Abstract
Bromodomain and extra-terminal (BET) proteins, including the ubiquitous BRD2/3/4 proteins, are epigenetic readers implicated in c-MYC transcription, cellular proliferation, cell-cycle progression, RNA elongation and DNA damage response. Using shRNA screening and BRD inhibitors, BRD4 has been established as a promising therapeutic target in acute leukemia (Zuber, Nature 2011). In the present study, we investigated the in vitro anti-leukemic effects of the small-molecule BRD2/3/4 inhibitor OTX015 (Oncoethix, Lausanne, Switzerland).
Expression of BRD2/3/4 and c-MYC was assessed by RQ-PCR in 5 myeloid (HL60, KG1, KG1a, K562, NOMO1) and 4 lymphoid (Jurkat, RS4-11, BV173, TOM1) leukemia cell lines and by Western blotting (WB) using commercial antibodies in the HL60, K562, Jurkat and RS4-11 lines. Nineteen AML and ten ALL patient banked leukemic cells were assayed by RQ-PCR only. Cell viability and IC50 values were assessed in cell lines by MTT assays after exposure to OTX015 (0.1nM-10µM) for 72h. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation. Induction of apoptosis was evaluated in cell lines and patient cells by outer membrane phosphatidylserine exposure and PI incorporation at 72 hours with increasing doses of OTX015 (25nM-500nM). Caspase-3 activation and mitochondrial cytochrome c release were studied by immunofluorescence (IF). Maturation was assessed by morphological studies after MGG staining and detection of CD11b by FACS analysis. Modulation of BRD2/3/4 proteins was investigated by WB.
OTX015 IC50 values were in the submicromolar range for KG1 and the MLL-driven NOMO1 cell lines (198.3 and 229.1nM, respectively), while K562 was the most resistant myeloid line, with an IC50 of 11.3µM. In contrast, in lymphoid cell lines tested, IC50 values ranged from 34.2 to 249.7nM, with the MLL-driven cell line RS4-11 being the most sensitive. Cell cycle arrest in subG1/G1 to S transition was observed in 8/9 cell lines and was most pronounced in RS4-11 and BV173. Significant apoptosis (up to 88% Annexin V positive cells) was only observed in KG1a and NOMO1 among myeloid cell lines, while OTX015 induced apoptosis in all lymphoid cell lines tested, ranging from 57% in RS4-11 to 90% in the BCR-ABL+ TOM1 cells. Similarly, OTX015 triggered caspase-dependent cell death, as NOMO1 and RS4-11 displayed significant caspase-3 activation and cytochrome c release, when compared to the resistant K562 cell line. Seven primary patient fresh samples (5 AML, 2ALL) were also analyzed. Ex vivo treatment induced apoptosis ranged from 35% to 87% in 6/7 patients. Exposure to OTX015 at 500nM for 7 days induced maturation in 51% and 65% of HL60 cells as detected by CD11b expression and morphology, respectively. Baseline expression of BRD2/3/4 varied among cell lines or patient samples, lower BRD2/3/4 expression levels were observed in the BCR-ABL+ K562 and BV173 cell lines, as well as in the 4 BCR-ABL+ ALL samples analyzed. Upon OTX015 exposure, down-regulation of the BRD4 target gene c-MYC was observed in all cell lines, without clear correlation with the proliferation inhibition rate and/or the intensity of induced apoptosis while no consistent BRD2/3/4 mRNAs down-regulation was seen. Interestingly, BRD2 protein was down-regulated in HL60, Jurkat and RS4-11 cell lines, but not in the K562 cell line.
OTX015 affects cell viability, induces cell cycle arrest in G1/S phase, and is able to induce significant apoptosis in leukemic cell lines and fresh AML and ALL samples at submicromolar drug concentrations. These concentrations were achieved in the serum of healthy volunteers after safe administration of the drug. With such characteristics, OTX015 appears to be an attractive anti-leukemic therapy, currently under early evaluation in a Phase Ib dose-escalation trial conducted in relapsed/refractory AML/ALL patients.
Riveiro:Oncology Therapeutic Development: Employment. Herait:Oncoethix: Employment. Dombret:Oncoethix: Research Funding.
Author notes
Asterisk with author names denotes non-ASH members.
