Role of HDAC8 Upregulation in Resistance of FLT3-ITD AML Stem Cells to FLT3 Inhibitors
Authors: Long Jun , Jia Mingyuan , Fang Weiyue , Chen Xinjie , Wang Zhongyu , Mu Lili , Xiang Rufang , LI Jun-Min , Hong Dengli , Liang Aibin , Hu Jiong 
Published: November 2019

The FLT3-ITD mutation occurs in 25-30% of acute myeloid leukemia (AML) incidence, and is associated with higher relapse rate and inferior survival. FLT3 inhibitors have shown potent antileukemic effect, but the responses are not sustained in most FLT3-ITD AML patients. Histone deacetylases (HDACs) are crucial for cancer stem cell maintenance through epigenetically regulating gene expression, modulating the function of non-histone proteins via post-translational modification, and maintaining genome stability. Here we investigated the potential role of HDACs in FLT3 inhibitors resistance and FLT3-ITD AML stem cells maintenance.
We firstly evaluated mRNA expression alterations of 11 HDACs in FLT3-ITD+ MV4-11 and MOLM-13 AML cells upon FLT3 inhibitor AC220 (Quizartinib) treatment, and found that HDAC8 was the most significantly upregulated gene. Inhibition of FLT3 signaling via siRNA-mediated knockdown of FLT3 also resulted in increased expression of HDAC8. Treatment of primary FLT3-ITD+ CD34+ AML cells with AC220 confirmed HDAC8 upregulation.
Targeting HDAC8 using specific HDAC8 inhibitor 22d or knockdown of HDAC8 readily resulted in significant apoptosis of cell lines and primary AML samples, and inhibited colony growth and replating activity in primary FLT3-ITD+ CD34+ AML cells. Moreover, these antileukemic effects were even more pronounced upon 22d and AC220 combination.
To explore the mechanism of HDAC8 mediated FLT3 inhibitor resistance, we investigated transcriptomic change of MV4-11 cells upon 22d treatment by RNA-seq. KEGG pathway analysis revealed that the p53 signaling pathway was significantly upregulated. Upstream regulator prediction by Ingenuity pathway analysis also showed p53 as the key activated upstream transcription factor. Western blot analysis on acetylated p53 (Lys382) demonstrated that targeting FLT3 signaling had little effect on the p53 acetylation, while suppression of HDAC8 markedly increased p53 acetylation, and this effect was even more significant when both FLT3 and HDAC8 were inhibited simultaneously. Knockdown of p53 could partially rescued FLT3-ITD AML cells from 22d and/or AC220 induced apoptosis, and offset the inhibitory effect on growth and colony formation.
We next studied the mechanism of HDAC8 upregulation upon FLT3 signally inhibition. We found FOXO1 and FOXO3 (FOXOs) binding motifs in HDAC8 promoter. FLT3 signaling inhibition promoted the binding of FOXOs to HDAC8 promoter. Knockdown both FOXOs abrogated HDAC8 upregulation upon AC220 treatment, and caused growth inhibition and apoptosis of FLT3-ITD AML cells.
Finally, to translate our experimental results into clinical usage, we tested the in vivo effect of 22d, AC220, and the combination of both on patient-derived xenograft (PDX) model engrafted with four primary FLT3-ITD AML samples. 22d or AC220 treatment reduced the percentage and total number of human CD45+ leukemic cells in murine bone marrow, and prolonged the survival of leukemic mice. The combination of 22d and AC220 significantly reduced AML cell engraftment compared with either 22d or AC220 alone, and further elongated the survival period. Human CD34+ cells in BM were markedly reduced in mice treated with 22d or AC220 alone, with further reduction seen in combination. Secondary transplantation of BM cells from mice receiving combination treatment resulted in significantly reduced engraftment compared with AC220 treatment alone, indicating reduced LSC capacity of residual cells. These results show that combination of 22d and AC220 enhances targeting of primitive AML stem cells in vivo.
In conclusion, upon FLT3 inhibitor treatment, FLT3-ITD AML cells may upregulate the expression of HDAC8 through FOXOs activation, which in turn inhibit the function of tumor suppressor gene p53, and promote the maintenance of FLT3-ITD AML stem cells and drug resistance. Targeting FLT3 and HDAC8 simultaneously may be promising approach to eliminating FLT3-ITD AML stem cells, and to cure this subset of leukemia.

Disclosures
No relevant conflicts of interest to declare.