Silencing of the DNA damage repair regulator PPP1R15A sensitizes acute myeloid leukemia cells to chemotherapy

Abstract

Acute Myeloid Leukemia (AML) is a life-threatening disease whose induction treatment consists of combination chemotherapy with Idarubicin and Cytarabine for fit patients. Treatment failures are frequent, urging the need for novel treatments for this disease. The DNA Damage Response Mechanism (DDR) comprises numerous molecules and pathways intended to arrest the cell cycle until DNA damage is repaired or else drive the cell to apoptosis. AML-derived cell lines after treatment with Idarubicin and Cytarabine were used for studying the expression profile of 84 DDR genes, through PCR arrays. Utilizing de novo AML patient and control samples we studied the expression of PPP1R15A, CDKN1A, GADD45A, GADD45G, and EXO1. Next, we performed PPP1R15A silencing in AML cell lines in two separate experiments using siRNA and CRISPR-cas9, respectively. Our findings highlight that DDR regulators demonstrate increased expression in patients with high cytogenetic risk possibly reflecting increased genotoxic stress. Especially, PPP1R15A is mainly involved in the recovery of the cells from stress and it was the only DDR gene upregulated in AML patients. The PPP1R15A silencing resulted in decreased viability of Idarubicin and Cytarabine-treated cell lines, in contrast to untreated cells. These findings shed light on new strategies to enhance chemotherapy efficacy and demonstrate that PPP1R15A is an important DDR regulator in AML and its downregulation might be a safe and effective way to increase sensitivity to chemotherapy in this disease.

Keywords: Acute myeloid leukemia; DNA damage response; GADD34; PPP1R15A; Resistance mechanisms.

Fig. 1

Heatmap of differentially expressed genes with at least one statistically significant fold change (p-value < 0.05) either in KASUMI-1 or MV4-11 live cells treated with Idarubicin or Cytarabine. Marked in bold are the selected genes whose expression was subsequently studied in bone marrow samples of AML patients

Fig. 2

(a) Differential expression of DDR genes in AML patients and controls. Brackets denote p < 0.05 (b) PPP1R15A expression in healthy BM mononuclear cell samples and AML samples based on data from the BEAT AML dataset. (c) Differential expression of DDR genes in different cytogenetic AML risk groups. Brackets denote p < 0.05 (d) Correlation between PPP1R15A and CDKN1A in AML patients

Fig. 3

Differential Expression of PPP1R15A in MOLM-13 and KASUMI-1 cells after treatment with Idarubicin and Cytarabine

Fig. 4

Cell viability after 24 h and 48 h of PPP1R15A silenced and scrambled cells. a: Kasumi-1, Idarubicin treated cell line, b: Kasumi-1, Cytarabine treated cell line, c: MOLM-13, Idarubicin treated cell line d: MOLM-13, Cytarabine treated cell line

Fig. 5

(a) TF-1 cells with PPP1R15A CRISPR-Cas9-mediated knockdown (clones 1 and 2) have a higher sensitivity to Cytarabine compared to control cells.(b) TF-1 cells with PPP1R15A CRISPR-Cas9-mediated knockdown (clones 1 and 2) have a higher sensitivity to Idarubicin compared to control cells. (c) TF-1 cells with PPP1R15A CRISPR-Cas9-mediated knockdown (clones 1 and 2) have lower IC50 dose for Cytarabine compared to control cells. (d) TF-1 cells with PPP1R15A CRISPR-Cas9-mediated knockdown (clones 1 and 2) have lower IC50 doses for Idarubicin compared to control cells