Bringing an Old Drug to a New Treatment Strategy in Treating FLT-ITD AML - Combination of Homoharringtonine and Sorafenib

Acute myeloid leukemia (AML) is a heterogeneous group of diseases characterized by an abnormal increase of myeloblasts in bone marrow (BM) and peripheral blood (PB). Conventional approach of treatment with standard induction chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT) has reached an impasse with a cure rate of only 30%. A gain-of-function internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) was found in 30% of AML and was associated with an inferior treatment response and clinical prognosis. Despite much interests in FLT3 inhibitors in clinical trials, the response was at best transient, limiting their clinical application. Homoharringtonine (HHT) is a natural alkaloid widely used in Mainland China for the treatment of AML. It is a protein translation inhibitor and affects primarily proteins with short half-lives, including many of the downstream effectors of FLT3 signaling. In this study, we evaluated if HHT can be used in combination with sorafenib in the treatment of FLT3-ITD+ AML and examined the mechanistic basis of their synergism.

The anti-leukemia effects of drugs on AML cell lines with or without FLT3-ITD mutation were evaluated by a high throughput PrestoBlue® fluorometric assay (a measure of viable cell number) after 3-day culture. Synergism between drugs in combination treatment was evaluated based on Excess Over Bliss Additivism (EOBA). The drug effects on leukemia initiating cells (LIC) activity were examined by xenotransplantation using NOD/SCID/IL2Rg^-/- NSG mice and engraftment was enumerated by the presence of human CD45⁺/mouse CD45.1^- cells 6 weeks after transplantation. The effects of HHT and FLT3 inhibitors on FLT3 signaling were examined by Western Blot and Phospho-flow analysis by flow cytometry.

HHT exhibited more potent growth inhibitory effect on FLT3-ITD⁺ AML cell lines, MV4-11 and MOLM-13 (IC50: 3.65 and 3.67 nM) than other AML cell lines (IC50: 7.7 - 32.3 nM). Combination of HHT and sorafenib (H+S) showed pronounced synergism in growth inhibition based on EOBA (29.7% ± 5%) at 3.65 nM (HHT) and 3.85 nM (S). H+S also induced significant increase in apoptosis in MV4-11 based on Annexin V+ population (H+S: 21.3% ± 2.3%; HHT: 11.7% ± 1.9%; S: 8.4% ± 1.0%). Synergism with HHT could also be demonstrated with other FLT3 inhibitors, quizartinib (EOBA: 16% ± 8%) and ponatinib (EOBA: 24% ± 6%) on MV4-11 cell lines. There was no synergism between sorafenib and conventional chemotherapy as exemplified by cytarabine. H+S in vitro significantly reduced engraftment of MV4-11 cells (Vehicle: 65.0% ± 9.7%; H+S: 21.8% ± 8.7%, p<0.01, n=6). HHT alone also inhibited leukemia growth of primary AML samples (IC₅₀: 38.4 ± 7.2 nM, n=88), that was below the peak plasma concentration of HHT (66 nM) in patients receiving treatment. Synergism between H+S was also seen in primary AML samples. Mechanistically, HHT treatment for 6 hours reduced total FLT3 and p-FLT3 protein levels in MV4-11 and MOLM-13. Protein levels of downstream effectors of FLT3 pathway including total Stat5, pStat5, pStat3, pErk were also reduced.

A phase II clinical trial of sorafenib (200-400 mg twice daily continuous) and HHT (1.5 mg/m² for 7 days every 28 days for the first cycle and for 4 days in subsequent cycles, until leukemia progression) combination treatment in patients with chemo-refractory FLT3-ITD+ AML has begun in Hong Kong since January 2014. Bone marrow was performed before and on day 21 after treatment and repeated every two cycles thereafter. Five patients have been treated, including two patients who were primarily refractory to sorafenib monotherapy. Complete remission (CR) was achieved in 1 patient and CR with incomplete hematological recovery (CRi) in four others after 1 cycle. The FLT3-ITD allelic burdens before treatment and at CR/CRi were 77.7 ± 9.6% and 20.0 ± 9.6% (p=0.007). As of 5^th August 2014, the two primary sorafenib refractory patients had leukemia progression at 65 and 95 days after treatment. The other three patients remained in remission at 113, 134 and 150 days after treatment.

In conclusion, HHT and sorafenib demonstrated significant synergistic effect in suppressing the growth of FLT3-ITD⁺ AML cells both in vitro and in vivo. It provides a promising strategy in improving treatment outcome of FLT3-ITD⁺ AML patients.