Activation of FMS-like tyrosine kinase 3 protects against isoprenaline-induced cardiac hypertrophy by improving autophagy and mitochondrial dynamics.

FMS-like receptor tyrosine kinase 3 (Flt3) expression was reported to increase in the heart in response to pathological stress, but the role of Flt3 activation and its underlying mechanisms remain poorly elucidated. This study was designed to investigate the role of Flt3 activation in sympathetic hyperactivity-induced cardiac hypertrophy and its mechanisms through autophagy and mitochondrial dynamics. In vivo, cardiac hypertrophy was established by subcutaneous injection of isoprenaline (6 mg/kg·day) in C57BL/6 mice for 7 consecutive days. The Flt3-ligand intervention was launched 2 h prior to isoprenaline each day. In vitro, experiments of cardiomyocyte hypertrophy, autophagy, and mitochondrial dynamics were performed in neonatal rat cardiomyocytes (NRCMs). Our results revealed that the expression level of Flt3 protein was significantly increased in the hypertrophic myocardium provoked by isoprenaline administration. Flt3-ligand intervention alleviated isoprenaline-induced cardiac oxidative stress, hypertrophy, fibrosis, and contractile dysfunction. Isoprenaline stimulation impaired autophagic flux in hypertrophic mouse hearts, supported by the accumulation of LC3II and P62 proteins, while Flt3-ligand restored the impairment of autophagic flux. Flt3 activation normalized the imbalance of mitochondrial fission and fusion in the hearts of mice evoked by isoprenaline as evidenced by the neutralization of elevated mitochondrial fission markers and reduced mitochondrial fusion markers. In NRCMs, Flt3-ligand treatment attenuated isoprenaline-stimulated hypertrophy, which was abolished by a Flt3-specific blocker AC220. Activating Flt3 reversed isoprenaline-induced autophagosome accumulation and impairment of autophagic flux probably by enhancing SIRT1 expression and consequently TFEB nuclear translocation. Flt3 activation improved the imbalance of mitochondrial dynamics induced by isoprenaline in NRCMs through the SIRT1/P53 pathway. Activation of Flt3 mitigated ISO-stimulated hypertrophy probably involves the restoration of autophagic flux and balance of mitochondrial dynamics. Therefore, activation of Flt3 attenuates isoprenaline-induced cardiac hypertrophy in vivo and in vitro, the potential mechanism probably attributes to SIRT1/TFEB-mediated autophagy promotion and SIRT1/P53-mediated mitochondrial dynamics balance. These findings suggest that activation of Flt3 may be a novel target for protection against cardiac remodeling and heart failure during sympathetic hyperactivity.

Design and Synthesis of 4-(Heterocyclic Substituted Amino)-1H-Pyrazole-3-Carboxamide Derivatives and Their Potent Activity against Acute Myeloid Leukemia (AML).

Fms-like receptor tyrosine kinase 3 (FLT3) has been emerging as an attractive target for the treatment of acute myeloid leukemia (AML). By modifying the structure of FN-1501, a potent FLT3 inhibitor, 24 novel 1H-pyrazole-3-carboxamide derivatives were designed and synthesized. Compound 8t showed strong activity against FLT3 (IC50: 0.089 nM) and CDK2/4 (IC50: 0.719/0.770 nM), which is more efficient than FN-1501(FLT3, IC50: 2.33 nM; CDK2/4, IC50: 1.02/0.39 nM). Compound 8t also showed excellent inhibitory activity against a variety of FLT3 mutants (IC50 < 5 nM), and potent anti-proliferative effect within the nanomolar range on acute myeloid leukemia (MV4-11, IC50: 1.22 nM). In addition, compound 8t significantly inhibited the proliferation of most human cell lines of NCI60 (GI50 < 1 µM for most cell lines). Taken together, these results demonstrated the potential of 8t as a novel compound for further development into a kinase inhibitor applied in cancer therapeutics.

Flt3 Activation Mitigates Mitochondrial Fragmentation and Heart Dysfunction through Rebalanced L-OPA1 Processing by Hindering the Interaction between Acetylated p53 and PHB2 in Cardiac Remodeling.

Recent studies have shown that FMS-like receptor tyrosine kinase 3 (Flt3) has a beneficial effect on cardiac maladaptive remodeling. However, the role and mechanism of Flt3 in mitochondrial dynamic imbalance under cardiac stress remains poorly understood. This study aims to investigate how Flt3 regulates p53-mediated optic atrophy 1 (OPA1) processing and mitochondrial fragmentation to improve cardiac remodeling. Mitochondrial fragmentation in cardiomyocytes was induced by isoprenaline (ISO) and H2O2 challenge, respectively, in vitro. Cardiac remodeling in mice was established by ligating the left anterior descending coronary artery or by chronic ISO challenge, respectively, in vivo. Our results demonstrated that the protein expression of acetylated-p53 (ac-p53) in mitochondria was significantly increased under cell stress conditions, facilitating the dissociation of PHB2-OPA1 complex by binding to prohibitin 2 (PHB2), a molecular chaperone that stabilizes OPA1 in mitochondria. This led to the degradation of the long isoform of OPA1 (L-OPA1) that facilitates mitochondrial fusion and resultant mitochondrial network fragmentation. This effect was abolished by a p53 K371R mutant that failed to bind to PHB2 and impeded the formation of the ac-p53-PHB2 complex. The activation of Flt3 significantly reduced ac-p53 expression in mitochondria via SIRT1, thereby hindering the formation of the ac-p53-PHB2 complex and potentiating the stability of the PHB2-OPA1 complex. This ultimately inhibits L-OPA1 processing and leads to the balancing of mitochondrial dynamics. These findings highlight a novel mechanism by which Flt3 activation mitigates mitochondrial fragmentation and dysfunction through the reduction of L-OPA1 processing by dampening the interaction between ac-p53 and PHB2 in cardiac maladaptive remodeling.

Activated FMS-like tyrosine kinase 3 ameliorates angiotensin II-induced cardiac remodelling.

AIM: FMS-like receptor tyrosine kinase 3 (Flt3) has been reported to be increased in cardiomyocytes responding to ischaemic stress. This study was to determine whether Flt3 activation could ameliorate pressure overload-induced heart hypertrophy and fibrosis, and to elucidate the mechanisms of action. METHODS: In vivo cardiac hypertrophy and remodelling experiments were conducted by infusing angiotensin II (Ang II) chronically in male C57BL/6 mice. Flt3-specific ligand (FL) was administered intraperitoneally every two days (5 µg/mouse). In vitro experiments on hypertrophy, apoptosis and autophagy mechanism were performed in neonatal rat cardiomyocytes (NRCMs) and H9c2 cells with adenovirus vector-mediated overexpression of Flt3. RESULTS: Our results demonstrated that following chronic Ang II infusion for 4 weeks, the mice exhibited heart hypertrophy, fibrosis, apoptosis and contractile dysfunction. Meanwhile, Ang II induced autophagic responses in mouse hearts, as evidenced by increased LC3 II and decreased P62 expression. These pathological alterations in Ang II-treated mice were significantly ameliorated by Flt3 activation with FL administration. In NRCMs and Flt3-overexpressed H9c2 cells, FL attenuated Ang II-induced pathological autophagy and inactivated AMPK/mTORC1/FoxO3a signalling, thereby efficiently mitigating cell hypertrophy and apoptosis. Conversely, the AMPK activator metformin or the mTORC1 inhibitor rapamycin reversed the effects of FL on the alterations of autophagy, hypertrophy and apoptosis in cardiomyocytes induced by Ang II. CONCLUSION: Flt3 activation ameliorates cardiac hypertrophy, fibrosis and contractile dysfunction in the mouse model of chronic pressure overload, most likely via suppressing AMPK/mTORC1/FoxO3a-mediated autophagy. These results provide new evidence supporting Flt3 as a novel therapeutic target in maladaptive cardiac remodelling.

Comprehensive kinase profile of pacritinib, a nonmyelosuppressive Janus kinase 2 inhibitor.

Pacritinib, potent inhibitor of Janus kinase 2 (JAK2), JAK2V617F, and fms-like receptor tyrosine kinase 3, is in Phase III development in myelofibrosis. Among type 1 inhibitors, pacritinib shows a lack of myelosuppression at doses that both inhibit JAK2/signal transducer and activator of transcription 3 pathway and demonstrate clinical efficacy. To elucidate these mechanisms and identify other disease targets, a kinome analysis screened 439 recombinant kinases at 100 nM pacritinib concentration. For kinases with >50% inhibition, pacritinib was titrated from 1 to 100 nM. JAK2, JAK2V617F, FLT3, colony-stimulating factor 1 receptor, and interleukin-1 receptor-associated kinase 1 achieved half-maximal inhibitory concentrations <50 nM. Pacritinib did not inhibit JAK1 (82% control at 100 nM). Lack of myelosuppression may stem from inhibiting JAK2 without affecting JAK1 and reducing hematopoietic inhibitory cytokines by suppressing interleukin-1 receptor-associated kinase 1 or colony-stimulating factor 1 receptor. The pacritinib kinome suggests therapeutic utility in acute myeloid leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, solid tumors, and inflammatory conditions.