Fig 2: 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 ß 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.

Fig 3: PIM kinase inhibition decreased tumor growth in a xenograft model of hepatoblastoma(A) Human hepatoblastoma cells, HuH6 (2.5 x 106 cells), were injected into the right flank of female athymic nude mice. When tumor sized reached an average of 150 mm3, mice were randomized to receive vehicle or AZD1208 by oral gavage for a total of 14 days (n = 15 per group). Tumor growth was followed and tumors were measured twice weekly with calipers to calculate tumor volumes. Mice treated with AZD1208 had significantly smaller tumors compared with vehicle-treated mice, reported as fold change tumor volume ± SEM. (B) Formalin-fixed, paraffin-embedded tumor xenografts were stained for Ki67, a marker of cell proliferation, and representative photomicrographs of vehicle-treated (top panel) and AZD1208-treated (bottom panel) xenografts are shown with IgG control in the bottom left corner (top panel). (C) The number of Ki67 cells per 500 cells in a representative section of each tumor was counted and the mean was calculated and reported for each group ± SEM. AZD1208-treated xenografts had significantly decreased Ki67 staining compared to vehicle-treated xenografts. (D) Formalin-fixed, paraffin-embedded tumor xenografts were stained for phospho-BAD (S112). When BAD is dephosphorylated, apoptosis is believed to occur. Representative photomicrographs of vehicle-treated (top panel) and AZD1208-treated (bottom panel) xenografts are shown with IgG control in the bottom left corner (top panel). (E) Slides stained for phospho-BAD were scored based upon the intensity of the stain and the percentage of tumor cells staining positive. AZD1208-treated xenografts had significantly decreased phospho-BAD staining compared to vehicle-treated xenografts, indicated an increase in apoptosis with AZD1208 treatment, reported as mean stain score ± SEM.

Fig 4: PIM kinase inhibition with AZD1208 decreased survival, proliferation, migration, and invasion, affected phosphorylation of p21, and induced apoptosis in the hepatoblastoma PDX, COA67(A) Following 24 hours of treatment with AZD1208, the survival of COA67 cells was assessed with the alamarBlue® Assay. Cells treated with AZD1208 had significantly decreased viability compared to the control cells. (B) Following 24 hours of treatment with AZD1208, the proliferation of COA67 cells was assessed. Cells treated with AZD1208 had significantly decreased proliferation compared to the control cells, reported as fold change ± SEM. (C) COA67 cells treated with increasing doses of AZD1208 were allowed to migrate through a micropore membrane for 72 hours. Migration was reported as fold change. COA67 cells treated with AZD1208 exhibited significantly decreased migration compared to untreated cells. (D) For invasion, AZD1208-treated cells were allowed to invade for 72 hours. Cells treated with AZD1208 exhibited significantly decreased invasion compared to untreated cells. All experiments were repeated at least in triplicate, with results reported as fold change ± SEM. (E) Immunoblotting for phospho-p21 (T145) and total p21 in COA67 cells showed a decrease in phospho-p21 with AZD1208 treatment with no accompanying change in total p21. (F) Immunoblotting for cleaved PARP and cleaved caspase 3, markers of apoptosis, in COA67 cells revealed an increase in both markers with increasing concentrations of AZD1208.

Fig 5: PRKACA confers resistance to anti-HER2 therapy and impairs apoptosis. A. Relative viability of screened BT474 cells containing each ORF and treated with AEE788 (top panel) or an shRNA targeting HER2 (bottom panel). B. PRKACA confers resistance to lapatinib. Cells were lentivirally-transduced with the indicated ORFs, treated with lapatinib at the indicated doses, and cell viability was assessed by ATP-based luminescence assay. Results are normalized to the DMSO control for each ORF and represent the mean and standard deviation (SD) of 6 replicates. C. PRKACA expression prevents lapatinib-induced cell death. BT474 cells were transduced with LACZ or PRKACA constructs and subsequently treated with lapatinib 5 uM or DMSO. Viable cells were counted by trypan blue exclusion 5 days after the addition of drug. Results show the mean and standard deviation of 3 replicates. D. Lapatinib treatment results in apoptosis of BT474 (top panel) and ZR-75-30 (bottom panel) cells, and this is partially rescued by PRKACA, PIM1, and BCL-XL. Cells were transduced with the indicated ORFs. Two days later lapatinib 10 uM or DMSO was added, and caspase 3/7 cleavage was measured 24h later by luminescent assay. Results represent the mean and SD of 5 replicates per cell line. For BT474, LACZ vs. PRKACA p = 0.0002, LACZ vs. PIM1 p = 0.000007. For ZR-75-30, LACZ vs. PRKACA p = 0.0003, LACZ vs. PIM1 p = 0.0006.