Myc Is Required for Activation of the ATM-Dependent Checkpoints in Response to DNA Damage

Background: The MYC protein controls cellular functions such as differentiation, proliferation, and apoptosis. In response to genotoxic agents, cells overexpressing MYC undergo apoptosis. However, the MYC-regulated effectors acting upstream of the mitochondrial apoptotic pathway are still unknown. Principal Findings: In this study, we demonstrate that expression of Myc is required to activate the Ataxia telangiectasia mutated (ATM)-dependent DNA damage checkpoint responses in rat cell lines exposed to ionizing radiation (IR) or the bacterial cytolethal distending toxin (CDT). Phosphorylation of the ATM kinase and its downstream effectors, such as histone H2AX, were impaired in the myc null cell line HO15.19, compared to the myc positive TGR-1 and HOmyc3 cells. Nuclear foci formation of the Nijmegen Breakage Syndrome (Nbs) 1 protein, essential for efficient ATM activation, was also reduced in absence of myc. Knock down of the endogenous levels of MYC by siRNA in the human cell line HCT116 resulted in decreased ATM and CHK2 phosphorylation in response to irradiation. Conversely, cell death induced by UV irradiation, known to activate the ATR-dependent checkpoint, was similar in all the cell lines, independently of the myc status. Conclusion: These data demonstrate that MYC contributes to the activation of the ATM-dependent checkpoint responses, leading to cell death in response to specific genotoxic stimuli.Swedish Cancer SocietySwedish Research Counci

MYCMI-7 : A Small MYC-Binding Compound that Inhibits MYC: MAX Interaction and Tumor Growth in a MYC-Dependent Manner

Deregulated expression of MYC family oncogenes occurs frequently in human cancer and is often associated with aggressive disease and poor prognosis. While MYC is a highly warranted target, it has been considered "undruggable," and no specific anti-MYC drugs are available in the clinic. We recently identified molecules named MYCMIs that inhibit the interaction between MYC and its essential partner MAX. Here we show that one of these molecules, MYCMI-7, efficiently and selectively inhibits MYC:MAX and MYCN:MAX interactions in cells, binds directly to recombinant MYC, and reduces MYC-driven transcription. In addition, MYCMI-7 induces degradation of MYC and MYCN proteins. MYCMI-7 potently induces growth arrest/apoptosis in tumor cells in a MYC/MYCN-dependent manner and downregulates the MYC pathway on a global level as determined by RNA sequencing. Sensitivity to MYCMI-7 correlates with MYC expression in a panel of 60 tumor cell lines and MYCMI-7 shows high efficacy toward a collection of patient-derived primary glioblastoma and acute myeloid leukemia (AML) ex vivo cultures. Importantly, a variety of normal cells be- come G1 arrested without signs of apoptosis upon MYCMI-7 treatment. Finally, in mouse tumor models of MYC-driven AML, breast cancer, and MYCN-amplified neuroblastoma, treatment with MYCMI-7 downregu- lates MYC/MYCN, inhibits tumor growth, and prolongs survival through apoptosis with few side effects. In conclusion, MYCMI-7 is a potent and selective MYC inhibitor that is highly relevant for the development into clinically useful drugs for the treatment of MYC-driven cancer.Significance: Our findings demonstrate that the small-molecule MYCMI-7 binds MYC and inhibits interaction between MYC and MAX, thereby ham- pering MYC-driven tumor cell growth in culture and in vivo while sparing normal cells

Role of MYC in the regulation of the DNA damage checkpoint responses.

<p>In response to IR or CDT, MYC contributes to activation of the ATM-dependent responses, leading to induction of apotosis. These pathways are delayed upon MYC homozygote deletion or iRNA-mediated knock down.</p

Activation of ATM and ATM-dependent responses upon induction of DNA damage is Myc dependent.

<p>TGR-1, HOmyc3, and HO15.19 cells were left untreated or exposed to IR (20Gy), and further incubated in complete medium for 2h. The levels of phospho-ATM and phospho-H2AX (γH2AX) were assessed by western blot analysis. Actin and total ATM were used as internal loading controls. One of three experiments is shown.</p

<i>myc</i> deletion does not alter cell death kinetics upon UV irradiation.

<p>TGR-1, the <i>myc</i> reconstituted cells HOmyc3, and the <i>myc</i> null HO15.19 cells were left untreated or exposed to UV irradiation and further incubated in complete medium for the indicated periods of time. <b>A</b>) Analysis of the cell cycle distribution assessed by PI staining and flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008924#s2" target="_blank">Material and Methods</a>. <b>B</b>) Phase contrast micrographs of the cells taken at the indicated time points (Magnification 40×). One out of three independent experiments is shown.</p

<i>myc</i> deletion impairs Nbs1 foci formation in response to DNA damage.

<p>TGR-1, the <i>myc</i> reconstituted HOmyc3, and the <i>myc</i> null HO15.19 cells were exposed to bacterial lysates (1∶2000) expressing the mutant (CTR) or the wild type form (CDT) of <i>H. hepaticus</i> CDT, or irradiated (IR, 20 Gy) and further incubated in complete medium for the indicated periods of time. The Nbs1 protein was detected by indirect immunostaining, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008924#s2" target="_blank">Material and Methods</a>. The figure shows the sub-cellular distribution of Nbs1 at 2h post-treatment.</p

Knock down of MYC expression decreases activation of ATM, CHK2 and p53.

<p><b>A</b>) HCT116 cells were transfected either with the control GFP-specific (CTR) or MYC specific siRNA for 48h. The expression of MYC was assessed by immunoprecipitation and western-blot analysis. One out of three independent experiments is shown. <b>B</b>) HCT116 cells, transfected either with the GFP-specific (CTR) or MYC specific siRNA were left untreated or exposed to IR (20 Gy), and further incubated in complete medium for the indicated periods of time. The levels of phosphorylation of ATM, CHK2 and p53 were assessed by western blot analysis. Actin and total ATM were used as an internal loading control. One out of three independent experiments is shown.</p

<i>myc</i> deletion prevents p53 stabilization in response to DNA damage.

<p><b>A</b>) TGR-1 and the <i>myc</i> reconstituted HOmyc3 cells were left untreated or exposed to IR (20 Gy) for the indicated periods of time. The levels of p53 were assessed by western blot analysis. <b>B</b>) TGR-1, the <i>myc</i> reconstituted HOmyc3 and the HO15.19 cells were left untreated or exposed to IR (20 Gy) for the indicated periods of time. The levels of p53 were assessed by western blot analysis. Actin was used as an internal loading control. One out of three independent experiments is shown.</p

<i>myc</i> deletion delays cell death upon irradiation.

<p><b>A</b>) The levels of the endogenous Myc protein were assessed by immunoprecipitation followed by western blot analysis in TGR-1, the <i>myc</i> reconstituted cells HOmyc3, and the <i>myc</i> null HO15.19 cells using α-Myc antibodies. Expression of actin in total cell lysates was used as control. <b>B</b>) TGR-1, the <i>myc</i> reconstituted cells HOmyc3, and the <i>myc</i> null HO15.19 cells were left untreated or irradiated (20Gy) and further incubated in complete medium for the indicated periods of time. Analysis of the cell cycle distribution was assessed by PI staining and flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008924#s2" target="_blank">Material and Methods</a>. One out of four independent experiments is shown.</p