Preclinical Evaluation of NTX-301, a Novel DNA Hypomethylating Agent in Ovarian Cancer.

PURPOSE: DNA methylation causes silencing of tumor-suppressor and differentiation-associated genes, being linked to chemoresistance. Previous studies demonstrated that hypomethylating agents (HMA) resensitize ovarian cancer to chemotherapy. NTX-301 is a highly potent and orally bioavailable HMA, in early clinical development. EXPERIMENTAL DESIGN: The antitumor effects of NTX-301 were studied in ovarian cancer models by using cell viability, stemness and ferroptosis assays, RNA sequencing, lipidomic analyses, and stimulated Raman spectroscopy. RESULTS: Ovarian cancer cells (SKOV3, IC50 = 5.08 nmol/L; OVCAR5 IC50 = 3.66 nmol/L) were highly sensitive to NTX-301 compared with fallopian tube epithelial cells. NTX-301 downregulated expression of DNA methyltransferases 1-3 and induced transcriptomic reprogramming with 15,000 differentially expressed genes (DEG, P < 0.05). Among them, Gene Ontology enrichment analysis identified regulation of fatty acid biosynthesis and molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase, known features of cancer stem cells. Low-dose NTX-301 reduced the ALDH(+) cell population and expression of stemness-associated transcription factors. Stearoyl-coenzyme A desaturase 1 (SCD), which regulates production of unsaturated fatty acids (UFA), was among the top DEG downregulated by NTX-301. NTX-301 treatment decreased levels of UFA and increased oxidized lipids, and this was blunted by deferoxamine, indicating cell death via ferroptosis. NTX-301-induced ferroptosis was rescued by oleic acid. In vivo, monotherapy with NTX-301 significantly inhibited ovarian cancer and patient-derived xenograft growth (P < 0.05). Decreased SCD levels and increased oxidized lipids were detected in NTX-301-treated xenografts. CONCLUSIONS: NTX-301 is active in ovarian cancer models. Our findings point to a new mechanism by which epigenetic blockade disrupts lipid homeostasis and promotes cancer cell death.

R1441C and G2019S LRRK2 knockin mice have distinct striatal molecular, physiological, and behavioral alterations.

LRRK2 mutations are closely associated with Parkinson's disease (PD). Convergent evidence suggests that LRRK2 regulates striatal function. Here, by using knock-in mouse lines expressing the two most common LRRK2 pathogenic mutations-G2019S and R1441C-we investigated how LRRK2 mutations altered striatal physiology. While we found that both R1441C and G2019S mice displayed reduced nigrostriatal dopamine release, hypoexcitability in indirect-pathway striatal projection neurons, and alterations associated with an impaired striatal-dependent motor learning were observed only in the R1441C mice. We also showed that increased synaptic PKA activities in the R1441C and not G2019S mice underlie the specific alterations in motor learning deficits in the R1441C mice. In summary, our data argue that LRRK2 mutations' impact on the striatum cannot be simply generalized. Instead, alterations in electrochemical, electrophysiological, molecular, and behavioral levels were distinct between LRRK2 mutations. Our findings offer mechanistic insights for devising and optimizing treatment strategies for PD patients.

Autophagy Differentially Regulates Insulin Production and Insulin Sensitivity.

Autophagy, a stress-induced lysosomal degradative pathway, has been assumed to exert similar metabolic effects in different organs. Here, we establish a model where autophagy plays different roles in insulin-producing ß cells versus insulin-responsive cells, utilizing knockin (Becn1F121A) mice manifesting constitutively active autophagy. With a high-fat-diet challenge, the autophagy-hyperactive mice unexpectedly show impaired glucose tolerance, but improved insulin sensitivity, compared to mice with normal autophagy. Autophagy hyperactivation enhances insulin signaling, via suppressing ER stress in insulin-responsive cells, but decreases insulin secretion by selectively sequestrating and degrading insulin granule vesicles in ß cells, a process we term "vesicophagy." The reduction in insulin storage, insulin secretion, and glucose tolerance is reversed by transient treatment of autophagy inhibitors. Thus, ß cells and insulin-responsive tissues require different autophagy levels for optimal function. To improve insulin sensitivity without hampering secretion, acute or intermittent, rather than chronic, activation of autophagy should be considered in diabetic therapy development.