Figure 2.
Presence of NPM1c shifts FLT3-D835Y localization to the endoplasmic reticulum. (A) Flow cytometry analysis of FLT3 surface expression reveals reduced FLT3-D835Y expression on the surface in NPM1c+ cells (blue) compared with NPM1 wt cells (green). FLT3-D835Y expression in NPM1c+ cells is comparable to FLT3-ITD surface expression (red). Gray peaks represent isotype controls. (B) Statistical analysis of FLT3-D835Y surface mean fluorescence intensity in NPM1c+ cells compared with NPM1 wt cells (n = 3). (C) Immunofluorescence staining shows colocalization of FLT3-D835Y with the ER marker Calnexin in murine NPM1c+ EGFP+ BM. (D) Immunofluorescence staining shows colocalization of FLT3-D835Y with the ER marker PDI in a NPM1c+ AML cell line (OCI-AML3), but not in NPM1 wt expressing HL-60 cells. (E) Mander’s coefficient analysis for colocalization of FLT3 with Calnexin in murine FLT3-D835Y+ NPM1c+ BM cells (n = 9 images with 49 cells) and in FLT3-D835Y+ NPM1 wt BM cells (n = 6 images with 189 cells). (F) Mander’s coefficient analysis for colocalization of FLT3-D835Y with PDI in OCI-AML3 (n = 6 images with 169 cells) and HL-60 cells (n = 4 images with 57 cells). (G) Immunoblot analysis of AML cell line lysates reveals expression of underglycosylated ER-localized 130-kDa form of FLT3-D835Y and FLT3-ITD in OCI-AML3, but not in HL-60, cells. (H) Co-immunoprecipitation of FLT3 shows interaction with NPM1c in OCI-AML3 FLT3-D835Y cells, low interaction in OCI-AML3 FLT3-ITD and no interaction in HL-60 FLT3-D835Y (negative control) cells. (I) Intracellular flow cytometric analysis of pSTAT5 in c-kit+ BM blasts from representative patients with AML reveals a significantly increased STAT5 activation in FLT3-TKD/NPM1c cells compared with FLT3-TKD/NPM1 wt cells (FLT3-TKD/NPM1c: n = 4 patients; FLT3-TKD/NPM1 wt: n = 4 patients). (J) Representative immunohistochemical staining of pSTAT5 in BM from patients with AML with cooccurred FLT3-TKD and NPM1c mutations shows increased positivity for pSTAT5 (red color) compared with samples from patients harboring FLT3-TKD together with NPM1 wt. (K) Flow cytometric analysis of BM cells from patients with AML reveals significantly decreased FLT3-TKD surface levels in NPM1c+ compared with NPM1 wt patient cells (FLT3-TKD/NPM1c: n = 5 patients; FLT3-TKD/NPM1 wt: n = 6 patients). *P < .05 and ***P < .001, by unpaired, 2-tailed Student t test (panels B, E, F, I and K).

Presence of NPM1c shifts FLT3-D835Y localization to the endoplasmic reticulum. (A) Flow cytometry analysis of FLT3 surface expression reveals reduced FLT3-D835Y expression on the surface in NPM1c+ cells (blue) compared with NPM1 wt cells (green). FLT3-D835Y expression in NPM1c+ cells is comparable to FLT3-ITD surface expression (red). Gray peaks represent isotype controls. (B) Statistical analysis of FLT3-D835Y surface mean fluorescence intensity in NPM1c+ cells compared with NPM1 wt cells (n = 3). (C) Immunofluorescence staining shows colocalization of FLT3-D835Y with the ER marker Calnexin in murine NPM1c+ EGFP+ BM. (D) Immunofluorescence staining shows colocalization of FLT3-D835Y with the ER marker PDI in a NPM1c+ AML cell line (OCI-AML3), but not in NPM1 wt expressing HL-60 cells. (E) Mander’s coefficient analysis for colocalization of FLT3 with Calnexin in murine FLT3-D835Y+ NPM1c+ BM cells (n = 9 images with 49 cells) and in FLT3-D835Y+ NPM1 wt BM cells (n = 6 images with 189 cells). (F) Mander’s coefficient analysis for colocalization of FLT3-D835Y with PDI in OCI-AML3 (n = 6 images with 169 cells) and HL-60 cells (n = 4 images with 57 cells). (G) Immunoblot analysis of AML cell line lysates reveals expression of underglycosylated ER-localized 130-kDa form of FLT3-D835Y and FLT3-ITD in OCI-AML3, but not in HL-60, cells. (H) Co-immunoprecipitation of FLT3 shows interaction with NPM1c in OCI-AML3 FLT3-D835Y cells, low interaction in OCI-AML3 FLT3-ITD and no interaction in HL-60 FLT3-D835Y (negative control) cells. (I) Intracellular flow cytometric analysis of pSTAT5 in c-kit+ BM blasts from representative patients with AML reveals a significantly increased STAT5 activation in FLT3-TKD/NPM1c cells compared with FLT3-TKD/NPM1 wt cells (FLT3-TKD/NPM1c: n = 4 patients; FLT3-TKD/NPM1 wt: n = 4 patients). (J) Representative immunohistochemical staining of pSTAT5 in BM from patients with AML with cooccurred FLT3-TKD and NPM1c mutations shows increased positivity for pSTAT5 (red color) compared with samples from patients harboring FLT3-TKD together with NPM1 wt. (K) Flow cytometric analysis of BM cells from patients with AML reveals significantly decreased FLT3-TKD surface levels in NPM1c+ compared with NPM1 wt patient cells (FLT3-TKD/NPM1c: n = 5 patients; FLT3-TKD/NPM1 wt: n = 6 patients). *P < .05 and ***P < .001, by unpaired, 2-tailed Student t test (panels B, E, F, I and K).

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