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Letters to the Editor
FAM122A promotes acute myeloid leukemia cell growth through inhibiting PP2A activity and sustaining MYC expression
Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of hematopoietic stem and progenitor cells (HSPC), characterized by uncontrolled proliferation, differentiation blockage and reduced apoptosis. Although progressive advances have been made in AML treatment over the last decades, the 5-year survival rates of AML patients are still low, especially for elderly patients.1,2 It is, therefore, necessary to understand the molecular patho- logical mechanisms and develop more effective treat- ment strategies for this disease. Given its frequent inacti- vation in AML cases, the importance of protein phos- phatase 2A (PP2A) as a tumor suppressor and promising target for therapy has been highlighted recently.3 The loss of PP2A activation occurs at different levels in AML, either with the mutation and downregulation of PP2A subunits or overexpression of PP2A inhibitors SET, CIP2A and SET interacting protein.4,5 Pharmacological restoration of PP2A activity by its activator (FTY720) effectively antagonizes leukemogenesis.6 It was shown that FAM122A (family with sequence similarity 122a), a highly conserved protein among a variety of mammalian species, interacts with PP2A-Aa and -B55a (a scaffold and regulatory subunit of the PP2A complex) and pro- motes the polyubiquitination and degradation of its Ca subunit,7 and FAM122A sumoylation increases the degradation of PP2A-Ca protein together with the reduced phosphatase activity of PP2A.8 Recently, we demonstrated that FAM122A maintains the growth of hepatocellular carcinoma cells through promoting MAPK/AKT signaling.9 However, the normal function and pathophysiological significance of FAM122A are still largely unknown. Here we investigated whether FAM122A has a role in the growth of AML cells and AML development.
Besides FAM122A, the FAM122 family has two other members, FAM122B and FAM122C, which share 60% and 30% amino acid sequence identity, respectively, with FAM122A. We examined the mRNA expression levels of these three FAM122 members in a panel of samples from patients with AML and acute lymphoblastic leukemia as well as human CD34+ HSPC by analyzing the public RNA-sequencing database (http://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE48173). As shown in Figure 1A, the levels of mRNA expression of FAM122A, but not of FAM122B and FAM122C, were significantly increased in patients with AML or acute lymphoblastic leukemia compared to the levels in normal HSPC. A similar pattern of higher expression of FAM122A was also found in bone marrow cells from patients with four subtypes of AML with different karyotypes, that is, t(8;21), t(15;17), inv(16)/t(16;16) and t(11q23)/MLL, compared with the expression in hematopoietic stem cells enriched for Lin−CD34+CD38−CD90+CD45RA− in normal bone mar- row cells (Online Supplementary Figure S1A) (http://servers.binf.ku.dk/hemaexplorer/). The Cancer Genome Atlas datasets also showed that AML patients with high FAM122A mRNA expression had shorter over- all survival (Figure 1B), suggesting a potentially oncogenic role of FAM122A in AML. Moreover, FAM122A protein expression was also higher in AML cell lines, compared to the level in peripheral blood mononuclear cells from three healthy individuals (Figure 1C).
To assess the function of FAM122A in AML, three human AML cell lines – (i) NB4, an acute promyelocytic
leukemia cell line carrying the t(15;17), (ii) U937 and (iii) THP1 with the MLL-AF9 fusion gene – were transfected with lentiviruses expressing short hairpin RNA (shRNA) targeting two distinct regions of FAM122A mRNA (desig- nated shFAM122A#1 and shFAM122A#2) together with a non-specific shRNA (shScramble). The results showed that FAM122A could be effectively knocked down with these two pairs of shRNA in all three AML cells (upper panels, Figure 1D), and knockdown of FAM122A caused a substantial decrease in cell growth compared to that of shScramble-expressing NB4 cells (bottom panels, Figure 1D), which could be significantly rescued by re-expres- sion of FAM122A (Figure 1E). Meanwhile, FAM122A silencing also suppressed the growth of normal CD34+ cells to some degree (Online Supplementary Figure S1B-D), suggesting that there is a differential requirement of FAM122A for the growth of normal HSPC and AML cells.
We continued to investigate whether FAM122A knock- down induces cell death and/or altered cell cycle distribu- tion. Examining NB4 cells, we found that fewer live cells were visible under light microscopy among cells trans- duced with shFAM122A-lentivirus for 4 days, compared with their control cells (upper panels, Figure 1F). NB4 cells transfected with shFAM122A#2 and especially shFAM122A#1 displayed significant increases in cells double-positive for annexin V and propidium iodide without significant alteration of the cell cycle distribution (bottom panels of Figure 1F and data not shown), indicat- ing that FAM122A silencing led to cell apoptosis. Similar results were also observed in U937 and THP1 cells (Figure 1G), although these effects became obvious after transduction for 6 days. The role of FAM122A in leukemic cell growth in vivo was further investigated using a mouse model of AML driven by MLL-AF9, which is produced by t(9;11)(p22;q23), frequently found in infant AML and associated with the M5 subtype of AML.10 MLL-AF9 leukemic cells with a yellow fluores- cent protein (YFP) tag from bone marrows of leukemic mice were engineered to express either green fluorescent protein (GFP)-tagged shControl or shRNA that target murine Fam122a (designated shFam122a#3 and shFam122a#4) with a lentivirus system (Figure 2A). GFP+/YFP+ cells in each condition purified by sorting were assessed for infection efficiency and colony formation, as well as AML development through injecting the sorted leukemic cells into lethally irradiated mice (all mice were used and cared for according to Shanghai Jiao Tong University School of Medicine and national guidelines). The efficient silencing effects of FAM122A were con- firmed in both shFam122a#3- and shFam122a#4-express- ing cells and FAM122A inhibition significantly abrogated colony-forming capacity in methylcellulose (Figure 2B and C). The purified MLL-AF9 leukemic cells expressing two Fam122a-shRNA together with shControl in equal numbers (2,000 or 5,000 cells) were transplanted into lethally irradiated recipient mice. As shown in Figure 2D, the recipient mice transplanted with shFam122a#3 and shFam122a#4 cells had significantly longer disease laten- cy and extended survival, as determined by mean sur- vival (50 or 53 days in shFam122a#3- or shFam122a#4- expressing mice compared to 25 days in control mice with 2,000 cells; 43 or 50 days in shFam122a#3- or shFam122a#4-expressing mice compared to 22 days in controls with 5,000 cells). Flow assisted cell sorting analysis of hematopoietic organs and peripheral blood further showed that mice transplanted with both Fam122a-shRNA leukemic cells displayed significantly lower percentages of YFP+ cells in peripheral blood, bone
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