ASP210 - a Potent Oligonucleotide-Based Inhibitor for Combating Both BCR-ABL1 Dependent and Independent TKI-Resistant CML Cells

Background: Tyrosine kinase inhibitors (TKIs) have remarkably changed the management of Ph+ chronic myeloid leukemia (CML) and have dramatically contributed to substantial prolongation of survival rates and superior quality of life. However, long-term clinical experience with TKIs shows that despite the apparent therapeutic benefit, ~35% of CML patients exhibit primary resistance or intolerance to TKIs, and ~25% of those treated is forced to switch TKI at least once during therapy due to acquired resistance. Mutations in the kinase domain of BCR-ABL1 are the most comprehensively described mechanism of TKI-resistance but fail to explain ~40% of resistant cases. Nevertheless, due to the availability of several TKIs, the issue of TKI-resistance has de facto reduced to the mutation T315I and BCR-ABL1 independent resistance. TKI-resistance thus represents a persistent clinical problem to be solved and ultimately calls for alternative treatment solutions for CML patients with leukemic clones escaping TKI therapy. Oligonucleotide therapeutics have generated high expectation stemming from their potency and the field has seen great advancements leading to an apparent progress in clinical trials. Given the vastly different molecular target compared to protein inhibitors (RNA versus protein) as well as principally different mechanism of action, oligonucleotide therapeutics might represent rational and potent tools to combat TKI-resistant CML cells. Progress beyond the state-of-the art: The design of standard oligonucleotides is relatively straightforward and is sequence driven. However, the promiscuity of oligonucleotides places severe limitations on this approach since sequence-dependent off-target effects deteriorate their toxicological profile. Unconventionally, we considered additional parameters during the design of an oligonucleotide-based inhibitor, including steric and thermodynamic aspects of its binding to the target RNA, which significantly improved its safety. Here we report on a potent, safe, and highly specific oligonucleotide-based modality against BCR-ABL1 mRNA, ASP210, which effectively induces apoptosis in both BCR-ABL1 dependent and independent TKI-resistant CML cells. Methods:BCR-ABL1 positive cell lines MOLM-7 a CML-T1, both sensitive and resistant to TKIs imatinib and dasatinib were used to investigate the efficacy of ASP210, a biotinylated 34mer phosphorothioate DNA with an internal poly(ethylene glycol) linker. Briefly, MOLM-7 and CML-T1 cells and their resistant sub-lines were incubated with defined concentrations of ASP210 ranging between 0.25-5.0 µM for 10 days (applied once daily). Cell viability was monitored by trypan blue staining every day. The results were normalized to control, i.e., to untreated cells on the respective day. Of note, MOLM-7 TKI-resistant cells did not possess any mutation in the kinase domain, while both resistant sub-lines derived from CML-T1 harbored the clinically relevant mutation T315I. BCR-ABL1 negative cell line HL-60 was used as target negative control in the same experimental setup. Results:In vitro functional tests of ASP210 revealed its ability to induce cell death of all BCR-ABL1 positive cell types studied. A continuous decrease in the viability of ASP210-exposed cells has been observed already after 24 hours in all cell types including both TKI-sensitive CML cells and the derived TKI-resistant sub-lines irrespective of their T315I status. The efficacy of ASP210 was confirmed to be dose-dependent, with ASP210 causing a ~90% reduction of CML cells by day 5. Importantly, no effect on the viability of BCR-ABL1 negative HL-60 cells was observed (viability remained ≥95%) upon exposure to ASP210. Conclusions: The present study demonstrates the cytoreductive potential of ASP210 in terms of its ability to induce apoptosis in both BCR-ABL1 dependent and independent TKI-resistant CML cells, with no effect on BCR-ABL1 negative cells.