Identification of drugs acting synergistically with an SHMT inhibitor. (A) IC₅₀ of SHIN1 in different BL cell lines measured by MTT assay. Cells were treated for 4 days (n = 4; mean ± SEM). The presence/absence of reported oncogenic ID3 and TCF3 mutations for the individual cell lines is indicated by + or −. (B) Stable cell lines isolated from murine Eµ:Myc (M2121, M452) and DMBC (BSQ12, BSQ27) tumors were treated with varying doses of SHIN2 for 72 hours, and viability was measured by CellTiterGlo assay (n = 3). Error bars represent standard deviation (SD). For each concentration, both Eµ:Myc lines were compared with both DMBC lines by Welch t test. *P < .05 for all comparisons at that concentration. (C) Results from “spiked-in,” quantitative high-throughput drug screening utilizing a mechanistically annotated library (MIPE 5.0), either synergizing or antagonizing SHIN1 treatment. (D) Selected hits from “spiked-in,” quantitative high-throughput drug screening that are synergistic with SHIN1 treatment. (E) Combined treatment of SHIN1 and methotrexate (MTX) shows synergy in MTT assay (n = 3; 1 representative analysis is shown). eob, excess over bliss. (F) Western blot of BL60 wild-type cells treated with either SHIN1 (concentrations ranging from 0.5-5 µM) or MTX (10-100 nM) for 3 days. GAPDH served as loading control. Cropped blots of representative experiments are shown with quantification for SHIN1-treated cells (n = 3-5; P = .002 in Student t test for altered TCF3 levels in 2.5 µM concentration and P = .003 for SHP-1 levels, respectively). (G) Annexin V staining in CD19⁺ cells derived from bone marrow of a 27-year-old patient with BL after in vitro treatment with SHIN1 (5 µM) and MTX (20 nM) for 96 hours. Cells were normalized to DMSO control. ID3 mutations (L64F, V55fs) were detected by exome sequencing (supplemental Figure 7A; supplemental Table 4). F, phenylalanine; fs, frame shift; L, leucin; V, valine.