Histone Acetyltransferase Pcaf Is Required for ATRA-Induced Granulocytic Differentiation in Acute Promyelocytic Leukemia Cells

Differentiation therapy with All-trans retinoic acid (ATRA) improves the treatment outcome of acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML); however, its molecular mechanism remains elusive. We have previously reported that inhibition of NAD-dependent histone deacetylase SIRT2 induces granulocytic differentiation in APL cells (PLOS ONE. 2013; 8(2): e57633), suggesting a possible involvement of protein acetylation in the differentiation of APL cells by ATRA.

To assess this possibility, in the present study, we examined expression of major histone-acetyltrasnferases such as GCN5, PCAF, and CBP/p300 upon ATRA-treatment, and found that PCAF-expression was dramatically increased at mRNA (1.8 to 7 fold, Figure 1A) as well as protein levels in NB4, HL-60, and APL primary cells. Consistent with this, PCAF mRNA expression was greatly induced (10 to 40 fold) in the bone marrow of APL patients who received ATRA-containing treatment (Figure 1B). A search in the public expression database revealed that PCAF-expression in bone marrow was generally reduced in not only APL but also other acute hematologic malignancies such as AML and acute lymphoblastic leukemia (ALL) (healthy donor; n = 74, APL; n=37, AML; n=505, ALL; n=750, healthy donor vs. AML, p < 0.001, healthy donor vs. APL, p < 0.001, healthy donor vs. ALL, p < 0.001, adopted from oncomine database). Interestingly, the reduction of PCAF-expression was much more evident in acute than chronic hematologic malignancies such as chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS) (CML; n = 76, MDS; n=206), suggesting that PCAF reduction is related to a blockade of proper differentiation in malignant cells. Based on these findings we hypothesized that PCAF plays an important role in ATRA-induced APL cell differentiation.

To prove this, we performed a loss-of-function assay using lentivirus vectors expressing 3 independent shRNA against PCAF or non-targeting shRNA (control) (Figure 1C). When PCAF was knocked down in HL-60 cells, these cells failed to differentiate into granulocytes with ATRA-treatment (31.9±11.9% vs. 4.9±2.2%, 5.2±0.7%, 8.6±3.7%, CD11b positive cells, control vs. 3 independent PCAF shRNA) (Figure 1C). Moreover, transduction of PCAF shRNA or non-target shRNA to primary APL cells made cells resistant to ATRA-induced granulocytic differentiation (data not shown), suggesting that PCAF is required for the ATRA-induced granulocytic differentiation in APL cells. To further investigate how PCAF promotes APL cell differentiation upon ATRA-treatment, we performed an acetylome analysis to identify a downstream molecule whose activity is regulated by PCAF through acetylation. We pulled-down acetylated proteins using anti-acetylated lysine antibody from cell extracts prepared 3 or 24 hours after either mock- or ATRA-treatment, and identified 5 proteins preferentially acetylated by ATRA-treatment including histone H3, which is a known acetylation target of PCAF. These results strongly support our hypothesis that upon ATRA-treatment, PCAF induction and subsequent acetylation of PCAF substrates promotes APL cell differentiation. More detailed understanding of PCAF-dependent APL cell differentiation may lead to the development of differentiation therapy against not only APL but also other AML.

Figure 1

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(A) PCAF mRNA induction in ATRA-treated NB4, HL-60, and APL primary cells. (B) PCAF induction in the bone marrow of APL patients during the ATRA-containing chemotherapy. Day 1 represents the mRNA level prior to the treatment. (C) PCAF knocked-down in HL-60 cells by 3 independent shRNA against PCAF or non-targeting shRNA (control) (Upper left). PCAF knocked-down cells lack a capacity to differentiate into granulocytes (CD11b positive cells) by ATRA-treatment (***; p<0.001, n=3) (lower left, and right).

Figure 1

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(A) PCAF mRNA induction in ATRA-treated NB4, HL-60, and APL primary cells. (B) PCAF induction in the bone marrow of APL patients during the ATRA-containing chemotherapy. Day 1 represents the mRNA level prior to the treatment. (C) PCAF knocked-down in HL-60 cells by 3 independent shRNA against PCAF or non-targeting shRNA (control) (Upper left). PCAF knocked-down cells lack a capacity to differentiate into granulocytes (CD11b positive cells) by ATRA-treatment (***; p<0.001, n=3) (lower left, and right).

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