Optimized Induction of Mitochondrial Apoptosis By Combination Therapies with Venetoclax for Chemotherapy-Free Treatment of BCR-ABL+ Acute Lymphoblastic Leukemia in Preclinical Models
Authors: Eder Matthias , Kirchhoff Hanna , Battmer Karin , Wohlan Katharina , Ricke-Hoch Melanie , Erschow Sergej , Law Edward , Kloos Arnold , Heuser Michael , Ganser Arnold , Hilfiker-Kleiner Denise , Heidenreich Olaf , Scherr Michaela 
Published: October 2019

Abstract
Background: BCR-ABL+ acute lymphoblastic leukemia (ALL) in adults has a poor prognosis with allogeneic stem cell transplantation (SCT) considered the best curative option for suitable patients. BH3 mimetics induce mitochondrial outer membrane permeabilization (MOMP) linked to apoptosis induction by releasing BH3-only proteins BIM and/or BID from the anti-apoptotic factors BCL2, BCL-XL, MCL1, BCLW and BFL1. The BCL2-specific BH3 mimetic venetoclax (ABT-199) may provide an opportunity to improve pharmacotherapy of BCR-ABL+ ALL in particular for elderly patients not suitable for SCT.
Aim: We aimed to rationally design optimized combination therapies for BCR-ABL+ ALL based on the molecular mechanisms of apoptosis induction by BH3 mimetics.
Methods: We first biochemically characterized binding of BIM, a BH3 only activator of mitochondrial apoptosis, to BCL2, BCLXL and MCL1 and its release by BH3 mimetics in two BCR-ABL+ ALL cell lines. We next visualized and quantitated MOMP-induction by BH3 mimetics in viable cells. We then characterized the effects of dexamethasone and tyrosine kinase inhibitors (TKI) imatinib and dasatinib on BIM expression and calculated dose-effect combination indices (CI) for combination therapies in cell lines and two BCR-ABL+ ALL primograft models co-cultured with mesenchymal stem cells ex vivo. We finally used in vivo bioluminescence and survival analyses in murine xenotransplantation models to evaluate therapeutic efficacy in vivo.
Results: In BCR-ABL+ BV173 and SUPB15 cells BIM but not BID binds to BCL2. BIM is rapidly released from BCL2 by venetoclax in a time and dose dependent manner. Release of BIM induces both MOMP (as defined by a decrease in mitochondrial membrane potential) and apoptosis (as defined by PARP cleavage and propidium iodide staining). Furthermore, BIM is strongly required for cytotoxicity of venetoclax, dasatinib and dexamethasone. Primary BCR-ABL+ ALL cells are more resistant against MOMP induction by venetoclax than BCR-ABL-negative ones, and BIM expression is reduced in these cells. Both, TKIs and dexamethasone augment BIM expression in BV173 and SUPB15 cells and act synergistically with venetoclax in cell lines and two BCR-ABL+ primografts (CI for the triple combination therapy of venetoclax, dexamethasone and dasatinib between 0.1 and 0.15, CI< 1.0 considered synergistic). Triple combinations with venetoclax, dexamethasone and TKIs efficiently attenuate leukemia progression in xenotransplantation models and, notably, the dasatinib- but not the imatinib-containing combination led to treatment- and leukemia-free long-term survival in a BCR-ABL+ mouse model.
Conclusions: These data demonstrate efficacy of venetoclax in ALL. Although BCR-ABL inhibits venetoclax cytotoxicity, this inhibition can be overcome by triple combination therapy with venetoclax, dexamethasone and dasatinib. Since the triple combination therapy can be curative in preclinical xenotranplatation models clinical studies with oral chemotherapy-free regimens may be considered in particular for elderly patients not suitable for allogeneic SCT.

Disclosures
Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees.