High Mobility Group AT-hook 2 (HMGA2) is a chromatin modifier and its overexpression has been found in a subset of patients with myelodysplastic syndrome (MDS). The high level of HMGA2 expression appears to predict poor prognosis in various tumors; however, it remains unclear how HMGA2 dysregulates expression of target genes to facilitate the transformation. To elucidate the mechanisms by which the overexpression of Hmga2 promotes the development of MDS, we generated an Hmga2-expressing Tet2-deficient (Hmga2-Tet2Δ/Δ) mouse model showing the progressive phenotype of MDS. We found that Hmga2-Tet2Δ/Δ mice had neutropenia and anemia, but variable platelet counts, accompanied by elevated frequencies of mutant cells in myeloid cells. Hmga2-Tet2Δ/Δ mice showed a similar median survival to Tet2Δ/Δ mice (274 days vs 290 days), but shorter survival than Hmga2-Tet2wt/wt mice (274 days vs undetermined). Moribund Hmga2-Tet2Δ/Δ mice showed progressive leukopenia and anemia, accompanied by the emergence of dysplastic neutrophils, myeloblasts and anisocytosis in the PB and BM and dysplastic megakaryocytes in the BM. Hmga2-Tet2Δ/Δ mice had mildly increased spleen weights, and expanded myeloid cells and HSPCs in the spleen without the deposition of fibrosis. During a 12-month observation, we found that Hmga2-Tet2Δ/Δ mice developed lethal MDS/MPN overlap disease (47%), MDS (33%), MPN (13%), and AML (7%), while 6 out of 11 Tet2Δ/Δ mice developed MPN (55%). Hmga2-Tet2wt/wt mice subsequently showed similar blood counts in PB and died without the expansion of leukemic or dysplastic blood cells. Therefore, Hmga2 overexpression did not transform wild-type HSCs but promoted the development of MDS in the absence of Tet2 in vivo.
In order to elucidate the molecular mechanisms underlying the transformation of Hmga2-Tet2Δ/Δ cells, we initially performed gene expression profiling by a RNA sequencing analysis in LSK HSPCs isolated from WT, Hmga2-Tet2wt/wt, Tet2Δ/Δ, and Hmga2-Tet2Δ/Δ mice at a pre-disease stage and those isolated from two Hmga2-Tet2Δ/Δ MDS/MPN and AML mice. Hmga2-Tet2Δ/Δ leukemic cells were placed closer to one out of two Hmga2-Tet2Δ/Δ cells at the pre-disease stage, but clearly apart from the other genotype cells, indicating that Hmga2 overexpression and Tet2 loss result in the accumulation of alterations in the transcriptional program during the development of MDS.In order to clarify the mechanisms by which the overexpression of Hmga2 alters the transcriptional program in Tet2-deficient cells, we performed the ChIP-sequencing of FLAG-tagged Hmga2 in bone marrow progenitor cells isolated from WT, Hmga2-Tet2wt/wt, and Hmga2-Tet2Δ/Δ mice. The numbers of Hmga2-binding peaks were markedly lower in Tet2-deficient cells than in Hmga2-Tet2wt/wt cells (2227 peaks versus 11500 peaks). Furthermore, annotated genes adjacent to Hmga2-binding sites partially overlapped in both genotype cells, whereas 2965 out of 3843 genes identified in Tet2 wild-type cells lost the binding peaks of Hmga2 upon the deletion of Tet2. Based on the DNA-binding capacity of Hmga2, the loss of Tet2 remodeled the binding sites of Hmga2 via change in DNA methylation in Hmga2-binding flanking regions, which were not observed in the presence of Tet2, leading to significant enrichments in genes involved in cell-to-cell adhesion and cell morphogenesis in Hmga2-Tet2Δ/Δ cells. Furthermore, we found that the overexpression of Hmga2 and loss of Tet2 resulted in the activation of oncogenic pathways (e.g. TGF-b, TNF-a), but suppressed the expression of genes in the unfolded protein response. Notably, the inhibition of bile acid metabolism to reactivate the unfolded protein response markedly attenuated the proliferation of Hmga2-Tet2Δ/Δ cells. These combinatory effects on the transcriptional program and cellular functions were not redundant to those in either single mutant cell, supporting Hmga2 being a proto-oncogene because its overexpression alone was not sufficient to develop MDS in vivo.
Thus, Hmga2 overexpression exerts synergistic, but also gain-of-function effects with the loss of Tet2 to target these key biological pathways and promotes the transformation of Tet2-deficient stem cells. This study also provides a new rationale for targeting the unfolded protein response in MDS cells expressing HMGA2.
Disclosures
No relevant conflicts of interest to declare.
Dependence of Endoplasmic Reticulum-associated Degradation on the Peptide Binding Domain and Concentration of BiP
