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Letters to the Editor
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Figure 3. The effect of FAM122A silencing on phosphorylatios of PP2A substrates and MYC expression. (A) Acute myeloid leukemia (AML) cells with FAM122A silencing and reexpression (shFAM122A#1-FAM122A-6m) were subjected to western blot for detection of the indicated proteins. (B) The indicated cells were treated with or without 50 nM okadaic acid (OA) for 6 h (NB4 cells), or 20 nM OA for 12 h (U937 cells), and then western blotting was performed to examine protein levels. (C) The MYC mRNA expression level was examined in FAM122A knockdown AML cells. Data are means with the bar indicating the standard devi- ation in an independent experiment. ***P<0.001 compared with shScramble cells. (D, E) FAM122A knockdown NB4 cells, together with shScramble cells, were treated with or without 20 mΜ MG132 for 4 h (D), or cyclohexamide at a dose of 100 mg/mL at different time points (E), followed by western blot to examine the indicated proteins. CHX: cyclohexamide.
marrow and spleen, compared with those of shControl counterparts (Figure 2E). The sizes and weights of livers and spleens from FAM122A-silenced recipients appeared much smaller than those of control recipients (Figure 2F and G), accompanied by less leukemic cell infiltration (Figure 2H). Collectively, these observations strongly sug- gest that FAM122A is a critical regulator of AML cell growth and development in vitro and in vivo.
Many key substrates of PP2A are involved in the regu- lation of cell growth and apoptosis, including pro-prolif- eration regulators AKT, ERK, MYC11 and antiapoptotic factors Bcl-212 and BAD.13 Considering that FAM122A was previously identified as a PP2A inhibitor, we further examined the phosphorylation states of a series of PP2A substrates. FAM122A silencing significantly decreased the phosphorylation of several PP2A substrates, includ- ing MYC at Ser62, ERK at Thr202/Tyr204, BAD at Ser112 and Bcl-2 at Ser70, but not the substrate of tyro- sine phosphatase SHP-1 such as JAK2 (Figure 3A). The effect of decreased phosphorylation could be abolished when FAM122A was re-expressed in shFAM122A#1 cells, indicating the altered phosphorylation events are specifically regulated by FAM122A, although we did not find a change in AKT phosphorylation. FAM122A knock- down also reduced total MYC and Bcl-2 protein levels (Figure 3A). Treatment with okadaic acid, a PP2A inhibitor, significantly abrogated FAM122A silencing- decreased phosphorylation of ERK and BAD, but not that of MYC, suggesting that FAM122A maintains AML cell growth and survival possibly by modulating PP2A activi- ty (Figure 3B). Indeed, PP2A-Cα silencing partially res- cued FAM122A knockdown-triggered apoptosis (Online Supplementary Figure S2).
MYC is essential for the occurrence and progression of AML,14 and there is mounting evidence that Ser62 phos- phorylation is critical for maintaining the stability of
MYC protein.15 However, we found that treatment with- okadaic acid or PP2A-Ca knockdown (Online Supplementary Figure S2B) did not affect phosphorylated MYC in FAM122A knockdown cells, suggesting that the reduction in phosphorylated MYC was not due directly to PP2A. FAM122A knockdown resulted in decreased MYC mRNA levels (Figure 3C), but did not affect the sta- bility or degradation of the MYC protein (Figure 3D and E). AF9-induced leukemic cells with FAM122A silencing also demonstrated a decrease in MYC protein (Figure 2B). These results strongly imply that FAM122A promotes AML cell growth also by maintaining MYC transcription- al expression.
In summary, our study shows that FAM122A is critical for maintaining AML cell growth and survival by modu- lating PP2A activity and sustaining MYC expression, findings which warrant further investigation into its potential role in the treatment of AML.
Man-Hua Liu,1 Jing Chen,1 Yun-Sheng Yang,1 Yin-Qi Wang,1 Guo-Qiang Chen,1 Yu Zhang2 and Ying Huang1
1Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education and Chinese Academy of Medical Sciences Research Unit (NO.2019RU043), Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine (SJTU-SM) and 2Department of Obstetrics and Gynecology, Ren-Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Correspondence:
YING HUANG - huangying@shsmu.edu.cn YU ZHANG - rjzhangyu@163.com doi:10.3324/haematol.2020.251462 Disclosures: No conflicts of interest to disclose.
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haematologica | 2021; 106(3)