A Novel Dual ATM/DNA-PK Inhibitor, XRD-0394, Potently Radiosensitizes and Potentiates PARP and Topoisomerase I Inhibitors.

A majority of patients with cancer receive radiotherapy as part of their treatment regimens whether using external beam therapy or locally-delivered radioisotopes. While often effective, some tumors are inadequately controlled with radiation and radiotherapy has significant short-term and long-term toxicities for cancer survivors. Insights into molecular mechanisms involved in cellular responses to DNA breaks introduced by radiation or other cancer therapies have been gained in recent years and approaches to manipulate these responses to enhance tumor cell killing or reduce normal tissue toxicity are of great interest. Here, we report the identification and initial characterization of XRD-0394, a potent and specific dual inhibitor of two DNA damage response kinases, ATM and DNA-PKcs. This orally bioavailable molecule demonstrates significantly enhanced tumor cell kill in the setting of therapeutic ionizing irradiation in vitro and in vivo. XRD-0394 also potentiates the effectiveness of topoisomerase I inhibitors in vitro. In addition, in cells lacking BRCA1/2 XRD-0394 shows single-agent activity and synergy in combination with PARP inhibitors. A phase Ia clinical trial (NCT05002140) with XRD-0394 in combination with radiotherapy has completed. These results provide a rationale for future clinical trials with XRD-0394 in combination with radiotherapy, PARP inhibitors, and targeted delivery of topoisomerase I inhibitors.

Participation of ATM, SMG1, and DDX5 in a DNA Damage-Induced Alternative Splicing Pathway.

Altered cellular responses to DNA damage can contribute to cancer development, progression, and therapeutic resistance. Mutations in key DNA damage response factors occur across many cancer types, and the DNA damage-responsive gene, TP53, is frequently mutated in a high percentage of cancers. We recently reported that an alternative splicing pathway induced by DNA damage regulates alternative splicing of TP53 RNA and further modulates cellular stress responses. Through damage-induced inhibition of the SMG1 kinase, TP53 pre-mRNA is alternatively spliced to generate TP53b mRNA and p53b protein is required for optimal induction of cellular senescence after ionizing radiation-induced DNA damage. Herein, we confirmed and extended these observations by demonstrating that the ATM protein kinase is required for repression of SMG1 kinase activity after ionizing radiation. We found that the RNA helicase and splicing factor, DDX5, interacts with SMG1, is required for alternative splicing of TP53 pre-mRNA to TP53b and TP53c mRNAs after DNA damage, and contributes to radiation-induced cellular senescence. Interestingly, the role of SMG1 in alternative splicing of p53 appears to be distinguishable from its role in regulating nonsense-mediated RNA decay. Thus, ATM, SMG1, and DDX5 participate in a DNA damage-induced alternative splicing pathway that regulates TP53 splicing and modulates radiation-induced cellular senescence.

Uncovering inherent cellular plasticity of multiciliated ependyma leading to ventricular wall transformation and hydrocephalus.

Specialized, differentiated cells often perform unique tasks that require them to maintain a stable phenotype. Multiciliated ependymal cells (ECs) are unique glial cells lining the brain ventricles, important for cerebral spinal fluid circulation. While functional ECs are needed to prevent hydrocephalus, they have also been reported to generate new neurons: whether ECs represent a stable cellular population remains unclear. Via a chemical screen we found that mature ECs are inherently plastic, with their multiciliated state needing constant maintenance by the Foxj1 transcription factor, which paradoxically is rapidly turned over by the ubiquitin-proteasome system leading to cellular de-differentiation. Mechanistic analyses revealed a novel NF-κB-independent IKK2 activity stabilizing Foxj1 in mature ECs, and we found that known IKK2 inhibitors including viruses and growth factors robustly induced Foxj1 degradation, EC de-differentiation, and hydrocephalus. Although mature ECs upon de-differentiation can divide and regenerate multiciliated ECs, we did not detect evidence supporting EC's neurogenic potential.

Convergence of Parkin, PINK1, and α-Synuclein on Stress-induced Mitochondrial Morphological Remodeling.

Mutations in PARKIN (PARK2), an ubiquitin ligase, cause early onset Parkinson disease. Parkin was shown to bind, ubiquitinate, and target depolarized mitochondria for destruction by autophagy. This process, mitophagy, is considered crucial for maintaining mitochondrial integrity and suppressing Parkinsonism. Here, we report that under moderate mitochondrial stress, parkin does not translocate to mitochondria to induce mitophagy; rather, it stimulates mitochondrial connectivity. Mitochondrial stress-induced fusion requires PINK1 (PARK6), mitofusins, and parkin ubiquitin ligase activity. Upon exposure to mitochondrial toxins, parkin binds α-synuclein (PARK1), and in conjunction with the ubiquitin-conjugating enzyme Ubc13, stimulates K63-linked ubiquitination. Importantly, α-synuclein inactivation phenocopies parkin overexpression and suppresses stress-induced mitochondria fission, whereas Ubc13 inactivation abrogates parkin-dependent mitochondrial fusion. The convergence of parkin, PINK1, and α-synuclein on mitochondrial dynamics uncovers a common function of these PARK genes in the mitochondrial stress response and provides a potential physiological basis for the prevalence of α-synuclein pathology in Parkinson disease.

Glycolysis-dependent histone deacetylase 4 degradation regulates inflammatory cytokine production.

Activation of the inflammatory response is accompanied by a metabolic shift to aerobic glycolysis. Here we identify histone deacetylase 4 (HDAC4) as a new component of the immunometabolic program. We show that HDAC4 is required for efficient inflammatory cytokine production activated by lipopolysaccharide (LPS). Surprisingly, prolonged LPS treatment leads to HDAC4 degradation. LPS-induced HDAC4 degradation requires active glycolysis controlled by GSK3ß and inducible nitric oxide synthase (iNOS). Inhibition of GSK3ß or iNOS suppresses nitric oxide (NO) production, glycolysis, and HDAC4 degradation. We present evidence that sustained glycolysis induced by LPS treatment activates caspase-3, which cleaves HDAC4 and triggers its degradation. Of importance, a caspase-3-resistant mutant HDAC4 escapes LPS-induced degradation and prolongs inflammatory cytokine production. Our findings identify the GSK3ß-iNOS-NO axis as a critical signaling cascade that couples inflammation to metabolic reprogramming and a glycolysis-driven negative feedback mechanism that limits inflammatory response by triggering HDAC4 degradation.

Microtubule acetylation amplifies p38 kinase signalling and anti-inflammatory IL-10 production.

Reversible acetylation of α-tubulin is an evolutionarily conserved modification in microtubule networks. Despite its prevalence, the physiological function and regulation of microtubule acetylation remain poorly understood. Here we report that macrophages challenged by bacterial lipopolysaccharides (LPS) undergo extensive microtubule acetylation. Suppression of LPS-induced microtubule acetylation by inactivating the tubulin acetyltransferase, MEC17, profoundly inhibits the induction of anti-inflammatory interleukin-10 (IL-10), a phenotype effectively reversed by an acetylation-mimicking α-tubulin mutant. Conversely, elevating microtubule acetylation by inhibiting the tubulin deacetylase, HDAC6, or stabilizing microtubules via Taxol stimulates IL-10 hyper-induction. Supporting the anti-inflammatory function of microtubule acetylation, HDAC6 inhibition significantly protects mice from LPS toxicity. In HDAC6-deficient macrophages challenged by LPS, p38 kinase signalling becomes selectively amplified, leading to SP1-dependent IL-10 transcription. Remarkably, the augmented p38 signalling is suppressed by MEC17 inactivation. Our findings identify reversible microtubule acetylation as a kinase signalling modulator and a key component in the inflammatory response.

A mechanism of COOH-terminal binding protein-mediated repression.

The E2F4 and E2F5 proteins specifically associate with the Rb-related p130 protein in quiescent cells to repress transcription of various genes encoding proteins important for cell growth. A series of reports has provided evidence that Rb-mediated repression involves both histone deacetylase (HDAC)-dependent and HDAC-independent events. Our previous results suggest that one such mechanism for Rb-mediated repression, independent of recruitment of HDAC, involves the recruitment of the COOH-terminal binding protein (CtBP) corepressor, a protein now recognized to play a widespread role in transcriptional repression. We now find that CtBP can interact with the histone acetyltransferase, cyclic AMP--responsive element--binding protein (CREB) binding protein, and inhibit its ability to acetylate histone. This inhibition is dependent on a NH2-terminal region of CtBP that is also required for transcription repression. These results thus suggest two complementary mechanisms for E2F/p130-mediated repression that have in common the control of histone acetylation at target promoters.

MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation.

The tumor suppressor p53 is stabilized and activated in response to cellular stress through post-translational modifications including acetylation. p300/CBP-mediated acetylation of p53 is negatively regulated by MDM2. Here we show that MDM2 can promote p53 deacetylation by recruiting a complex containing HDAC1. The HDAC1 complex binds MDM2 in a p53-independent manner and deacetylates p53 at all known acetylated lysines in vivo. Ectopic expression of a dominant-negative HDAC1 mutant restores p53 acetylation in the presence of MDM2, whereas wild-type HDAC1 and MDM2 deacetylate p53 synergistically. Fibroblasts overexpressing a dominant negative HDAC1 mutant display enhanced DNA damage-induced p53 acetylation, increased levels of p53 and a more pronounced induction of p21 and MDM2. These results indicate that acetylation promotes p53 stability and function. As the acetylated p53 lysine residues overlap with those that are ubiquitylated, our results suggest that one major function of p53 acetylation is to promote p53 stability by preventing MDM2-dependent ubiquitylation, while recruitment of HDAC1 by MDM2 promotes p53 degradation by removing these acetyl groups.