Exploration Of Targeting NOTCH1 By HSP90 Inhibition In T-ALL And Mechanism Of MWCNTS' Inhibition Of Pgp And MRP4

1 Effects and mechanism of targeting NOTCH1 by HSP90 inhibition in acute T-cell lymphoblastic leukemiaObjective:Acute T-cell lymphoblastic leukemia (T-ALL) is a malignant disorder of thymocyte progenitors, affecting both children and adults. The overall prognosis of T-ALL remains unsatisfied in comparison with B cell ALL and the outcome of T-ALL patients with primary resistant or relapsed leukemia remains poor. The frequent NOTCH1 activating mutations and/or FBW7 loss-of-function mutations has vaulted dysregulated NOTCH signaling to the center of T-ALL pathogenesis. Yet the strategy of blocking the NOTCH signaling by y-secretase inhibitors (GSIs) against T-ALL have not made substantial progresses in clinical treatments due to intolerable toxicity and/or insignificant efficacy. In order to circumvent the limitation of direct targeting of NOTCH1 that is currently used, we have sought alternative approaches for NOTCH1 inhibition by targeting HSP90 based on its function to chaperone protein stability.Methods:We have evaluated the effects of HSP90 inhibitors on intracellular NOTCH1 protein amounts and its downstream target gene expression by immunoblotting and RT-qPCR analysis. Interaction between NOTCH1 and HSP90 (or STUB1) was determined by co-immunoprecipitation (Co-IP). The effect of STUB1 on NOTCH1 activity was assessed by luciferase reporter assays. Lentiviral infection of shRNA in T-ALL cells was used to silence the expression of HSP90 or STUB1. HSP90 inhibitors-induced cytotoxicity was evaluated in vitro by cell viability (CCK8) and apoptosis (Annexin V/PI). A human T-ALL xenograft model and a secondary transplant from Kras-driven murine T-ALL with mutant NOTCH1 alleles were established to evaluate the potential anti-leukemic effects of HSP90 inhibitors in vivo.Results:HSP90 inhibitors effectively impeded proliferation and induced apoptosis of NOTCH1-dependent T-ALL cells and enforced expression of nuclear NOTCH1 partially rescued HSP90 inhibitor-mediated cell death. Moreover, we found that HSP90 inhibitors had great anti-leukemic effects in murine T-ALL derived from Kras-transgenic mouse models with enforced expression of NOTCH 1 mutant alleles. Similarly, xenograft mice with human T-ALL were sensitive to HSP90 inhibition. Mechanistically, NOTCH 1 was identified as a novel client protein of HSP90, inhibition of which leads to protesome-mediated intracellular NOTCH 1 (ICN1) degradation. In this process, we reveal an unreported NOTCH 1 E3 ubiquitin ligase STUB1 that mediates polyubiquitination of ICN1 and directs its rapid protein turnover.Conclusion:Herein, we have revealed a novel regulatory mechanism whereby the chaperon protein HSP90 is required for intracellular NOTCH1 stability and HSP90 inhibition renders STUB1-mediated polyubiquitination of ICN1, resulting in rapid protein decay. Identification of nuclear NOTCH1 as a novel HSP90 client has also provided a rationale of applying HSP90 inhibitors as alternative therapeutic regimen to benefit patients with T cell leukemia or other NOTCH 1-addicted tumors.2 Mechanism of MWCNTs'inhibition of Pgp and MRP4 medicated efflux function in caco-2 cellsObjective:ABC transporters, a large family of integral membrane proteins, of which over-expression can decrease cellular drug accumulation through enhanced active extrusion, thus results in cancer multidrug resistance. Preliminary study has shown the inhibiting effect on the efflux of rhodamine 123(R123) and fluorescein, which are substrates of P-glycoprotein (Pgp) and multidrug resistance related protein 4(MRP4), thus increased there intracellular retention. However the intrinsic mechanism remains under-investigated. Our research focused on how and why MWCNTs affect the function of Pgp and MRP4, so as to provide scientific basis for MWCNTs'potential application in anti-multidrug resistance delivery system.Methods:We revaluated MWCNTs'effects on the effux function of Pgp and MRP4 by FACS analysis of the absorption and retention of there substrats R123 and fluorescein in caco-2 cells; Real-time qPCR and Western blot were used to assess Pgp and MRP4 gene expression alteration under MWCNTs treatment; Combined application with Immunofluorescent labling and FACS were to examine MWCNTs'effects on Pgp and MRP4 distribution. Interaction between MWCNTs and caco-2 cells was observed through scanning electron microscopy (SEM) and transmission electron microscopy (TEM); Retrovirus transfection was adopted to discuss the intermediary role of transcription factor c-Myc between MWCNTs and membrane transporters.Results:In our research, we discovered that 1) MWCNTs impeded the transport activity of Pgp and MRP4.2) MWCNTs blocked the gene expression of P-gp, MRP4 as well as c-Myc.4) Over-expression of c-Myc rescued the mRNA and protein decay of Pgp and MRP4 by MWCNTs.5) MWCNTs interacted with caco-2 cell membrane by derectly insertion or endocytosis.6) MWCNTs increased cellular ROS production and membrane peroxidation.7) Anti-oxidants failed to antagonize MWCNTs'effect on Pgp and MRP4.Conclusion:The data suggests that MWCNTs impeds the transport activity of Pgp and MRP4 via gene expression inhibition. Oncogene c-Myc may serves as an up-regulator of Pgp and MRP4, inhibition of which by MWCNTs gives rise to the ultimate efflux blockage in colon cancer. The mechanisms shed light on MWCNTs' novel cellular effects that may be utilized to establish CNTs-based drug delivery systems to overcome ABC transporter-mediated drug resistance as well as to antagonize cancer.