Asxl1 deficiency in embryonic fibroblasts leads to cellular senescence via impairment of the AKT-E2F pathway and Ezh2 inactivation

Abstract

Although ASXL1 mutations are frequently found in human diseases, including myeloid leukemia, the cell proliferation-associated function of ASXL1 is largely unknown. Here, we explored the molecular mechanism underlying the growth defect found in Asxl1-deficient mouse embryonic fibroblasts (MEFs). We found that Asxl1, through amino acids 371 to 655, interacts with the kinase domain of AKT1. In Asxl1-null MEFs, IGF-1 was unable to induce AKT1 phosphorylation and activation; p27Kip1, which forms a ternary complex with ASXL1 and AKT1, therefore remained unphosphorylated. Hypophosphorylated p27Kip1 is able to enter the nucleus, where it prevents the phosphorylation of Rb; this ultimately leads to the down-regulation of E2F target genes as confirmed by microarray analysis. We also found that senescence-associated (SA) genes were upregulated and that SA β-gal staining was increased in Asxl1 −/− MEFs. Further, the treatment of an AKT inhibitor not only stimulated nuclear accumulation of p27Kip1 leading to E2F inactivation, but also promoted senescence. Finally, Asxl1 disruption augmented the expression of p16Ink4a as result of the defect in Asxl1-Ezh2 cooperation. Overall, our study provides the first evidence that Asxl1 both activates the AKT-E2F pathway and cooperates with Ezh2 through direct interactions at early embryonic stages, reflecting that Asxl1 disruption causes cellular senescence.The additional sex comb (Asx) gene was originally identified in Drosophila as an enhancer of trithorax group (TrxG) and Polycomb group (PcG) proteins 1 . Three paralog Asx-like (Asxl) genes have been found in mammals, encoding ASXL1, ASXL2, and ASXL3 2-4 . We recently reported that ASXL1 is mainly located in the nucleus and acts as a dual-function regulator of nuclear receptors, either cooperating with SRC1 for activation or with HP1 for repression in the presence of a ligand 5,6 . ASXL1 also seems to function as a tumor suppressor. Mutations of ASXL1 are often found in human diseases and are mostly linked to acute myeloid leukemia 7 , myelodysplastic syndromes and chronic myelomonocytic leukemia 8 , and Bohring-Opitz syndrome 9 . Despite the somatic ASXL1 mutations frequently reported in leukemia patients, the mechanisms by which ASXL1 mutations cause cancer are not fully understood. Recent studies using leukemia cells from human patients with ASXL1 mutations showed that ASXL1 interacts with histone methyltransferase EZH2, one of PRC2 members, to increase histone H3K27 tri-methylation (me3) 10 . In addition, Asxl1 deletion in mice was accompanied with reduction of H3K27me3. In contrast, loss of Bap1, one of Asxl1 binding partners, resulted in enhanced H3K27me3 level and EZH2-dependent transformation 11 , suggesting distinct, independent roles of Asxl1 and Bap1 in myeloid leukemogenesis.AKT, also called protein kinase B, was identified as the cellular counterpart of a viral oncogene. Amplified AKT isoforms has been found in several types of human cancers [12][13][14] . Not only is AKT a key regulator of cell

Hcfc1a regulates neural precursor proliferation and asxl1 expression in the developing brain.

“…However, RNA-sequencing of brain homogenates provided us a list of potential downstream candidate effectors of hcfc1a. Of those candidates, the asxl1 gene was afforded high priority because of its known role regulating cellular proliferation (24,44,56) and for its documented function in mouse embryonic stem cells and neural differentiation (23). Interestingly, the hcfc1a co60/+ allele causes a 14fold induction of asxl1 expression.…”

“…regulates the cell cycle via AKT-E2F (24). Based upon these data, we hypothesized that overexpression of asxl1 in hcfc1a co60/+ larvae promotes proliferation of NPCs.…”

“…Using literature analysis, we identified the asxl1 gene as one a possible downstream effector of Hcfc1a in the developing brain. asxl1 encodes a transcriptional regulator that is essential for proper cell proliferation and whose deletion causes cellular senescence (24,44). More importantly, mutations in ASXL1 have been associated with Boring Opitz Syndrome (605039), which has been characterized by profound intellectual disability (45).…”

“…However, this function can be inhibited by decreasing the activity of PI3K/AKT. Therefore, we treated hcfc1a co60/+ larvae and their wildtype siblings with LY294002, as described in (24). hcfc1a co60/+ embryos were treated at 24HPF with 12uM LY294002 or vehicle control (DMSO).…”

“…Our results demonstrate that the hcfc1a co60/+ allele results in increased numbers of proliferating NPCs (Sox2+) and hypomotility. Subsequent RNA sequencing on whole brain homogenates obtained from the hcfc1a co60/+ allele identified increased expression of asxl1, which encodes a transcription factor known to modulate the cell cycle (23,24). Furthermore, inhibition of asxl1 expression in larvae carrying the hcfc1a co60/+ allele restored the number of NPCs to normal levels.…”