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Tumor Biology

, Volume 37, Issue 9, pp 12513–12523 | Cite as

INPP4B-mediated DNA repair pathway confers resistance to chemotherapy in acute myeloid leukemia

  • Ping Wang
  • Dan Ma
  • Jishi WangEmail author
  • Qin Fang
  • Rui Gao
  • Weibing Wu
  • Lu Cao
  • Xiuying Hu
  • Jiangyuan Zhao
  • Yan Li
Original Article
  • 438 Downloads

Abstract

INPP4B has been recently shown to be a poor prognostic marker and confer chemo- or radio-resistance in AML cells, whereas, the underlying mechanisms remain unclear. Herein, we aimed to explore the possible mechanisms mediated the resistance to chemotherapy in AML. We found that INPP4B-mediated resistance to genotoxic drug, cytarabine, was accompanied by lower p-H2AX accumulation in KG-1 cells, and INPP4B knockdown evidently sensitized KG-1 cells to cytarabine, meanwhile, p-H2AX expression was increased dramatically. Then, we observed that INPP4B knockdown inhibited the loss of p-H2AX expression after cytarabine removal in INPP4B-silenced KG-1 cells, whereas, in control KG-1 cells, the expression of p-H2AX was reduced in a time-dependent manner. Next, INPP4B knockdown can significantly downregulate ATM expression and subsequently inhibit the activation of ATM downstream targets of p-ATM, p-BRCA1, p-ATR, and p-RAD51. Furthermore, nuclear localization of p65 was inhibited after INPP4B knockdown, and reactivation of p65 can rescue the INPP4B knockdown-induced inhibition of ATM, p-ATM, p-BRCA1, p-ATR, and p-RAD51. Finally, INPP4B expression was positively correlated with ATM expression in AML cells, both INPP4B knockdown and KU55933 can significantly sensitize primary myeloid leukemic cells to cytarabine treatment.

Collectively, these data suggest that enhanced ATM-dependent DNA repair is involved in resistance to chemotherapy in INPP4Bhigh AML, which could be mediated by p65 nuclear translocation, combination chemotherapy with INPP4B or DNA repair pathway inhibition represents a promising strategy in INPP4Bhigh AML.

Keywords

INPP4B Cytarabine Resistance DNA repair 

Introduction

INPP4B (inositol polyphosphate 4-phosphatase II) is an important regulator in cancer cell survival. Its role in tumorigenesis is controversial. INPP4B can act as a tumor suppressor in many solid tumors, however, increased INPP4B expression and promoting disease progression have also been demonstrated in some cancers, including acute myeloid leukemia (AML) [1]. Its expression showed different tissue distribution pattern, including the lung, spleen and testis and brain, and in hematopoietic cell types of B cells, NK cells and macrophages, differential INPP4B expression was also reported to be detected [2]. It is originally considered as a tumor suppressor in breast cancer, prostate cancer, and ovarian cancer due to its function of catalyzing PI(3,4)P2 to PI(3)P and inhibiting the PI3K/Akt signaling [3, 4, 5, 6]. In hematological cancers, INPP4B is lost in 12 % of Down-Syndrome associated acute lymphoblastic leukemia [7], and its expression in malignant erythroleukemia proerythroblasts is usually silenced [8]. As an oncogene, INPP4B was also reported to enhance oncogenic activity of the kinase SGK3 in breast cancer and promote resistance by upregulation of hexokinase 2 in laryngeal cancer cells [9, 10], and in nasopharyngeal cancer Hep2 cells, induction of INPP4B triggers the development of a tumor-resistance phenotype and targeting INPP4B can resolve the radioresistance of cancer cells [11]. Interestingly, INPP4B has been recently reported to be functioned as a poor prognostic maker, and resist to radiotherapy and chemotherapy in AML, including cytarabine, daunorubicin, etoposide, and irradiation [12, 13, 14]. At present, there is little reports about the underlying mechanisms mediated INPP4Bhigh leukemic cells to genotoxic treatment, and the downstream targets of INPP4B are unclear, however, an enhanced ability to resist genotoxic exposure might explain this clinical observation in AML, leading us to focus on the DNA damage repair pathway in INPP4Bhigh AML cells.

DNA damage is a common phenomenon during cell survival, it can be triggerred by various factors, such as chemotherapeutics, ionizing radiation, UV as well as reactive oxygen species (ROS) and so on [15], it includes single-strand DNA breaks (SSBs) and, double strend breaks (DSBs) and so on, and the DSBs is the most deleterious DNA lesions, because a single unrepaired DSB is enough to induce cell death [16]. There are six primary DNA repair pathways in human cells: base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination (HR), Fanconi anemia/BRCA pathway (HR), nonhomologous end-joining (NHEJ), and translesion DNA synthesis (TLS). Each pathway repairs a different type of lesion [17], of which homologous recombination (HR) and nonhomologous end-joining (NHEJ) pathway are participated in DSBs repair [18].

DNA repair pathway is necessary to maintain the stability and integrity of genomic DNA for normal cellular survival, otherwise, the frequency of cell death, gene mutations, or malignant transformation to tumor cells is increasing [19]. However, cancer cells usually have DNA repair pathway deficiencies, which render cancer cells more vulnerable to genotoxic treatment but can also drive resistance if the DNA repair pathway is excessively activated [20]. Increased DNA repair contributes to drug resistance and the risk of disease relapse [21]. In hematologic malignancies, inhibition of DNA repair in FLT3-ITD AML patients can reduce the risk of disease relapse by impairing the ability of leukemic cells to survive chemotherapy-induced DNA damage [22]. Moreover, a growing number of reports have suggested that upregulation of DNA damage response (DDR) genes can provide AML cells with escaping from mechanisms to the DDR anticancer barrier and induce chemotherapy resistance [23]. Given that INPP4B loss is reported to be concomitant loss of ATM, ATR, and BRCA1 in ovarian tumors [24], and its role of resistance to genotoxic treatment in AML, we therefore speculate that INPP4B-mediated resistance to genotoxic treatment is correlated with DNA repair pathway in AML.

Therefore, this study aimed to investigate whether INPP4B expression is correlated with DNA repair pathway, and if so, how INPP4B expression influences the DNA repair, which will highlight the mechanisms underlying genotoxic treatment resistance in AML with INPP4Bhigh.

Materials and methods

Patients

Bone marrow samples were obtained from AML patients (n = 61) after written informed consent. The patients were diagnosed by using FAB classification and patient characteristics are listed in Table 1. Bone marrow mononuclear cells (MNCs) were isolated by Ficoll density centrifugation. The study was approved by the Institutional Review Board of the Affiliated Hospital of Guizhou Medical University.
Table 1

Patient characteristics

 

Overall population

INPP4B high

INPP4B low

Total number

 No. (%)

61

22 (36 %)

39 (64 %)

Sex

 Male (%)

32 (52 %)

9 (15 %)

23 (38 %)

 Female (%)

29 (48 %)

13 (21 %)

16 (26 %)

Age, years

 Median (range)

39 (9–78)

41 (9–74)

37 (9–78)

WBC, ×109/L

 Median (range)

64.7 (15.8–180.3)

61.7 (15.8–164.5)

66.5 (17.8–180.3)

Bone marrow blasts, %

 Median (range)

47 (30–81.1)

50 (30–81)

45.65 (30–81.1)

FAB classification

 M0

0

0

0

 M1

1

1

0

 M2

30

14

16

 M3

0

0

0

 M4

6

2

4

 M5

2

0

2

 M6

1

1

0

 M7

0

0

0

 Unclassificated

21

4

17

Cytogenetics

 Normal

27

11

16

 Abnormal

30

9

21

 Unknown

4

2

2

Cytogenetic risk

 Favorable

13

3

10

 Intermediate

29

11

18

 Adverse

15

5

10

 Unknown

4

2

2

FAB French‐American‐British classification, WBC white blood cell count

Cell lines and cell culture conditions

Human AML cell lines, including KG-1, HL60, SKM-1, U937 cells, were cultured in 1640 medium supplemented with 15 % fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. The medium and antibiotics were bought from Hyclone (Logan, UT, USA). All cells were maintained at 37 °C in a 5 % CO2 humidified atmosphere.

Chemicals and antibodies

Cytarabine (Ara-c) was purchased from Pfizer, Inc. (New York City, NY, USA).

Antibodies used included p-H2AX (5438S), INPP4B (4039S), ATM (2873S), BRCA1 (14823S), ATR (13934S), Rad51 (8875S), p-ATM (5883S), p-BRCA1 (9009S), p-ATR (2853S), p65 (8242S), and PCNA (13110S); these antibodies were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). p-Rad51 (SAB4504264) was purchased from Sigma. β-Actin (AA128-1) and secondary antibodies were purchased from Beyotime (Shanghai, China).

siRNA delivery

siRNA targeting INPP4B (Viewsolid Biotech, Beijing, China) was used for knockdown of INPP4B gene, and control siRNA was applied as a control. INPP4B-siRNA sense: CGAUGUCAGUGACACUUGATT, INPP4B-siRNA anti-sense: UCAAGUGUCACUGACAUCGAT were transfected into 1 × 105 KG-1 cells using lipofectamine according to the manufacturer’s instructions. Cells were maintained for 48 h and the depletion of INPP4B proteins was determined by western blotting.

Cell proliferation assay

Cell proliferation was measured using MTT assay. Cells were seeded at a density of 1 × 103 cells per well in 96-well plates. After overnight incubation at 37 °C, serial dilutions of cytarabine were added for indicated time. Twenty microliters of 5 mg/mL of MTT (Amresco, USA) were then added into each well and the cells cultured at 37 °C for an additional 4 to 6 h. The resulting formazan crystals were solubilized by the addition of 150 mL of DMSO to each well. The optical density level under 570 nm was measured and the percentage of cell viability was ultimately calculated.

Western blot analysis

Western blot analysis was carried out to analyze protein expression and activation after cells were treated with cytarabine or not. Briefly, total proteins were extracted by lysing cells in buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 0.5 % NP-40 Nonidet P-40, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 25 mg/ml leupeptin, and 25 mg/ml aprotinin. The lysates were cleared by centrifugation, and the supernatants were collected. Equal amounts of protein lysate were used for western blot analyses. Chemiluminescence was detected by exposure to X-OMAT BT film.

Quantitative real-time PCR

Total RNA was isolated and purified from cells using the RNeasy Kit (Qiagen, Hilden, Germany) and reverse-transcribed using the Omniscript Reverse Transcription Kit (Qiagen). cDNAs were analyzed by quantitative real-time PCR using primers (Generay Biotechnology Corp, Shanghai, China) and SYBR Green qPCR Mix (Beijing Zoman Biotechnology, Beijing, China). Human INPP4B primers: antisense 5′-CGAATTCGCATCCACTTATTG-3′, sense: 5′-GGAAAGTGTGAGCGGAAAAG-3′; Human β-actin primers used as internal control.

Statistical analysis

Each experiment or assay was performed at least three times, and representative examples are shown. Datas were reported as means ± SEM. Differences between the treated groups were calculated using Student’s t test. Differences were considered significant when p < 0.05 (*) or p < 0.01 (**).

Results

INPP4B-mediated resistance to chemotherapy is correlated with lower DSBs formation

To address the question of whether INPP4B expression has an impact on the genotoxic drug-induced DNA damage, we firstly detected the basal INPP4B expression in KG-1, HL60, SKM-1, U937 cell lines, and CD34+ cells. Only KG-1 cells showed a relatively higher expression of INPP4B mRNA [Fig. 1a]. Then, we used KG-1 cells with basal INPP4Bhigh and HL60 cells with basal INPP4Blow to substantiate the effects of INPP4B on cytarabine efficacy, and different concentrations of cytarabine (0, 2, 4, 6, 8 μM) were incubated in KG-1 and HL60 cells for 24 h. Consistent with the previous reports, MTT analysis results showed that KG-1 cells with INPP4Bhigh were less sensitive to cytarabine compared with HL60 cells [Fig. 1b]. As is reported, p-H2AX expression can be increased with the incidence of DSBs (Podhorecka et al. 2010), then we compared the DSBs marker, p-H2AX, between the two cell lines before or after cytababine treatment, and we found that cytarabine treatment resulted in higher p-H2AX expression in HL60 than in KG-1 cells (p < 0.01) [Fig. 1c]. To further confirm the relation between INPP4B and p-H2AX expression, INPP4B expression was silenced by siRNA in KG-1 cells, INPP4B gene and protein were both downregulated (p < 0.05) [Fig. 1d], combination therapy of INPP4B siRNA and cytarabine significantly decreased KG-1 cell viabilities compared with cytarabine treatment alone (p < 0.05) [Fig. 1e], and next p-H2AX expression was detected again, interestingly, combination therapy of cytarabine and INPP4B knockdown significantly increased p-H2AX expression compared with cytarabine treatment alone (p < 0.05) [Fig. 1f].
Fig. 1

INPP4B-mediated resistance to chemotherapy is accompanied by lower DSBs formation. a The INPP4B mRNA expression was detected among AML cell lines, KG-1, HL60, SKM-1, U937, and CD34+ derived from healthy person by real-time PCR. b Cell viabilities were examined by MTT assay after exposure to cytarabine (0, 2, 4, 6, 8 μM) in KG-1 and HL60 cells for 24 h. c p-H2AX protein expression was measured by western blot before or after cytarabine (4 μM) treatment. d Real-time PCR and western blot analysis of INPP4B expression following INPP4B siRNA transfection for 48 h. e MTT analysis of the effects of INPP4B knockdown on cytarabine-induced decreasement of cell viabilies. f Western blot analysis of the p-H2AX protein expression after INPP4B knockdown. Performed in triplicate. *p < 0.05, **p < 0.01

INPP4B knockdown inhibits the loss of p-H2AX expression in cytarabine-damaged KG-1 cells

As a higher p-H2AX expression in HL60 with INPP4Blow and in INPP4B-silenced KG-1 cells after cytarabine exposure, we wondered if this increase in p-H2AX expression could be attributed to fewer DSB repair. To address the question of whether INPP4B expression has an impact on the repair of DSBs, p-H2AX expression was quantified in control KG-1 cells and in INPP4B-silenced KG-1 cells after exposure cytarabine (4 μM) for 0, 4, 8, 16, and 24 h seperately, and results showed that p-H2AX expression was increased in a time-dependent manner, and the increase in p-H2AX expression is more evident in INPP4B-silenced KG-1 cells over time (p < 0.05) [Fig. 2a]. Then, we removed the cytarabine, p-H2AX expression was examined again after a 8-, 16-, and 24-h repair period separately in KG-1 cells and INPP4B-silenced KG-1 cells. Interestingly, the loss of INPP4B expression was evident in KG-1 cells (p < 0.05); however, we did not observe significant reduction of p-H2AX expression in INPP4B-silenced KG-1 cells, indicating that INPP4B expression may be positively correlated with DNA lesion repair [Fig. 2b].
Fig. 2

The effects of INPP4B knockdown on the inhibition of p-H2AX loss in KG-1 cells. a KG-1 cells were incubated with cytarabine (4 μM) in the absence or presence of INPP4B-siRNA for 0, 4, 8, 16, and 24 h, respectively, p-H2AX expression was detected by western blot. b KG-1 cells were treated with cytarabine (4 μM) in the absence or presence of INPP4B-siRNA for 24 h, and then the cytarabine was removed with fresh medium for 8, 16, and 24 h, respectively, p-H2AX expression was detected by western blot assay. The p-H2AX phosphorylation value at t = 8, 16, and 24 h declined over time in control KG-1 cells, whereas in INPP4B-silenced KG-1 cells, there are no obvious changes of H2AX phosphorylation. Repeated in triplicate. *p < 0.05. When compared with INPP4B-silenced KG-1 cells, the statistical differences in KG-1 cells were indicated by # p < 0.05, ## p < 0.01

Expression of INPP4B is involved in ATM-dependent HR repair on DNA damage

To further explore whether INPP4B-mediated DNA damage repair is associated with HR repair signaling or not, we first detected mRNA expression of HR repair-related molecular, ATM, BRCA1, ATR, and RAD51, after INPP4B knockdown, and only ATM mRNA expression was inhibited by INPP4B siRNA, and followed by inhibition of ATM protein expression (p < 0.01); however, we did not find obvious inhibition of BRCA1, ATR, and RAD51 [Fig. 3a, b]. Then, the ATM and the phosphorylation of ATM, BRCA1, ATR, and RAD51 were detected again after cytarabine treatment combined with INPP4B knockdown or not, surprisingly, ATM, p-ATM, p-BRCA1, p-ATR, and p-RAD51 were all downregulated after combination treatment with INPP4B knockdown and cytarabine compared with cytarabine exposure alone (p < 0.05) [Fig. 3b, c]. Finally, to indentify whether DNA repair response is dependent on ATM activity, a specifical ATM inhibitor, KU55933 (10 μM), and its activator Chloroquine (20 μg/ml) were used, and MTT analysis results showed that KU55933 significantly sensitized KG-1 cells to cytarabine (p < 0.05), similar to the role of INPP4B siRNA, but chloroquine attenuated this sensitizing effects of INPP4B siRNA on cytarabine treatment (p < 0.05), pointing to an involvement of the ATM-dependent DNA repai pathway [Fig. 3d].
Fig. 3

INPP4B expression is involved in the activation of ATM-dependent HR DNA repair pathway, and inhibition of DNA repair response restores the sensitivity to cytarabine in KG-1 cells. a mRNA expressions of ATM, ATR, RAD51, and BRCA1 after INPP4B knockdown were detected by real-time PCR analysis. b ATM, ATR, RAD51, and BRCA1 protein expressions were examined by western blot in control, control siRNA-transduced, and INPP4B-siRNA-transduced KG-1 cells. c ATM, p-ATM, p-ATR, p-RAD51, and p-BRCA1 protein expressions were examined by western blot in control, control siRNA-transduced, and INPP4B-siRNA-transduced KG-1 cells after cytarabine treatment. d KG-1 cells were treated with cytarabine and INPP4B siRNA or KU55933 (10 μM) or Chloroquine (20 μg/ml) alone or combination for 24 h. Cell viability was measured by MTT. Three separate experiments were performed. *p < 0.05, **p < 0.01

INPP4B-mediated enchanced DNA repair signaling requires nuclear localization of p65 protein

To further probe how INPP4B influences the DNA repair response, we examined the role of NF-kB signaling in this process, and the functional subunit of p65 was targeted. We found that INPP4B knockdown showed no evident changes in p65 mRNA expression [Fig. 4a]. Next, the effects of INPP4B on the nuclear translocation of p65 protein were detected, and we found that INPP4B significantly increased the nuclear translocation of p65 protein (p < 0.05), in concordance with the decrease of cytoplasmic p65 protein [Fig. 4b]. To make sure that the increase in the nuclear translocation of p65 is related to the INPP4B-mediated DNA repair, TNF-α, the activator of p65, was used. Western blot assay showed that TNF-α decreased the INPP4B knockdown-induced p-H2AX accumulation following cytarabine treatment (p < 0.05) [Fig. 4c]. Next, we tested the ATM, p-ATM, p-BRCA1, p-ATR, and p-RAD51 expression in the presence of INPP4B knockdown and TNF-α simultaneously as well as cytarabine exposure, and results showed that ATM, p-ATM, p-BRCA1, p-ATR, and p-RAD51 expressions were decreased after TNF-α and INPP4B knockdown combination compared with INPP4B knockdown alone (p < 0.05) [Fig. 4d]. Subsequently, MTT assay showed TNF-α rescued INPP4B knockdown-induced the decrease of cell viabilities, resulting in higher cell viabilities compared with INPP4B knockdown treatment in the presence of cytarabine (p < 0.05) [Fig. 4e].
Fig. 4

INPP4B-mediated enchanced DNA repair signaling is correlated with nuclear localization of p65 protein. a p65 mRNA expression was detected in control, control siRNA-transduced, and INPP4B siRNA-tranduced KG-1 cells by real-time PCR. b p65 protein expression of nucleus and cytoplasm were tested respectively by western blot analysis. PCAN as a loading control for the nucleus, and β-actin as a loading control for the cytoplasm. c KG-1 cells were treated with INPP4B siRNA or TNF-α alone or combination prior to cytarabine treatment. p-H2AX protein expression was detected by western blot. d ATM, p-ATM, p-ATR, p-RAD51, and p-BRCA1 protein expressions were detected in INPP4B siRNA or TNF-α treatment alone or combination in a condition of cytarabine exposure. e Before cytarabine treatment for 24 h, KG-1 cells were incubated with control siRNA, INPP4B siRNA, TNF-α alone or combination, then cell viabilities were measured by MTT assay. Repeated in triplicate. *p < 0.05, **p < 0.01

ATM is positively correlated with INPP4B expression in AML patients

To validate the clinical relevance of our findings that INPP4B expression is participated in DNA damage repair, bone marrow mononuclear cells (BMNCs) derived from 61 AML patients were collected (Table 1). Firstly, INPP4B mRNA expressions were measured by real-time PCR, results indicated that INPP4Bhigh often accompanied by high mRNA expression of ATM with the value of Pearson r is 0.6428 (p < 0.0001) [Fig. 5a]. Next, we examined ATM protein expression between INPP4Bhigh and INPP4Blow AML patients, in concordance with our study, a positive correlation between INPP4B and ATM expression in AML patients were observed. INPP4Bhigh AML patients showed a relative higher expression of ATM compared that of INPP4Blow AML patients (p < 0.05) [Fig. 5b]. At the same time, we tested the efficacy of KU55933 and INPP4B knockdown on primary leukemic cells derived from three INPP4Bhigh AML patients at relapse, and results showed that both INPP4B knockdown and KU55933 can significantly sensitize these primary myeloid leukemic cells to cytarabine treatment (p < 0.05) [Fig. 5c].
Fig. 5

The correlation between INPP4B and ATM in AML patients. a INPP4B and ATM mRNA expressions were detected in primary leukemic cells derived from 61 AML samples. The relation between INPP4B and ATM mRNA expression was analyzed by correlation analysis. b INPP4B and ATM protein expressions were detected by western blot in INPP4Bhigh and INPP4Blow AML patients. c Primary leukemic cells were treated with combination treatment of cytarabine with INPP4B siRNA or KU55933 for 24 h, and then cell viabilities were examined by MTT assay. Repeated in triplicate. *p < 0.05, **p < 0.01

Discussion

Recently, INPP4B has been becoming an interesting marker due to its oncogenic role of predicting poor survival and its link to resistance to chemotherapy and ionizing radiation in AML [1]. However, the underlying mechanism of INPP4B mediated-genotoxic remains unclear in AML, so we focus on the DNA repair pathway in INPP4Bhigh AML cells and explore the possible mechanisms.

Firstly, we detected the INPP4B expression among the myeloid leukemic cell lines and found KG-1 cells showed a higher INPP4B base line. As a different INPP4B base between KG-1 and HL60 cells, then we observed the higher cell viabilities of KG-1 with INPP4Bhigh than HL60 with INPP4Blow following cytarabine treatment. p-H2AX is a well-known maker of DNA double-strand breaks, H2AX is phosphorylated at the sites of DSBs and the level of p-H2AX has been shown to give a sensitive and accurate estimation of the number of DSBs within DNA. The presence of p-H2AX in chromatin can be detected shortly after induction of DSBs in the form of discrete nuclear foci [20]. To explore if the difference of this cell viabilities is associated with the distinction of DSBs formation, then we detected the p-H2AX expression between KG-1 and HL60 cells after cytarabine treatment, and observed p-H2AX expression is higher in HL60 than KG-1 cells. Next, we silenced INPP4B expression by INPP4B siRNA in KG-1 cells and identified that INPP4B-mediated resistance to genotoxic treatment is along with lower p-H2AX expression and INPP4B knockdown increased DSBs formation. However, INPP4B knockdown sensitized KG-1 to cytarabine, and p-H2AX expression also sharply increased after INPP4B knockdown, indicating that INPP4B knockdown is correlated with the increase in DSBs formation. It has been established that DSBs formation is subsequently accompanied by DSB repair [25], and the loss of p-H2AX has been shown to closely correlate to DSB repair [26]. To further understand how INPP4B influences the DSBs formation, whether the enhanced DSBs formation after INPP4B knockdown could be attributed to lower DNA damage repair, we next targeted the loss of p-H2AX expression. Firstly, we demonstrated that p-H2AX expression of both control KG-1 and INPP4B-silenced KG-1 was increased in a time-dependent manner in condition of continuous exposure to cytababine for 0, 4, 8, 16, and 24 h, and the increasement was more obvious in INPP4B-silenced KG-1 cells than in control KG-1 cell. However, when we freshed the cytarabine and made the cells to repair for 8, 16, and 24 h, we observed a time-dependent loss of p-H2AX expression in control KG-1 cell, while in INPP4B-silenced KG-1 cells, there is no obvious p-H2AX loss. The inhibition of p-H2AX loss (no DSB repair) by INPP4B knockdown confirmed the DSB repair pathway as a downstream target of INPP4B. NHEJ and HR repair pathways are participated in DSBs repair, and HR DNA repair pathway is more accurate and critical for DSBs repair [27]. ATM, ATR, BRCA1, and RAD51 are important components of HR repair pathway and play a vital role in HR repair [28, 29]. And it has been reported that INPP4B loss results in DNA repair deficiency via loss of BRCA1, ATM, and ATR in ovarian cancer cells [24]. In primary AML, higher BRCA1 mRNA level, decreased PARP1 mRNA, and p-H2AX expression can be the markers to predict resistance to response [30]. Thus, we detected the ATM, ATR, BRCA1, and RAD51 expression in our study, and only ATM expression was inhibited after INPP4B knockdown, whereas, there showed no significant changes of ATR, BRCA1, and RAD51 expression. However, besides ATM inhibition, the phosphorylation of ATM, ATR, BRCA1, and RAD51 were all downregulated after INPP4B knockdown under treatment of cytarabine. As is reported, ATM is a protein kinase that phosphorylates several key proteins involved in the DDR. It can be activated by its autophosphorylation at Ser1981 position [20], and it can also phosphorylate its downstreams of ATR, BRCA1, and RAD51 to activate the HR DNA repair [19, 31], so we speculated that the inhibition of p-ATM, p-ATR, p-BRCA1, and p-RAD51 may be ascribed to ATM inhibition. To further identify whether INPP4B-mediated DNA repair is ATM-dependent or not, we then evaluated the influences of KU55933 and Chloroquine on INPP4B knockdown-induced sensitizing effect on cytarabine treatment. Similar to INPP4B siRNA, KU55933 can evidently sensitize to cytarabine, and upregulation of ATM by Chloroquine decreased the INPP4B siRNA-induced sensitizing effects in the presence of cytarabine, demonstrating that INPP4B-mediated DNA repair is ATM-dependent. However, other moleculars may also participate in INPP4B-mediated HR repair, so our focus on ATM, BRCA1, ATR, and RAD51 may be limited. In addition, we also found INPP4B knockdown can inhibit the nuclear translocation of p65, activation of p65 nuclear translocation by TNF-α can reverse the INPP4B knockdown-induced upregulation of p-H2AX expression and downregulation of ATM, p-ATM, p-ATR, p-BRCA1, and p-RAD51. Concomitantly, TNF-α can also reverse INPP4B knockdown-induced sensitizing effect, with a higher cell viabilities compared with INPP4B siRNA alone in condition of cytarabine treatment, suggesting the NF-KB subunit, p65, nuclear translocation may function a critical role in INPP4B-mediated DNA repair. At present, the attention to the relation between NF-KB signaling and DNA repair is increasing, It is reported that NF-kB-dependent DNA damage-signaling regulates DNA double-strand break repair mechanisms in mature peripheral blood lymphocytes [32], and NF-KB subunit, p50, can regulate FANCD2 expression in multiple myeloma [33]. Other literatures also showed that DNA damage can activate NF-KB pathway via ATM activation [34], however, in our study, we found ATM expression was regulated by NF-KB activation. Except NF-KB pathway, inhibition of the MAPK-ERK pathway reduced the levels of phosphorylated ATM foci [35], and cells lacking STAT3 showed decreased DNA repair through ATM and ATR [36], HO-1/CO signaling is also critical in DNA repair through upregulation of p-ATM, p-ATR, p-BRCA1, and p-RAD51 [37]. Besides, EGFR nuclear translocation is associated with DNA repair [38]. Therefore, maybe there exists cross interaction between NF-KB and ATM, there still remain more research to indentify the role of p65 nuclear translocation in the process of INPP4B-mediated DNA repair, and other repair signaling may also be participated in this process. INPP4B contains a functional N-terminal C2-lipid binding domain, which is involved in the regulation of AKT activation, besides, it also contains a Nervy homology 2 domain known to mediate oligomerization (i.e., AML1-ETO oligomerization) or protein-protein interaction [39], our data (Supplemental Figure 1) and the previous study [14] both showed a no evident relationship between the AKT activation and INPP4B-mediated resistance to chemotherapy, so does it possible INPP4B directly interacts with ATM? This remains to be explored.

In human CD34+ cells, downregulation of DNA repair corresponds to increased drug sensitivity and apoptotic response [40]. And in our research, we first found a positive correlation between INPP4B and ATM expression, and the p-ATM, p-ATR, p-BRCA1, and p-RAD51 were also upregulated in INPP4Bhigh AML patients. Then, the ATM-specific inhibitor, KU55933 and INPP4B knockdown both showed an obvious sensitizing effects on cytarabine treatment in INPP4Bhigh AML cells. However, the detected samples were limited, a large number of patient samples are necessary.

In conclusion, our results demonstrate that INPP4B expression is associated with enhanced ATM-dependent DSB repair, which could be mediated by p65 nuclear translocation, INPP4B or DNA repair pathway inhibition represents a promising strategy to reverse resistance to chemotherapeutics in INPP4Bhigh AML.

Notes

Acknowledgments

This study was supported, in part, by the National Natural Science Foundation of China (nos. 81070444, 81270636, 81360501, and 81470006), International Cooperation Project of Guizhou Province (no. 2011-7010), Social Project of Guizhou Province (no. 2011-3012), Provincial Government Special Fund of Guizhou Province (no. 2010-84), and Project of Science and Technology Bureau of Guiyang City (no. [2012103-36]).

Supplementary material

13277_2016_5111_MOESM1_ESM.doc (212 kb)
ESM 1 (DOC 211 kb)

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Ping Wang
    • 1
    • 2
    • 3
  • Dan Ma
    • 1
    • 2
    • 4
  • Jishi Wang
    • 1
    • 2
    • 3
    Email author
  • Qin Fang
    • 4
    • 5
  • Rui Gao
    • 1
    • 2
    • 3
  • Weibing Wu
    • 1
    • 2
    • 3
  • Lu Cao
    • 5
  • Xiuying Hu
    • 1
    • 2
    • 3
  • Jiangyuan Zhao
    • 1
    • 2
    • 3
  • Yan Li
    • 1
    • 2
    • 3
  1. 1.Department of HematologyAffiliated Hospital of Guizhou Medical UniversityGuiyangChina
  2. 2.Key Laboratory of Hematological Disease Diagnostic and Treat Centre of GuiZhou ProvinceGuiyangChina
  3. 3.GuiZhou Province Hematopoietic Stem Cell Transplantation CenterAffiliated Hospital of Guizhou Medical UniversityGuiyangChina
  4. 4.Department of PharmacyAffiliated BaiYun Hospital of Guizhou Medical UniversityGuiyangChina
  5. 5.Department of PharmacyAffiliated Hospital of Guizhou Medical UniversityGuiyangChina

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