Primary cell isolation, cell proliferation/viability, and CFU experiments

All mouse breeding/maintenance and treatment procedures were performed with full IACUC approval. Bone marrow cells were isolated from freshly sacrificed mice and enriched for stem/progenitor cells using CD117 MicroBeads and separated on an AutoMACS Pro separator (Miltenyi) according to manufacturer specifications. For cell proliferation and viability assays, 20 thousand 32D or c-Kit⁺ cells from bone marrow were seeded in 96 well plate in 200 μl of RPMI medium supplemented with 10% FBS in a titration of Ibrutinib. Ibrutinib concentrations ranged from 5 nM-5000 nM in a final concentration of 0.1% DMSO for 32D cells and 1 nM-1000 nM in a final concentration of 0.1% DMSO for c-Kit⁺ cells incubated at 37C and 5% CO₂ for 24 h. Cell proliferation and viability was determined using Trypan Blue live/dead hemocytometer cell counting. For clonogenic assays, c-Kit⁺ cells were plated in triplicate at 2,000 cells/mL in M3434 (Stemcell Technologies) supplemented with 40 ng/mL G-CSF and either 0.1% DMSO, 100 nM Ibrutinib, or 1 μM Ruxolitinib and the total colony number was determined 7 days later. For human cell viability assays, human CD34⁺ cells were plated at 20,000 cells/100 μL in IMDM supplemented with 20% BIT9500 (Stemcell Technologies), 55 μM β-mercaptoethanol, and 40 ng/mL G-CSF with 0.1% DMSO or a dose titration of Ibrutinib or Ruxolitinib. For synergy assays, sorted human CD34⁺ cells were plated in triplicate at 1,000 cells/mL in H4434 supplemented with 40 ng/mL G-CSF with 0.1% DMSO, 100 μM Ibrutinib or 100 μM Ruxolitinib and the total colony number was determined 7 days later. Additional details of the primary cell isolation and analysis are given in Supplementary Methods.