Expression of 67-Kda Laminin Receptor (67LR) Relates to the Aggressiveness and Poor Prognosis of AML: Modulation of the Expression of GM-CSF Receptor by 67LR.
Authors: Ando Koji , Miyazaki Yasushi , Imanishi Daisuke , Iwanaga Masako , Tsushima Hideki , Taguchi Jun , Fukushima Takuya , Yoshida Shinichiro , Hata Tomoko , Tomonaga Masao 
Published: October 2019

Background: The 67-kDa laminin receptor (67LR) is a non-integrin cell surface receptor for laminin, one of the major components of extracellular matrix. Previously (at ASH 2007, PS 2845), we reported that the high expression of 67LR on CD34+AML cells was related with a poor prognosis of AML, and that the forced expression of 67LR resulted in the enhanced proliferation and the resistance against apoptosis of leukemia cells by increasing phosphorylated STAT5. However, there was no information about how the expression of 67LR modulated the signaling of STAT5 pathway.

Objective: To elucidate how 67LR enhances the signaling of STAT5 pathway and the role of 67LR in clinical course of AML.

Methods: The cDNA or short interfering RNA for LR cloned into various kinds of plasmids was expressed in TF-1 and AML193 leukemia cell lines via stable transfection. As overexpressors of LR, TF-1LR and AML193LR were generated, and as a suppressor of LR, TF-1si. Because it is well known that the signaling of STAT5 pathway was enhancing by GM-CSF signaling, the expression of the GM-CSF receptor α subunit (GM-CSFRα) was analyzed by flowcytometry. To test the interaction between 67LR with V5-tag and GM-CSFRα with Flag-tag protein, we transiently transfected expression plasmids containing cDNA for these protein into human 293T cells. Lysates of these transfected cells were subjected to the immunoprecipitation using anti-V5 or anti-Flag Ab, followed by immunoblotting with HRP conjugated antibodies. From 44 AML patients (M0[2], M1[4], M2[18], M4[11], M5[3], M6[3], MDS/AML[3]), CD34-positive cells were isolated by column method to examine the surface expression of 67LR with flowcytometry. To assess the clinical significance of 67LR expression, 44 pattients with AML were divided into two groups by the surface-expression level of 67LR: 20 cases in the high-expression group (positive in >25% of cells, LR-H) and 24 cases in the low-expression group (LRL). Clinical factors including WBC, LDH, and overall survival were compared between two groups.

Results: The surface expression of 67LR was 64% in parental TF-1, 92% in TF-1LR, and 36% in TF-1si, and that was 65% in AML193 and 91% in AML193LR. The surface expression of GM-CSFRα was lower in TF-1si (MFI: 5.1) than its control (MFI:7.3), whereas that was higher in TF-1LR (MFI:19.2) than its control (MFI:7.3). In AML193LR, GM-CSFRα was also higher than the control (MFI:34.7 and 18.9, respectively). These results demonstrated that the modulation of 67LR expression contributed to changes in the level of GM-CSFRα on cell surface. Immunoprecipitation assays indicated that 67LR and GM-CSFRα protein were present in the same protein complex in vivo. In clinical samples, median surface expression of 67LR on CD34 positive AML cells was 17%, and the median intensity of GM-CSFRα was 5.8. We found a significant positive relationship between the surface expression of 67LR and the median intensity of GM-CSFRα on CD34 positive AML cells (chi-square value was 0.04). WBC counts and LDH levels at diagnosis were significantly higher in LR-H group than that in LH-L (p=0.04, p=0.02, respectively). There was a significant difference in survival between LR-L and LR-H groups (median survival 803 and 239 days, respectively, p=0.009).

Conclusion: We found the level of 67LR could modulate the expression of GM-CSFRα, thereby it might enhance the phosphorylation of STAT5. Among AML patients, high level of 67LR expression was related to the higher WBC count, elevated LDH and shorter survival. The expression of 67LR and GM-CSFRα in clinical samples also showed significant correlation in amount. These data suggested that the high expression of 67LR resulted in the proliferation of AML cells by increasing the expression of GM-CSFRα. These features could contribute, at least in part, to a poor prognosis of AML.