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Introduction Acute myeloid leukemia (AML) with mutated TP53 (TP53MT) is resistant to chemotherapy and has a poor prognosis. Prior reports suggest that DNA methyltransferase inhibitors (DNMTi), such as decitabine and azacitidine, form covalent DNA-DNMT1 adducts and invoke a DNA damage response (DDR) characterized by activation of the ATR-CHK1 pathway, including in TP53MT AML samples. This study evaluates whether decitabine exhibits cytotoxic synergy with replication checkpoint modulators (ATRi, CHK1/2i, WEE1i) in pre-clinical models of AML. Methods Parental cell lines (U937, KG1a, Molm13, MV-4-11) were obtained commercially and genomically validated. The Molm13 TP53-/- cell line was generated via CRISPR/Cas9 knockout. Cells were maintained in RPMI-1640 medium with 10% FBS, 2 mM glutamine, and antibiotics at 37 ºC, 5% CO2. Decitabine, ceralasertib (ATRi), prexasertib (CHK1/2i), MK-8776 (CHK1i), and adavosertib (WEE1i) were reconstituted at 10 mM in DMSO. For drug treatments, cells were cultured for 48 h in medium with 0.2% (v/v) DMSO or drug. Western blots were stained with commercial antibodies followed by peroxidase-coupled secondary antibodies and imaged by chemiluminescence. Apoptosis assays were performed via flow cytometry (BD FACSCanto II) with annexin V-APC (BD Biosciences) and/or propidium iodide (Sigma) staining. Peripheral blood and bone marrow samples were obtained from AML patients after informed consent at Mayo Clinic and Johns Hopkins University. Mononuclear cells were isolated via Histopaque-1077 (Sigma) centrifugation and used immediately. Colony forming assays were performed in Methocult H4435 (Stem Cell Technologies) supplemented with vehicle or drug; leukemic colonies were counted after 12-16 days. Combination indices (CI) were determined by the Chou-Talalay method (CalcuSyn) where values <1 indicate synergy and >1 antagonism. This work was supported in part by R01 CA289285. Results Western blots of TP53MT AML cell lines (U937, KG1a) and primary samples treated with decitabine (≤200 nM) for 24 h demonstrated increased phosphorylation of H2AX and S296/S345 CHK1, consistent with activation of the ATR-CHK1 DDR pathway. These changes were less pronounced in TP53 wild type cell lines (Molm13, MV-4-11). In subdiploid (SubG1) assays, U937 cells exhibited resistance to monotherapy with decitabine (EC50 = 330 ± 200 nM) and variable sensitivity to ceralasertib (250 - 500 nM), prexasertib (1 - 10 nM), MK-8776 (250 - 1,000 nM), and adavosertib (50 - 400 nM). When combined, however, marked synergy was observed between decitabine and both ceralasertib (mean CI 0.15) and adavosertib (CI 0.59). Synergy was also observed with the selective CHK1i, MK-8776 (CI 0.56), but less-so with the dual CHK1/2i, prexasertib (CI 0.93), consistent with the hypothesis of an ATR-CHK1 DNA damage response. Similar results were obtained from decitabine (EC50 = 300 ± 100 nM) and ceralasertib (CI 0.54), adavosertib (CI 0.67), MK-8776 (CI 0.52), and prexasertib (CI 0.81) in annexin V assays. These combinations were further evaluated (SubG1 assays) in an isogenic pair of Molm13 and Molm13 TP53-/- cell lines. In Molm13 cells, decitabine (EC50 = 23 ± 11 nM) was again synergistic with adavosertib (CI 0.60), but not ceralasertib (CI 1.3) or prexasertib (CI 1.2). In Molm13 TP53-/- cells, however, very little synergy was observed between decitabine (EC50 = 53 ± 22 nM) and ceralasertib (CI 1.5), prexasertib (CI 4.3), or adavosertib (CI 1.0) except for at higher concentrations of adavosertib (400 nM, CI 0.89). Thus, while biallelic TP53 inactivation conferred resistance to DNMTi and checkpoint inhibitors, the degree of resistance was context-dependent and could be partially overcome by WEE1 inhibition. The combinations of decitabine with ceralasertib (n = 2), prexasertib (n = 4), or adavosertib (n = 3) were evaluated in colony forming assays using primary AML samples from newly-diagnosed patients with diverse clinicomolecular profiles. Combination treatment was antagonistic with ceralasertib (CI 2.2), additive with prexasertib (CI 1.0), and synergistic with adavosertib (CI 0.88). Conclusions In multiple cell lines and diverse primary AML samples, decitabine activated the ATR-CHK1 DDR pathway and synergized with ATRi, CHK1/2i, and WEE1i. Amongst these, only the combination of decitabine plus adavosertib exhibited synergy across all three models, highlighting WEE1 as a potential target in TP53MT AML.

Abstract Senescent cells drive tissue dysfunction through the senescence-associated secretory phenotype (SASP). We uncovered a central role for mitochondria in the epigenetic regulation of the SASP, where mitochondrial-derived metabolites, specifically citrate and acetyl-CoA, fuel histone acetylation at SASP gene loci, promoting their expression. We identified the mitochondrial citrate carrier (SLC25A1) and ATP-citrate lyase (ACLY) as critical for this process. Inhibiting these pathways selectively suppresses SASP without affecting cell cycle arrest, highlighting their potential as therapeutic targets for age-related inflammation. Notably, SLC25A1 inhibition reduces systemic inflammation and extends healthspan in aged mice, establishing mitochondrial metabolism as pivotal to the epigenetic control of aging.

Abstract INTRODUCTION Alzheimer's disease (AD) is the most prevalent neurodegenerative disease, yet our comprehension predominantly relies on studies within non‐Hispanic White (NHW) populations. Here we provide an extensive survey of the proteomic landscape of AD across diverse racial/ethnic groups. METHODS Two cortical regions, from multiple centers, were harmonized by uniform neuropathological diagnosis. Among 998 unique donors, 273 donors self‐identified as African American, 229 as Latino American, and 434 as NHW. RESULTS While amyloid precursor protein and the microtubule‐associated protein tau demonstrated higher abundance in AD brains, no significant race‐related differences were observed. Further proteome‐wide and focused analyses (specific amyloid beta [Aβ] species and the tau domains) supported the absence of racial differences in these AD pathologies within the brain proteome. DISCUSSION Our findings indicate that the racial differences in AD risk and clinical presentation are not underpinned by dramatically divergent patterns in the brain proteome, suggesting that other determinants account for these clinical disparities. Highlights We present a large‐scale proteome (∼10,000 proteins) of DLPFC (998) and STG (244) across AD cases. About 50% of samples were from racially and ethnically diverse brain donors. Key AD proteins (amyloid and tau) correlated with CERAD and Braak stages. No significant race‐related differences in amyloid and tau protein levels were observed in AD brains. AD‐associated protein changes showed a strong correlation between the brain proteomes of African American and White individuals. This dataset advances understanding of ethnoracial‐specific AD pathways and potential therapies.

Abstract Glioblastoma (GBM) remains one of the most therapy-resistant malignancies with frequent local failures despite aggressive surgery, chemotherapy, and ionizing radiation (IR). Small molecule inhibitors of DNA-dependent protein kinase (DNA-PKi) are potent radiosensitizers currently in clinical trials. Determining which patients may benefit from radiosensitization with DNA-PKi is critical to avoid unnecessary increased risk of normal tissue toxicity. In this study, we used GBM patient-derived xenografts (PDX) in orthotopic murine models to study the relationship between molecular features, pharmacokinetics, and the radiosensitizing potential of the DNA-PKi peposertib. We show that peposertib radiosensitizes established and PDX GBM lines in vitro at 300 nmol/L and above, with a significant increase in radiosensitization by maintaining post-IR exposure for >12 hours. Radiosensitization by peposertib is mediated by catalytic inhibition of DNA-PK, and knockdown of DNA-PK by short hairpin RNA (shRNA) largely abolished the radiosensitizing effect. Peposertib decreased auto-phosphorylation of DNA-PKcs after IR in a dose-dependent manner with a delay in resolution of γH2AX foci at 24 hours. The addition of peposertib to IR significantly increased survival in GBM120 orthotopic xenografts, but not in GBM10. There was no difference in plasma or average tumor concentrations of peposertib in the two cohorts. Although the mechanism underpinning this discordant effect in vitro versus in vivo is not clear, there was an association for greater sensitization in TP53 mutant lines. Transfection of a dominant-negative TP53 mutant in baseline TP53 wild-type GBM lines significantly delayed growth and decreased nonhomologous end joining efficiency (but not homologous recombination), after peposertib exposure. See related commentary by Buchsbaum, p. 840

Abstract BACKGROUND Metabolic dysregulation is a hallmark of neurodegenerative diseases, including Alzheimer's disease (AD) and progressive supranuclear palsy (PSP). Although metabolic dysregulation is a common link between these two tauopathies, a comprehensive brain metabolic comparison of the diseases has not yet been performed. METHODS We analyzed 342 postmortem brain samples from the Mayo Clinic Brain Bank and examined 658 metabolites in the cerebellar cortex and the temporal cortex between the two tauopathies. RESULTS Our findings indicate that both diseases display oxidative stress associated with lipid metabolism, mitochondrial dysfunction linked to lysine metabolism, and an indication of tau‐induced polyamine stress response. However, specific to AD, we detected glutathione‐related neuroinflammation, deregulations of enzymes tied to purines, and cognitive deficits associated with vitamin B. DISCUSSION Our findings underscore vast alterations in the brain's metabolome, illuminating shared neurodegenerative pathways and disease‐specific traits in AD and PSP. Highlights First high‐throughput metabolic comparison of Alzheimer's diesease (AD) versus progressive supranuclear palsy (PSP) in brain tissue. Cerebellar cortex (CER) shows substantial AD‐related metabolic changes, despite limited proteinopathy. AD impacts both CER and temporal cortex (TCX); PSP's changes are primarily in CER. AD and PSP share metabolic alterations despite major pathological differences.

Neuropathic pain is a debilitating chronic condition that lacks effective treatment. The role of cytokine- and chemokine-mediated neuroinflammation in its pathogenesis has been well documented. Follistatin (FST) is a secreted protein known to antagonize the biological activity of cytokines in the transforming growth factor–β (TGF-β) superfamily. The involvement of FST in neuropathic pain and the underlying mechanism remain largely unknown. Here, we report that FST was up-regulated in A-fiber sensory neurons after spinal nerve ligation (SNL) in mice. Inhibition or deletion of FST alleviated neuropathic pain and reduced the nociceptive neuron hyperexcitability induced by SNL. Conversely, intrathecal or intraplantar injection of recombinant FST, or overexpression of FST in the dorsal root ganglion (DRG) neurons, induced pain hypersensitivity. Furthermore, exogenous FST increased neuronal excitability in nociceptive neurons. The biolayer interferometry (BLI) assay and coimmunoprecipitation (co-IP) demonstrated direct binding of FST to the insulin-like growth factor–1 receptor (IGF1R), and IGF1R inhibition reduced FST-induced activation of extracellular signal–regulated kinase (ERK) and protein kinase B (AKT), as well as neuronal hyperexcitability. Further co-IP analysis revealed that the N-terminal domain of FST exhibits the highest affinity for IGF1R, and blocking this interaction with a peptide derived from FST attenuated Nav1.7-mediated neuronal hyperexcitability and neuropathic pain after SNL. In addition, FST enhanced neuronal excitability in human DRG neurons through IGF1R. Collectively, our findings suggest that FST, released from A-fiber neurons, enhances Nav1.7-mediated hyperexcitability of nociceptive neurons by binding to IGF1R, making it a potential target for neuropathic pain treatment.