Arnovitz, Stephen

Abstract Activation of β-catenin in CD4+CD8+ double-positive (DP) thymocytes halts development before the thymic selection stage and predisposes to transformation. Leukemogenesis, but not the developmental block, depends on TCF-1, β-catenin’s DNA-binding partner. In this study, we show that β-catenin activation directs the DNA-binding protein HEB to block DP thymocyte development. Conditional loss of HEB in DP thymocytes with stabilized β-catenin restores the frequencies of postselection TCRβhi/CCR7+ and TCRβhi/CD69+ DPs and their cell-cycle profile. This recovery is associated with significant reversal of β-catenin-induced expression changes, particularly those related to the CD69+ DP cell signature and to cell-cycle pathways. Stabilizing β-catenin in DP thymocytes directs HEB binding to ≈11,000 novel DNA sites throughout the genome. Novel HEB sites mark most CD69+ DP cell signature genes that change expression upon activation of β-catenin and then revert after loss of HEB. Moreover, many of the novel HEB sites occupy promoter regions of genes enriched in mitotic cell cycle pathways. HEB binding to those regions correlates with downregulation of the associated genes, and HEB inactivation restores expression to physiologic levels. These findings highlight a molecular interplay between HEB and β-catenin that can impair thymic development.

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

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.

Abstract The molecular and functional diversity of regulatory T-cells (Tregs) in health and in disease remains unclear. We previously described in colorectal cancer (CRC) patients a subpopulation of RORγt+ Tregs with elevated expression of β-catenin and pro-inflammatory properties. Here we observed progressive expansion of RORγt+ Tregs in inflammatory bowel disease (IBD) patients during inflammation and early dysplasia. Activating Wnt/β-catenin signaling in human and murine Tregs was sufficient to recapitulate the disease-associated increase in frequencies of RORγt+ Tregs expressing IL-17, IFN-γ, and TNFa. We found that binding of the β-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt/β-catenin activation induced newly accessible chromatin sites in these genes and upregulated their expression. These findings indicate that TCF-1 and Foxp3 together limit the expression of pro-inflammatory genes in Tregs. Activation of ꞵ-catenin signaling interferes with this function and promotes the disease-associated RORγt+ Treg phenotype.