Tcf-1 promotes genomic instability and T cell transformation in response to aberrant ß-catenin activation.

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 ß-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 ß-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 ß-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/ß-catenin signaling.

Asymmetrical forward and reverse developmental trajectories determine molecular programs of B cell antigen receptor editing.

During B lymphopoiesis, B cell progenitors progress through alternating and mutually exclusive stages of clonal expansion and immunoglobulin (Ig) gene rearrangements. Great diversity is generated through the stochastic recombination of Ig gene segments encoding heavy and light chain variable domains. However, this commonly generates autoreactivity. Receptor editing is the predominant tolerance mechanism for self-reactive B cells in the bone marrow (BM). B cell receptor editing rescues autoreactive B cells from negative selection through renewed light chain recombination first at Igκ then Igλ loci. Receptor editing depends on BM microenvironment cues and key transcription factors such as NF-κB, FOXO, and E2A. The specific BM factor required for receptor editing is unknown. Furthermore, how transcription factors coordinate these developmental programs to promote usage of the λ chain remains poorly defined. Therefore, we used two mouse models that recapitulate pathways by which Igλ light chain-positive B cells develop. The first has deleted J kappa (Jκ) genes and hence models Igλ expression resulting from failed Igκ recombination (Igκdel). The second models autoreactivity by ubiquitous expression of a single-chain chimeric anti-Igκ antibody (κ-mac). Here, we demonstrated that autoreactive B cells transit asymmetric forward and reverse developmental trajectories. This imparted a unique epigenetic landscape on small pre-B cells, which opened chromatin to transcription factors essential for Igλ recombination. The consequences of this asymmetric developmental path were both amplified and complemented by CXCR4 signaling. These findings reveal how intrinsic molecular programs integrate with extrinsic signals to drive receptor editing.

Wnt-ß-catenin activation epigenetically reprograms Treg cells in inflammatory bowel disease and dysplastic progression.

The diversity of regulatory T (Treg) cells in health and in disease remains unclear. Individuals with colorectal cancer harbor a subpopulation of RORγt+ Treg cells with elevated expression of ß-catenin and pro-inflammatory properties. Here we show progressive expansion of RORγt+ Treg cells in individuals with inflammatory bowel disease during inflammation and early dysplasia. Activating Wnt-ß-catenin signaling in human and murine Treg cells was sufficient to recapitulate the disease-associated increase in the frequency of RORγt+ Treg cells coexpressing multiple pro-inflammatory cytokines. Binding of the ß-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt-ß-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 Treg cells. Activation of ß-catenin signaling interferes with this function and promotes the disease-associated RORγt+ Treg phenotype.

TCF-1 and HEB cooperate to establish the epigenetic and transcription profiles of CD4+CD8+ thymocytes.

Thymocyte development requires a complex orchestration of multiple transcription factors. Ablating either TCF-1 or HEB in CD4+CD8+ thymocytes elicits similar developmental outcomes including increased proliferation, decreased survival, and fewer late Tcra rearrangements. Here, we provide a mechanistic explanation for these similarities by showing that TCF-1 and HEB share ~7,000 DNA-binding sites genome wide and promote chromatin accessibility. The binding of both TCF-1 and HEB was required at these shared sites for epigenetic and transcriptional gene regulation. Binding of TCF-1 and HEB to their conserved motifs in the enhancer regions of genes associated with T cell differentiation promoted their expression. Binding to sites lacking conserved motifs in the promoter regions of cell-cycle-associated genes limited proliferation. TCF-1 displaced nucleosomes, allowing for chromatin accessibility. Importantly, TCF-1 inhibited Notch signaling and consequently protected HEB from Notch-mediated proteasomal degradation. Thus, TCF-1 shifts nucleosomes and safeguards HEB, thereby enabling their cooperation in establishing the epigenetic and transcription profiles of CD4+CD8+ thymocytes.

Superenhancer reprogramming drives a B-cell-epithelial transition and high-risk leukemia.

IKAROS is required for the differentiation of highly proliferative pre-B-cell precursors, and loss of IKAROS function indicates poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL). Here we show that IKAROS regulates this developmental stage by positive and negative regulation of superenhancers with distinct lineage affiliations. IKAROS defines superenhancers at pre-B-cell differentiation genes together with B-cell master regulators such as PAX5, EBF1, and IRF4 but is required for a highly permissive chromatin environment, a function that cannot be compensated for by the other transcription factors. IKAROS is also highly enriched at inactive enhancers of genes normally expressed in stem-epithelial cells. Upon IKAROS loss, expression of pre-B-cell differentiation genes is attenuated, while a group of extralineage transcription factors that are directly repressed by IKAROS and depend on EBF1 relocalization at their enhancers for expression is induced. LHX2, LMO2, and TEAD-YAP1, normally kept separate from native B-cell transcription regulators by IKAROS, now cooperate directly with them in a de novo superenhancer network with its own feed-forward transcriptional reinforcement. Induction of de novo superenhancers antagonizes Polycomb repression and superimposes aberrant stem-epithelial cell properties in a B-cell precursor. This dual mechanism of IKAROS regulation promotes differentiation while safeguarding against a hybrid stem-epithelial-B-cell phenotype that underlies high-risk B-ALL.

ß-Catenin promotes colitis and colon cancer through imprinting of proinflammatory properties in T cells.

The density and type of lymphocytes that infiltrate colon tumors are predictive of the clinical outcome of colon cancer. High densities of T helper 17 (T(H)17) cells and inflammation predict poor outcome, whereas infiltration by T regulatory cells (Tregs) that naturally suppress inflammation is associated with longer patient survival. However, the role of Tregs in cancer remains controversial. We recently reported that Tregs in colon cancer patients can become proinflammatory and tumor-promoting. These properties were directly linked with their expression of RORγt (retinoic acid-related orphan receptor-γt), the signature transcription factor of T(H)17 cells. We report that Wnt/ß-catenin signaling in T cells promotes expression of RORγt. Expression of ß-catenin was elevated in T cells, including Tregs, of patients with colon cancer. Genetically engineered activation of ß-catenin in mouse T cells resulted in enhanced chromatin accessibility in the proximity of T cell factor-1 (Tcf-1) binding sites genome-wide, induced expression of T(H)17 signature genes including RORγt, and promoted T(H)17-mediated inflammation. Strikingly, the mice had inflammation of small intestine and colon and developed lesions indistinguishable from colitis-induced cancer. Activation of ß-catenin only in Tregs was sufficient to produce inflammation and initiate cancer. On the basis of these findings, we conclude that activation of Wnt/ß-catenin signaling in effector T cells and/or Tregs is causatively linked with the imprinting of proinflammatory properties and the promotion of colon cancer.

ß-Catenin induces T-cell transformation by promoting genomic instability.

Deregulated activation of ß-catenin in cancer has been correlated with genomic instability. During thymocyte development, ß-catenin activates transcription in partnership with T-cell-specific transcription factor 1 (Tcf-1). We previously reported that targeted activation of ß-catenin in thymocytes (CAT mice) induces lymphomas that depend on recombination activating gene (RAG) and myelocytomatosis oncogene (Myc) activities. Here we show that these lymphomas have recurring Tcra/Myc translocations that resulted from illegitimate RAG recombination events and resembled oncogenic translocations previously described in human T-ALL. We therefore used the CAT animal model to obtain mechanistic insights into the transformation process. ChIP-seq analysis uncovered a link between Tcf-1 and RAG2 showing that the two proteins shared binding sites marked by trimethylated histone-3 lysine-4 (H3K4me3) throughout the genome, including near the translocation sites. Pretransformed CAT thymocytes had increased DNA damage at the translocating loci and showed altered repair of RAG-induced DNA double strand breaks. These cells were able to survive despite DNA damage because activated ß-catenin promoted an antiapoptosis gene expression profile. Thus, activated ß-catenin promotes genomic instability that leads to T-cell lymphomas as a consequence of altered double strand break repair and increased survival of thymocytes with damaged DNA.