Abstract
Background and Rationale: Maternal embryonic leucine zipper kinase (MELK) is a Ser/Thr protein kinase that is aberrantly expressed in many tumor types and demonstrated to be important in formation and maintenance of cancer stem cells. We have previously reported that MELK is also aberrantly expressed in AML cell lines and primary patient leukemia cells and is associated with a poor prognosis (Alachkar et. al., Oncotarget, 2014). MELK knockdown resulted in leukemia cell growth inhibition and apoptosis. Based on these preclinical insights, a small molecule inhibitor of MELK kinase activity, OTS167, (OncoTherapy Sciences, Tokyo, Japan) has been developed and is being tested in an early phase clinical trial in advanced myeloid malignancies at the University of Chicago (IRB#15-1373). Here, we report the activity of MELK signaling blockade in AML with FLT3 mutations, which are associated with drug resistance and poor clinical outcomes, and demonstrate synergy with known FLT3 kinase inhibitors.
Methods: Both MELK and FLT3-ITD activity were targeted by treating the human AML cell lines MV4:11, MOLM-13, KOCL-48 and KG-1 which aberrantly express MELK as well as a FLT3-ITD, FLT3-TKD or FLT3-WT with the small molecule MELK inhibitor OTS167 alone or in combination with FLT3 kinase inhibitors Crenolanib or Gilteritinib.
Results: OTS167 induced cell death of FLT3 mutant cell lines in a dose dependent manner and with IC50 similar to known FLT3 kinase inhibitors (OTS167 IC50 1.8-18.2nM; Crenolanib IC50 2.31-13.8nM; Gilteritinib IC50 4.9-36.3nM). Using isobologram analysis, OTS167 in combination with either Gilteritinib or Crenolanib induced synergistic cell death in FLT3 mutant leukemia cells at 48hrs (Figure 1A), but not in FLT3-WT leukemia cells. We found that OTS167 alone had the effect of inhibiting auto-phosphorylation of FLT3-ITD, as well as inhibiting MELK activity resulting in downregulation of MELK protein expression. Treatment of cells with 50nM OTS167 resulted in loss of FLT3-ITD (Y591) phosphorylation within 1hr, and total FLT3-ITD protein expression within 24 hours (Figure 1B). Loss of FLT3-ITD protein expression occurred in the presence of FLT3 mRNA and was not due to decreased stability of the protein, as treatment with OTS167 in the presence of cycloheximide did not decrease FLT3-ITD lifespan. Notably, the loss of FLT3-ITD protein expression observed with OTS167 treatment was not observed with the same concentration of FLT3 inhibitors Crenolanib or Gilteritinib. In vitro kinase assays using recombinant FLT3-ITD confirmed that OTS167 could act directly to inhibit FLT3-ITD kinase activity at an IC50 of 4.9nM, compared to a reported MELK IC50 of 0.11-1.1nM (Chung, et. al., Oncotarget, 2012). In MV4:11 cells, the constitutive activation of the MAPK, PI3K, and STAT5 signaling pathways by FLT3-ITD is inhibited by 50nM OTS167, similar to inhibition by 50nM FLT3 inhibitors Crenolanib and Gilteritinib (Figure 1C).
In addition, treatment with either OTS167 or FLT3 inhibitor resulted in loss of MYC mRNA and protein expression at 3hr, suggesting that more effective downregulation of MYC expression by the combination may increase the susceptibility of FLT3 mutant AML to apoptosis. The inhibition of FLT3-ITD activation and expression after OTS167 treatment was not observed when MELK protein activity was downregulated using siRNA, suggesting the inhibitor may simultaneously target two independent signaling pathways active in cellular transformation. These results indicate that OTS167 may have a dual specificity in FLT3 mutant AML through targeting both aberrant MELK and FLT3 activity.
Conclusion: In summary, the MELK inhibitor, OTS167, may be an attractive therapeutic agent for FLT3 mutant AML, with dual targeting resulting in decreased levels of FLT3-ITD protein and kinase activity. These data provide a rationale for testing dual MELK and FLT3 kinase inhibitors in patients with FLT3 mutant AML.
Odenike: CTI/Baxalta: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria. Stock: Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.
