Search

Identifying new drug targets and developing safe and effective drugs is both challenging and risky. Furthermore, characterizing drug development risk, the probability that a drug will eventually receive regulatory approval, has been notoriously hard given the complexities of drug biology and clinical trials. This inherent risk is often misunderstood and mischaracterized, leading to inefficient allocation of resources and, as a result, an overall reduction in R&D productivity. Here we argue that the recent resurgence of Machine Learning in combination with the availability of data can provide a more accurate and unbiased estimate of drug development risk.

The discovery of the radiation-induced bystander effect has expanded knowledge of radiobiological mechanisms of ionizing radiation. A counterpart in vivo phenomenon is the radiation-induced abscopal effect (RIAE). It is not known how radiation settings affect non-targeted normal tissues and therefore the risk of adverse RIAE. At the Australian Synchrotron we examined systemic effects of microbeam radiotherapy (MRT) and broad beam (BB) configurations, in mice that were locally exposed to a very short pulse of a high dose-rate synchrotron beam (49 Gy/sec). We determined how radiation volume and dose impact the RIAE. C57BL/6 mice were irradiated with 10 or 40 Gy incident dose of MRT or BB in an 8x8, 8x1, or 2x2-mm area of the right hind leg. Blood samples, irradiated skin and a variety of normal unirradiated tissues were collected for DNA damage analysis of double-strand breaks (DSBs) quantified as gamma-H2AX foci and oxidatitive clustered DNA lesions (OCDL). OCDLs elevated in a wide variety of unirradiated normal tissues. In out-of-field duodenum, a trend for elevated apoptotic cells was observed, however DSBs elevated only after exposure to lower doses. These genotoxic events were accompanied by changes in concentrations of MDC, CCL2/MCP1, Eotaxin, IL-10, TIMP-1, VEGF, TGFβ-1 and TGFβ-2 plasma cytokines and by changes in frequencies of macrophages, neutrophils and T-lymphocytes in duodenum. Overall, systemic radiation responses were dose-independent (1). The MRT irradiations was repeated in immune-deficient mice: NSG, CCL2 knock-outs, and in C57BL/6 mice treated with anti-CSF1R antibody which effectively depletes macrophages. Strikingly, these effects and the abscopal innate and adaptive immune effector responses were completely or partially abrogated in the mice with various immune deficiencies, highlighting the role of the functional immune system in propagation of systemic genotoxic effects of radiation. These findings have implications for the planning of therapeutic and diagnostic radiation to reduce the risk of radiation-related adverse systemic effects. 1. Ventura et al, Cancer Research, e-pub (2017). Citation Format: Jessica Ventura, Pavel Lobachevsky, Jason Palazzolo, Helen Forrester, Nicole Haynes, Alesia Ivashkevich, Andrew Stevenson, Christopher Hall, Vassilis Gorgoulis, John Hamilton, Alexandros Georgakilas, Calr Sprung, Olga A. Martin. Localized synchrotron radiation in mice induces persistent systemic genotoxic events mediated by the functional immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4573.

The response of eukaryotic cells to DNA damage includes the activation of phosphatidylinositol-3 kinase–related kinases (PIKK), such as ATM, ATR, and DNA-dependent protein kinase (DNA-PK). These three kinases have very similar substrate specificities in vitro, but in vivo, their substrates overlap only partially. Several in vivo substrates of ATM and ATR have been identified and almost all of them are involved in DNA damage–induced cell cycle arrest and/or apoptosis. In contrast, few in vivo substrates of DNA-PK have been identified. These include histone H2AX and DNA-PK itself. We identify here valosin-containing protein (VCP) as a novel substrate of DNA-PK and other PIKK family members. VCP is phosphorylated at Ser784 within its COOH terminus, a region previously shown to target VCP to specific intracellular compartments. Furthermore, VCP phosphorylated at Ser784 accumulated at sites of DNA double-strand breaks (DSBs). VCP is a protein chaperone that unfolds and translocates proteins. Its phosphorylation in response to DNA damage and its recruitment to sites of DNA DSBs could indicate a role of VCP in DNA repair.

TP53 mutants (mutp53) are involved in the pathogenesis of most human cancers. Specific mutp53 proteins gain oncogenic functions (GOF) distinct from the tumor suppressor activity of the wild-type protein. Tumor-associated-macrophages, a hallmark of solid tumors, are typically correlated with poor prognosis. Here we report a non-cell-autonomous mechanism whereby mutp53 cancer cells reprogram TAM to a tumor supportive and anti-inflammatory state. The colon cancer cells harboring GOF mutp53 selectively shed miR-1246-enriched exosomes. Uptake of these exosomes by neighboring macrophages triggers their miR-1246 dependent reprogramming into a cancer-promoting state. Mutp53-reprogammed TAM favor anti-inflammatory immunosuppression with increased activity of TGF-β. These findings, observed also in colon cancer patients, strongly support a microenvironmental GOF role for mutp53 in actively engaging the immune system to promote cancer progression and metastasis. Citation Format: Tomer Cooks, Ioannis S. Pateras, Lisa M. Jenkins, Keval M. Patel, Ana I. Robles, James Morris, Tim Forshew, Ettore Appella, Vassilis G. Gorgoulis, Curtis C. Harris. Mutant p53 cancers reprogram tumor-associated macrophages via exosomal miR-1246 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3701. doi:10.1158/1538-7445.AM2017-3701

Oncogene-induced DNA replication stress is thought to drive genomic instability in cancer. In particular, replication stress can explain the high prevalence of focal genomic deletions mapping within very large genes in human tumors. However, the origin of single-nucleotide substitutions (SNS) in nonfamilial cancers is strongly debated. Some argue that cancers have a mutator phenotype, whereas others argue that the normal DNA replication error rates are sufficient to explain the number of observed SNSs. Here, we sequenced the exomes of 24, mostly precancerous, colon polyps. Analysis of the sequences revealed mutations in the APC, CTNNB1, and BRAF genes as the presumptive cancer-initiating events and many passenger SNSs. We used the number of SNSs in the various lesions to calculate mutation rates for normal colon and adenomas and found that colon adenomas exhibit a mutator phenotype. Interestingly, the SNSs in the adenomas mapped more often than expected within very large genes, where focal deletions in response to DNA replication stress also map. We propose that single-stranded DNA generated in response to oncogene-induced replication stress compromises the repair of deaminated cytosines and other damaged bases, leading to the observed SNS mutator phenotype. Cancer Res; 72(23); 6279–89. ©2012 AACR.

Introduction: TP53 mutants are involved in the pathogenesis of most solid tumors and are known to gain oncogenic functions distinct from their original wild-type form. The existence of such gain-of-function (GOF) activities is supported by ample evidence, however only in a cell-autonomous fashion. Since tumor-associated-macrophages (TAM) are also a hallmark of solid tumors typically correlated with poor prognosis, we investigated the link between mutations in the TP53 gene (mutp53) occurring in epithelial tumor cells and the formation of a surrounding TAM population in-situ. Methods: By designing a co-culture system we incubated human primary monocytes together with colorectal cancer (CRC) cells differing in their p53 status. Relevant macrophages markers were evaluated on RNA level and protein level. In addition, co-cultured macrophages were subjected to various functional assays (phagocytosis, migration, and invasion). In attempt to confirm clinical relevance, samples from a cohort of human CRC patients were analyzed using genomic and immunohistochemical methods. To identify the interaction between the tumor cells and the macrophages, we isolated exosomes from the CRC cells and subjected them to a Nanostring analysis to learn about their microRNAs composition. Results: In this study, we discovered that mutp53 exerts a non-cell-autonomous effect over neighboring macrophages by using specific microRNAs (miRs) which are shuttled through an exosomal transfer resulting with a phenotype change of the affected macrophages. Mutp53-specific exosomes containing cargoes such as miR-1246 were shown to be used by macrophages at the receiving end, thus promoting the formation of TAM subset also observed in surgical specimens resected from cancer patients. Conclusions: Altogether, these findings are consistent with a microenvironmental role for specific “hot-spot” p53 mutants tightening the interaction between the tumor cell and the immune compartment. Additionally, this study is the first to show a non-cell-autonomous role played by mutant p53 - the most common form of mutation found in human cancers. Deciphering the intricate regulation shared by the tumor cell and its surrounding macrophages may give rise to novel prognostic and diagnostic tools as well as to therapeutic approaches targeting TAM, specific tumor-promoting miRs and mutp53-specific subsets of exosomes. Citation Format: Tomer Cooks, Ioannis S. Pateras, Keval M. Patel, Tim Forshew, Vassilis G. Gorgoulis, Curtis C. Harris. Exosomes from mutant TP53 cancer cells polarize tumor associated macrophages. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 906.

Mutations in the p53 tumor suppressor are very frequent in human cancer. Often, such mutations lead to the constitutive overproduction of mutant p53 proteins, which may exert a cancer-promoting gain of function. We now report that cancer-associated mutant p53 can augment the induction of nuclear factor κB (NFκB) transcriptional activity in response to the cytokine tumor necrosis factor α (TNFα). Conversely, down-regulation of endogenous mutant p53 sensitizes cancer cells to the apoptotic effects of TNFα. Analysis of human head and neck tumors and lung tumors reveals a close correlation between the presence of abundant mutant p53 proteins and the constitutive activation of NFκB. Together, these findings suggest that p53 mutations may promote cancer progression by augmenting NFκB activation in the context of chronic inflammation. [Cancer Res 2007;67(6):2396–401]

Activating transcription factor (ATF)-2 is a member of the ATF/cyclic AMP-responsive element binding protein family of transcription factors. It has been shown, in vitro, to possess growth factor-independent proliferation and transformation capacity. The information concerning the involvement of ATF-2 in carcinogenesis is rather limited. In a previous report, we showed a progressive increase in the levels of various activator protein (AP)-1 components, including phosphorylated ATF-2, in a series of mouse skin cell lines that represented developmental stages of the mouse skin carcinogenesis system. In the present study, we examined in detail the role of ATF-2 in the development of mouse skin spindle cells A5 and CarB, which correspond to the late and most aggressive stage of the mouse skin carcinogenesis model. To address this issue, we overexpressed a dominant negative form of ATF-2 in the A5 and CarB cell lines and examined their behavior in vitro and in vivo at the molecular and cellular level. The stable transfectants expressed decreased levels of phosphorylated ATF-2 and c-Jun. Subsequently, we observed that dominant negative ATF-2 affected the composition and reduced the activity of AP-1. The above biochemical changes were followed, both in vitro and in vivo in BALB/c severe combined immunodeficient mice, by suppression of the aggressive characteristics of the A5 and CarB mouse skin spindle cells. We attributed this behavior to the significant down-regulation of cyclin D1, cyclin A, and ATF-3, known AP-1 targets implicated in cell cycle control and promotion. In conclusion, our findings underscore a key regulatory role of ATF-2 in tumor growth and progression of mouse skin tumors.