SETD2 alteration impairs the DNA damage response and increases mutation rate at sites of diminished H3K36me3. (A) MOLM-13 SETD2 clone 1 and MOLM-13 isogenic control 3 were treated with cytarabine (100 nM), 6-TG (200 nM), or l-asparaginase (0.001 IU/μL) and assessed for apoptosis by flow cytometry after staining for AnnexinV and propidium iodide (PI) 24 to 48 hours later. (B) Western blots for Chk1 phospho-S345 of MOLM-13 SETD2 clone 1 and MOLM-13 isogenic control 3 treated with vehicle, 6-TG, (200 nM) or cytarabine (100 nM) for 4 hours. (C) MOLM-13 isogenic lines were treated with cytarabine or vehicle and examined for γ-H2A.X foci. Representative images shown. Primary stain: rabbit anti-gamma H2AX phospho S139; secondary stain: anti-rabbit Alexa647; counterstain: DAPI. Original magnification ×40. (D) The number of foci per cell were quantified in ImageJ for each condition. The red threshold is twice the standard deviation above the mean number of foci in the vehicle-treated isogenic control. A Student t test was performed between each pair of treatments. (E) Schema of experimental approach. MOLM-13 SETD2 clone 1 or isogenic control 3 cells were treated with 6-TG (200 nM) or vehicle for 14 days and then single-cell sorted to obtain subclones with clonal mutations. Whole exome sequencing (WES) was performed on 10 6-TG–treated subclones (4 SETD2-mutant and 6 isogenic control) and 1 each of vehicle-treated SETD2-mutant and isogenic control subclones. The pretreatment SETD2-mutant or isogenic control clones were subjected to WES, H3K36me3 ChIP-Seq, and total RNAseq. (F) The number of novel mutations for each 6-TG–treated MOLM-13 SETD2-mutant or isogenic control subclone compared with its matched pretreatment clone was determined based on WES. The total mutations and expected GA/CT transitions are shown. (G) Exons were ordered by the amount of normalized H3K36me3 for both the SETD2-mutated and isogenic control clones. Mutation rates were calculated using a moving window average, dividing the total number of mutations by the number of base pairs for each window. The average H3K36me3 level for each window was also determined, in each clone. A Pearson’s correlation was used to calculate the R² value for each clone. (H) Moving window average of H3K36me3 levels arranged by gene expression level on RNAseq. (I) Mutation rates were calculated using a moving window average of genes by expression profile. The average expression level for each window was also determined and a Pearson’s correlation was used to calculate the R² value for the MOLM-13 isogenic control and the MOLM-13 SETD2-mutant clone.