Oncogenic stress sensitizes murine cancers to hypomorphic suppression of ATR
David W. Schoppy, Ryan L. Ragland, Oren Gilad, Nishita Shastri, Ashley A. Peters, Matilde Murga, Oscar Fernandez-Capetillo, J. Alan Diehl, Eric J. Brown
David W. Schoppy, Ryan L. Ragland, Oren Gilad, Nishita Shastri, Ashley A. Peters, Matilde Murga, Oscar Fernandez-Capetillo, J. Alan Diehl, Eric J. Brown
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Research Article

Oncogenic stress sensitizes murine cancers to hypomorphic suppression of ATR

Abstract

Oncogenic Ras and p53 loss-of-function mutations are common in many advanced sporadic malignancies and together predict a limited responsiveness to conventional chemotherapy. Notably, studies in cultured cells have indicated that each of these genetic alterations creates a selective sensitivity to ataxia telangiectasia and Rad3-related (ATR) pathway inhibition. Here, we describe a genetic system to conditionally reduce ATR expression to 10% of normal levels in adult mice to compare the impact of this suppression on normal tissues and cancers in vivo. Hypomorphic suppression of ATR minimally affected normal bone marrow and intestinal homeostasis, indicating that this level of ATR expression was sufficient for highly proliferative adult tissues. In contrast, hypomorphic ATR reduction potently inhibited the growth of both p53-deficient fibrosarcomas expressing H-rasG12V and acute myeloid leukemias (AMLs) driven by MLL-ENL and N-rasG12D. Notably, DNA damage increased in a greater-than-additive fashion upon combining ATR suppression with oncogenic stress (H-rasG12V, K-rasG12D, or c-Myc overexpression), indicating that this cooperative genome-destabilizing interaction may contribute to tumor selectivity in vivo. This toxic interaction between ATR suppression and oncogenic stress occurred without regard to p53 status. These studies define a level of ATR pathway inhibition in which the growth of malignancies harboring oncogenic mutations can be suppressed with minimal impact on normal tissue homeostasis, highlighting ATR inhibition as a promising therapeutic strategy.

Authors

David W. Schoppy, Ryan L. Ragland, Oren Gilad, Nishita Shastri, Ashley A. Peters, Matilde Murga, Oscar Fernandez-Capetillo, J. Alan Diehl, Eric J. Brown

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Figure 7

ATR inhibition synergizes with H-rasG12V, K-rasG12D, and c-Myc overexpression to cause increased genomic instability and cell synthetic lethality.

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ATR inhibition synergizes with H-rasG12V, K-rasG12D, and c-Myc overexpre...
(A) Phosphorylation of H2AX in response to ATR inhibition in the context of oncogene expression. NIH3T3 cells were transduced with retroviruses expressing the indicated oncogenes or empty control vector (pBabe-puro). Following drug resistance marker selection, cell lines were expanded and treated with 1 μM ATR-45 inhibitor for 7 hours. Cells were then harvested for Western blot detection of the indicated proteins. (B) Cell-cycle distribution of γH2AX following ATR inhibition. Oncogene-expressing and control cell lines were treated with 4 μM ATR-45 inhibitor for 24 hours and detected for phospho-S139 H2AX and DNA content (propidium iodide staining). Aphidicolin (0.5 μM) was added to control cells to induce exogenous replication stress and serve as a positive control for its effects. (C) Chromatid breaks following short-term ATR inhibition in oncogene-expressing and control cell lines. Cell lines were treated with 2 μM ATR-45 inhibitor for 7 hours, as described in A, were harvested for mitotic spreads, and chromatid breaks were quantified. Nocodazole (0.5 μM) was added 4 hours prior to harvest. Data represent mean ± SEM.