Hemizygous Uba1M41L expression confers increased sensitivity to on-target UBA1 inhibition by TAK243. (A) Proliferation of 32D cells with hemizygous expression of Uba1WT and Uba1M41L over 3 days in the presence of DMSO or a range of TAK243 concentrations. A single representative experiment (left, mean ± SD of 3 technical replicates) and calculated 50% inhibitory concentration (IC50) values (right, mean ± SD of 4 biological replicates) are shown. Statistical significance was determined using a Mann-Whitney test. (B) Competition between 32D Uba1WT and Uba1M41L cells in the presence of DMSO (open symbols) or 10 nM TAK243 (closed symbols) over 4 days. Uba1M41L variant allele frequency (VAF) was determined by quantitative analysis of Sanger sequencing data (mean ± SD of 3 technical replicates).21 Statistical significance was determined using a 2-tailed unpaired t test with Welch correction. (C) Quantification of apoptotic cells by annexin V–propidium iodide (PI) flow cytometry after 16-hour treatment of 32D Uba1WT and Uba1M41L cells with DMSO or 10 nM TAK243. Bars depict live (annexin V–negative/PI–negative), early apoptotic (annexinV+/PIlow-mid), and late apoptotic (annexinV+/PIhigh) cells (mean ± SD of 3 technical replicates). (D) Immunoblot for PARP1, H2AX, and H2AX pS139 proteins after 16-hour treatment of 32D Uba1WT and Uba1M41L cells with DMSO or 10 nM TAK243. Immunoblot for vinculin (VINC) was performed as a protein loading control. (E) Schematic depiction of the knock-in strategy used for generation of 32D cells expressing the TAK243 drug-binding mutation Uba1A580S. (F) Proliferation of 32D cells with hemizygous expression of Uba1WT, Uba1M41L, or Uba1M41L;A580S over 3 days in the presence of DMSO or a range of TAK243 concentrations. A single representative experiment (left, mean ± SD of 3 technical replicates) and calculated IC50 values from 3 independent biological replicates (right, mean ± SD) are shown. Statistical significance was determined using a 2-tailed unpaired t test with Welch correction.
