Most tumors initially respond to treatment, yet refractory clones subsequently develop owing to resistance mechanisms associated with cancer cell plasticity and heterogeneity. We used a chemical biology approach to identify protein targets in cancer cells exhibiting diverse driver mutations and representing models of tumor lineage plasticity and therapy resistance. An unbiased screen of a drug library was performed against cancer cells followed by synthesis of chemical analogs of the most effective drug. The cancer subtype target range of the leading drug was determined by PRISM analysis of over 900 cancer cell lines at the Broad Institute, MA. RNA-sequencing and enrichment analysis of differentially expressed genes, as well as computational molecular modeling and pull-down with biotinylated small molecules were used to identify and validate RPS6KB1 (p70S6K or S6K1) as an essential target. Genetic restoration was used to test the functional role of S6K1 in cell culture and xenograft models. We identified a novel derivative of the antihistamine drug ebastine, designated Super-ebastine (Super-EBS), that inhibited the viability of cancer cells representing diverse KRAS and EGFR driver mutations and models of plasticity and treatment resistance. Interestingly, PRISM analysis indicated that over 95% of the diverse cancer cell lines tested were sensitive to Super-EBS and the predicted target was the serine/threonine kinase S6K1. S6K1 is upregulated in various cancers relative to counterpart normal/benign tissues and phosphorylated-S6K1 predicts poor prognosis for cancer patients. We noted that inhibition of S6K1 phosphorylation was necessary for tumor cell growth inhibition, and restoration of phospho-S6K1 rendered tumor cells resistant to Super-EBS. Inhibition of S6K1 phosphorylation by Super-EBS induced caspase-2 dependent apoptosis inhibition of the Cdc42/Rac-1/p-PAK1 pathway that led to actin depolymerization and caspase-2 activation. The essential role of S6K1 in the action of Super-EBS was recapitulated in xenografts, and knockout of S6K1 abrogated tumor growth in mice. S6K1 is a therapeutic vulnerability in tumors exhibiting intrinsic and/or acquired resistance to treatment.

Caspase-2 is a unique and conserved cysteine protease that is involved in several cellular processes, including different forms of cell death, maintenance of genomic stability, and the response to reactive oxygen species. Despite advances in caspase-2 research in recent years, the mechanisms underlying its activation remain largely unclear. Although caspase-2 is activated in the PIDDosome complex, its processing could occur even in the absence of PIDD1 and/or RAIDD, suggesting the existence of an alternative platform for caspase-2 activation. Here, we show that caspase-2 undergoes ubiquitination and interacts with scaffolding protein p62/sequestosome-1 (SQSTM1) under normal conditions and in response to DNA damage. p62 promotes proteasomal but not autophagic caspase-2 degradation as well as its dimerization and activation that triggers the caspase cascade and, subsequently, cell death. Inhibition of p62 expression attenuates cisplatin-induced caspase-2 processing and apoptosis. Notably, the ZZ domain of p62 is critical for caspase-2 binding, whereas the UBA domain is seemingly required to stabilize the p62-caspase-2 complex. Thus, we have uncovered the dual role of p62 in regulating caspase-2 activity: it can foster the degradation of caspase-2 in the proteasome or facilitate its activation by acting as a scaffold platform.

Caspase-2, a highly conserved member of the caspase family, is considered an initiator caspase that triggers apoptosis in response to some cellular stresses. Previous studies suggest that an intracellular multi-protein complex PIDDosome, induced by genotoxic stress, serves as a platform for caspase-2 activation. Due to caspase-2's inability to process effector caspases, however, the mechanism underlying caspase-2-mediated cell death upon PIDDosome activation remains unclear. Here, we conducted an unbiased genome-wide genetic screen and identified that the Bcl2 family protein BID is required for PIDDosome-induced, caspase-2-mediated apoptosis. PIDDosome-activated caspase-2 directly and functionally processes BID to signal the mitochondrial pathway for apoptosis induction. In addition, a designed chemical screen identified a compound, HUHS015, which specifically activates caspase-2-mediated apoptosis. HUHS015-stimulated apoptosis also requires BID but is independent of the PIDDosome. Through extensive structure-activity relationship efforts, we identified a derivative with a potency of ~60 nmol/L in activating caspase-2-mediated apoptosis. The HUHS015-series of compounds act as efficient agonists that directly target the interdomain linker in caspase-2, representing a new mode of initiator caspase activation. Human and mouse caspase-2 differ in two crucial residues in the linker, rendering a selectivity of the agonists for human caspase-2. The caspase-2 agonists are valuable tools to explore the physiological roles of caspase-2-mediated cell death and a base for developing small-molecule drugs for relevant diseases.

PIDDosome formation followed by caspase-2 activation is critical for genotoxic stress-induced apoptotic cell death. Failure of proper caspase-2 activation causes a neurodevelopmental disorder and intellectual disability. R815W, R862W, and Q863stop mutations in p53-induced protein with a death domain (PIDD), a component of the PIDDosome, also lead to this disorder. However, the molecular mechanisms underlying this pathogenesis remain elusive. In this study, we analyzed the molecular mechanisms underlying the pathogenesis of the PIDD DD pathogenic variants R815W, R862W, and Q863stop. We determined that these mutations prevented the interaction between PIDD and RIP-associated Ich-1/Ced-3 homologous protein with a death domain (RAIDD), a molecule that mediates PIDDosome formation. The disruption of this interaction affects PIDDosome formation and caspase-2 activation.

We investigated the ability of graphene quantum dot (GQD) nanoparticles to protect SH-SY5Y human neuroblastoma cells from oxidative/nitrosative stress induced by iron-nitrosyl complex sodium nitroprusside (SNP). GQD reduced SNP cytotoxicity by preventing mitochondrial depolarization, caspase-2 activation, and subsequent apoptotic death. Although GQD diminished the levels of nitric oxide (NO) in SNP-exposed cells, NO scavengers displayed only a slight protective effect, suggesting that NO quenching was not the main protective mechanism of GQD. GQD also reduced SNP-triggered increase in the intracellular levels of hydroxyl radical (OH), superoxide anion (O), and lipid peroxidation. Nonselective antioxidants, OH scavenging, and iron chelators, but not superoxide dismutase, mimicked GQD cytoprotective activity, indicating that GQD protect cells by neutralizing OH generated in the presence of SNP-released iron. Cellular internalization of GQD was required for optimal protection, since a removal of extracellular GQD by extensive washing only partly diminished their protective effect. Moreover, GQD cooperated with SNP to induce autophagy, as confirmed by the inhibition of autophagy-limiting Akt/PRAS40/mTOR signaling and increase in autophagy gene transcription, protein levels of proautophagic beclin-1 and LC3-II, formation of autophagic vesicles, and degradation of autophagic target p62. The antioxidant activity of GQD was not involved in autophagy induction, as antioxidants N-acetylcysteine and dimethyl sulfoxide failed to stimulate autophagy in SNP-exposed cells. Pharmacological inhibitors of early (wortmannin, 3-methyladenine) or late stages of autophagy (NHCl) efficiently reduced the protective effect of GQD. Therefore, the ability of GQD to prevent the in vitro neurotoxicity of SNP depends on both OH/NO scavenging and induction of cytoprotective autophagy.

Hypomyelinating leukodystrophy 17 is an autosomal recessive disease affecting myelin-forming oligodendroglial cells in the central nervous system. The gene responsible for HLD17 encodes aminoacyl-tRNA synthase complex-interacting multifunctional protein 2, whose product proteins form a scaffold that supports aminoacyl-tRNA synthetases throughout the cell body. Here we show that the HLD17-associated nonsense mutation (Tyr35-to-Ter [Y35X]) of AIMP2 localizes AIMP2 proteins as aggregates into the Golgi bodies in mouse oligodendroglial FBD-102b cells. Wild type AIMP2 proteins, in contrast, are distributed throughout the cell body. Expression of the Y35X mutant proteins, but not the wild type proteins, in cells upregulates Golgi stress signaling involving caspase-2 activation. Cells expressing the wild type proteins exhibit differentiated phenotypes with web-like structures bearing many processes following the induction of differentiation, whereas cells expressing the Y35X mutant proteins fail to differentiate. Furthermore, CASP2 knockdown but not control knockdown reverses the phenotypes of cells expressing the mutant proteins. These results suggest that HLD17-associated AIMP2 mutant proteins are localized in the Golgi bodies where their proteins stimulate Golgi stress-responsive CASP2 to inhibit differentiation; this effect is ameliorated by knockdown of CASP2. These findings may reveal some of the molecular and cellular pathological mechanisms underlying HLD17 and possible approaches to ameliorating the disease's effects.

Activation of the endoplasmic reticulum stress sensor, IRE1α, is required for effective immune responses against bacterial infection and is associated with human inflammatory diseases in which neutrophils are a key immune component. However, the specific role of IRE1α in regulating neutrophil effector function has not been studied. In this study, we show that infection-induced IRE1α activation licenses neutrophil antimicrobial capacity, including IL-1β production, formation of neutrophil extracellular traps (NETs), and methicillin-resistant (MRSA) killing. Inhibition of IRE1α diminished production of mitochondrial reactive oxygen species and decreased CASPASE-2 activation, which both contributed to neutrophil antimicrobial activity. Mice deficient in CASPASE-2 or neutrophil IRE1α were highly susceptible to MRSA infection and failed to effectively form NETs in the s.c. abscess. IRE1α activation enhanced calcium influx and citrullination of histone H3 independently of mitochondrial reactive oxygen species production, suggesting that IRE1α coordinates multiple pathways required for NET formation. Our data demonstrate that the IRE1α-CASPASE-2 axis is a major driver of neutrophil activity against MRSA infection and highlight the importance of IRE1α in neutrophil antibacterial function.

PIDD1 encodes p53-Induced Death Domain protein 1, which acts as a sensor surveilling centrosome numbers and p53 activity in mammalian cells. Early results also suggest a role in DNA damage response where PIDD1 may act as a cell-fate switch, through interaction with RIP1 and NEMO/IKKg, activating NF-κB signaling for survival, or as an apoptosis-inducing protein by activating caspase-2. Biallelic truncating mutations in CRADD-the protein bridging PIDD1 and caspase-2-have been reported in intellectual disability (ID), and in a form of lissencephaly. Here, we identified five families with ID from Iran, Pakistan, and India, with four different biallelic mutations in PIDD1, all disrupting the Death Domain (DD), through which PIDD1 interacts with CRADD or RIP1. Nonsense mutations Gln863* and Arg637* directly disrupt the DD, as does a missense mutation, Arg815Trp. A homozygous splice mutation in the fifth family is predicted to disrupt splicing upstream of the DD, as confirmed using an exon trap. In HEK293 cells, we show that both Gln863* and Arg815Trp mutants fail to co-localize with CRADD, leading to its aggregation and mis-localization, and fail to co-precipitate CRADD. Using genome-edited cell lines, we show that these three PIDD1 mutations all cause loss of PIDDosome function. Pidd1 null mice show decreased anxiety, but no motor abnormalities. Together this indicates that PIDD1 mutations in humans may cause ID (and possibly lissencephaly) either through gain of function or secondarily, due to altered scaffolding properties, while complete loss of PIDD1, as modeled in mice, may be well tolerated or is compensated for.

Centrosome amplification results into genetic instability and predisposes cells to neoplastic transformation. Supernumerary centrosomes trigger p53 stabilization dependent on the PIDDosome (a multiprotein complex composed by PIDD1, RAIDD and Caspase-2), whose activation results in cleavage of p53's key inhibitor, MDM2. Here, we demonstrate that PIDD1 is recruited to mature centrosomes by the centriolar distal appendage protein ANKRD26. PIDDosome-dependent Caspase-2 activation requires not only PIDD1 centrosomal localization, but also its autoproteolysis. Following cytokinesis failure, supernumerary centrosomes form clusters, which appear to be necessary for PIDDosome activation. In addition, in the context of DNA damage, activation of the complex results from a p53-dependent elevation of PIDD1 levels independently of centrosome amplification. We propose that PIDDosome activation can in both cases be promoted by an ANKRD26-dependent local increase in PIDD1 concentration close to the centrosome. Collectively, these findings provide a paradigm for how centrosomes can contribute to cell fate determination by igniting a signalling cascade.

Caspase-2 is a unique and conservative cysteine protease which plays an important role in several cellular processes including apoptotic cell death. Although the molecular mechanisms of its activation remain largely unclear, a major role belongs to the architecture of the caspase-2 active center. We demonstrate that the substitution of the putative phosphorylation site of caspase-2, Serine-384 to Alanine, blocks caspase-2 processing and decreases its enzymatic activity. Strikingly, in silico analysis using molecular dynamics simulations has shown that Serine-384 is crucially involved in interactions within the caspase-2 active center. It stabilizes Arginine-378, which forms a crucial hydrogen bond with the aspartate residue of a substrate. Hence, Serine-384 is essential for supporting a proper architecture of the active center of caspase-2. Moreover, molecular modeling strongly proved steric inaccessibility of Ser-384 to be phosphorylated. Importantly, a multiple alignment has demonstrated that both Serine-384 and Arg-378 residues are highly conservative across all members of caspase family, which allows us to suggest that this diade is indispensable for caspase processing and activity. Spontaneous mutations in this diade might influence oncosuppressive function of caspases, in particular of caspase-2. Likewise, the mutation of Ser-384 is associated with the development of lung squamous cell carcinoma and adenocarcinoma. Taken together, we have uncovered a central feature of the caspase-2 activation mechanism which is crucial for the regulation of its signaling network.