SETD2 inhibition phenocopies methionine depletion inducing cell death in AML samples. (A-B) Relative cell counts and relative viability of MOLM13 cells grown for 72 hours with and without methionine and with increasing concentrations of the SETD2 inhibitor, SETD2-IN-1 TFA (n = 3). (C) Western blot for H3K36me3 levels in MOLM13 cells following 24 hours of exposure to SETD2-IN-1. (D) Western blot for H3K36me3 levels in MOLM13 cells following 72 hours of exposure to SETD2-IN-1. Unmodified histone H3 was used as loading control. (E-N) Relative viable cell counts and viability (DAPI⁻), both shown as percentages, in 5 primary AML samples (AML#18-22) grown for 7 days in liquid culture with a concentration range of SETD2-IN-1 TFA. The proliferation index of 4 of these 5 AMLs can be seen in supplemental Figure 9M-P, where the control conditions were the same and performed at the same time. (O) Relative cell counts for MOLM13 cells grown for 48 hours with or without methionine, with and without 1 mM homocysteine (Hcy), with SETD2-IN-1 both alone and in combination with Hcy supplementation. n = 3-4 experiments combined from the Seahorse experiments and independent homocysteine rescue experiments). (P) Western blot for H3K36me3 levels in a MOLM13 Hcy rescue experiment, with and without exposure to SETD-IN-1 TFA. β-Actin and unmodified histone H3 were used as loading controls. (Q) Basal OCR in a MOLM13 Hcy rescue of methionine depletion with and without SETD-IN-1 TFA exposure. Forty-eight-hour experiment: (R) Max OCR in a MOLM13 Hcy rescue of methionine depletion with and without SETD-IN-1 TFA exposure. All experiments: error bars represent mean ± SEM. Statistical analysis by ordinary 1-way ANOVA, ∗P < .05, ∗∗P < .01, ∗∗∗∗P < .0001.