Expression of Kindlins in Acute Myeloid Leukemia

Abstract

Abstract 4910The Kindlin family of intracellular proteins has recently emerged as key regulators of cellular functions and cell-matrix interactions. They comprise of three evolutionarily conserved members, kindlin-1, kindlin-2 and kindlin-3, they share considerable sequence and structural similarities. A few of study revealed that Kindlin-2 influences solid tumor cell invasion and resistance. With regard to AML, the influence of Kindlins is still unknown. To evaluate the clinical significance of Kindlin-2 in acute myeloid leukemia (AML), we investigated the expression of Kindlin-2, kindling-3 in AML cells. 1. Materials and methodsK562, KG-1a, HL60, U937, Jurkat cell lines were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS, GIBCO) at 37°C in a humidified atmosphere of 5% CO2. Bone marrow (BM) samples were obtained from 88 patients with de novo AML from Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC). Samples of 9 normal donors and ITP were used as the control group.Bone marrow mononuclear cells (BMMCs) were prepared by Ficoll-Hypaque density gradient centrifugation. Expressions of Kindlin-2, Kindlin-3 were detected by RQ-PCR. The following primers for real-time PCR were used: (a) Kindlin-2 sense primer, 5’-CCGCTCGAGCTATGCGTATCCCCGTAG-3’; (b) Kindlin-2 antisense primer, 5’-CGACGCGTCTAGCGAGGGGTTGTC-3’; (c) Kindlin-3 sense primer, 5’-CCGCTCGAGCTATGCGTATCCCCGTAG-3’; (d) Kindlin-3 antisense primer, 5’-CGACGCGTCTAGCGAGGGGTTGTC-3’; (e) GAPDH sense primer, 5’-GAAGGTGAAGGTCGGAGTC-3’; (f) GAPDH antisense primer, 5’-GAAGATGGTGATGGGATTTC-3’. Analysis was performed using ABI 7500 Sequence Detection software (Applied Biosystems). The expression of Kindlin-2 and Kindlin-3 were showed as RQ value calculated through ΔΔCt method [ΔΔCt = (CtKindlin □ CtGAPDH)sample □ (CtKindlin □ CtGAPDH)calibrator]. The ΔCt (CtKindlin □ CtGAPDH) of K562 was defined as calibrator, and the RQ of calibrator was 1.000. Relationships between Kindlin-2, Kindlin-3 and the patients’ clinical data were analyzed.2. Results Expression of Kindlins in newly diagnosis AMLTable 1Characteristics of the AML patients and expression of KindlinsCharacteristicNo. of Patients (%)Kindlin-2 (RQ-In)Kindlin-3 (RQ-In)SexMale46(52.3%)0.307±1.6590.066±0.738Female42(47.7%)0.005±1.6760.117±0.942p-value0.3980.776WBC<30×109/L22(25.3%)0.409±1.534−0.013±0.753≥30×109/L65(74.7%)-0.547±1.8930.394±1.021p-value0.0200.049WHO typeAML with t(8;21)(q22;q22)16(18.2%)0.842±1.730−0.207±0.811AML with t(15;17)(q22;q12)14(15.9%)Acute myelomonocytic leukaemia5(5.7%)−0.240±1.4730.243±0.790Acute monoblastic and monocytic leukaemia31(35.2%)AML without maturation3(3.4%)−0.148±1.6300.267±0.869AML with maturation13(14.8%)Acute erythroid leukaemia6(6.8%)p-value0.0160.046NCCN risk status(APL excluded)Better-risk20(27.0%)0.446±1.8880.057±1.056Intermediate-risk41(55.4%)0.027±1.4690.17±0.741Poor-risk13(17.6%)-0.376±1.5000.347±0.794p-value0.3470.630Induction chemotherapy39Pretherapy390.216±1.602−0.114±0.628Postremission391.555±1.1020.372±0.633p-value<0.0010.004The level of Kindlin-2 in AML (0.163±1.665) was significantly lower than that in non-AML (1.683±1.395) controls (p=0.010). No significant difference was found between the AML and controls in levels of Kindlin-3 (p=0.216). Out of the 79 patients who accepted treatment, 61 patients achieved complete remission (CR) and 18 patients were NR. Patients with higher expression of Kindlin-2 had a higher CR rate (86.8% vs 68.3%) (p=0.050). Expression of kindling 3 was unrelated to CR rate. Both of kindling-2 and kindling-3 increased after CR. This finding implicates Kindlin-2 as a potential prognostic factor of AML. Disclosures:No relevant conflicts of interest to declare.