Inhibiting WEE1 Selectively Kills Histone H3K36me3-Deficient Cancers by dNTP Starvation

Abstract

Histone H3K36 trimethylation (H3K36me3) is frequently lost in multiple cancer types, identifying it as an important therapeutic target. Here we identify a synthetic lethal interaction in which H3K36me3-deficient cancers are acutely sensitive to WEE1 inhibition. We show that RRM2, a ribonucleotide reductase subunit, is the target of this synthetic lethal interaction. RRM2 is regulated by two pathways here: first, H3K36me3 facilitates RRM2 expression through transcription initiation factor recruitment; second, WEE1 inhibition degrades RRM2 through untimely CDK activation. Therefore, WEE1 inhibition in H3K36me3-deficient cells results in RRM2 reduction, critical dNTP depletion, S-phase arrest, and apoptosis. Accordingly, this synthetic lethality is suppressed by increasing RRM2 expression or inhibiting RRM2 degradation. Finally, we demonstrate that WEE1 inhibitor AZD1775 regresses H3K36me3-deficient tumor xenografts.

Graphical abstract

Figure 1

WEE1 Inhibition Selectively Kills H3K36me3-Deficient Cancer Cells (A) Western blot analysis of SETD2 and H3K36me3 levels in A498, LB996, RCC4, and U2OS cells. A498 + SETD2 are A498 cells stably expressing a SETD2 cDNA. (B) Viability curves of SETD2 wild-type (RCC4, U2OS) and SETD2-deficient (A498, LB996) cells after exposure to WEE1 inhibitor AZD1775 (5 days). (C) Viability of A498 cells expressing either an empty vector (A498 + C) or SETD2 cDNA (A498 + SETD2) after exposure to AZD1775 (200 nM) (72 hr). (D) Western blot analysis of SETD2, KDM4A, and H3K36me3 in U2OS cells transfected with either control siRNA (siNT) or SETD2 siRNAs (siSETD2#3, siSETD2#5), and in U2OS cells stably expressing H3.3K36M or KDM4A. (E) Viability curves of U2OS cells transfected with either control siRNA (siNT) or SETD2 siRNAs (siSETD2#3 and siSETD2#5) (48 hr) and exposed to AZD1775 (5 days). (F) Viability curves of U2OS cells expressing either an empty vector, H3.3K36M, or KDM4A after exposure to AZD1775 (5 days). (G) Western blot analysis of SETD2 and H3K36me3 levels in U2OS parental cells or U2OS cells with CRISPR knockout of SETD2. (H) Viability curves of U2OS parental cells and U2OS SETD2 CRISPR knockout cells after exposure to AZD1775 (5 days). (I) Percentage of apoptotic cells after exposure to AZD1775 (48 hr). For all graphs in Figure 1, data are presented as mean ± SEM, n = 3 independent experiments. ^∗∗∗p < 0.001, unpaired and two-tailed t test was used. See also Figure S1.

Figure 2

WEE1 Inhibitor AZD1775 Abolishes DNA Replication in SETD2-Deficient Cells (A) BrdU FACS analysis of the cell cycle distribution of U2OS wild-type and U2OS SETD2 CRISPR knockout cells after exposure to DMSO (0.02%) or AZD1775 (200 nM) (48 hr). (B) DNA fiber analysis of replication fork velocity in U2OS cells transfected with control siRNA (siNT) or SETD2 siRNAs (si#3 and si#5) (48 hr) prior to treatment with DMSO or AZD1775 (200 nM) (48 hr). Representative images of the replication track after treatments; scale bar represents 5 μm (left). The distribution of fork velocity was analyzed from >100 ongoing replication forks in each condition (right). Data are presented as mean ± SEM, n = 3 independent experiments. (C and D) U2OS (C) and A498 (D) cells were treated with DMSO or AZD1775 (200 nM, 24 hr), pulsed with EdU (10 min), and harvested either immediately or after thymidine chase (Thd Chase) (90 min) and analyzed by western blots (left). Graphical representations of the iPOND data are shown on the right. Click reaction was used to conjugate biotin to nascent DNA. No click acted as control with biotin azide omitted. See also Figure S2.

Figure 3

H3K36me3 Regulates RRM2 Expression (A) Western blot analysis of RRM2, RRM1, and P53R2 protein levels in U2OS cells transfected with control (siNT) or SETD2 siRNA (siSETD2) (48 hr) and exposed to either DMSO or AZD1775 (200 nM) (24 hr). (B) qRT-PCR analysis of RRM2 mRNA levels in siSETD2 U2OS cells (normalized to siNT) and in CRISPR SETD2-knockout U2OS cells (normalized to parental cells). (C) qRT-PCR analysis of RRM2 mRNA levels in SETD2-deficient (A498, LB996) and proficient cells (U2OS, RCC4) (left). Western blot analysis of RRM2 protein levels in these cells (right). (D) ChIP analysis of the enrichment of H3K36me3, TAF6, RNA-Pol2, and phospho-Pol2 (Ser5) at the RRM2 promoter in U2OS cells transfected with control siRNA (siNT) or SETD2 siRNA (siSETD2). The qPCR data are presented as percentage of input. (E) Viability curves of U2OS cells transfected with either control siRNA (siNT) or RRM2 siRNA (siRRM2) (48 hr) and exposed to AZD1775 (5 days) (left). Western blot analysis of RRM2 and RRM1 protein levels (right). (F) Viability curves of U2OS cells treated with DMSO, HU, or GM combined with indicated concentrations of AZD1775 (5 days). U2OS CRISPR SETD2-knockout cells were used as a control. For all graphs in Figure 3, data are presented as mean ± SEM, n = 3 independent experiments. ^∗∗∗p < 0.001, ^∗∗p < 0.01; ^∗p < 0.05; n.s., not significant. Unpaired and two-tailed t tests were used for (D), and column statistics (one sample t test) were used for (B) and (C). See also Figure S3.

Figure 4

WEE1 Inhibition Promotes RRM2 Degradation and Aberrant Origin Firing (A) U2OS and U2OS CRISPR SETD2-knockout cells were treated with cycloheximide (50 μg/ml) in the presence of DMSO, AZD1775 (400 nM), MG132 (10 μM) or AZD1775 + MG132. Cells were collected at the indicated times, lysed, and immunoblotted as indicated. (B) A498 cells were treated with AZD1775 (200 nM) combined with either DMSO, neddylation inhibitor MLN4924 (NAEi) (0.1 mM), CDK1 inhibitor RO3306 (CDK1i) (10 μM) or CDK2 inhibitor (CVT-313) (3 μM). Western blot analysis of phospho-CDK1 (Y15) and RRM2 levels after indicated treatment (24 hr) (top). Viability of A498 cells after indicated treatment (72 hr) (bottom). (C) U2OS cells expressing either an empty vector (Vector) or a degradation-resistant mutant RRM2 (T33A) were transfected with control siRNA (siNT) or SETD2 siRNA (siSETD2) (48 hr) prior to treatment with either DMSO or AZD1775 (200 nM). Western blot analysis of RRM2 levels after indicated treatment (24 hr) (top), and viability of these cells after indicated treatment (96 hr) (bottom). (D) dATP levels in U2OS cells stably expressing either an empty vector (Vector) or a degradation-resistant mutant RRM2 (T33A), transfected with control siRNA (siNT) or SETD2 siRNA (siSETD2) (48 hr) prior to treatment with either DMSO or AZD1775 (200 nM) (24 hr). Data are normalized to the control (siNT + DMSO) of each cell line. (E) Viability curves of U2OS CRISPR SETD2-knockout cells transfected with either control siRNA (siNT) or CDC6 siRNA (siCDC6) (48 hr) and exposed to AZD1775 (5 days) (left). Western blot analysis of CDC6 protein levels (right). (F) Viability of U2OS CRISPR SETD2-knockout cells transfected with control siRNA (siNT), RRM2 siRNA (siRRM2), CDT2 siRNA (siCDT2), or both (siRRM2 + siCDT2) (48 hr) (left). Western blot analysis of RRM2 and CDT2 protein levels (right). For all graphs in Figure 4, data are presented as mean ± SEM, n = 3 independent experiments. ^∗∗∗p < 0.001; ^∗∗p < 0.01; ^∗p < 0.05; n.s., not significant. Unpaired and two-tailed t tests were used for (B), (C), and (F), and column statistics (one sample t test) were used for (D). See also Figure S4.

Figure 5

WEE1 Inhibitor AZD1775 Regresses SETD2-Deficient Tumors In Vivo (A) Tumor volumes for each treatment group, with arrows indicating the days when the inhibitors were given. Data are presented as mean ± SEM, n = 7 mice for A498 and U2OS, n = 5 mice for LB996. (B) Representative tumors in mice treated with either AZD1775 or vehicle (day 13). (C–F) Representative images of tumors generated from A498 cells (C and E) and quantification results (D and F) of immunohistochemistry analysis of pan-nuclear γH2AX (C and D) and cleaved-Caspase-3 (CC3) (E and F) levels in tumors (day 13). Scale bar represents 50 μm. Data are presented as mean ± SEM, n = 3 tumors. ^∗∗∗p < 0.001; ^∗p < 0.05; unpaired and two-tailed t tests were used. (G) Immunohistochemistry staining with anti-H3K36me3 antibody to distinguish SETD2-deficient from SETD2-proficient xenografts. Scale bar represents 50 μm. (H) Representative images of H3K36me3-negative and H3K36me3-positive tumors identified by immunohistochemistry staining of human renal cancer patient tissue microarray. Scale bar represents 50 μm. See also Figure S5.

Figure 6

Schematic Overview of the Synthetic Lethal Interaction between H3K36me3 Loss and WEE1 Inhibition RRM2 is regulated by two pathways. In the first, SETD2 catalyzes histone H3K36me3, which promotes RRM2 expression. In the second, WEE1 negatively regulates CDK activity and upon WEE1 inhibition, hyperactive CDK promotes RRM2 degradation and aberrant origin firing. Therefore, WEE1 inhibition in H3K36me3-deficient cells leads to critically reduced dNTP pool levels, resulting in replication stress and cell death.