Preclinical Evaluation of Bisantrene As Single Agent and in Combination with Decitabine for Acute Myeloid Leukemia

Anthracyclines, particularly doxorubicin and idarubicin, together with cytarabine, have comprised standard of care induction chemotherapy in acute myeloid leukemia (AML) for almost 4 decades. Although 60% of patients achieve remission, the majority relapse within 1-2 years. As such, outcomes in AML remain poor, with overall 5-year survival around 27%. In elderly AML patients the situation is worse, with 80% dying within one year of diagnosis. Toxicity of induction therapy is a major barrier to treatment success, precluding many unfit and elderly patients. Hypomethylating agents (HMA; azacytidine, decitabine) provide a less toxic alternative and have improved treatment options for the unfit. However, only 50% of patients respond to HMA therapy and most eventually acquire resistance, resulting in relapse. Approaches that reduce the toxicity of induction therapy and improve the efficacy of HMA treatment are emerging, e.g. combinations with Venetoclax. Nevertheless, novel combination strategies that improve outcomes in certain mutation subtypes need to be explored. Bisantrene, an anthracene derivative developed over 4 decades ago as a less cardiotoxic chemotherapy alternative to anthracyclines, shows broad activity across multiple solid and haematological malignancies. Several clinical studies in the 1980s showed bisantrene is an effective AML salvage therapy, producing response rates up to 50% without accompanying cardiotoxicity (Rothman J., 2017 Int J Cancer Res Ther;2(2):1-10) In a recent investigator-led Phase II trial of heavily pre-treated relapsed/refractory AML patients, 1/10 achieved a complete remission and 3/10 patients achieved partial remission. Interestingly, all responders were characterised by extramedullary disease (Cannani et al., 2020 Eur J Haematol 2021;106:260-266). We postulate that bisantrene may represent a suitable candidate for combination with HMA in patients unfit for standard induction chemotherapy. To explore this idea, we evaluated the in vitro and in vivo effects of bisantrene both alone and in combination with decitabine. The in vitro activity of bisantrene was initially assessed in a range of human and mouse AML cell lines covering the major molecular and clinical subtypes. All cells were sensitive to bisantrene, showing IC 50 values in the range 16-563 nM. FLT3-ITD + cells in particular were sensitive, while NRAS, KRAS and KIT mutant lines were also responsive. When tested against primary patient AML samples, 15/49 (30.6%) responded strongly to bisantrene treatment ex vivo over the dose range 0.1-1 µM. Bisantrene was shown to induce G1 accumulation and dose-dependent apoptosis in the FLT3-ITD + cell line, MOLM13. When tested in combination with decitabine, pre-treatment with decitabine for 24 h followed by decitabine plus bisantrene for 3 days produced synergistic cytotoxicity in MOLM13, MV4-11 and OCI-AML3 cells. Bisantrene and decitabine together resulted in S phase accumulation in MOLM13 cells and synergistic induction of apoptosis. The in vivo efficacy of bisantrene alone and in combination with decitabine were examined using the MOLM13- luc cell line and a patient derived xenograft (PDX; AML16) (FLT3-ITD +, NPM1, IDH2 and WT1 mutant) in NSG mice. Bisantrene (10 mg/kg i.v. twice/week) significantly reduced leukemic burden and increased median survival compared to vehicle control in MOLM-13- luc engrafted mice, and significantly reduced leukemic burden and increased median survival in a dose and schedule-dependent manner (2.5mg/kg and 5mg/kg, twice or three times weekly) in the PDX model. The combination of bisantrene (5 or 10 mg/kg twice weekly) and decitabine (0.2 mg/kg i.p. 5 days/week) significantly improved survival in MOLM13- luc (Figure 1) and PDX AML16 (Figure 2) engrafted mice, respectively, relative to vehicle control and each single agent. The combination also reduced leukemic infiltration to extramedullary sites (spleen, liver, uterus, brain). These data support that addition of bisantrene to HMA backbone therapy may provide an important new treatment strategy in AML. A prospective Phase Ib/II trial is being planned to evaluate this new therapeutic approach.