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Oncogenic stress sensitizes murine cancers to hypomorphic suppression of ATR
David W. Schoppy, … , J. Alan Diehl, Eric J. Brown
David W. Schoppy, … , J. Alan Diehl, Eric J. Brown
Published December 1, 2011
Citation Information: J Clin Invest. 2012;122(1):241-252. https://doi.org/10.1172/JCI58928.
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Research Article

Oncogenic stress sensitizes murine cancers to hypomorphic suppression of ATR

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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 4

ATR reduction potently suppresses the growth of p53-null fibrosarcomas driven by H-rasG12V and increases genomic instability.

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ATR reduction potently suppresses the growth of p53-null fibrosarcomas d...
(A) Quantification of correctly spliced mouse (ATRfl) and human (ATRseckel) ATR transcript in p53–/–ATRfl/seckelCreERT2+ cell lines expressing H-rasG12V. Relative transcript quantity was measured through qRT-PCR analysis of RNA isolated from the indicated cultures (n = 4 independent cell lines). Data represent mean ± SEM. (B) Representative images of tamoxifen-treated ATRfl/+CreERT2+ (top panel) or ATRfl/seckelCreERT2+ (bottom panel) tumors 10 days following the initiation of treatment. TAM, tamoxifen. (C) Measurement of fibrosarcoma growth following ATRfl deletion. Tumors were allowed to grow to 100–200 mm3 before initiation of tamoxifen treatment to recombine the ATRfl allele. Tamoxifen treatment days are indicated by arrows. *P < 0.01 (n = 4–13 tumors per genotype, per measurement). Percentages listed above at specific time points indicate the abundance of the ATRfl-recombined allele (ATRΔ) in tumors isolated from that time point (n = 1–3 tumors per genotype/measurement). Data represent mean ± SEM. (D) H&E-stained sections (left panels) or γH2AX/DAPI–stained sections (right panels; γH2AX, green; DAPI, blue) of H-rasG12V–expressing tumors (A–C) isolated 10 days after initial ATRfl deletion. Enlarged cells with aberrant nuclei are indicated with arrows. Original magnification, ×200. (E) Quantification of γH2AX-positive cells in H-rasG12V–expressing tumors (A–C) isolated 9–10 days after initial ATRfl deletion. The abundance of γH2AX-positive cells was determined from 10 high power field images (HPF, ×200) from each tumor type (n = 4–6 tumors per genotype). Only nucleated (DAPI-positive) cells were scored in this analysis. Data represent mean ± SEM.
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