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. 2012 Jan;122(1):241-52.
doi: 10.1172/JCI58928. Epub 2011 Dec 1.

"V体育2025版" Oncogenic stress sensitizes murine cancers to hypomorphic suppression of ATR

David W Schoppy  1 , Ryan L Ragland, Oren Gilad, Nishita Shastri, Ashley A Peters, Matilde Murga, Oscar Fernandez-Capetillo, J Alan Diehl, Eric J Brown
Affiliations

Oncogenic stress sensitizes murine cancers to hypomorphic suppression of ATR (V体育官网)

David W Schoppy et al. J Clin Invest. 2012 Jan.

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 VSports手机版. 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. .

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Figures

Figure 1
Figure 1. A genetic system to conditionally reduce ATR expression to hypomorphic levels in adult mice.
(A) Illustration of the 5′ region of the humanized ATRseckel allele. (B) Quantification of mouse (ATRfl) and human (ATRseckel) ATR transcript containing exons 8–9 in adult (6 to 8 week old) bone marrow and intestinal epithelium. Relative transcript quantity was measured through qRT-PCR analysis of RNA isolated from the indicated tissues as described in Supplemental Figure 2 (n = 5 mice per genotype, per tissue). Data show mean ± SEM. (C) Treatment used to achieve conditional ATR suppression in adult tissues. (D) Western blot analysis of ATR expression in either adult bone marrow (top panel) or intestinal epithelium (bottom panel) isolated 10 days following initial tamoxifen treatment/ATR suppression. MCM3 is shown as a loading control.
Figure 2
Figure 2. ATR hypomorphic suppression has a minimal impact on normal tissue homeostasis.
(A) H&E-stained sections of humeral bones from tamoxifen-treated mice at the indicated time points after initial treatment. Original magnification, ×200. (B) Absolute number of myeloid cells (Mac1+Gr1+) obtained from 4 hind limb bones of mice at the indicated time points (n = 5–15 mice per genotype, per time point). (C) H&E-stained sections of intestines from tamoxifen-treated mice at the indicated time points after initial treatment. Original magnification, ×200. (D) Weight of mice at the indicated time points. (E and F) Abundance of the ATRfl-recombined allele (ATRΔ) in the bone marrow (E) and intestines (F) of tamoxifen-treated mice. Time points represent the number of days after tamoxifen treatment. The frequency of ATRfl recombination was determined by qPCR amplification of the ATRfl allele from genomic DNA isolated from each tissue (n = 5–15 mice per genotype, per time point). Data show mean ± SEM.
Figure 3
Figure 3. ATR hypomorphic suppression is more limiting to MLL-ENL– and N-rasG12D–transformed AMLs than to normal bone marrow cells.
(A) The amount of correctly spliced ATR transcript from the ATRseckel allele. Transcript levels relative to the murine ATRfl allele are shown. These levels were quantified through qRT-PCR as described in Figure 1 and Methods. Data represent mean ± SEM. (B) Experimental approach to suppressing ATR expression in murine AML. AML cells were transplanted from a primary recipient to secondary recipients, and tamoxifen was given upon detection of substantial representation (>20%) of GFP+ AML cells in peripheral white blood cells. (C) ATR hypomorphic suppression is competitively disadvantageous to AMLs to a greater degree than to normal bone marrow. tamoxifen-treated secondary (AML) recipient mice were sacrificed at various days following initiation of treatment. Genomic DNA was isolated from leukemic bone marrow, and Cre-mediated ATRfl recombination (ATRΔ) was quantified through qPCR. For comparison, bone marrow from systemically treated ATRfl/seckel mice was quantified similarly for the persistence of ATRΔ/seckel cells. No significant differences were observed between p53+/+ and p53–/– backgrounds. Data represent mean ± SEM.
Figure 4
Figure 4. ATR reduction potently suppresses the growth of p53-null fibrosarcomas driven by H-rasG12V and increases genomic instability.
(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.
Figure 5
Figure 5. H-rasG12V-transformed p53+/+ fibrosarcomas that express low levels of p53R175H are also sensitive to hypomorphic ATR reduction.
(A) Quantification of correctly-spliced mouse (ATRfl) and human (ATRseckel) ATR transcript in p53+/+ATRfl/seckelCreERT2+ cell lines ectopically expressing p53R175H and H-rasG12V. Data represent mean ± SEM. (B) Measurement of p53R175H-expressing tumor growth following ATRfl deletion. Tamoxifen treatment was performed on days 1 through 5, as shown in Figure 4. Data represent mean ± SEM. (C) Quantification of γH2AX-positive cells in H-rasG12V–expressing tumors (B) following ATRfl deletion. Tumors were isolated 9–10 days after initial tamoxifen treatment and quantified for γH2AX-positive cells as described in Figure 4E. Data represent mean ± SEM.
Figure 6
Figure 6. Genomic instability caused by ATR suppression is significantly enhanced by H-rasG12V expression.
(A) Western blot analysis of immortalized MEF cultures 48 hours following initial 4-OHT treatment. Separate blots from similarly prepared protein lysates were detected for ATR and MCM3 (top panels) or γH2AX, phospho-ERK, and actin (bottom panels). S-phase content was quantified through EdU incorporation/detection in cultures harvested 48 hours after initial 4-OHT treatment. Cumulative doublings were obtained through cell counting at 48 hours after initial 4-OHT treatment. (B) Chromosomal breakage analysis of MEF cultures from A. Cultures were collected for metaphase analysis 48 hours after initial 4-OHT treatment. Gaps/breaks per metaphase were scored in readily assessable metaphase spreads, while metaphase spreads with more than 15 breaks were scored separately and weighted into this analysis. The percentage of metaphase spreads with more than 15 breaks is listed in the lower panel. PCC was also scored, and the percentage of metaphase spreads marked by PCC is listed in the lower panel. Data represent mean ± SEM.
Figure 7
Figure 7. ATR inhibition synergizes with H-rasG12V, K-rasG12D, and c-Myc overexpression to cause increased genomic instability and cell synthetic lethality.
(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.
Figure 8
Figure 8. ATR inhibition selectively suppresses the expansion of H-rasG12V– and c-Myc–transformed fibroblasts and increases cell death.
(A) Effects of ATR inhibition on the proliferation of oncogene-expressing and control cell lines. Asynchronously growing cell lines were treated with ATR inhibitor (2 μM); medium and inhibitor were replaced at replating every 2 days. Cells were counted at replating, and cumulative doublings were quantified. Data represent mean ± SEM. (B) Cell death following ATR inhibition. Sub-G1 DNA content was quantified by propidium iodide staining and flow cytometry (left panels) both 2 days (right bar graph) and 4 days (left bar graph) after continuous ATR inhibitor treatment as described in A. Peak levels of cell death were observed in the respective oncogene-transformed cell lines at these time points. SEM bars are indicated from 3–5 independent experiments.
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