Program: Oral and Poster Abstracts
Session: 617. Acute Myeloid Leukemia: Biology, Cytogenetics and Molecular Markers in Diagnosis and Prognosis: Poster II
METHODS: (A) Five AML cell lines (OTX015 IC50 60 – 10,000 nM) were studied: OCI-AML3, NOMO-1, HL-60, KG1a and K562. After 24h starvation, OTX015 or vehicle (DMSO) was added daily to the culture medium for 3 days at various concentrations. After 96h, cells were assessed for gene expression by RT-qPCR and seeded in methycellulose. Colonies were scored after 14 days. (B) Bone-marrow mononuclear cells (BMNC) from AML patients obtained at diagnosis after informed consent were cultured for three weeks in a niche-like hypoxic milieu shown to maintain leukemic stem cells (Griessinger et al. Stem Cells Transl Med. 2014). OTX015 200 nM or DMSO was added weekly. This concentration is in the range of trough concentrations achievable at the MTD of OTX015 in phase I trials. Residual leukemic cells were sorted and plated on methylcellulose. Colonies were scored after 14 days. The resulting L-LTC-IC frequency was reported relative to the number of BMNC initially seeded.
RESULTS: (A) To dissect the effect of OTX015 on AML progenitors from that on the leukemic bulk, we determined for each cell line a maximal OTX015 concentration that could be administered repeatedly for 3 days without significantly impairing proliferation or viability (MTT) at day 4 of culture (referred as low-dose concentration). As expected, this target concentration, ranging from 50 to 500 nM, was lower in cell lines with low OTX015 IC50. This prolonged low-dose exposure to OTX015 recapitulated BETi-associated gene expression changes including CMYC downregulation and HEXIM1 upregulation in all cell lines, and significantly reduced clonogenicity compared to DMSO in 4/5 cell lines, but not in NPM1-mutated OCI-AML3 cells (IC50: 60 nM, target concentration 50 nM), despite modulation of CMYC and HEXIM1 expression. Overall, there was no correlation between the level of CMYC repression and clonogenicity. Transcriptome analyses are ongoing to identify gene expression changes specifically associated with inhibition of clonogenicity.
(B) L-LTC-IC frequency after prolonged exposure to 200 nM OTX015 was determined in specimens from 11 AML patients with variable oncogenetics. L-LTC-IC frequency was reduced in 5/11 patients, reaching statistical significance in 3 cases; OTX015 reduced L-L-LTC-IC in 3 of 4 NPM1-mutated samples, but not in any of the 3 patients with high-risk cytogenetics. No clear correlation was found between induction of apoptosis on primary blasts after short-term, and L-LTC-IC reduction after long-term 200nM OTX015 exposure respectively. Patients’ samples number is being extended to identify oncogenetic predictors of L-LTC-IC reduction.
CONCLUSION: Our results suggest that in AML cell lines or primary samples, prolonged exposure to low concentrations of the clinically-available BET inhibitor OTX015 results in activity against leukemic progenitors independent of induction of proliferation arrest or apoptosis in blasts. Molecular mechanisms and oncogenic markers of this activity are being investigated. These results warrant clinical investigation of the anti-leukemic properties of prolonged low-dose OTX015 administration.
Disclosures: Riveiro: Oncoethix: Research Funding ; OTD: Employment . Herait: Oncoethix: Other: shareholder ; Oncoethix: Other: Chief medical officer ; Oncoethix: Other: shareholder . Dombret: Oncoethix: Research Funding . Itzykson: Oncoethix: Research Funding .
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