treg cell-specific TCF-1 deficiency promoted tumor growth. Consistently, tumor-infiltrating Treg cells of colorectal cancer patients showed lower TCF-1 expression and increased TH17 expression signatures compared to adjacent normal tissue and circulating T-cells. Thus, Treg cell-specific TCF-1 expression differentially regulates TH17-mediated inflammation and T-cell cytotoxicity, and can determine colorectal cancer outcome. Supported by NIH R01 AI 108682 (FG & KK), NIH RO1 AI 147652 (FG), NIH R35GM138283 (MK), and Praespero Innovation Award Alberta, Canada (FG & KK)

Abstract 20 to 30% of breast cancers overexpress ERRB2, encoding the human epidermal growth factor-2 receptor (HER2). Overexpression of HER2 is associated with poorer prognosis, particularly in metastatic disease. Although HER2 therapeutics exists many patients eventually develop uncontrolled tumor growth. The BALB/c-neuT mouse has been a popular model for investigating HER2-positive breast cancer. We have found that immune responsiveness and MHC genotype are important factors in spontaneous tumor development and tumor rejection in the neuT model. To assess the influence of immune response potential on tumor biology we introduced the skg ZAP-70 mutation or the H-2 b MHC haplotype into the neuT model. Spontaneous tumors appeared with decreased frequency and metastasis or increased frequency and metastasis, respectively, in comparison to the BALB/c model. H-2 b tumors were readily transplantable into the breast fat pads of syngeneic BALB.B-neuT hosts which developed systemic immune abnormalities very similar to those observed in BALB/c - neuT or BALB/c WT mice bearing neuT tumors. However, BALB.B WT hosts initially accepted the grafts, but subsequently rejected them after they were established. Adoptive transfer of splenocytes from BALB.B WT mice which had rejected tumor conferred resistance to the tumor grafts, as well as to spontaneous tumors developing in BALB.B neuT littermates. The strong H-2b-mediated antitumor response in WT mice indicates efficient antigen presentation, and presumably leads to strong tolerance in neuT+ animals. The importance of an intact T cell signaling and MHC genotype indicate a critical role for T cell-mediated immunity and tolerance in the biology of the spontaneously developing tumor and metastasis.

Abstract Checkpoint blockade has improved patient outcome through reinvigorating T cells against cancer. Despite this success, many patients don’t respond, leaving them with few options. Tumors utilize many immunosuppressive mechanisms, including altering the function of innate immune cells. Cancer cells can promote differentiation of macrophages into tolerogenic “M2-like” macrophages, which support tumor growth. Macrophage differentiation by cancer cells is influenced by many receptors, including Siglec-E. Siglec-E binds to α2,8-linked disialic acids, generated by the enzyme ST8Sia6, on O-linked glycoproteins, and functions to inhibit activation of innate immune cells. Cancer patients with high expression of ST8Sia6 exhibit reduced survival. Therefore, ST8Sia6 overexpressing tumors may engage Siglec-E, leading to immune suppression and accelerating tumor growth. Mice injected with ST8Sia6 overexpressing MC38 and B16-F10 tumors had increased tumor burden, reduced survival and an increase M2 macrophage polarization. The acceleration in tumor growth was abrogated when injected into Siglec-E knockout mice. Thus, the primary function of ST8Sia6 in tumors is an extrinsic alteration of the immune response, rather than an intrinsic effect on cell growth. Similarly, in a spontaneous colon cancer model, ST8Sia6 overexpression in a murine colon cancer model accelerated the development of tumors, decreasing survival to 2.5 months instead of 6 months. The overexpression of ST8Sia6 on tumors leads to increased Arg-1 expression by CD11b+ CD11c+Ly6C−Ly6G− cells, which suppresses the immune response. Therefore, ST8Sia6 expression in tumors inhibits the immune response to promote tumor growth in a Siglec-E dependent manner.

Abstract Circadian clocks regulate numerous physiological processes that vary across the day-night (diurnal) cycle, but whether the circadian clock regulates the adaptive immune response to cancer is mostly unclear. In mouse models of polyposis, and human colorectal cancer patients, we showed that tumor infiltrating Tregs overexpressed b-catenin and RORgt, and secreted IL-17. These Tregs suppressed tumor reactive cytotoxic T cells, and promoted tumor growth. Therefore, we examined how the expression of a dominant negative ClockΔ19 gene affects inflammation and susceptibility to cancer in the APCΔ468 mouse model of hereditary polyposis. ClockΔ19 mutant mice had high expression of RORgt+ and b-catenin+ Tregs. These mice showed a notable expansion of Th17 inflammation in vivo upon injection of aCD3. Functionally these Tregs showed pro-inflammatory properties. In addition to these effects, APCΔ468 ClockΔ19 mice had increased numbers of CD11b+ cell, Ly6C+CXCR1− monocytes, and Ly6C+Ly6G+ myeloid derived suppressor cells (MDSC) as compared with APCΔ468 mice. APCΔ468 ClockΔ19 mice had higher tumor burden in the small intestine and colon than the APCΔ468 mice. Both T-cells and tumors had intense stabilization of nuclear b-catenin and were more aggressive as indicated by abundance of stromal mMCP6+ mast cells (MC), which typically appear at the invasive borders of tumors. Our finding indicating increase in tumor load and worsening inflammation caused by direct ClockΔ19 effects in the epithelium and enhanced TH17 bias in the immune system.

Abstract Regulatory T-cells (Tregs) play a dual role in colon cancer, promoting immune tolerance and suppressing inflammation. Two types of Tregs infiltrate the gut. Thymic derived Helios+CD4+Foxp3+ Tregs have high affinity for self-antigens, including tumor-antigens and protect the tumor, while Helios−CD4+Foxp3+ extra-thymic Tregs that are generated in the gut have microbial specificity and suppress harmful inflammation. In colon cancer, there is a preferential expansion of a population of Helios−CD4+Foxp3+RORγt+ Tregs with enhanced T-cell suppressive properties and pro-inflammatory properties that promote tumor growth. We showed that this population of Tregs in cancer patients expresses elevated levels of β-catenin. To test the role of canonical Wnt signaling in the generation of pro-inflammatory Tregs, we stabilized β-catenin or deleted its DNA binding partner TCF-1 in Tregs. In both instances expression of RORγt was upregulated. While the stabilization of β-catenin led to loss of Treg functions, ablation of TCF-1 reproduced the phenotype and functional characteristics of Tregs in colon cancer. The TCF-1 deficient Tregs were pro-inflammatory, potent T-cell suppressive, and tumor promoting.

AbstractBackgroundDisruption of central circadian rhythms likely mediated by changes in microbiota and a decrease in gut-derived metabolites like short chain fatty acids (SCFAs) negatively impacts colonic barrier homeostasis. We aimed to explore the effects of isolated peripheral colonic circadian disruption on the colonic barrier in a mouse model of colitis and explore the mechanisms, including intestinal microbiota community structure and function.MethodsColon epithelial cell circadian rhythms were conditionally genetically disrupted in mice: TS4Cre-BMAL1lox (cBMAL1KO) with TS4Cre as control animals. Colitis was induced through 5 days of 2% dextran sulfate sodium (DSS). Disease activity index and intestinal barrier were assessed, as were fecal microbiota and metabolites.ResultsColitis symptoms were worse in mice with peripheral circadian disruption (cBMAL1KO). Specifically, the disease activity index and intestinal permeability were significantly higher in circadian-disrupted mice compared with control animals (TS4Cre) (P < .05). The worsening of colitis appears to be mediated, in part, through JAK (Janus kinase)-mediated STAT3 (signal transducer and activator of transcription 3), which was significantly elevated in circadian-disrupted (cBMAL1KO) mice treated with DSS (P < .05). Circadian-disrupted (cBMAL1KO) mice also had decreased SCFA metabolite concentrations and decreased relative abundances of SCFA-producing bacteria in their stool when compared with control animals (TS4Cre).ConclusionsDisruption of intestinal circadian rhythms in colonic epithelial cells promoted more severe colitis, increased inflammatory mediators (STAT3 [signal transducer and activator of transcription 3]), and decreased gut microbiota–derived SCFAs compared with DSS alone. Further investigation elucidating the molecular mechanisms behind these findings could provide novel circadian directed targets and strategies in the treatment of inflammatory bowel disease.

Understanding the mechanisms promoting chromosomal translocations of the rearranging receptor loci in leukemia and lymphoma remains incomplete. Here we show that leukemias induced by aberrant activation of β-catenin in thymocytes, which bear recurrent Tcra/Myc-Pvt1 translocations, depend on Tcf-1. The DNA double strand breaks (DSBs) in the Tcra site of the translocation are Rag-generated, whereas the Myc-Pvt1 DSBs are not. Aberrantly activated β-catenin redirects Tcf-1 binding to novel DNA sites to alter chromatin accessibility and down-regulate genome-stability pathways. Impaired homologous recombination (HR) DNA repair and replication checkpoints lead to retention of DSBs that promote translocations and transformation of double-positive (DP) thymocytes. The resulting lymphomas, which resemble human T cell acute lymphoblastic leukemia (T-ALL), are sensitive to PARP inhibitors (PARPis). Our findings indicate that aberrant β-catenin signaling contributes to translocations in thymocytes by guiding Tcf-1 to promote the generation and retention of replication-induced DSBs allowing their coexistence with Rag-generated DSBs. Thus, PARPis could offer therapeutic options in hematologic malignancies with active Wnt/β-catenin signaling.