Targeting Leukemic Stem Cells in Acute Myeloid Leukemia Using a CpG-Linked Anti-Mir-126 Oligonucleotide

Introduction: Acute myeloid leukemia (AML) is a lethal hematopoietic malignancy, characterized by the accumulation of clonal myeloid progenitor cells arrested in development. AML patients have dismal survival odds with a 5-year overall survival (2011 - 2017) of 29.5% (SEER). The main cause of AML treatment failure is the persistence of leukemia stem cells (LSCs). Despite recent approval of new drugs, LSCs are not specifically targeted by AML therapies. MicroRNA (miR)-126 is a non-coding regulatory RNA that is necessary for the maintenance of LSCs in the bone marrow (BM) niche, where vascular endothelial cells (ECs) supply LSCs with miR-126 and promote LSC quiescence. Several lines of evidence validate miR-126 as a target in AML. Preclinical development of CpG-anti-miR126 oligonucleotide, termed miRisten, an inhibitor of miR-126, as a drug to treat AML is described.

Hypothesis: Systemic administration of miRisten will result in pharmacological depletion of miR-126 levels in the ECs, AML blast cells and LSCs, leading to the eradication of LSCs.

Objective: To investigate the pharmacological properties of miRisten to establish a dosing strategy that would result in prolonged knockdown of miR-126.

Experimental Plan: Pharmacokinetic/pharmacodynamic (PK/PD) studies were carried out in healthy wild type mice. miRisten was administered as a bolus or 30 min continuous intravenous infusion (CIVI) at 50 mg/kg (mpk) b.i.d. and the resulting PK parameters compared in plasma. miR126 expression was evaluated in BM mononuclear cells (MNC), lung, liver, kidney, heart and stomach/intestines prior to the fourth dose of miRisten (-30 min; start of infusion = 0 min) and at 30, 60, 90, 120, and 160 min after initiation of infusion. A 21-day preliminary toxicology study was carried out in mice using the same dosing regimen.

Results: Plasma miRisten levels revealed a C_max = 4098 nM (bolus) and 556.1 nM (CIVI), t_½ = 0.16h (bolus) and 0.4h (CIVI) and area under the curve (AUC) of 465.0 nMxhr (bolus) and 508.4 nMxhr (CIVI). Lung, which contains a high concentration of ECs, had the highest relative levels of miR-126 normalized by Sno234 "housekeeping gene", followed by the heart and kidney. BMMNC had the lowest relative levels. In all cases, the nadir of miR-126 levels occurred approximately 1 hour after start of infusion. In BMMNC, the nadir of miR-126 was lower than 0.1% of basal level when miRisten was given CIVI while as a bolus, the nadir was ~33% of basal levels. In all tissues except lung and stomach, the level of miR-126 prior to the 4th dose was lower than at basal levels prior to miRisten exposure. The recovery of miR-126 levels varied by organ but was more pronounced in the lung and stomach, possibly reflecting a more dynamic regulation of miR-126 in these tissues and shorter intracellular t_½ of the miR-126. No significant toxicities were observed by cage-side observation of behavior, monitoring of food/water intake and weight or by histopathological examination of tissues following 21 days of treatment.

Conclusion: These data are consistent with widespread tissue distribution of miRisten and supports administering as CIVI at 50 mpk b.i.d.as miRisten effectively sustains knockdown of miR-126 with this regimen. The AUC was comparable for bolus vs CIVI but the C_max was ~7.5 fold lower with the CIVI which therefore might be safer. When given for 21 day,s miRisten showed no overt or histologically evident toxicity.

Marcucci:Lynx: Membership on an entity's Board of Directors or advisory committees; Abbvie: Other: Speaker and advisory scientific board meetings.