Inhibition of poly(ADP-ribose) polymerase 1 protects against acute myeloid leukemia by suppressing the myeloproliferative leukemia virus oncogene

Abstract

An abnormal expression of poly(ADP-ribose) polymerase 1 (PARP-1) has been described in many tumors. PARP-1 promotes tumorigenesis and cancer progression by acting on different molecular pathways. PARP-1 inhibitors can be used with radiotherapy or chemotherapy to enhance the susceptibility of tumor cells to the treatment. However, the specific mechanism of PARP-1 in acute myeloid leukemia (AML) remains unknown. Our study showed that expression of PARP-1 was upregulated in AML patients. PARP-1 inhibition slowed AML cell proliferation, arrested the cell cycle, induced apoptosis in vitro and improved AML prognosis in vivo. Mechanistically, microarray assay of AML cells with loss of PARP-1 function revealed that the myeloproliferative leukemia virus oncogene (MPL) was significantly downregulated. In human AML samples, MPL expression was increased, and gain-of-function and loss-of-function analysis demonstrated that MPL promoted cell growth. Moreover, PARP-1 and MPL expression were positively correlated in AML samples, and their overexpression was associated with an unfavorable prognosis. Furthermore, PARP-1 and MPL consistently acted on Akt and ERK1/2 pathways, and the anti-proliferative and pro-apoptotic function observed with PARP-1 inhibition were reversed in part via MPL activation upon thrombopoietin stimulation or gene overexpression. These data highlight the important function of PARP-1 in the progression of AML, which suggest PARP-1 as a potential target for AML treatment.

Keywords: MPL; PARP-1; acute myeloid leukemia; prognosis.

Figure 1. Aberrant expression of poly(ADP-ribose) polymerase 1 (PARP-1) in acute myeloid leukemia (AML) patients and effect of PARP-1 inhibition on proliferation and cell cycle in AML cell lines

A. qRT-PCR analysis of PARP-1 mRNA in bone marrow from AML patients (n = 30) and controls (n = 15). Each point represents one sample. Horizontal bars represent the means, the whiskers represent SEM. **P < 0.01, AML vs. control. B. Cell viability of Kasumi-1 and THP-1 cells treated with 0, 5, 10, 20, 30, or 40 μM PARP-1 inhibitor PJ34. **P < 0.01, 40 vs. 0 μM. C. Flow cytometry and D. cell cycle quantification of AML cell G2/M arrest with PARP-1 inhibition. *P < 0.05, PJ34 vs. control. E. Western blot analysis of cyclin B1, CDK1, and P27 expression with PARP-1 inhibitor PJ34 or control treatment and F. quantification. *P < 0.05 and **P < 0.01, PJ34 vs. control.

Figure 2. Effect of PARP-1 inhibition on apoptosis and molecular pathways in AML cell lines

A. Flow cytometry and B. quantification of apoptotic AML cells stained with Annexin V-FITC and PI. *P < 0.05 and **P < 0.01, compared to 0 μM. C. Western blot analysis and D. quantification of PAR, p-Akt, t-Akt, p-ERK, t-ERK, Bcl-2, and Bcl-xL expression. *P < 0.05 and **P < 0.01, PJ34 vs. control. Data represent the mean ± SEM.

Figure 3. PARP-1 inhibition improves AML prognosis in vivo in AML mice

A. Fluorescent microscopy and flow cytometry of C1498 cells transduced by a lentivirus with a GFP reporter. B. Western blot analysis of PAR expression in AML mouse tissue with and without PARP-1 inhibitor PJ34. **P < 0.01, PJ34 vs. normal saline (NS). C. Appearance, liver, and spleen of representative AML mice treated with and without PARP-1 inhibitor PJ34. Scale bar: 10 mm. D. Body weights of mice treated with and without PARP-1 inhibitor PJ34. **P < 0.01, PJ34 vs. NS. E. Survival of mice treated with and without PARP-1 inhibitor PJ34. **P < 0.01, PJ34 vs. NS. F. Flow cytometry analysis of GFP-positive cells in total peripheral blood leukocytes (*P < 0.05, PJ34 vs. NS) and G. liver monoplast suspension. H. Hematoxylin and eosin staining of hepatic tissues. Scale bars: 200 μm (top panels) and 100 μm (bottom panels). Data represent the mean ± SEM.

Figure 4. Microarray analysis of gene expression with PARP-1 inhibition

A. Microarray assay of genes with >2.0-fold upregulation or ≥ 0.5-fold downregulation in C1498 mouse AML cells treated with PARP-1 inhibitor PJ34. B. qRT-PCR of 5 genes related to leukemia for validation of the microarray results. C. Gene ontology enrichment analysis of differentially expressed genes. The number of genes with a significantly changed expression is shown in parentheses. D. Top 15 enrichment pathways based on the KEGG database.

Figure 5. High expression of the myeloproliferative leukemia virus oncogene (MPL) in AML patients and MPL sustained malignant proliferation

A. qRT-PCR analysis of MPL mRNA level in bone marrow from AML patients (n = 27) and controls (n = 11). Each point represents one sample. Horizontal bars represent the means, the whiskers represent SEM. **P < 0.01, AML vs. control. B. Western blot analysis and quantification of MPL protein expression by lentiviral infection in Kasumi-1 and THP-1 cells. NC(−), negative control of MPL knockdown; LV-MPL(−), MPL knockdown; NC(+), negative control of MPL overexpression; LV-MPL(+), MPL overexpression. *P < 0.05 and **P < 0.01, LV-MPL vs. NC. C. Cell viability of Kasumi-1 and THP-1 cells with MPL knockdown or overexpression. *P < 0.05 and **P < 0.01, LV-MPL vs. NC. D. Western blot analysis and E. quantification of p-Akt, t-Akt, p-ERK, t-ERK, p-JNK, t-JNK, p-P38, and t-P38 protein expression with MPL knockdown in Kasumi-1 and THP-1 cells. *P < 0.05, **P < 0.01, LV-MPL vs. NC. Data represent the mean ± SEM.

Figure 6. PARP-1 acts on MPL expression

A. Positive correlation between PARP-1 and MPL expression in patient bone marrow samples (n = 27, P < 0.01). B. Western blot analysis of PAR and MPL protein levels and C. qRT-PCR analysis of MPL mRNA level with or without PARP-1 inhibitor PJ34. *P < 0.05, **P < 0.01, PJ34 vs. PBS. D–F. qRT-PCR and western blot analysis of PARP-1 expression upon lentivirus interference. NC, negative control of PARP-1 knockdown; LV-PARP-1(−), PARP-1 knockdown. *P < 0.05 and **P < 0.01, LV-PARP-1 vs. NC. E, F. Western blot analysis of MPL expression with PARP-1 gene silencing. *P < 0.05 and **P < 0.01, LV-PARP-1 vs. NC. Data represent the mean ± SEM.

Figure 7. Enforced expression or activation of MPL partially rescues the effect of PARP-1 inhibition in AML cells

A. Cell viability of Kasumi-1 and THP-1 cells with thrombopoietin (TPO) and/or PARP-1 inhibitor PJ34. *P < 0.05 and **P < 0.01, compared to control. B. Cell viability, C. flow cytometry of apoptosis and D. its quantification of AML cell lines overexpressing MPL and incubated with PARP-1 inhibitor PJ34 for 48 h. *P < 0.05 and **P < 0.01, LV-MPL vs. NC. Data represent the mean ± SEM.

Figure 8. Schematic representation of the role of PARP-1 in the regulation of AML cell survival and proliferation

PARP-1 is overexpressed in AML, which may be caused by DNA damage when excessive division and proliferation of tumor cells. As a transcriptional coregulator, PARP-1 upregulates MPL expression by polyADP-ribosylation, which further activates Akt and ERK1/2 pathways, and allows malignant cells to unlimited proliferation and escaping apoptosis.