HMGA2 As a Potential Molecular Target in MLL-AF4 Positive Infant Acute Lymphoblastic Leukemia
Authors: Eguchi-Ishimae Minenori , Eguchi Mariko , Wu Zhouying , Ming Wen , Iwabuki Hidehiko , Inukai Takeshi , Sugita Kanji , Ishii Eiichi 
Published: October 2019

Leukemic cells of acute lymphoblastic leukemia (ALL) in infants are frequently characterised by chromosome translocations involving 11q23, resulting in the rearrangement of the mixed-lineage leukemia (MLL) gene and subsequent generation of MLL fusion gene. Among more than 50 genes which have been identified as the fusion partner of the MLL gene, fusion with AF4 is characteristically observed in infant ALL representing a hallmark of poor prognosis. Although recent progress of intensive chemotherapy with or without stem cell transplantation has improved its treatment outcome, the treatment is often accompanied by long-term side effects. Less toxic molecular targeting therapies are therefore necessary for infant ALL.

We have previously reported that in infant ALL with MLL fusion gene, microRNA let-7b is significantly downregulated by DNA hypermethylation of its promoter region. The downregulation of let-7b is one of the consequences of oncogenic MLL fusion proteins contributing to leukemogenesis possibly through upregulation of let-7b-regulated target genes with oncogenic potential such as high mobility group AT-hook 2 (HMGA2). HMGA2 is a chromatin-remodelling factor, which alters chromatin architecture by binding to AT-rich regions in the DNA, either promoting or inhibiting the expression of its target genes. One of the targets of HMGA2 is CDKN2A gene which encodes 2 cell cycle regulators p16INK4A and p14ARF. This let-7b-HMGA2-CDKN2A axis regulates cellular growth and senescence of stem cells both in normal and pathological state such as cancer.

We initially examined the expression of HMGA2 in leukemic cells obtained from 35 MLL-rearranged infant ALL patients (MLL-AF4, n = 26; MLL-AF9, n = 4; MLL-ENL, n = 5) using quantitative RT-PCR. As results, HMGA2 was highly expressed in most of the patients with MLL fusion gene, especially in MLL-AF4-positive cases, compared to those without the fusion. These results indicate that deregulation of let-7b-HMGA2 axis by MLL fusion may contribute to leukemogenesis and could be a possible target of molecular therapy against MLL-rearranged ALL.

As let-7b is downregulated by promoter hypermethylation, demethylating agents such as 5-azacytidine could be applied to recover the expression of the gene in leukemic cells with MLL fusion gene. To test this possibility, leukemic cell lines with MLL-AF4 fusion gene were used. The administration of 5-azacytidine alone was able to restore the expression of suppressed let-7b as well as p16INK4A gene in the leukemic cells, but the effects was incomplete, showing persistent partial promoter methylation. In addition, the recovered expression disappeared when 5-azacytidine was removed. On the other hand, when HMGA2 inhibitor was combined with 5-azacytidine, the expression of let-7b was upregulated and sustained resulting in suppression of HMGA2 protein itself. This upregulation of let-7b and suppression of HMGA2 protein persisted even after the removal of 5-azacytidine, possibly through maintaining of the demethylating status by HMGA2 inhibitor. Inhibition of HMGA2 by either siRNA or HMGA2 inhibitor suppressed the growth of MLL-AF4-positive leukemic cells when analysed by MTT assay. The effects of HMGA2 inhibitor on cell growth inhibition became more prominent in combination with demethylating agent 5-azacytidine.

Our results revealed the functional significance of let-7b and HMGA2 in controlling MLL-AF4-positive leukemic cell growth and the therapeutic potential of combining demethylating agent and the HMGA2 inhibitor in the treatment of MLL-AF4-positive ALL.

Disclosures

No relevant conflicts of interest to declare.