AbstractIntroduction - The Myc proteins are transcription factors that have essential roles in cell growth and proliferation by both positively and negatively regulating gene expression. Mutation, amplification, or activation of the MYC oncogene family is one of the most frequent events associated with cancer. In acute myeloid leukemia (AML), c-Myc is commonly activated and plays an important role in the initiation and maintenance of the disease. In particular, c-Myc is upregulated by activating mutations of the Flt3 receptor tyrosine kinase, one of the most prevalent types of mutations in AML, and also by the AML-associated fusion proteins AML1-ETO, PML/RARα, and PLZF/RARα. It has also been shown that c-Myc is negatively regulated by C/EBPα, a transcription factor essential for granulocytic differentiation, and that c-Myc expression is elevated in myeloid leukemias in which C/EBPα is mutated. Additionally, c-Myc is stabilized in AML with mutations leading to aberrant cytoplasmic localization of nucleophosmin (NPM), the most frequent genetic alteration in AML without karyotypic aberrations. Importantly, the MYC gene itself, located at 8q24, has been found to be one of the most commonly amplified regions in AML. Lastly, the importance of c-Myc in myeloid leukemogenesis has been further demonstrated by the induction of myeloid leukemias in mouse models overexpressing c-Myc in bone marrow progenitors.Methods - In the current study, we disrupt MYC transcription in AML blasts, by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains, specifically BRD4) of putative coactivator proteins implicated in transcriptional initiation and elongation.Results - Following extensive chemical optimization and biological characterization, we identified two advanced pre-clinical candidates, EP11313 and EP11336. These compounds have excellent affinity for BRD4 and achieve high in-vivo exposure in animal models. Both agents possess drug-like physical characteristics, and their pharmacologic properties compared favorably to the BRD4 inhibitor IBET-762 (GSK525762A). Respectively for IBET-762, EP11313 and EP11336 we calculated BRD4 IC50 (23, 7, 5 nM), BRD4 KD (55, 2, 6 nM), molecular weight (424, 415, 445 Da), solubility (194, 20, 85 μM), cLogP (1.8, 3.7, 3.0), PSA (81, 78, 98 Ǻ2), MDR-MDCK Efflux ratio BA/AB (27.9, 1.3, 1.9) and MDR-MDCK Papp (1.1, 7.3, 10.2). In vitro, both molecules have nanomolar anti-leukemic activity across a panel c-myc dysregulated AML cell lines (OCI-AML2, OCI-AML3, KG-1a, HL-60, MV-411 and NB-4). Growth inhibition was variable and context dependent (GI50 range: 265 nM - 1618 nM). Following a brief incubation in-vitro, both EP11313 and EP11336 lead to activation of caspase 3 and initiation of apoptotic cell death. Over-expression of c-myc is known to impair myeloid differentiation in response to the retinoid all-trans-retinoic acid (ATRA) in AML, considering this and the research interests of our group in broadening the anti-leukemic effects of ATRA, we showed that ATRA combined with bromodomain inhibition led to greater cell kill in vitro than was observed for ATRA, or either bromodomain inhibitor tested alone (see figure).Conclusions - Both EP11313 and EP11336 are currently in late pre-clinical development, and are being optimized for first in man studies in AML and other tumor types. Updated information on the in-vivo activity of these compounds and mechanism of action in AML will be presented.Figure 1. The BET bromodomain inhibitors EP11313 and EP11336, impair the viability of AML blasts in vitro and have synergistic activity when combined with the retinoid all-trans -retinoic acid (AtRA). MV-411 cells were incubated with the indicated concentrations of EP11313, EP11336 and AtRA for 48 hours. Cell viability was measured using a bioluminescence assay (CellTiter-Glo¨, left) and induction of caspase 3, was measured by FACS (right). Growth inhibition (GI) in OCI-AML3 cells was calculated using linear extrapolation (top). Figure 1. The BET bromodomain inhibitors EP11313 and EP11336, impair the viability of AML blasts in vitro and have synergistic activity when combined with the retinoid all-trans -retinoic acid (AtRA). MV-411 cells were incubated with the indicated concentrations of EP11313, EP11336 and AtRA for 48 hours. Cell viability was measured using a bioluminescence assay (CellTiter-Glo¨, left) and induction of caspase 3, was measured by FACS (right). Growth inhibition (GI) in OCI-AML3 cells was calculated using linear extrapolation (top).DisclosuresJohnstone: Epigenetix: Employment. Albert:Epigenetix: Employment. Collard:Epigenetix: Employment.
