Faculty Bibliography

Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSCs), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia.

Aberrant DNA methylation is a characteristic feature of cancer including blood malignancies. Mutations in the DNA methylation regulators DNMT3A, TET1/2 and IDH1/2 are recurrent in leukemia and lymphoma. Specific and distinct DNA methylation patterns characterize subtypes of AML and lymphoma. Regulatory regions such as promoter CpG islands, CpG shores and enhancers show changes in methylation during transformation. However, the reported poor correlation between changes in methylation and gene expression in many mouse models and human studies reflects the complexity in the precise molecular mechanism for why aberrant DNA methylation promotes malignancies. This review will summarize current concepts regarding the mechanisms behind aberrant DNA methylation in hematopoietic malignancy and discuss its importance in cancer prognosis, tumor heterogeneity and relapse.

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.

The cohesin complex (consisting of Rad21, Smc1a, Smc3, and Stag2 proteins) is critically important for proper sister chromatid separation during mitosis. Mutations in the cohesin complex were recently identified in a variety of human malignancies including acute myeloid leukemia (AML). To address the potential tumor-suppressive function of cohesin in vivo, we generated a series of shRNA mouse models in which endogenous cohesin can be silenced inducibly. Notably, silencing of cohesin complex members did not have a deleterious effect on cell viability. Furthermore, knockdown of cohesin led to gain of replating capacity of mouse hematopoietic progenitor cells. However, cohesin silencing in vivo rapidly altered stem cells homeostasis and myelopoiesis. Likewise, we found widespread changes in chromatin accessibility and expression of genes involved in myelomonocytic maturation and differentiation. Finally, aged cohesin knockdown mice developed a clinical picture closely resembling myeloproliferative disorders/neoplasms (MPNs), including varying degrees of extramedullary hematopoiesis (myeloid metaplasia) and splenomegaly. Our results represent the first successful demonstration of a tumor suppressor function for the cohesin complex, while also confirming that cohesin mutations occur as an early event in leukemogenesis, facilitating the potential development of a myeloid malignancy.

The methylcytosine dioxygenase TET1 ('ten-eleven translocation 1') is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.

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.

Specific combinations of acute myeloid leukemia (AML) disease alleles, including FLT3 and TET2 mutations, confer distinct biologic features and adverse outcome. We generated mice with mutations in Tet2 and Flt3, which resulted in fully penetrant, lethal AML. Multipotent Tet2(-/-);Flt3(ITD) progenitors (LSK CD48(+)CD150(-)) propagate disease in secondary recipients and were refractory to standard AML chemotherapy and FLT3-targeted therapy. Flt3(ITD) mutations and Tet2 loss cooperatively remodeled DNA methylation and gene expression to an extent not seen with either mutant allele alone, including at the Gata2 locus. Re-expression of Gata2 induced differentiation in AML stem cells and attenuated leukemogenesis. TET2 and FLT3 mutations cooperatively induce AML, with a defined leukemia stem cell population characterized by site-specific changes in DNA methylation and gene expression.

Hematopoietic stem cells (HSCs) possess the ability to generate all hematopoietic cell types as well as to self-renew over long periods, but the mechanisms that regulate their unique properties are incompletely understood. Herein, we show that homozygous deletion of the miR-29a/b-1 bicistron results in decreased numbers of hematopoietic stem and progenitor cells (HSPCs), decreased HSC self-renewal, and increased HSC cell cycling and apoptosis. The HSPC phenotype is specifically due to loss of miR-29a, since miR-29b expression is unaltered in miR-29a/b-1 null HSCs, and only ectopic expression of miR-29a restores HSPC function both in vitro and in vivo. HSCs lacking miR-29a/b-1 exhibit widespread transcriptional dysregulation and adopt gene expression patterns similar to normal committed progenitors. A number of predicted miR-29 target genes, including Dnmt3a, are significantly upregulated in miR-29a/b-1 null HSCs. The loss of negative regulation of Dnmt3a by miR-29a is a major contributor to the miR-29a/b-1 null HSPC phenotype, as both in vitro Dnmt3a shRNA knockdown assays and a genetic haploinsufficiency model of Dnmt3a restored the frequency and long-term reconstitution capacity of HSCs from miR-29a/b-1 deficient mice. Overall, these data demonstrate that miR-29a is critical for maintaining HSC function through its negative regulation of Dnmt3a.