Efficacy of OR21, a Novel Oral Demethylating Agent in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disease induced by t(9;22)(q34;q11) translocation. The prognosis of patients with CML has dramatically improved since tyrosine kinase inhibitors (TKIs) were introduced, and recent studies show that approximately 40 to 50 % of CML patients achieved deep molecular response (DASISION and ENESTnd trial) within several years. However, therapeutic options for patients with CML who are resistant for TKIs are limited. Besides BCR-ABL kinase domain mutation, somatic mutations associated with epigenetic gene alteration (e.g. TET, DNMT3A) are reportedly involved in TKI resistance and disease progression. Thus, we investigated the efficacy of DNA demethylating agents in CML. Azacitidine (AZA) and decitabine (DAC), currently available as DNA demethylating agents, have low bioavailability for oral administration because they are easily degraded by cytidine deaminase. Then, we are developing a novel demethylating agent, OR21, with possible oral absorbability as a prodrug of DAC. In vivo analysis using cynomolgus monkeys, the area under the curve of DAC after intraduodenally administration of DAC (1.5mg/kg, 6.6µmol/kg) or OR21 (2.25mg/kg, 6.6µmol/kg) was 0.01 µM·h, 0.298 µM·h respectively, indicating OR21 have high oral absorbability.

To assess the demethylating activity of OR21 for CML, we performed western blot analysis and bisulfite pyrosequencing assay to measure LINE1 methylation, using CML cell-lines (K562, BV173). OR21 decreased DNMT1 protein level as a result of demethylating and LINE1 methylation in K562 and BV173 comparable to DAC. Next, we performed cell growth inhibition and cell apoptosis assay after 72 hours exposure of OR21 to assess anti-tumor effect in vitro. OR21 inhibited cell growth comparable to DAC in CML cell-lines (K562, BV173, KCL22, MYL) in a dose-dependent manner. Notably, OR21 inhibited the cell growth and apoptosis against BV173 with an extremely low concentration (IC50; 5nM) than that of AZA (IC50; 122nM) via significant accumulation of reactive oxygen species. Whereas, OR21 weakly induced cell apoptosis against K562. OR21 induced G₂/M phase cell-cycle arrest in CML cell-lines except for BV173 via pRb downregulation. These results indicated mechanisms of anti-tumor effect in OR21 were induction of cell apoptosis (BV173) or cell cycle arrest (K562, KCL22, MYL). Because of the different mechanism of action, we assessed whether OR21 and TKIs combination can enhance the anti-tumor effect of CML. We investigated the combination effects of OR21 with imatinib (IM) or dasatinib (DAS) in K562. Combination index values at IC₈₀ calculated by Calucusyn software showed 0.642±0.129 (with IM), 1.182±0.2 (with DAS), respectively. Accordingly, OR21 combined with TKIs showed at least additive or synergistic effect for K562.

TKI resistance, which can be associated with somatic mutations leading to epigenetic gene alteration or loss of function of p53, is an obstacle for molecular remission in patients with CML, thus we examined the effects of OR21 in TKI resistant cell lines or efficacy on p53 mutational status. OR21 inhibited the cell growth in IM-resistant cell-line MYL-R, a derivative of MYL, which had overexpression of Lyn, and Ba/F3 BCR-ABL^T315I, which exogenously expressed Bcr-Abl (T315I), indicating OR21 could overcome TKI resistance in CML. OR21 or cytarabine did not enhance cell apoptosis against K562, MYL and KCL22 (p53 deficient or mutant cell lines) combination with nutlin-3a (MDM-2 inhibitor), while increased cell apoptosis was observed in BV173 (p53 wild type) treated with cytarabine and nutlin-3a, but not with OR21 and nutlin-3a. These results suggested the effects of OR21 did not depend on p53 mutational status.

Finally, we used a mouse xenograft model to evaluate anti-tumor effect of OR21 in vivo. BALB/c Rag-2/JAK3 double-deficient (BRJ) mice were injected intravenously via tail vein with　5 ×10⁶ BV173 cells. OR21 were administered at a dose of 2.7mg/kg (equivalent to DAC 1.0mg/kg in AUC) and PBS (vehicle) twice weekly. OR21 significantly prolonged survival in a xenograft mice model (median 35 days vs not reached, P<0.01).

In conclusion, a novel orally available demethylating agent OR21 is effective for CML cells including TKI resistant clones. The efficacy and safety of OR21 for CML is expected to be verified by early-phase clinical trials.