PAF/PAFR signaling pathway is partially responsible for in vivo sensitization to TKI treatment upon Pafah1b3 loss. (A) Line graph showing the percentage of PAFR-expressing cells, of total leukemia cells, in mouse PB, BM, and SPL during dasatinib treatment. Mice were injected with 10⁶BCR-ABL1⁺ BCP-ALL cells expressing tdtomato fluorescent protein, and were then treated with dasatinib (20 mg/kg q.d. for 3 days, starting at 11 days postinjection). Samples of mouse blood, BM, and SPL were taken from 3 mice/day starting on day 11 postinjection prior to dasatinib treatment, and were assessed for percentage of cells expressing PAFR on their surface via flow cytometry by staining cells with an anti-PAFR antibody conjugated to FITC and measuring the percentage of FITC⁺ cells of tdtomato⁺ cells. (B) Bar graph summarizing total percentage of leukemia cells expressing surface PAFR from before treatment (−) and 1 day after dasatinib treatment (+) time points from experiment shown in panel A. At least 3 mice were used per time point. (C-D) Bar graphs showing cell viability of BCR-ABL1⁺ BCP-ALL cells cultured in vitro either alone (C) or with BMSCs (D) after treatment with dasatinib alone, PAF alone, or a combination or dasatinib + PAF. Viability is normalized to untreated cells cultured in parallel in all cases, and is measured via flow cytometry after 48 hours of treatment by staining with 4′,6-diamidino-2-phenylindole (DAPI) as a cell exclusion stain. Drugs were used at an ∼LD85 for dasatinib and ∼LD55 for PAF when dosed alone, with the same dose of each drug used for the combination treatment; higher doses are needed to achieve equivalent killing when leukemia cells are cocultured with BMSCs, so dosage is adjusted accordingly. Three culture plates are used per condition. (E) Survival analysis of dasatinib-treated mice receiving 10⁴ KO −cDNA (top) or KO +cDNA (bottom) cells in the presence of absence of the PAFR antagonist WEB-2086. WEB-2086 was given 3 times per week intraperitoneally (i.p.) at 5 mg/kg starting 9 days postinjection; dasatinib was given at 20 mg/kg q.d. for 3 days starting 11 days postinjection. Significance was calculated using the Mantel-Cox test; 3 to 5 mice were used per condition and genotype. (F) Scatterplots showing fold change in leukemic burden of mice from survival curves in depicted in panel E of mice receiving transplants of KO −cDNA (top) or KO +cDNA (bottom) cells as measured by in vivo bioluminescent imaging (total flux = photons per second) from start of treatment to 1 day post end of treatment. The posttreatment burden for each mouse is normalized to the pretreatment burden of that same mouse. Bioluminescent images were collected using a Xenogen IVIS system and analyzed using Living Image version 4.4 software (Caliper Life Sciences). Error bars represent standard deviation; P values were calculated using the Student t test.