Fig 2: CCRL2 knockdown alters the efficacy of JAK2 inhibition by the selective inhibitor fedratinib.(A) CCRL2 knockdown increases the antiproliferative effect of 0.25 μΜ fedratinib (P = 0.033-sh1, P = 0.001-sh2) in MDS-L cells. (B) CCRL2 knockdown increases the antiproliferative effect of 0.25 μΜ fedratinib in TF-1 cells (P < 0.001-sh1, P = 0.002-sh2). (C) CCRL2 knockdown increases the antiproliferative effect of 0.25 μΜ fedratinib (P < 0.001-sh1, P = 0.001-sh2) in DAMI cells. (D) CCRL2 knockdown increases the anticlonogenic effect of 0.25 μΜ fedratinib (P < 0.001) in MDS-L cells. (E) CCRL2 knockdown increases the anticlonogenic effect of 0.25 μΜ (P < 0.001) and 0.5 μΜ fedratinib (P < 0.002) in TF-1 cells. (F) CCRL2 knockdown increases the anticlonogenic effect of 0.25 μΜ (P = 0.002) and 0.5 μΜ fedratinib (P < 0.001) in DAMI cells. (G) The clonogenicity inhibitory effect of fedratinib is decreased in MDS-L cells with suppressed CCRL2 (P < 0.001). (H) The clonogenicity inhibitory effect of 0.25 μΜ fedratinib is increased in TF-1 cells with suppressed CCRL2 (P = 0.006). (I) The clonogenicity inhibitory effect of 0.25 μΜ fedratinib is increased in TF-1 cells with suppressed CCRL2 (P = 0.020). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 3: CCRL2 is up-regulated in MDS and sAML cells.(A) Comparison of the mRNA levels of the five AR-regulated genes CCRL2, C3AR1, GYK, SOCS3, and FLOT2 between healthy stem and progenitor cells, de novo AML (AMLs without MDS-related chromosomal abnormalities), MDS, and sAML (AMLs with MDS-related chromosomal abnormalities) based on data extracted from the BloodSpot database. CCRL2 is the only gene that is up-regulated in MDS and sAML compared with both healthy cells and de novo AMLs (P < 0.001). (B) Patients with AML with high CCRL2 expression (based on median expression) have significantly worse overall survival (P = 0.031) compared with patients with AML with low CCRL2 expression based on the TCGA dataset. (C) Analysis of the CCRL2 protein levels by flow cytometry showed that CD34+ cells from patients with MDS and MDS/MPN express relatively higher levels of CCRL2 (P = 0.071) compared with CD34+ cells from healthy controls. CD34+ blasts from patients with sAML express higher levels of CCRL2 compared with CD34+ blasts from patients with de novo AML (P < 0.001). (D) CCRL2 expression is higher in CD34+CD38− cells from patients with MDS compared with CD34+CD38− cells from healthy individuals (P = 0.001). Graphs show the mean value and SD of the mean value.

Fig 4: CCRL2 knockdown affects the growth and clonogenicity of erythroleukemia cell lines.(A) CCRL2 knockdown with two different lentiviruses significantly suppresses the growth of TF-1 cells in the presence (P < 0.001) and absence of GM-CSF (P < 0.001). CCRL2 knockdown with two different lentiviruses suppresses at a lower extent the growth of DAMI cells (P = 0.009-sh1, P = 0.058-sh2). (B) CCRL2 knockdown with two different lentiviruses decreases the colony formation of TF-1 cells in the presence (P < 0.001) and absence of GM-CSF (P < 0.001). CCRL2 knockdown with two different lentiviruses decreases the colony formation of DAMI cells (P = 0.002-sh1, P = 0.015-sh2) at a lower extent compared with TF-1 cells. (C) CCRL2 knockdown with sh1 increased the percentage of apoptotic TF-1 cells (P = 0.015-sh1, P = 0.097-sh2), and CCRL2 knockdown with the two lentiviruses decreased the percentage of TF-1 cells in the G2-S phase (P < 0.001). (D) CCRL2 knockdown with two different lentiviruses increases the expression of CD41 (P < 0.001), CD71 (P < 0.001), and CD235a (P = 0.002-sh1, P = 0.025-sh2). CCRL2 knockdown does not affect the expression of CD41 (P = 0.230-sh1, P = 0.912-sh2). CCRL2 knockdown increases the expression of CD71 (P < 0.001-sh1, P = 0.026-sh2) and CD235a (P = 0.002-sh1, P = 0.067-sh2). (E) Western blotting showing the effect of CCRL2 knockdown in the JAK2/STAT signaling in TF-1 and DAMI cells. CCRL2 knockdown suppresses the phosphorylation of JAK2, STAT3, and STAT5 in TF-1 cells. CCRL2 knockdown decreases the phosphorylation of JAK2 but does not affect the phosphorylation of STAT3 and STAT5 in DAMI cells.

Fig 5: CCRL2 affects JAK2/STAT signaling in MDS cells.(A) Representative Western blotting showing CCRL2 knockdown effect by two different shRNAs (sh1 and sh2) on JAK2 (Tyr1007/1008), STAT3 (Tyr105), and STAT5 (Tyr694) phosphorylation in MDS92 and MDS-L. (B) CCRL2 knockdown decreases the RNA levels of the JAK2/STAT target genes: MYC (MDS92: P = 0.001-sh1, P = 0.002-sh2; MDS-L: P = 0.001-sh1, P = 0.010-sh2), PIM1 (MDS92: P = 0.003-sh1, P = 0.010-sh2; MDS-L: P = 0.002-sh1, P = 0.040-sh2), BCL2 (MDS92: P = 0.020-sh1, P = 0.060-sh2; MDS-L: P = 0.008-sh1, P = 0.005-sh2), MCL1 (MDS92: P = 0.025-sh1, P = 0.004-sh2; MDS-L: P = 0.004-sh1, P = 0.019-sh2), and DNMT1 (MDS92: P = 0.009-sh1, P = 0.010-sh2; MDS-L: P = 0.008-sh1, P = 0.040-sh2), n = 3. (C) Western blotting showing that CCRL2 knockdown suppresses the phosphorylation of JAK2, STAT3, and STAT5 at 30 min and 6 hours of IL-3 (20 ng/ml) treatment following 48 hours of IL-3 starvation. (D) Coimmunoprecipitation assay showing that CCRL2 precipitates with JAK2 and that CCRL2 knockdown does not affect the interaction between JAK2 and the common β signal transducing subunit of CD123 (CSF2RB) but decreases the interaction between JAK2 and STAT3/5 proteins. IgG, immunoglobulin G. (E) Representative images from immunofluorescence staining (×40 and ×60 magnification) showing localization of CCRL2 (green) in the cytoplasm and membrane of MDS-L cells. Confocal microscopy reveals areas of colocalization with JAK2 (red). DAPI, 4′,6-diamidino-2-phenylindole. (F) The mean fluorescence intensity (MFI) of phosphorylated STAT3 (P-STAT3) is positively associated with the MFI of CCRL2 in CD34+ cells from patients with MDS and CD34+ blasts from patients with AML (Coef, 0.15; P = 0.001), n = 16.