Faculty Bibliography

Little is known on post-transcriptional regulation of stem cell maintenance and differentiation. Here we characterize the role of Ddb1, a component of the CUL4-DDB1 ligase complex. Ddb1 is highly expressed in hematopoietic stem cells and its deletion leads to abrogation of hematopoiesis, targeting specifically transiently amplifying progenitor subsets. Ddb1 deletion in non-dividing lymphocytes had no discernible phenotypes. Ddb1 silencing activated the p53 pathway and lead to apoptosis. The abrogation of hematopoietic progenitor cells can be partially rescued by simultaneous deletion of p53. Interestingly, depletion of DDB1 in embryonic stem cell (ESC) does not affect survival or cell cycle progression but leads to loss of pluripotency, suggesting distinct roles of DDB1 in adult and embryonic stem cells. Mass-spectrometry revealed distinct interactions between DDB1 and DCAFs, the substrate-recognizing components of the CUL4 complex between cell types. Our studies identify the CUL4-DDB1 complex as a novel post-translational regulator of stem maintenance and differentiation.

Protein degradation plays key roles in diverse pathways in cell division, growth and differentiation. Aberrant stabilization of crucial proteins participating in oncogenic pathways is often observed in cancer. The importance of proper protein turnover is exemplified by the SCFFbxw7 ubiquitin ligase, which is frequently mutated in human cancer, including T cell acute lymphoblastic leukemia. Recent studies have revealed novel substrates of Fbxw7 and shed light on its role on differentiation of stem cells and expansion of stem-cell-like cells driving tumorigenesis. Detailed understanding of the contribution of the Fbxw7-regulated network of proteins in initiation and progression of cancer will facilitate the identification of candidate intervention targets in human cancer.

The role of the microenvironment in T cell acute lymphoblastic leukemia (T-ALL), or any acute leukemia, is poorly understood. Here we demonstrate that T-ALL cells are in direct, stable contact with CXCL12-producing bone marrow stroma. Cxcl12 deletion from vascular endothelial, but not perivascular, cells impeded tumor growth, suggesting a vascular niche for T-ALL. Moreover, genetic targeting of Cxcr4 in murine T-ALL after disease onset led to rapid, sustained disease remission, and CXCR4 antagonism suppressed human T-ALL in primary xenografts. Loss of CXCR4 targeted key T-ALL regulators, including the MYC pathway, and decreased leukemia initiating cell activity in vivo. Our data identify a T-ALL niche and suggest targeting CXCL12/CXCR4 signaling as a powerful therapeutic approach for T-ALL.

Activating NOTCH1 mutations occur in ~60% of human T-cell acute lymphoblastic leukemias (T-ALLs), and mutations disrupting the transcription factor IKZF1 (IKAROS) occur in ~5% of cases. To investigate the regulatory interplay between these driver genes, we have used a novel transgenic RNA interference mouse model to produce primary T-ALLs driven by reversible Ikaros knockdown. Restoring endogenous Ikaros expression in established T-ALL in vivo acutely represses Notch1 and its oncogenic target genes including Myc, and in multiple primary leukemias causes disease regression. In contrast, leukemias expressing high levels of endogenous or engineered forms of activated intracellular Notch1 (ICN1) resembling those found in human T-ALL rapidly relapse following Ikaros restoration, indicating that ICN1 functionally antagonizes Ikaros in established disease. Furthermore, we find that IKAROS mRNA expression is significantly reduced in a cohort of primary human T-ALL patient samples with activating NOTCH1/FBXW7 mutations, but is upregulated upon acute inhibition of aberrant NOTCH signaling across a panel of human T-ALL cell lines. These results demonstrate for the first time that aberrant NOTCH activity compromises IKAROS function in mouse and human T-ALL, and provide a potential explanation for the relative infrequency of IKAROS gene mutations in human T-ALL.

Mutations affecting spliceosomal proteins are the most common mutations in patients with myelodysplastic syndromes (MDS), but their role in MDS pathogenesis has not been delineated. Here we report that mutations affecting the splicing factor SRSF2 directly impair hematopoietic differentiation in vivo, which is not due to SRSF2 loss of function. By contrast, SRSF2 mutations alter SRSF2's normal sequence-specific RNA binding activity, thereby altering the recognition of specific exonic splicing enhancer motifs to drive recurrent mis-splicing of key hematopoietic regulators. This includes SRSF2 mutation-dependent splicing of EZH2, which triggers nonsense-mediated decay, which, in turn, results in impaired hematopoietic differentiation. These data provide a mechanistic link between a mutant spliceosomal protein, alterations in the splicing of key regulators, and impaired hematopoiesis.

Epigenetic regulation of lineage-specific genes is important for the differentiation and function of T cells. Ten-eleven translocation (Tet) proteins catalyze 5-methylcytosine (5mC) conversion to 5-hydroxymethylcytosine (5hmC) to mediate DNA demethylation. However, the roles of Tet proteins in the immune response are unknown. Here, we characterized the genome-wide distribution of 5hmC in CD4(+) T cells and found that 5hmC marks putative regulatory elements in signature genes associated with effector cell differentiation. Moreover, Tet2 protein was recruited to 5hmC-containing regions, dependent on lineage-specific transcription factors. Deletion of Tet2 in T cells decreased their cytokine expression, associated with reduced p300 recruitment. In vivo, Tet2 plays a critical role in the control of cytokine gene expression in autoimmune disease. Collectively, our findings suggest that Tet2 promotes DNA demethylation and activation of cytokine gene expression in T cells.