The 3-Drug Combination Treatment ART838, ABT199 Plus Sorafenib Is Effective Against AML Xenografts, Possibly Via Cooperative Targeting of MCL1
Authors: Moses Blake S , Fox Jennifer , Chen Xiaochun , McCullough Samantha , Tran Sang Ngoc , Kirkpatrick Allison , Kumar Kalyani , Tyner Jeffrey W. , Posner Gary H. , Civin Curt I. 
Published: October 2019

Abstract
Antimalarial artemisinins have broad antineoplastic activity in vitro, are well tolerated and inexpensive, and can be parenterally or orally administered in humans. Artemisinin-derived trioxane diphenylphosphate dimer 838 (ART838; a potent artemisinin-derivative) inhibited acute leukemia growth in vivo and in vitro, at doses where normal human CD34+ hematopoietic stem-progenitor cell clonogenicity was essentially unaffected (Fox et al, Oncotarget 2016, PMID: 26771236). In our focused drug combination screen for drugs that synergize with ART838, the only BCL2 inhibitors in the screen library of 111 emerging antineoplastic compounds, navitoclax (ABT737) and venetoclax (ABT199; FDA-approved), were identified as 2 of the top 3 candidates. Synergies between ART838 and BCL2 inhibitors were validated in multiple acute leukemia cell lines and primary cases. This ART838-BCL2 inhibitor synergy may be due to reduced levels of MCL1 protein that we and others have observed in multiple acute leukemia cell lines and primary cases treated with artemisinins (Budhraja et al, Clin Cancer Res 2017, PMID: 28974549).
Treatment of acute leukemia xenografts with the ART838 plus ABT199 combination reduced leukemia growth rates and prolonged survivals, compared to vehicle or either ART838 or ABT199 alone. To add to the efficacy of this ART838 plus ABT199 treatment regimen, we sought to rationally add a third low-toxicity active antileukemic agent. Sorafenib (SOR; FDA-approved) inhibits multiple kinases which may mediate its antileukemic activity, with the importance of the targets varying from case to case; e.g. FLT3 is an important target in many AMLs. In addition, several reports have found that SOR reduces MCL1 protein stability and translation through inhibition of the ERK and PI3K pathways (Wang et al, Clin Cancer Res 2016, PMID: 26459180; Huber et al, Leukemia 2011, PMID: 21293487). In all acute leukemia cell lines tested, we observed large reductions in MCL1 protein levels with SOR treatment, which may further rationalize the addition of SOR to our ART838 plus ABT199 antileukemic regimen. We had previously observed strong in vitro synergy between ART838 and SOR (PMID: 26771236). Treatment of acute leukemia xenografts with the ART838 plus SOR combination reduced leukemia xenograft growth rates and prolonged survivals, compared to single drugs.
Mice bearing luciferase-labelled acute leukemia xenografts were treated (PO daily x5) with single drug or 2-drug or 3-drug combinations of ART838, ABT199, and SOR, each at their individual maximally tolerated doses. Treatment with this 3-drug combination caused rapid regression of luciferase-labelled MV4;11 AML xenografts (Fig 1A). The 5-day treatment cycles were repeated every other week, and mice receiving this 3-drug combination survived >4 times longer than vehicle-treated mice (Fig 1B). Mouse body weights were stable during treatment. Although myelosuppression is the human clinical dose-limiting toxicity of each of these 3 drugs, mouse blood cell counts during 3-drug combination treatment were in the normal range. Treatment of a luciferase-labelled primary AML leukemia xenograft with this 3-drug combination reduced leukemia growth more than the single drugs or 2-drug combinations (Fig 1C). Assessment of efficacy and pharmacokinetics-pharmacodynamics against diverse acute leukemia xenografts will test this combination of ART838, ABT199 plus SOR as a rational low-toxicity drug triad for treatment of acute leukemias and potentially other cancers.

Disclosures
Fox: Intrexon Corporation: Employment. Tyner:Genentech: Research Funding; Janssen: Research Funding; AstraZeneca: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Constellation: Research Funding; Array: Research Funding; Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aptose: Research Funding. Civin:ConverGene LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GPB Scientific LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 3DBioWorks Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BD (Becton Dickinson): Honoraria.