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Supplemental Figures 1-5. Supplemental Figure 1. Kinase selectivity profile of ATRi. Supplemental Figure 2. Screening of inhibitors in cellular γ-H2AX assay in U2OS cells. Supplemental Figure 3. Flow cytometry profiles assessing γ-H2AX response to compare antimetabolite combinations with a fixed concentration of ATRi in U2OS cells. Supplemental Figure 4. γ-H2AX response and cell viability following ATRi treatment in U2OS cells. Supplemental Figure 5. In vivo pharmacokinetics and γ-H2AX pharmacodynamics of ATRi in GRANTA-519 (ATM mut) MCL tumor xenograft model.

Supplemental Table 2. SiRNA sequences (Supplemental data)

Supplemental Table 1. DiscoveRx ATR inhibitor profile

Supplemental Figure legends. Supplemental Figure 1. Kinase selectivity profile of ATRi. Supplemental Figure 2. Screening of inhibitors in cellular γ-H2AX assay in U2OS cells. Supplemental Figure 3. Flow cytometry profiles assessing γ-H2AX response to compare antimetabolite combinations with a fixed concentration of ATRi in U2OS cells. Supplemental Figure 4. γ-H2AX response and cell viability following ATRi treatment in U2OS cells. Supplemental Figure 5. In vivo pharmacokinetics and γ-H2AX pharmacodynamics of ATRi in GRANTA-519 (ATM mut) MCL tumor xenograft model.

Mechanisms to maintain genomic integrity are essential for cells to remain viable. Not surprisingly, disruption of key DNA damage response pathway factors, such as ataxia telangiectasia-mutated (ATM)/ataxia telangiectasia and RAD3-related (ATR) results in loss of genomic integrity. Here, a synthetic lethal siRNA-screening approach not only confirmed ATM but identified additional replication checkpoint proteins, when ablated, enhanced ATR inhibitor (ATRi) response in a high-content γ-H2AX assay. Cancers with inactivating ATM mutations exhibit impaired DNA double-stranded break (DSB) repair and rely on compensatory repair pathways for survival. Therefore, impairing ATR activity may selectively sensitize cancer cells to killing. ATR inhibition in an ATM-deficient context results in phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs) and leads to induction of γ-H2AX. Using both in vitro and in vivo models, ATR inhibition enhanced efficacy in ATM loss-of-function mantle cell lymphoma (MCL) compared with ATM wild-type cancer cells. In summary, single-agent ATR inhibitors have therapeutic utility in the treatment of cancers, like MCL, in which ATM function has been lost.Implications: These data suggest that single-agent ATR inhibitors have therapeutic utility and that ATR uses a complex and coordinated set of proteins to maintain genomic stability that could be further exploited. Mol Cancer Res; 13(1); 120–9. ©2014 AACR.

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