RESUMEN
PURPOSE: The JAK1/2 inhibitor ruxolitinib has demonstrated significant benefits for patients with myeloproliferative neoplasms (MPN). However, patients often lose response to ruxolitinib or suffer disease progression despite therapy with ruxolitinib. These observations have prompted efforts to devise treatment strategies to improve therapeutic efficacy in combination with ruxolitinib therapy. Activation of JAK-STAT signaling results in dysregulation of key downstream pathways, notably increased expression of cell-cycle mediators including CDC25A and the PIM kinases. EXPERIMENTAL DESIGN: Given the involvement of cell-cycle mediators in MPNs, we sought to examine the efficacy of therapy combining ruxolitinib with a CDK4/6 inhibitor (LEE011) and a PIM kinase inhibitor (PIM447). We utilized JAK2-mutant cell lines, murine models, and primary MPN patient samples for these studies. RESULTS: Exposure of JAK2-mutant cell lines to the triple combination of ruxolitinib, LEE011, and PIM447 resulted in expected on-target pharmacodynamic effects, as well as increased apoptosis and a decrease in the proportion of cells in S-phase, compared with ruxolitinib. As compared with ruxolitinib monotherapy, combination therapy led to reductions in spleen and liver size, reduction of bone marrow reticulin fibrosis, improved overall survival, and elimination of disease-initiating capacity of treated bone marrow, in murine models of MPN. Finally, the triple combination reduced colony formation capacity of primary MPN patient samples to a greater extent than ruxolitinib. CONCLUSIONS: The triple combination of ruxolitinib, LEE011, and PIM447 represents a promising therapeutic strategy with the potential to increase therapeutic responses in patients with MPN.
Asunto(s)
Trastornos Mieloproliferativos , Neoplasias , Mielofibrosis Primaria , Animales , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina , Humanos , Janus Quinasa 1 , Janus Quinasa 2/metabolismo , Ratones , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Nitrilos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de SeñalRESUMEN
Botulism is caused by a potent neurotoxin that blocks neuromuscular transmission, resulting in death by asphyxiation. Currently, the therapeutic options are limited and there is no antidote. Here, we harness the structural and trafficking properties of an atoxic derivative of botulinum neurotoxin (BoNT) to transport a function-blocking single-domain antibody into the neuronal cytosol where it can inhibit BoNT serotype A (BoNT/A1) molecular toxicity. Post-symptomatic treatment relieved toxic signs of botulism and rescued mice, guinea pigs, and nonhuman primates after lethal BoNT/A1 challenge. These data demonstrate that atoxic BoNT derivatives can be harnessed to deliver therapeutic protein moieties to the neuronal cytoplasm where they bind and neutralize intracellular targets in experimental models. The generalizability of this platform might enable delivery of antibodies and other protein-based therapeutics to previously inaccessible intraneuronal targets.
Asunto(s)
Toxinas Botulínicas Tipo A , Botulismo , Anticuerpos de Dominio Único , Animales , Botulismo/tratamiento farmacológico , Cobayas , Ratones , Modelos Animales , NeurotoxinasRESUMEN
Genetic studies have identified recurrent somatic mutations in acute myeloid leukemia (AML) patients, including in the Wilms' tumor 1 (WT1) gene. The molecular mechanisms by which WT1 mutations contribute to leukemogenesis have not yet been fully elucidated. We investigated the role of Wt1 gene dosage in steady-state and pathologic hematopoiesis. Wt1 heterozygous loss enhanced stem cell self-renewal in an age-dependent manner, which increased stem cell function over time and resulted in age-dependent leukemic transformation. Wt1-haploinsufficient leukemias were characterized by progressive genetic and epigenetic alterations, including those in known leukemia-associated alleles, demonstrating a requirement for additional events to promote hematopoietic transformation. Consistent with this observation, we found that Wt1 depletion cooperates with Flt3-ITD mutation to induce fully penetrant AML. Our studies provide insight into mechanisms of Wt1-loss leukemogenesis and into the evolutionary events required to induce transformation of Wt1-haploinsufficient stem/progenitor cells.
Asunto(s)
Epigénesis Genética , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Mutación , Proteínas Represoras/genética , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Autorrenovación de las Células , Eliminación de Gen , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Leucemia Mieloide Aguda/patología , Leucopoyesis , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/metabolismo , Células Mieloides/patología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Proteínas WT1 , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
The Frank-Starling mechanism, whereby increased diastolic filling leads to increased cardiac output, depends on increasing the sarcomere length (Ls) of cardiomyocytes. Ventricular stiffness increases with advancing age, yet it remains unclear how such changes in compliance impact sarcomere dynamics in the intact heart. We developed an isolated murine heart preparation to monitor Ls as a function of left ventricular pressure and tested the hypothesis that sarcomere lengthening in response to ventricular filling is impaired with advanced age. Mouse hearts isolated from young (3-6 mo) and aged (24-28 mo) C57BL/6 mice were perfused via the aorta under Ca(2+)-free conditions with the left ventricle cannulated to control filling pressure. Two-photon imaging of 4-{2-[6-(dioctylamino)-2-naphthalenyl]ethenyl}1-(3-sulfopropyl)-pyridinium fluorescence was used to monitor t-tubule striations and obtain passive Ls between pressures of 0 and 40 mmHg. Ls values (in µm, aged vs. young, respectively) were 2.02 ± 0.04 versus 2.01 ± 0.02 at 0 mmHg, 2.13 ± 0.04 versus 2.23 ± 0.02 at 5 mmHg, 2.21 ± 0.03 versus 2.27 ± 0.03 at 10 mmHg, and 2.28 ± 0.02 versus 2.36 ± 0.01 at 40 mmHg, indicative of impaired sarcomere lengthening in aged hearts. Atomic force microscopy nanoindentation revealed that intact cardiomyocytes enzymatically isolated from aged hearts had increased stiffness compared with those of young hearts (elastic modulus: aged, 41.9 ± 5.8 kPa vs. young, 18.6 ± 3.3 kPa; P = 0.006). Impaired sarcomere lengthening during left ventricular filling may contribute to cardiac dysfunction with advancing age by attenuating the Frank-Starling mechanism and reducing stroke volume.