Identification of benzodiazepine Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput screen against RUNX1-CBF interaction

Oncogenesis and Development Section, Zebrafish Core, National Human Genome Research Institute, National Center for Advancing Translational Sciences, Molecular Virology Section, National Institute of Allergy and Infectious Diseases, and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 08/2012; 109(36):14592-7. DOI: 10.1073/pnas.1200037109
Source: PubMed


Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for ∼24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia (ALL). Current treatments for CBF leukemias are associated with significant morbidity and mortality, with a 5-y survival rate of ∼50%. We hypothesize that the interaction between RUNX1 and CBFβ is critical for CBF leukemia and can be targeted for drug development. We developed high-throughput AlphaScreen and time-resolved fluorescence resonance energy transfer (TR-FRET) methods to quantify the RUNX1-CBFβ interaction and screen a library collection of 243,398 compounds. Ro5-3335, a benzodiazepine identified from the screen, was able to interact with RUNX1 and CBFβ directly, repress RUNX1/CBFB-dependent transactivation in reporter assays, and repress runx1-dependent hematopoiesis in zebrafish embryos. Ro5-3335 preferentially killed human CBF leukemia cell lines, rescued preleukemic phenotype in a RUNX1-ETO transgenic zebrafish, and reduced leukemia burden in a mouse CBFB-MYH11 leukemia model. Our data thus confirmed that RUNX1-CBFβ interaction can be targeted for leukemia treatment and we have identified a promising lead compound for this purpose.

Download full-text


Available from: Paul Liu
  • Source
    • "We began with a preliminary attempt at evaluating the roles of roles of RUNX1 in blast cell transformation and the activation of inducible cytokine genes associated with pDHSs. For this purpose, we prepared T B in the presence of either the inhibitor Ro5-3335, which is reported to suppress RUNX1 function (Cunningham et al, 2012), or with DMSO as a control. The inhibitor was included during the activation of CD4 T cells by ConA, after which the cells were cultured for 24 h with the inhibitor and IL-2. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Immunological memory is a defining feature of vertebrate physiology, allowing rapid responses to repeat infections. However, the molecular mechanisms required for its establishment and maintenance remain poorly understood. Here, we demonstrated that the first steps in the acquisition of T‐cell memory occurred during the initial activation phase of naïve T cells by an antigenic stimulus. This event initiated extensive chromatin remodeling that reprogrammed immune response genes toward a stably maintained primed state, prior to terminal differentiation. Activation induced the transcription factors NFAT and AP‐1 which created thousands of new DNase I‐hypersensitive sites (DHSs), enabling ETS‐1 and RUNX1 recruitment to previously inaccessible sites. Significantly, these DHSs remained stable long after activation ceased, were preserved following replication, and were maintained in memory‐phenotype cells. We show that primed DHSs maintain regions of active chromatin in the vicinity of inducible genes and enhancers that regulate immune responses. We suggest that this priming mechanism may contribute to immunological memory in T cells by facilitating the induction of nearby inducible regulatory elements in previously activated T cells.
    Full-text · Article · Jan 2016 · The EMBO Journal
  • Source
    • "Whether this results from cooperating events that substitute for the AML1‐ ETO signal or is due to incomplete loss of AML1‐ETO expression upon doxycycline removal remains to be determined. This is an important question, given the efforts that are focused on targeting the AML1‐ETO protein directly (Cunningham et al, 2012). "

    Full-text · Article · Dec 2013 · EMBO Molecular Medicine
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.
    Preview · Article · Aug 2013 · PLoS ONE
Show more

We use cookies to give you the best possible experience on ResearchGate. Read our cookies policy to learn more.