Tumor suppressor function of Gata2 in acute promyelocytic leukemia.

Most patients with acute promyelocytic leukemia (APL) can be cured with combined all-trans retinoic acid (ATRA) and arsenic trioxide therapy, which induces the destruction of PML-RARA, the initiating fusion protein for this disease. However, the underlying mechanisms by which PML-RARA initiates and maintains APL cells are still not clear. Therefore, we identified genes that are dysregulated by PML-RARA in mouse and human APL cells and prioritized GATA2 for functional studies because it is highly expressed in preleukemic cells expressing PML-RARA, its high expression persists in transformed APL cells, and spontaneous somatic mutations of GATA2 occur during APL progression in mice and humans. These and other findings suggested that GATA2 may be upregulated to thwart the proliferative signal generated by PML-RARA and that its inactivation by mutation (and/or epigenetic silencing) may accelerate disease progression in APL and other forms of acute myeloid leukemia (AML). Indeed, biallelic knockout of Gata2 with CRISPR/Cas9-mediated gene editing increased the serial replating efficiency of PML-RARA-expressing myeloid progenitors (as well as progenitors expressing RUNX1-RUNX1T1, or deficient for Cebpa), increased mouse APL penetrance, and decreased latency. Restoration of Gata2 expression suppressed PML-RARA-driven aberrant self-renewal and leukemogenesis. Conversely, addback of a mutant GATA2R362G protein associated with APL and AML minimally suppressed PML-RARA-induced aberrant self-renewal, suggesting that it is a loss-of-function mutation. These studies reveal a potential role for Gata2 as a tumor suppressor in AML and suggest that restoration of its function (when inactivated) may provide benefit for AML patients.

Remethylation of Dnmt3a-/- hematopoietic cells is associated with partial correction of gene dysregulation and reduced myeloid skewing.

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine Dnmt3a-/- bone marrow cells. To determine whether the hypomethylation phenotype of Dnmt3a-/- hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a-/- mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic "state" created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.

Sequencing a mouse acute promyelocytic leukemia genome reveals genetic events relevant for disease progression.

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML). It is characterized by the t(15;17)(q22;q11.2) chromosomal translocation that creates the promyelocytic leukemia-retinoic acid receptor α (PML-RARA) fusion oncogene. Although this fusion oncogene is known to initiate APL in mice, other cooperating mutations, as yet ill defined, are important for disease pathogenesis. To identify these, we used a mouse model of APL, whereby PML-RARA expressed in myeloid cells leads to a myeloproliferative disease that ultimately evolves into APL. Sequencing of a mouse APL genome revealed 3 somatic, nonsynonymous mutations relevant to APL pathogenesis, of which 1 (Jak1 V657F) was found to be recurrent in other affected mice. This mutation was identical to the JAK1 V658F mutation previously found in human APL and acute lymphoblastic leukemia samples. Further analysis showed that JAK1 V658F cooperated in vivo with PML-RARA, causing a rapidly fatal leukemia in mice. We also discovered a somatic 150-kb deletion involving the lysine (K)-specific demethylase 6A (Kdm6a, also known as Utx) gene, in the mouse APL genome. Similar deletions were observed in 3 out of 14 additional mouse APL samples and 1 out of 150 human AML samples. In conclusion, whole genome sequencing of mouse cancer genomes can provide an unbiased and comprehensive approach for discovering functionally relevant mutations that are also present in human leukemias.