NRF2-driven miR-125B1 and miR-29B1 transcriptional regulation controls a novel anti-apoptotic miRNA regulatory network for AML survival

Transcription factor NRF2 is an important regulator of oxidative stress. It is involved in cancer progression, and has abnormal constitutive expression in acute myeloid leukaemia (AML). Posttranscriptional regulation by microRNAs (miRNAs) can affect the malignant phenotype of AML cells. In this study, we identified and characterised NRF2-regulated miRNAs in AML. An miRNA array identified miRNA expression level changes in response to NRF2 knockdown in AML cells. Further analysis of miRNAs concomitantly regulated by knockdown of the NRF2 inhibitor KEAP1 revealed the major candidate NRF2-mediated miRNAs in AML. We identified miR-125B to be upregulated and miR-29B to be downregulated by NRF2 in AML. Subsequent bioinformatic analysis identified putative NRF2 binding sites upstream of the miR-125B1 coding region and downstream of the mir-29B1 coding region. Chromatin immunoprecipitation analyses showed that NRF2 binds to these antioxidant response elements (AREs) located in the 5′ untranslated regions of miR-125B and miR-29B. Finally, primary AML samples transfected with anti-miR-125B antagomiR or miR-29B mimic showed increased cell death responsiveness either alone or co-treated with standard AML chemotherapy. In summary, we find that NRF2 regulation of miR-125B and miR-29B acts to promote leukaemic cell survival, and their manipulation enhances AML responsiveness towards cytotoxic chemotherapeutics

A universal bioluminescence tomography system for pre-clinical image-guided radiotherapy research

CBCT-guided small animal irradiators encounter challenges in localizing soft-tissue targets due to low imaging contrast. Bioluminescence tomography (BLT) offers a promising solution, but they have largely remained in laboratorial development, limiting accessibility for researchers. In this work, we develop a universal, commercial-graded BLT-guided system (MuriGlo) designed to seamlessly integrate with commercial irradiators and empower researchers for translational studies. We demonstrate its capabilities in supporting in vitro and in vivo studies. The MuriGlo comprises detachable mouse bed, thermostatic control, mirrors, filters, and CCD, enabling multi-projection and multi-spectral imaging. We evaluate that the thermostatic control effectively sustains animal temperature at 37{\deg}C throughout imaging, and quantify that the system can detect as few as 61 GL261-AkaLuc cells in vitro. To illustrate how the MuriGlo can be utilized for in vivo image-guided research, we present 3 strategies, BLT-guided 5-arc, 2-field box, and BLI-guided single-beam, ranging from complicated high-conformal to simplest high-throughput plans. The high conformal BLT-guided 5-arc plan fully covers the gross tumor volume (GTV) at prescribed dose with minimal normal tissue exposure (3.9%), while the simplified, high-throughput BLT-guided 2-field box achieves 100% GTV coverage but results in higher normal tissue exposure (13.1%). Moreover, we demonstrate that the localization accuracy of MuriGlo for both widely-used SARRP and SmART irradiators is within1 mm, and the tumor coverage reaches over 97% with 0.75mm margin. The universal BLT-guided system offers seamless integration with commercial irradiators, achieving comparable localization accuracy, expected to supporting high-precision radiation research

A universal bioluminescence tomography system for pre-clinical image-guided radiotherapy research

CBCT-guided small animal irradiators encounter challenges in localizing soft-tissue targets due to low imaging contrast. Bioluminescence tomography (BLT) offers a promising solution, but they have largely remained in laboratorial development, limiting accessibility for researchers. In this work, we develop a universal, commercial-graded BLT-guided system (MuriGlo) designed to seamlessly integrate with commercial irradiators and empower researchers for translational studies. We demonstrate its capabilities in supporting in vitro and in vivo studies. The MuriGlo comprises detachable mouse bed, thermostatic control, mirrors, filters, and CCD, enabling multi-projection and multi-spectral imaging. We evaluate that the thermostatic control effectively sustains animal temperature at 37{\deg}C throughout imaging, and quantify that the system can detect as few as 61 GL261-AkaLuc cells in vitro. To illustrate how the MuriGlo can be utilized for in vivo image-guided research, we present 3 strategies, BLT-guided 5-arc, 2-field box, and BLI-guided single-beam, ranging from complicated high-conformal to simplest high-throughput plans. The high conformal BLT-guided 5-arc plan fully covers the gross tumor volume (GTV) at prescribed dose with minimal normal tissue exposure (3.9%), while the simplified, high-throughput BLT-guided 2-field box achieves 100% GTV coverage but results in higher normal tissue exposure (13.1%). Moreover, we demonstrate that the localization accuracy of MuriGlo for both widely-used SARRP and SmART irradiators is within1 mm, and the tumor coverage reaches over 97% with 0.75mm margin. The universal BLT-guided system offers seamless integration with commercial irradiators, achieving comparable localization accuracy, expected to supporting high-precision radiation research

Mechanism of Chemical Activation of Nrf2

NF-E2 related factor-2 (Nrf2) promotes the transcription of many cytoprotective genes and is a major drug target for prevention of cancer and other diseases. Indeed, the cancer-preventive activities of several well-known chemical agents were shown to depend on Nrf2 activation. It is well known that chemopreventive Nrf2 activators stabilize Nrf2 by blocking its ubiquitination, but previous studies have indicated that this process occurs exclusively in the cytoplasm. Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and orchestrates Nrf2 ubiquitination, and it has been a widely-held view that inhibition of Nrf2 ubiquitination by chemopreventive agents results from the dissociation of Nrf2 from its repressor Keap1. Here, we show that while the activation of Nrf2 by prototypical chemical activators, including 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) and sulforaphane (SF), results solely from inhibition of its ubiquitination, such inhibition occurs predominantly in the nucleus. Moreover, the Nrf2 activators promote Nrf2 association with Keap1, rather than disassociation, which appears to result from inhibition of Nrf2 phosphorylation at Ser40. Available evidence suggests the Nrf2 activators may block Nrf2 ubiquitination by altering Keap1 conformation via reaction with the thiols of specific Keap1 cysteines. We further show that while the inhibitory effects of CPDT and SF on Nrf2 ubiquitination depend entirely on Keap1, Nrf2 is also degraded by a Keap1-independent mechanism. These findings provide significant new insight about Nrf2 activation and suggest that exogenous chemical activators of Nrf2 enter the nucleus to exert most of their inhibitory impact on Nrf2 ubiquitination and degradation

Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype

A duplex 21 nucleotide small interfering RNA (siRNA) against human Keap1 is described that represents a unique class of cancer chemopreventive agent. This siRNA can knockdown Keap1 mRNA and thereby relieve negative regulation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-mediated gene expression. The siRNA lowered endogenous Keap1 mRNA t

MDM2 promotes cell motility and invasiveness through a RING-finger independent mechanism

AbstractRecent studies connect MDM2 with increased cell motility, invasion and/or metastasis proposing an MDM2-mediated ubiquitylation-dependent mechanism. Interestingly, in renal cell carcinoma (RCC) p53/MDM2 co-expression is associated with reduced survival which is independently linked with metastasis. We therefore investigated whether expression of p53 and/or MDM2 promotes aggressive cell phenotypes. Our data demonstrate that MDM2 promotes increased motility and invasiveness in RCC cells (N.B. similar results are obtained in non-RCC cells). This study shows for the first time both that endogenous MDM2 significantly contributes to cell motility and that this does not depend upon the MDM2 RING-finger, i.e. is independent of ubiquitylation (and NEDDylation). Our data suggest that protein–protein interactions provide a likely mechanistic basis for MDM2-promoted motility which may constitute future therapeutic targets