SUMMARYOncogene activation has been shown to generate replication-born DNA damage, also known as replicative stress (RS). Notably, the ATR kinase -and not ATM-is the primary responder to RS. One limitation for the study of ATR is the lack of potent inhibitors. We here describe a cell-based screening strategy that has allowed us to identify compounds with ATR inhibitory activity in the nanomolar range. Pharmacological inhibition of ATR generates RS, leading to chromosomal breakage in the presence of conditions that stall replication forks. Moreover, ATR inhibition is particularly toxic for p53 deficient cells, this toxicity being exacerbated by RS-generating conditions such as the overexpression of cyclin E. Importantly, one of the compounds is NVP-BEZ235, a dual PI3K/mTOR inhibitor that is currently being tested for cancer chemotherapy, but which we now show is also very potent against ATM, ATR and DNA-PKcs.

Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy. Cancer Res; 70(15); 6268-76. ©2010 AACR.

Abstractp38 MAPK signaling has been implicated in the regulation of processes leading to cancer development and progression. Chronic inflammation is a known risk factor for tumorigenesis, yet the precise mechanism of this association remains largely unknown. The related p38aMAPK (MAPK14) proteins p38g (MAPK12) and p38d (MAPK13) were recently shown to modulate the immune response, although their role in tumorigenesis remains controversial and their function in inflammation-associated cancer has not been studied. We analyzed the role of p38g and p38d in colon cancer associated to colitis using the azoxymethane/dextran sodium sulphate (AOM/DSS) colitis-associated colon cancer model in wild-type (WT), p38g-, p38d-, and p38g/ d-deficient (p38g/d À/À ) mice. We found that p38g/d deficiency significantly decreased tumor formation, in parallel with a decrease in proinflammatory cytokine and chemokine production. Analysis of leukocyte populations in p38g/d À/À mouse colon showed less macrophage and neutrophil recruitment than in WT mice. Together, our results establish that p38g and p38d are central to colitis-associated colon cancer formation through regulation of hematopoietic cell response to injury, and validate p38g and p38d as potential targets for cancer therapy. Cancer Res; 74(21); 6150-60. Ó2014 AACR.

Candida albicans is a frequent aetiologic agent of sepsis associated with high mortality in immunocompromised patients. Developing new antifungal therapies is a medical need due to the low efficiency and resistance to current antifungal drugs. Here, we show that p38γ and p38δ regulate the innate immune response to C. albicans. We describe a new TAK1‐TPL2‐MKK1‐ERK1/2 pathway in macrophages, which is activated by Dectin‐1 engagement and positively regulated by p38γ/p38δ. In mice, p38γ/p38δ deficiency protects against C. albicans infection by increasing ROS and iNOS production and thus the antifungal capacity of neutrophils and macrophages, and by decreasing the hyper‐inflammation that leads to severe host damage. Leucocyte recruitment to infected kidneys and production of inflammatory mediators are decreased in p38γ/δ‐null mice, reducing septic shock. p38γ/p38δ in myeloid cells are critical for this effect. Moreover, pharmacological inhibition of p38γ/p38δ in mice reduces fungal burden, revealing that these p38MAPKs may be therapeutic targets for treating C. albicans infection in humans.

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The contribution of chronic skin inflammation to the development of squamous cell carcinoma (SCC) is poorly understood. While the mitogen-activated protein kinase p38α regulates inflammatory responses and tumour development, little is known about the role of p38γ and p38δ in these processes. Here we show that combined p38γ and p38δ (p38γ/δ) deletion blocked skin tumour development in a chemically induced carcinogenesis model. p38γ/δ deletion reduced TPA-induced epidermal hyperproliferation and inflammation; it inhibited expression of proinflammatory cytokines and chemokines in keratinocytes in vitro and in whole skin in vivo, resulting in decreased neutrophil recruitment to skin. Our data indicate that p38γ/δ in keratinocytes promote carcinogenesis by enabling formation of a proinflammatory microenvironment that fosters epidermal hyperproliferation and tumourigenesis. These findings provide genetic evidence that p38γ and p38δ have essential roles in skin tumour development, and suggest that targeting inflammation through p38γ/δ offers a therapeutic strategy for SCC treatment and prevention.

Patients with ESRD undergoing peritoneal dialysis develop progressive peritoneal fibrosis, which may lead to technique failure. Recent data point to Th17-mediated inflammation as a key contributor in peritoneal damage. The leukocyte antigen CD69 modulates the setting and progression of autoimmune and inflammatory diseases by controlling the balance between Th17 and regulatory T cells (Tregs). However, the relevance of CD69 in tissue fibrosis remains largely unknown. Thus, we explored the role of CD69 in fibroproliferative responses using a mouse model of peritoneal fibrosis induced by dialysis fluid exposure under either normal or uremic status. We found that cd69 mice compared with wild-type (WT) mice showed enhanced fibrosis, mesothelial to mesenchymal transition, IL-17 production, and Th17 cell infiltration in response to dialysis fluid treatment. Uremia contributed partially to peritoneal inflammatory and fibrotic responses. Additionally, antibody-mediated CD69 blockade in WT mice mimicked the fibrotic response of cd69 mice. Finally, IL-17 blockade in cd69 mice decreased peritoneal fibrosis to the WT levels, and mixed bone marrow from cd69 and Rag2c mice transplanted into WT mice reproduced the severity of the response to dialysis fluid observed in cd69 mice, showing that CD69 exerts its regulatory function within the lymphocyte compartment. Overall, our results indicate that CD69 controls tissue fibrosis by regulating Th17-mediated inflammation.

<p>Figure S1. Effect of p38gamma and p38delta deletion in early stages of colitis-associated colon cancer induced by AOM/DSS treatment. Figure S2. p38gamma and p38deltaexpression in AOM/DSS-treated and untreated mouse colon. WT, p38gamma /-, p38delta/- or p38gamma/delta/- mice were AOM/DSS-treated and untreated. At 9 weeks post-AOM injection, colon extracts were prepared for mRNA and protein analysis. Figure S3. p38gamma or p38deltadeletion has no significant effect on body weight and colon length in the course of DSS-induced colitis. WT and p38gamma/delta/- mice were treated with 3% DSS in drinking water for 5 days. Figure S4. p38gamma/delta-/- mice show increased IEC apoptosis after DSS treatment. Figure S5. ERK1/2 phosphorylation, but not IkappaBalpha proteolysis, was reduced in p38gamma/delta-/- colon cells compared to WT. Figure S6. Effect of p38gamma/deltadeletion in the expression of chemokine receptors and in the recruitment of neutrophils, macrophages and Treg cells. Table SI. Primer sequences used for gene expression.</p>