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. 2017 Sep 21;8(56):95038-95053.
doi: 10.18632/oncotarget.21119. eCollection 2017 Nov 10.

Protein Phosphatase 4 Regulatory Subunit 2 (PPP4R2) Is Recurrently Deleted in Acute Myeloid Leukemia and Required for Efficient DNA Double Strand Break Repair

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Free PMC article

Protein Phosphatase 4 Regulatory Subunit 2 (PPP4R2) Is Recurrently Deleted in Acute Myeloid Leukemia and Required for Efficient DNA Double Strand Break Repair

Julia K Herzig et al. Oncotarget. .
Free PMC article

Abstract

We have previously identified a recurrent deletion at chromosomal band 3p14.1-p13 in patients with acute myeloid leukemia (AML). Among eight protein-coding genes, this microdeletion affects the protein phosphatase 4 regulatory subunit 2 (PPP4R2), which plays an important role in DNA damage response (DDR). Investigation of mRNA expression during murine myelopoiesis determined that Ppp4r2 is higher expressed in more primitive hematopoietic cells. PPP4R2 expression in primary AML samples compared to healthy bone marrow was significantly lower, particularly in patients with 3p microdeletion or complex karyotype. To identify a functional role of PPP4R2 in hematopoiesis and leukemia, we genetically inactivated Ppp4r2 by RNAi in murine hematopoietic stem and progenitor cells and murine myeloid leukemia. Furthermore, we ectopically expressed PPP4R2 in a deficient human myeloid leukemic cell line. While PPP4R2 is involved in DDR of both hematopoietic and leukemic cells, our findings indicate that PPP4R2 deficiency impairs de-phosphorylation of phosphorylated key DDR proteins KRAB-domain associated protein 1 (pKAP1), histone variant H2AX (γH2AX), tumor protein P53 (pP53), and replication protein A2 (pRPA2). Potential impact of affected DNA repair processes in primary AML cases with regard to differential PPP4R2 expression or 3p microdeletion is also supported by our results obtained by gene expression profiling and whole exome sequencing. Impaired DDR and increased DNA damage by PPP4R2 suppression is one possible mechanism by which the 3p microdeletion may contribute to the pathogenesis of AML. Further studies are warranted to determine the potential benefit of inefficient DNA repair upon PPP4R2 deletion to the development of therapeutic agents.

Keywords: 3p; AML; DNA repair; PPP4R2; gene deletion.

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts interests.

Figures

Figure 1
Figure 1. Ppp4r2 is differentially expressed in hematopoiesis and neoplasia
Expression of Ppp4r2 in murine hematopoietic subpopulations and PPP4R2 in AML patients was determined by qRT-PCR. (A) Ppp4r2 single gene expression among murine hematopoietic subpopulations relative to the housekeeping gene Actin and normalized to Lin-/Sca1+/ckit+ (LSK) cells [LSK (n = 3); common lymphoid progenitors (CLP, n = 4); common myeloid progenitors (CMP, n = 5); megakaryocyte/erythrocyte progenitors (MEP, n = 8); granulocyte/monocyte progenitors (GMP, n = 4); macrophages (n = 6); granulocytes (n = 5)]. (B) PPP4R2 mRNA expression relative to the housekeeping gene beta-2 microglobulin (B2M) in a selected cohort of AML patients (n = 79) normalized to healthy bone marrow (BM; n = 8). (C) PPP4R2 mRNA expression relative to B2M in distinct AML subgroups [CN-AML with 3p CDR (n = 10), CN-AML (n = 24), CK-AML with 3p CDR (n = 23), CK-AML (n = 22)] normalized to healthy BM (n = 8). Data are represented by each individual data point and the mean. Statistical analyses were carried out using unpaired two-tailed t-test. A p-value ≤0.05 was considered significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Figure 2
Figure 2. Ppp4r2 suppression regulates DNA damage response in normal murine hematopoietic cells
(A) Impact of Ppp4r2 knockdown in murine Lin- bone marrow (BM) cells on clonogenic growth determined by colony forming cells in methylcellulose (CFC-Assay; n = 4). (B) Impact of Ppp4r2 knockdown in murine Lin- BM cells on DNA damage response upon ionizing radiation (IR) with 2 Gy determined by the mean fluorescence intensity (MFI) of phosphorylated KRAB-domain associated protein 1 [pKAP1(S824)] at the indicated time post IR (n = 2). Representative histogram depicts the MFI shift of pKAP1 (S824) at 0.5 h post IR in either murine Lin- BM cells with Ppp4r2 knockdown or control. (C) DNA damage at indicated time post IR with 2 Gy in murine Lin- BM cells upon Ppp4r2 knockdown determined by the MFI of phosphorylated histone variant H2AX [γH2AX (S139); n = 3]. Representative histogram depicts the MFI shift of γH2AX (S139) at 0.5 h post IR in murine Lin- BM cells with either Ppp4r2 knockdown or control. (D) Representative Western Blot displaying the effect of IR on phosphorylation of KAP1 (S824) and P53 (S15) in Lin- BM cells with either Ppp4r2 knockdown or control. Vertical lines have been inserted to indicate a repositioned gel lane. (E) Apoptosis induction upon IR with 2 Gy displayed as the percentage of AnnexinV+/7AAD- Lin- BM cells with either Ppp4r2 knockdown or control at the indicated time post IR (n = 3). Data are represented by the mean ± SD. Statistical analyses were carried out using unpaired two-tailed t-test or multiple t-tests corrected for multiple comparisons using the Holm-Sidak method. A p-value ≤ 0.05 was considered significant, *p ≤ 0.05.
Figure 3
Figure 3. Ppp4r2 loss-of-function enhances DNA damage in murine leukemic bone marrow cells
(A) Impact of Ppp4r2 knockdown in murine MLLT3-KMT2A transformed Lin- bone marrow (BM) cells on DNA damage response upon ionizing radiation (IR) with 2 Gy measured by the mean fluorescence intensity (MFI) of phosphorylated KRAB-domain associated protein 1 [pKAP1 (S824)] at the indicated time post IR (n = 3). Representative histogram depicts the MFI shift of pKAP1 (S824) at 2 h post IR in either murine MLLT3-KMT2A transformed Lin- BM cells with Ppp4r2 knockdown or control. (B) DNA damage at indicated time post IR with 2 Gy in murine MLLT3-KMT2A Lin- BM cells upon Ppp4r2 knockdown determined by the MFI of phosphorylated histone variant H2AX [γH2AX (S139); n = 3]. Representative histogram depicts the MFI shift of γH2AX at 2 h post IR in murine MLLT3-KMT2A transformed Lin- BM cells with either Ppp4r2 knockdown or control. (C) Representative Western Blot displaying the effect of IR on phosphorylation of the key DDR protein RPA2 (S33), P53 (S15), KAP1 (S824), and H2AX (S139) in murine MLLT3-KMT2A Lin- BM cells with either Ppp4r2 knockdown or control. Vertical lines have been inserted to indicate a repositioned gel lane. (D) Apoptosis induction upon IR with 2 Gy displayed as the percentage of AnnexinV+/7AAD- murine MLLT3-KMT2A Lin- BM cells with either Ppp4r2 knockdown or control at the indicated time post IR (n = 5). Impact of Ppp4r2 knockdown in murine MLLT3-KMT2A transformed Lin- BM cells on (E) clonogenic growth and replating capacity determined by colony forming cells in methylcellulose (CFC-Assay; n = 5), and (F) proliferation potential measured by MTS-Assay (n = 3). Data are represented by the mean ± SD. Statistical analyses were carried out using unpaired two-tailed t-test or multiple t-tests corrected for multiple comparisons using the Holm-Sidak method. A p-value ≤ 0.05 was considered significant, *p ≤ 0.05.
Figure 4
Figure 4. Re-expression of PPP4R2 restores DNA repair in leukemic cells with 3p microdeletion
(A) Impact of PPP4R2 restoration in human leukemic MEG-01 cells on DNA damage response upon ionizing radiation (IR) with 2 Gy determined by the mean fluorescence intensity (MFI) of phosphorylated KRAB-domain associated protein 1 [pKAP1 (S824)] at the indicated time post IR (n = 5). Representative histogram depicts the MFI shift of pKAP1 (S824) at 2 h post IR in either MEG-01 cells with PPP4R2 restoration or control. (B) DNA damage at indicated time post IR with 2 Gy in MEG-01 cells upon PPP4R2 re-expression determined by the MFI of phosphorylated histone variant H2AX [γH2AX (S139); n = 4]. Representative histogram depicts the MFI shift of γH2AX (S139) at 2 h post IR in MEG-01 cells with either PPP4R2 restoration or control. (C) Representative Western Blot displaying the effect of IR with 2 Gy on phosphorylation of the DDR protein RPA2 (S33), P53 (S15), KAP1 (S824), and H2AX (S139) in MEG-01 cells with either PPP4R2 restoration or control. (D) Effect of PPP4R2 re-expression and exposure to IR with 2 Gy on apoptosis induction displayed as the percentage of AnnexinV+/7AAD- cells in comparison to control (n = 4). Impact of PPP4R2 restoration on (E) clonogenic growth determined by colony forming cells in methylcellulose (CFC-Assay; n = 4), and (F) proliferation potential measured by MTS-Assay (n = 3). Data are represented by the mean ± SD. Statistical analyses were carried out using unpaired two-tailed t-test or multiple t-tests corrected for multiple comparisons using the Holm-Sidak method. A p-value ≤ 0.05 was considered significant, *p ≤ 0.05.
Figure 5
Figure 5. Proposed model for PPP4R2 and its involvement in DNA damage response in normal hematopoietic and leukemic cells

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