Samuel Sidi

Adenosine diphosphate (ADP) ribosylation (ADPr) regulates multiple stress responses, yet substrates in the apoptotic machinery remain elusive. We show that a single, DNA damage–induced ADPr event controls proapoptotic PIDDosome (PIDD1/RAIDD/caspase-2) formation in response to unresolved interstrand DNA cross-links (ICL). ADPr targets conserved E783 in the PIDD1 death domain (DD); is catalyzed by poly(ADP-ribose) polymerase 4 (PARP4), a phylogenetically orphan PARP of previously unknown function; is reversed by the ribosylhydrolase activity of PARP14; and is triggered by Ataxia Telangiectasia and RAD3-related (ATR) phosphorylation–induced, PIAS1-mediated SUMOylation of the PIDD1 DD, which enables PARP4 docking. PIDD1 ADPr is dispensable for the recruitments of RAIDD and caspase-2 but essential for the dimerization of the caspase. Hence, denying E783 ADPr spares the onset of PIDDosome assembly but blocks its completion, thus eliminating caspase-2 activation and ensuing apoptosis. Conversely, removal of PARP14 forces apoptosis, even in cells with tolerable damage. The data identify PARP4 as an ICL response effector and illuminate a three-step modification sequence of the PIDD1 DD that conducts PIDDosome assembly from initiation to completion.

SUMOylation regulates numerous cellular stress responses, yet targets in the apoptotic machinery remain elusive. We show that a single, DNA damage-induced monoSUMOylation event controls PIDDosome (PIDD1/RAIDD/caspase-2) formation and apoptotic death in response to unresolved DNA interstrand crosslinks (ICLs). SUMO-1 conjugation occurs on conserved K879 in the PIDD1 death domain (DD); is catalyzed by PIAS1 and countered by SENP3; and is triggered by ATR phosphorylation of neighboring T788 in the PIDD1 DD, which enables PIAS1 docking. Phospho/SUMO-PIDD1 proteins are captured by nucleolar RAIDD monomers via a SUMO-interacting motif (SIM) in the RAIDD DD, thus compartmentalizing nascent PIDDosomes for caspase-2 recruitment. Denying SUMOylation or the SUMO-SIM interaction spares the onset of PIDDosome assembly but blocks its completion, thus eliminating the apoptotic response to ICL repair failure. Conversely, removal of SENP3 forces apoptosis, even in cells with tolerable ICL levels. SUMO-mediated PIDDosome control is also seen in response to DNA breaks but not supernumerary centrosomes. These results illuminate PIDDosome formation in space and time and identify a direct role for SUMOylation in the assembly of a major pro-apoptotic device.

Interleukin-1 receptor (IL-1R)–associated kinases (IRAKs) are core effectors of Toll-like receptors (TLRs) and IL-1R in innate immunity. Here, we found that IRAK4 and IRAK1 together inhibited DNA damage–induced cell death independently of TLR or IL-1R signaling. In human cancer cells, IRAK4 was activated downstream of ATR kinase in response to double-strand breaks (DSBs) induced by ionizing radiation (IR). Activated IRAK4 then formed a complex with and activated IRAK1. The formation of this complex required the E3 ubiquitin ligase Pellino1, acting structurally but not catalytically, and the activation of IRAK1 occurred independently of extracellular signaling, intracellular TLRs, and the TLR/IL-1R signaling adaptor MyD88. Activated IRAK1 translocated to the nucleus in a Pellino2-dependent manner. In the nucleus, IRAK1 bound to the PIDD1 subunit of the proapoptotic PIDDosome and interfered with platform assembly, thus supporting cell survival. This noncanonical IRAK signaling pathway was also activated in response to other DSB-inducing agents. The loss of IRAK4, of IRAK4 kinase activity, of either Pellino protein, or of the nuclear localization sequence in IRAK1 sensitized p53-mutant zebrafish to radiation. Thus, the findings may lead to strategies for overcoming tumor resistance to conventional cancer treatments.

Interleukin-1 receptor (IL-1R)-associated kinases (IRAKs) are core effectors of Toll-like receptor (TLR) and IL-1R signaling, with no reported roles outside of innate immunity. We find that vertebrate cells exposed to ionizing radiation (IR) sequentially activate IRAK4 and IRAK1 through a phosphorylation cascade mirroring that induced by TLR/IL-1R, resulting in a potent anti-apoptotic response. However, IR-induced IRAK1 activation does not require the receptors or the IRAK4/1 adaptor protein MyD88, and instead of remaining in the cytoplasm, the activated kinase is immediately transported to the nucleus via a conserved nuclear localization signal. We identify: double-strand DNA breaks (DSBs) as the biologic trigger for this pathway; the E3 ubiquitin ligase Pellino1 as the scaffold enabling IRAK4/1 activation in place of TLR/IL-1R-MyD88; and the pro-apoptotic PIDDosome (PIDD1-RAIDD-caspase-2) as a critical downstream target in the nucleus. The data delineate a non-canonical IRAK signaling pathway derived from, or ancestral to, TLR signaling. This DSB detection pathway, which is also activated by genotoxic chemotherapies, provides multiple actionable targets for overcoming tumor resistance to mainstay cancer treatments.

The peptidyl-prolyl cis-trans isomerase, Pin1, acts as a unified signaling hub that is exploited in cancer to activate oncogenes and inactivate tumor suppressors, in particular through up-regulation of c-Myc target genes. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to discover covalent inhibitors targeting Pin1’s active site nucleophile - Cys113, leading to the development of Sulfopin, a double-digit nanomolar Pin1 inhibitor. Sulfopin is highly selective for Pin1, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement, and phenocopies genetic knockout of Pin1. Although Pin1 inhibition had a modest effect on viability in cancer cell cultures, Sulfopin induced downregulation of c-Myc target genes and reduced tumor initiation and tumor progression in murine and zebrafish models of MYCN-driven neuroblastoma. Our results suggest that Sulfopin is a suitable chemical probe for assessing Pin1-dependent pharmacology in cells and in vivo . Moreover, these studies indicate that Pin1 should be further investigated as a potential cancer target.

Antitumor immunity has emerged as a favorable byproduct of radiation therapy (RT), whereby tumor-associated antigens released from irradiated cells unleash innate and adaptive attacks on tumors located both within and outside the radiation field. RT-induced immune responses further provide actionable targets for overcoming tumor resistance to RT (R-RT); immunotherapy (IT) with checkpoint inhibitors or Toll-like receptor (TLR) agonists can markedly improve, if not synergize with, RT in preclinical models, and several of these drugs are currently investigated as radiosensitizers in patients. In an unbiased chemical-genetic screen in a zebrafish model of tumor R-RT, we unexpectedly found that Interleukin 1 Receptor-Associated Kinase 1 (IRAK1), a core effector of TLR-mediated innate immunity, also functions in live fish and human cancer models to counter RT-induced cell death mediated by the PIDDosome complex (PIDD-RAIDD-caspase-2). IRAK1 acting both as a driver of intrinsic tumor R-RT and as an effector of RT-induced antitumor immunity would, at first glance, pose obvious therapeutic conundrums. IRAK1 inhibitors would be expected to sensitize the irradiated tumor to RT but simultaneously thwart RT-induced antitumor immunity as initiated by stromal dendritic cells. Conversely, TLR agonist-based immunotherapy would be expected to intensify RT-induced antitumor immunity but at the expense of fueling IRAK1-mediated cell survival in the irradiated tumor. We discuss how IRAK1's differential reliance on catalytic activity in the radiation vs. TLR responses might help overcome these hurdles, as well as the crucial importance of developing IRAK1 inhibitors that lack activity against IRAK4, the kinase activity of which is essential for IRAK1 activation in both pathways.

Mitosis is controlled by a complex series of signaling pathways but mitotic control following DNA damage remains poorly understood. Effective DNA damage sensing and repair is integral to survival but is largely thought to occur primarily in interphase and be repressed during mitosis due to the risk of telomere fusion. There is, however, increasing evidence to suggest tight control of mitotic progression in the incidence of DNA damage, whether induced in mitotic cells or having progressed from failed interphase checkpoints. Here we will discuss what is known to date about signaling pathways controlling mitotic progression and resulting cell fate in the incidence of mitotic DNA damage.

Drug-based strategies to overcome tumour resistance to radiotherapy (R-RT) remain limited by the single-agent toxicity of traditional radiosensitizers (for example, platinums) and a lack of targeted alternatives. In a screen for compounds that restore radiosensitivity in p53 mutant zebrafish while tolerated in non-irradiated wild-type animals, we identified the benzimidazole anthelmintic oxfendazole. Surprisingly, oxfendazole acts via the inhibition of IRAK1, a kinase thus far implicated in interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) immune responses. IRAK1 drives R-RT in a pathway involving IRAK4 and TRAF6 but not the IL-1R/TLR–IRAK adaptor MyD88. Rather than stimulating nuclear factor-κB, radiation-activated IRAK1 prevented apoptosis mediated by the PIDDosome complex (comprising PIDD, RAIDD and caspase-2). Countering this pathway with IRAK1 inhibitors suppressed R-RT in tumour models derived from cancers in which TP53 mutations predict R-RT. Moreover, IRAK1 inhibitors synergized with inhibitors of PIN1 a prolyl isomerase essential for IRAK1 activation, in response to pathogens and as shown here, ionizing radiation. These data identify an IRAK1 radiation-response pathway as a rational chemoradiation therapy target.

Despite being frequently mutated or deregulated in acute myeloid leukemia (AML) and many other cancers, the mechanisms by which nucleophosmin (NPM1) regulates oncogenesis remain elusive. We found that NPM1 plays a direct and conserved role in DNA damage-induced assembly of the PIDDosome complex, the activating platform for caspase-2. This function is carried in the nucleolus and is essential for caspase-2-mediated apoptosis in response to a variety of DNA injuries.