Abstract
Pharmacologic inhibition of BCR-ABL by tyrosine kinase inhibitors (TKI) provides effective therapy for chronic phase but not blast crisis (BC) CML. The inability of TKIs to control BC CML is due to the reactivation of BCR-ABL through TKI resistance-conferring mutations, as well as the activation of alternative oncogenic pathways that mediate acquired leukemia stem cell (LSC) function and differentiation block in leukemic progenitors. Accordingly, the development of effective BC therapeutics will require reversing multiple contributors to the BC phenotype, and indicates that targeting BCR-ABL alone will not be sufficient to control BC CML. Indeed, despite the ability of third-generation TKIs to effectively inhibit the majority of clinically-relevant BCR-ABL kinase domain mutations, most patients with BC treated with these agents continue to relapse despite initial responses. Because our recent work has highlighted the importance of the MNK kinase-eIF4E axis in b-catenin-associated BC self-renewal (Lim et al., PNAS18;110(25):E2298-307, 2013), we set out to develop compounds capable of targeting BCR-ABL, TKI-resistant BCR-ABL mutants, as well as the MNK kinases simultaneously. Here, we report the discovery and characterization of two lead compounds ETC-219 and ETC-027 that inhibit clinically relevant BCR-ABL isoforms (E255K, T315I) as well as the MNK1/2 kinases at nanomolar potency (Table 1).Table 1In vitro kinase assays for ETC-219 and ETC-027.Compound IDIC50 MNK1 (µM)IC50 MNK2 (µM)IC50 ABL (µM)IC50 ABL (T315I) (µM)IC50 ABL (E255K) (µM)ETC-2190.2540.0150.0140.0130.148ETC-0275.0150.0240.2140.0913.19
Using a panel of 104 kinases, we showed that ETC-219 and ETC-027 inhibited 90% of the activity of 15 and 12 kinases respectively when tested at 1uM. ETC-219 and ETC-027 interact with only 27 and 20 of off-target kinases respectively, and offer significant selectivity over kinases outside of the MNK and BCR-ABL families.
ETC-219 and ETC-027 have favorable pharmacokinetic properties, and include the following ADME characteristics (Table 2).Table 2ADME characteristics of ETC-219 and ETC-027.ETC-219ETC-027ParametersUnitIVPOIVPODosemg/kg1050550C0ng/ml21241714Cmaxng/ml12122490Tmaxh12T1/2h22.21.82.1CLL/h/kg39.72.83.5VzL/kg8.831.37.410.8VssL/kg7.25.8AUC0-t(last)ng.h/ml32905128179514337AUC0-infng.h/ml33025133179814349Extrapolated area for AUC0-inf%0.37000.11000.16000.0800F%3180
Regarding targeting of BCR-ABL-dependent cellular proliferation and survival, we found that ETC-219 and ETC-027 exhibited potent anti-proliferative activity against a panel of BC CML cell lines (K562, BV-173, EM-2, KCL-22, JURL-MK1), as well as Ba/F3-BCR-ABL cells containing the T315I mutation. In an in vivo xenograft model, employing K562-eIF4E cell lines in NOD-SCID mice, we found that both compounds inhibited tumor growth in a dose-dependent manner, and a dose-dependent decrease in eIF4E phosphorylation in tumor explants were also observed. Tumor regression was observed at 100 mg/kg for ETC-219. Importantly, mice given ETC-219 and ETC-027 at 100 mg/kg and 200 mg/kg doses did not exhibit significant toxicity.
With respect to MNK-eIF4E-b-catenin axis inhibition, ETC-219 and ETC-027 inhibited eIF4E phosphorylation at IC50s of 0.186 nM and 0.28 nM respectively in HeLa cells. In parallel, both compounds inhibited Wnt/b-catenin reporter activity in BC CML cell lines, as well as the expression of a panel of Wnt-regulated genes. Using the serial replating assay as a readout for LSC function and self-renewal capacity, we found that ETC-219 and ETC-027 were effective at 1nM at preventing serial replating using primary CD34+ cells from patients in BC. Furthermore, ETC-219 and ETC-027 inhibited the serial replating efficacy of BC cells from patients with clinical resistance to the third generation TKI, ponatinib.
Conclusion
Our results demonstrate that: i) small molecule compounds with favorable ADME properties can be developed to potently and specifically inhibit the BCR-ABL and MNK kinases simultaneously; ii) Dual specific MNK and BCR-ABL inhibitors are effective at abrogating BCR-ABL-driven growth and proliferation, as well as the MNK-eIF4E-dependent self-renewal function of BC LSCs. We conclude that dual specific BCR-ABL and MNK inhibitors may warrant testing in patients with BC CML.
Disclosures:
Chuah: Novartis: Honoraria; BMS: Honoraria.
Pretreatment or Posttreatment with Aripiprazole Attenuates Methamphetamine-induced Stereotyped Behavior in Mice