Small-Molecule Inhibition of BRD4 Is a Novel Promising Approach to Therapeutically Target Leukemia Stem Cells in AML,

Abstract 3484

Acute myeloid leukemia (AML) is a stem cell-derived malignancy characterized by uncontrolled proliferation and accumulation of myeloblasts in hematopoietic tissues. The clinical course and prognosis in AML vary depending on deregulated genes, cell type(s) involved, and the biological properties of the clone. In most variants of AML, the complexity and heterogeneity of oncogenomes pose a challenge for the development of effective targeted therapeutics. However, diverse genetic aberrations in AML typically converge functionally to dysregulate the same cellular core processes. One key event is the corruption of myeloid cell-fate programs resulting in the generation of aberrantly self-renewing leukemia stem cells (LSC), which maintain and propagate the disease and are often resistant to conventional chemotherapy. Hence, strategies aimed at terminating aberrant self-renewal and eradicating LSC are considered as key for the development of more effective AML therapies. In an effort to systematically probe genes involved in chromatin regulation as potential therapeutic targets, we recently employed an unbiased screening approach combining AML mouse models and new in-vivo RNAi technologies, through which we identified the epigenetic ‘reader' BRD4 as new candidate drug target in AML (Zuber et al., Nature, in press). Inhibition of BRD4 using RNAi or a new small-molecule inhibitor (JQ1) blocking BRD4 binding to acetylated histones, showed profound antileukemic effects in AML mouse models, in all human AML cell lines tested (n=8) as well as in primary AML cells. In all models tested, BRD4 suppression was found to trigger apoptosis as well as terminal myeloid differentiation, and potently suppressed expression programs previously associated with LSC. As one key target, we observed a dramatic transcriptional repression of MYC, which recently has been discussed as core component of an LSC associated transcriptional module. To further evaluate suppression of BRD4 as a potential therapeutic approach to eradicate LSC in human AML, we analyzed the effects of JQ1 in primary AML cells obtained from 17 patients with freshly diagnosed or relapsed/refractory AML (females, n=5, males, n=12, median age: 54 years; range: 21–80 years). In unfractionated primary AML cells, submicromolar doses of JQ1 were found to induce major growth-inhibitory effects (IC₅₀ between 0.05 and 0.5 μM) in a broad spectrum of AML subtypes. No differences in IC₅₀ values were seen when comparing drug effects in AML cells kept in the presence or absence of growth-stimulating cytokines (G-CSF, IL-3, SCF). In addition, JQ1 treatment effectively triggered apoptosis in all patients tested, with similar anti-leukemic activities observed in newly diagnosed pts and refractory/relapsed AML. To further evaluate the clinical value of BRD4 as a clinically relevant target in AML, we analyzed the effect of JQ1 on AML LSC. In these experiments, JQ1 effectively induced apoptosis in CD34⁺/CD38⁺ progenitor cells as well as in CD34⁺/CD38⁻ AML stem cells in all donors examined as evidenced by combined surface/Annexin-V staining. Furthermore, JQ1 was found to induce morphologic signs of maturation in 6 of 7 patients examined, thereby confirming our previous data obtained in mouse AML cells. Finally, we were able to show that JQ1 synergizes with Ara-C in inducing growth inhibition in HL60 cells and KG-1 cells. In summary, our data show that small-molecule inhibition of BRD4 has strong anti-leukemic effects in a broad range of AML subtypes. Furthermore, our results support the notion that JQ1's ability to suppress LSC specific transcriptional modules may translate into a therapeutic entry point for eradicating LSC in primary AML. While a more extensive in vivo evaluation of these effects, as well as the development of pharmacologically improved compounds will be required, all existing data unambiguously highlight small-molecule inhibition of BRD4 as a new promising concept in AML therapy.