Gilteritinib and Venetoclax Synergize to Eliminate FLT3/ITD+ Leukemia Cells through BIM
Authors: Zhu Ruiqi , Li Li , Nguyen Bao , Duffield Amy S. , Small Donald 
Published: November 2019

Gilteritinib and Venetoclax synergize to eliminate FLT3/ITD+ leukemia cells through BIM
Abstract
Acute myeloid leukemia (AML) is characterized by a clonal proliferation of immature myeloid cells in the bone marrow or other tissues. The most commonly mutated gene in AML is FMS-like tyrosine kinase (FLT3). FLT3 downstream signaling pathways include PI3K/AKT, STAT5 and MAPK, which affect apoptosis, differentiation and cell proliferation. An internal tandem duplication mutation in FLT3 (FLT3/ITD) is identified in approximately 25% of patients with AML, and this mutation is associated with a particularly poor prognosis. A subset of AML also has a point mutation in the tyrosine kinase domain of FLT3 (FLT3/TKD); however, this mutation does not have the same pronounced impact on prognosis as the FLT3/ITD mutation.
Since tyrosine kinases are an attractive drug target, tyrosine kinase inhibitors (TKIs) that target FLT3 have been developed, including recent agents that show enhanced specificity, such as Gilteritinib. Despite these advances, TKI monotherapy continues to show limited success, indicating that combination therapy is likely necessary for the effective treatment of FLT3/ITD AML. As FLT3 signaling pathway activity is known to be anti-apoptotic, in this study we investigated the combinatorial effect of a FLT3-selective TKI and the BCL-2 inhibitor Venetoclax. The BCL-2 protein plays a key role in apoptosis, with anti-apoptotic/prosurvival effects. Venetoclax is a selective BCL-2 inhibitor, and is used clinically in the treatment of chronic lymphocytic leukemia and relapsed/refractory AML.
We first investigated the combinatorial effect of treatment with Gilteritinb and Venetoclax in a FLT3/ITD+ leukemia cell line (Molm14). Combined treatment with Gilteritinib (20nM) and Venetoclax (80nM) reduced cell proliferation by 83.7%, as compared to Gilteritinib (62.2%, P<0.05) or Venetoclax (37.3%, P<0.05) alone. The drug combination demonstrated synergy (CI=0.42). Combined treatment shows that these agents act synergistically to enhance apoptosis (88.2%), as compared with Gilteritinib (52.1%, P<0.05) or Venetoclax (12.1%, P<0.05) alone. Moreover, the combined treatment also significantly reduced cell proliferation in patient samples with FLT3/ITD+ and FLT3/TKD mutations. Further experiments with cell line (Molm14 cells resistant to 60nM CEP-701) showed that Venetoclax can re-sensitize FLT3 TKI-resistant cell lines to TKI treatment. Western blot analysis indicates that this effect is mediated by inhibiting MAKP pathway-whose reactivation is an important reason for TKI resistance in FLT3/ITD+ patients. These data demonstrate that combined treatment with Gilteritinb and Venetoclax reduces cell proliferation and enhances apoptosis in a FLT3/ITD leukemia cell line.
We next investigated the mechanism of action of this drug combination. BCL-2 and other antiapoptotic proteins in this subfamily (i.e. MCL-1, BCL-XL) exert their pro-survival effects by sequestering BIM. BIM is a proapoptotic protein, that, when released by BCL-2, can activate cell death mediators. These cell death mediators include BAX and BAK, which perforate the mitochondrial membrane, resulting in apoptosis. Venetoclax acts by binding to BCL-2 and displacing BIM, freeing BIM to associate with and activate cell death mediators, resulting in apoptosis. We found that in Molm14 cells, Venetoclax not only dissociates BIM from BCL2, but also decreases expression of BIM and enhances the binding of BIM and MCL-1. In contrast, we found that Gilteritinib increases expression of BIM, reduces the binding of BIM with MCL-1 via a reduction of MCL1 expression, and shows enhanced binding of BIM with BCL2. Cells treated with both Venetoclax and Gilteritinib show dissociation of BIM from both BCL2 and MCL-1, though the interaction between BIM and BCL-XL is not affected. Combination treatment also showed increased binding between BIM and the cell death mediator BAX, leading to increased apoptosis.
These studies provide evidence that the addition of Venetoclax may enhance TKI therapy in the treatment of FLT3/ITD leukemia. Additionally, these findings suggest that enhanced cell death in FLT3/ITD AML cells treated with combination therapy occurs because Venetoclax mitigates unintentded pro-survival effects of the TKI, including an increase in BIM expression and increased association between BIM and BCL-2.

Disclosures
Duffield: MedImmune: Consultancy; Boston Biomedical/Sumitomo Dainippon Pharma Co., Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Small:Pharos I, B & T: Consultancy, Research Funding; InSilico Medicine: Membership on an entity's Board of Directors or advisory committees.