mTOR inhibitors reduce CRT_Del52–mediated cytokine-independent proliferation and colony-forming capacity of CD34⁺ cells from patients with MPN. (A,C,E,G,I) Representative proliferation plots of Ba/F3-MPL (A,C,I) or Ba/F3-MPL-CRT_Del52 (E,G) cells treated with or without 50 nM rapamycin (A and E, n = 3), 100 nM rapamycin (A and E, n = 6; I, n = 3), or 100 nM everolimus (C, n = 4; G, n = 8; I, n = 3), either in the presence of IL-3 (A,C) or human TPO (I) or in the absence of any cytokine (cytokine-independent; E,G) as indicated. (B,D,F,H,J) Averaged relative proliferation corresponding to the conditions shown in A,C,E,G,I. Cell counts of untreated cells were set at 100% for all time points. Two-way analysis of variance (B,F,J) and multiple paired t tests (D,H) were used to determine statistical significance (indicated by P value) on different days. (K-L) Representative blots of lysates of Ba/F3-MPL CRT_Del52 cells treated with 100 nM rapamycin (K) or 100 nM everolimus (L) that were harvested at different time points during proliferation assays and compared with untreated cells. Two serial dilutions of each lysate were loaded onto consecutive lanes, as indicated. (M) Representative blot of secreted CRT_Del52 immunoprecipitated using anti-CRT(C_mut) antibody from the culture media of Ba/F3-MPL CRT_Del52 cells (n = 3), collected on different days of cytokine-independent proliferation assay with or without 100 nM everolimus treatment. Lysates of untreated Ba/F3-MPL and Ba/F3-MPL CRT_Del52 cells grown in the presence of IL-3 were loaded as controls in the specified lanes. CRT_Del52 blots (K,L,M) and MPL blots (K-L) were probed with anti-CRT(C_mut) and anti-MPL antibodies, respectively. GAPDH blots (K-L) show the relative loading of lysates, as indicated. LC3 blots (K-L) show autophagy activation upon rapamycin or everolimus treatment, as indicated by the LC3-II bands (arrow). (N-Q) CD34⁺ hematopoietic stem cells isolated from bone marrow samples of patients with MPN or mobilized leukopaks from healthy donors were plated on semisolid methylcellulose-containing media (N-O) or in liquid cultures containing StemSpan megakaryocyte expansion supplement (P-Q) with or without 50 nM (N-O) or 100 nM everolimus (P-Q). CD34⁺ cells were either treated with or without 50 nM everolimus for 24 hours before plating (pre-treatment; N) or everolimus was added to the plating media (continuous treatment; O). The number of colonies was counted 12 to 18 days after plating either manually or using ImageJ. Each dot indicates an individual healthy donor (4 and 3 healthy donors for pre-treatment (N) and continuous treatment (O), respectively) or a patient (6 independent collections from 5 patients), and lines connect untreated and treated values for each donor/patient. Cells growing in the megakaryocyte expansion supplement were collected on day 7 after seeding, stained for surface CD41 and CD42a markers, and analyzed by flow cytometry. The gating strategy used for the analysis of CD41⁺CD42a⁺ cells is shown (P). The percentage of CD41⁺CD42a⁺ cells under untreated and treated conditions measured on day 7 are plotted (Q). Each dot indicates an individual healthy donor (7 independent experiments with 4 healthy donor samples) or a patient (6 patients), and lines connect untreated and treated values for each donor/patient. Statistical significance was calculated using paired t tests and graphs were plotted using GraphPad Prism (N,O,Q). All P values ≤.05 are shown. The bands marked by an asterisk in panel K are nonspecific bands. FSC-A, Forward Scatter-Area; SSC-A, Side Scatter-Area; 7-AAD, 7-Aminoactinomycin D.