RESUMEN
Myeloproliferative neoplasms (MPN) are rare hematologic disorders characterized by clonal hematopoiesis. Familial clustering is observed in a subset of cases, with a notable proportion exhibiting heterozygous germline mutations in DNA double-strand break repair genes (e.g., BRCA1). We investigated the therapeutic potential of targeting BRCA1 haploinsufficiency alongside the JAK2V617F driver mutation. We assessed the efficacy of combining the PARP inhibitor olaparib with interferon-alpha (IFNα) in CRISPR/Cas9-engineered Brca1+/- Jak2V617F-positive 32D cells. Olaparib treatment induced a higher number of DNA double-strand breaks, as demonstrated by γH2AX analysis through Western blot (p = 0.024), flow cytometry (p = 0.013), and confocal microscopy (p = 0.071). RAD51 foci formation was impaired in Brca1+/- cells compared to Brca1+/+ cells, indicating impaired homologous recombination repair due to Brca1 haploinsufficiency. Importantly, olaparib enhanced apoptosis while diminishing cell proliferation and viability in Brca1+/- cells compared to Brca1+/+ cells. These effects were further potentiated by IFNα. Olaparib induced interferon-stimulated genes and increased endogenous production of IFNα in Brca1+/- cells. These responses were abrogated by STING inhibition. In conclusion, our findings suggest that the combination of olaparib and IFNα presents a promising therapeutic strategy for MPN patients by exploiting the synthetic lethality between germline BRCA1 mutations and the JAK2V617F MPN driver mutation.
Asunto(s)
Proteína BRCA1 , Trastornos Mieloproliferativos , Neoplasias , Humanos , Proteína BRCA1/genética , ADN , Células Germinativas , Haploinsuficiencia , Interferón-alfa/farmacología , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/genética , Neoplasias/tratamiento farmacológico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Reparación del ADN por Recombinación , Mutaciones Letales SintéticasRESUMEN
AIMS: In white adipose tissue (WAT) the cell cycle regulators CDK4 and CDK6 (CDK4/6) promote adipogenesis and maintain the adipocyte mature state. Here we aimed to investigate their role in the Ucp1-mediated thermogenesis of WAT depots and in the biogenesis of beige adipocytes. MAIN METHODS: We treated mice with the CDK4/6 inhibitor palbociclib at room temperature (RT) or cold and analyzed thermogenic markers in the epididymal (abdominal) and inguinal (subcutaneous) WAT depots. We also assessed the effect of in vivo palbociclib-treatment on the percentage of beige precursors in the stroma vascular fraction (SVF), and on its beige adipogenic potential. Finally, we treated SVFs and mature adipocytes from WAT depots with palbociclib in vitro to study the role of CDK4/6 in beige adipocytes biogenesis. KEY FINDINGS: In vivo CDK4/6 inhibition downregulated thermogenesis at RT and impaired cold-induced browning of both WAT depots. It also reduced the percentage of beige precursors and beige adipogenic potential of the SVF upon differentiation. A similar result was observed with direct CDK4/6 inhibition in the SVF of control mice in vitro. Importantly, CDK4/6 inhibition also downregulated the thermogenic program of beige differentiated- and depots-derived adipocytes. SIGNIFICANCE: CDK4/6 modulate Ucp1-mediated thermogenesis of WAT depots in basal and cold-stressing conditions controlling beige adipocytes biogenesis by adipogenesis and transdifferentiation. This shows a pivotal role of CDK4/6 in WAT browning that could be applied to fight obesity or browning-associated hypermetabolic conditions such as cancer cachexia.
Asunto(s)
Adipocitos , Tejido Adiposo Blanco , Animales , Ratones , Tejido Adiposo Blanco/metabolismo , Adipocitos/metabolismo , Diferenciación Celular , Adipogénesis , Termogénesis , Tejido Adiposo Pardo/metabolismo , Proteína Desacopladora 1/metabolismoRESUMEN
Resistance to therapy remains a major obstacle in cancer management. Although treatment with hormone and CDK4/6 inhibitors is successful in luminal breast cancer, resistance to these treatments is frequent, highlighting the need for novel therapeutic strategies to delay disease progression and improve patient survival. Here, we assessed the mechanisms of acquired resistance using T47D and MCF-7 tamoxifen- and palbociclib-resistant cell-line variants in culture and as xenografts, and patient-derived cells (PDCs) obtained from sensitive or resistant patient-derived xenografts (PDXs). In these models, we analyzed the effect of specific kinase inhibitors on survival, signaling and cellular aggressiveness. Our results revealed that mTOR inhibition is more effective than PI3K inhibition in overcoming resistance, irrespective of PIK3CA mutation status, by decreasing cell proliferation and tumor growth, as well as reducing cell migration and stemness. Moreover, a combination of mTOR and CDK4/6 inhibitors may prevent pathway reactivation downstream of PI3K, interfering with the survival of resistant cells and consequent tumor escape. In conclusion, we highlight the benefits of incorporating mTOR inhibitors into the current therapy in ER + breast cancer. This alternative therapeutic strategy not only enhances the antitumor response but may also delay the emergence of resistance and tumor recurrence.
Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos , Línea Celular Tumoral , Recurrencia Local de Neoplasia , Serina-Treonina Quinasas TOR/metabolismo , Hormonas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la CiclinaRESUMEN
Urine deposition by grazing livestock is the single largest source of ammonia (NH3) volatilisation losses in New Zealand. Urease inhibitors (UI) have been used to mitigate NH3 losses from fertiliser urea and animal urine. In previous trials, the UI effect in reducing NH3 emissions from urine has been measured by applying urine mixed with the UI to the pasture soil thus increasing the chances of better interaction of the UI in inhibiting the urease enzyme. However, these trials do not represent a realistic grazing scenario where only urine is deposited onto the soil. This current research aimed to identify the best time to spray nBTPT (a UI containing 0.025% N-(n-butyl) thiophosphoric triamide) onto pasture soil to reduce NH3 losses from urine patches. The treatments were: a control (without urine and nBTPT), urine alone at 530â kgâ Nâ ha-1 and urine plus nBTPT. The UI was applied to the chambers and soil plots 5 and 3 days prior to urine deposition, on the same day and 1, 3 and 5 after urine deposition in autumn. Ammonia losses were measured using the dynamic chamber method. The application of the inhibitor prior to urine deposition reduced NH3 losses with reductions of 27.6% and 17.5% achieved for UAgr-5 and UAgr-3, respectively. However, reductions in NH3 emission were 0.6-2.9% for inhibitor applied post urine deposition. There was also a reduction in both soil NH4 +-N concentration and soil pH in comparison with urine alone or with the treatments where nBTPT was applied after urine deposition.
Asunto(s)
Amoníaco , Ureasa , Amoníaco/análisis , Animales , Fertilizantes/análisis , Suelo , UreaRESUMEN
Improved efficacy of neoadjuvant endocrine-targeting therapies in luminal breast carcinomas could be achieved with optimal use of pathway targeting agents. In a mouse model of ductal breast carcinoma we identify a tumor regressive stromal reaction that is induced by neoadjuvant endocrine therapy. This reparative reaction is characterized by tumor neovascularization accompanied by infiltration of immune cells and carcinoma-associated fibroblasts that stain for phosphorylated ribosomal protein S6 (pS6), downstream the PI3K/Akt/mTOR pathway. While tumor variants with higher PI3K/Akt/mTOR activity respond well to a combination of endocrine and PI3K/Akt/mTOR inhibitors, tumor variants with lower PI3K/Akt/mTOR activity respond more poorly to the combination therapy than to the endocrine therapy alone, associated with inhibition of stromal pS6 and the reparative reaction. In human breast cancer xenografts we confirm that such differential sensitivity to therapy is primarily determined by the level of PI3K/Akt/mTOR in tumor cells. We further show that the clinical response of breast cancer patients undergoing neoadjuvant endocrine therapy is associated with the reparative stromal reaction. We conclude that tumor level and localization of pS6 are associated with therapeutic response in breast cancer and represent biomarkers to distinguish which tumors will benefit from the incorporation of PI3K/Akt/mTOR inhibitors with neoadjuvant endocrine therapy.