Samuel Sidi - US grants

DESCRIPTION (provided by applicant): Defects in p53 signaling eliminate apoptotic responses to radiation therapy in many human cancers. In head and neck squamous cell carcinoma (HNSCC), the fifth most common cancer worldwide, TP53 mutations cause locoregional recurrence of radioresistant tumors, an invariably fatal form of the disease. Thus there is an urgent need for agents that will bypass mutant TP53 to restore radiosensitivity in HNSCC. This proposal focuses on an emerging apoptotic pathway, designated 'Chk1-suppressed' (CS) apoptosis, whose activation by Chk1 inhibitors restores radiosensitivity in p53-deficient zebrafish, mouse, and human cancer cells (Sidi et al., Cell 2008). We propose that Chk1 inhibitors and associated CS pathway define a promising therapeutic opportunity for TP53 mutant HNSCC. Our work has elucidated the core backbone of the CS pathway, which comprises a novel ATM/ATR-caspase-2 axis that bypasses p53 and attendant mitochondrial and death-receptor signaling cascades. Recently, we identified the PIDDosome (PIDD-RAIDD-caspase-2 complex), but not the intrinsic apoptosome or extrinsic DISC, as the caspase-activation platform at work in the CS pathway. These results strengthen the notion that the CS axis defines a third apoptotic pathway in vertebrate cells and were published in the September 14th issue of Molecular Cell (Ando et al. Mol Cell 2012). While we hypothesize that the CS pathway will be therapeutically effective in TP53 mutant HNSCC, the extreme heterogeneity of this disease makes it essential that we develop biologic tools that predict or assess PIDDosome activity in tumors. However, our molecular understanding of PIDDosome biology is very limited. To both deepen our understanding of PIDDosome signaling and identify predictive and pharmacodynamic biomarkers of CS pathway therapy, we propose three specific aims. The first and second aims are designed to identify novel PIDDosome regulators and substrates, respectively, by elucidating the roles of four PIDD-interacting molecules we recently identified. The third aim integrates cutting-edge cancer genomics with in vivo functional genetics in zebrafish to identify genetic predictors of HNSCC response to CS pathway therapy. Candidate predictive genotypes will be functionally characterized using the functional CS pathway markers identified in Aim 1, Aim 2, or our previous studies, and validated in ex vivo cultures of primary HNSCC samples from the OR. In summary, we aim to make a significant impact in the newly emerging area of PIDDosome-mediated apoptotic signaling, thereby translating CS apoptosis into an effective HNSCC therapy.

Project Summary Interleukin-1 Receptor (IL-1R)-Associated Kinase 1, IRAK1, is a core transducer of Toll-like receptor (TLR) and IL-1R-mediated innate immune signaling from flies to humans. In response to pathogen infection, ligated TLR/IL-R receptors almost instantaneously activate IRAK1 via formation of the MyDDosome complex (IL-1R/TLR?MyD88-IRAK4-IRAK1) at the cell surface. Once phosphorylated by IRAK4 and itself, fully activated IRAK1 dissociates from the platform and engages NF-kB and other signaling cascades, culminating in the acute inflammatory response. Until recently, vertebrate IRAK1 had not been implicated in processes other than the microbial response. In an unbiased zebrafish screen, we recently identified IRAK1 as essential for cell survival in response to ionizing radiation (gIR) (Liu et al., Nat Cell Biol 2019; ref. 1). This function is conserved in human cells and drives cellular resistance to radiotherapy (R-RT) in tumor models. Rather than acting to stimulate NF-kB, IRAK1 drives cell survival by countering apoptosis mediated by the PIDDosome complex (PIDD-RAIDD- caspase-2). In further contrast with canonical IRAK1/4 immune signaling, our preliminary data indicate that the IRAK1 response to gIR: (i) fully requires its kinase activity; (ii) does not require the IL-1R/TLR?IRAK1/4 adaptor MyD88; and most strikingly, (iii) initiates in the nucleus of irradiated cells and not at the cell surface. gIR-induced IRAK1 activation does however occur within minutes of stimulus and absolutely requires IRAK4, suggesting the existence of a novel oligomeric platform responsible for orchestrating gIR-induced IRAK1 activation in place of the MyDDosome. While the evidence convincingly points to a novel IRAK1 stress response pathway in vertebrates, the cellular and molecular bases of gIR-induced IRAK1 activation remain to be defined. In Aim 1, we will monitor the localization of both active and native IRAK1 as a function of time after gIR; identify the cellular signal(s) that effectively trigger IRAK1 activation in irradiated cells, with DNA breaks, micronucleation, cytokines and danger- associated molecular patterns (DAMPs) as primary candidates; and explore whether environmental stresses (e.g., UV irradiation) can trigger the pathway. In Aim 2, we will dissect the molecular mechanism of IRAK1 activation in response to gIR, first by focusing on the roles of IRAK4 and IRAK1 itself; second by identifying the DD protein substituting for MyD88 as scaffold for the gIR-induced IRAK4/IRAK1 activation platform; and finally taking unbiased, larger scale proteomic approaches toward the unbiased identification of the upstream sensors, transducers and regulators that orchestrate IRAK1 activation in irradiated nuclei. Beyond illuminating a novel stress response pathway in vertebrates, our proposal explores a pathway implicated in tumor R-RT. Thus, an additional immediate impact of our work might be the discovery of novel drug targets for overcoming R-RT in the majority of cancer patients that receive RT as part of their treatment.

Project Summary Mutations in p53 and attendant apoptotic pathways impair tumor cell responses to radiation and chemotherapy in many human malignancies. Combining genetics, functional genomics and proteomics in mammalian cells and zebrafish embryos these past 6 years, we identified a novel apoptotic pathway that bypasses p53-dependent pathways altogether via activation of the PIDDosome (PIDD-RAIDD- caspase-2) complex (Sidi et al., Cell 2008; Ando et al., Mol Cell 2012; Thompson et al., Mol Cell 2015; Ando et al., J Cell Biol 2017). Unlike the mitochondrial apoptosome (cytc-Apaf1-caspase-9) and death receptor complex (FAS-FADD-caspase-8), the PIDDosome does not require p53 for activation or function. PIDDosome assembly can be activated by inhibiting its negative regulator, Chk1 kinase. As such, Chk1 inhibitors restore radiosensitivity in p53 mutant zebrafish embryos, MEF, and human cancer cell lines. The PIDDosome is also responsive to DNA damaging chemotherapies such as topoisomerase inhibitors. Altogether, the PIDDosome pathway defines both a novel apoptotic axis and a promising targeted strategy for overcoming treatment resistance in cancer. However, our molecular understanding of the PIDDosome remains very limited. To expand our knowledge of the pathway and identify novel diagnostic tools and drug targets therein, this proposal will focus on the mechanisms by which DNA damage triggers PIDDosome assembly in vertebrate cells. Thus far, we have shown that DNA damage triggers PIDDosome formation via (i) ATM/ATR-mediated phosphorylation of PIDD, which enables RAIDD recruitment to the platform (Mol Cell 2012); and (ii) the binding of PIDD to nucleophosmin (NPM1), which provides a scaffold for PIDDosome assembly (JCB 2017). In Aim 1, we will elucidate the mechanism by which a newly identified PIDD interactor, the DNA repair protein FANCI, recruits PIDD to DNA crosslinks and enables its phosphorylation by ATM at these lesions. Notably, these experiments may identify FANCI as the first biochemically described ?repair/death? switch in vertebrates. In Aim 2, we will elucidate the mechanism by which NPM1 and two newly identified nucleolar PIDD-binding proteins, NOLC1 and NCL, coordinately orchestrate PIDDosome formation in response to IR. These experiments may ultimately outline the major apoptotic branch in the nucleolar DNA damage response. Finally, using xenograft models of intrinsic tumor radioresistance (Liu et al., Nat Cell Biol, accepted in principle), we will assess for the first time the potential of PIDDosome targeting as a strategy to overcome radioresistance in TP53 mutant cancers. Altogether, these studies integrate the PIDDosome in the cellular responses to DNA repair failure, replication stress and nucleolar stress. Our proposal is thus ideally positioned to reveal the role of the PIDDosome in cancer etiology, one of the most hotly debated questions in the field of apoptosis.