Sorafenib (BAY 43-9006) Directly Targets FLT3-ITD in Acute Myelogenous Leukemia.
Authors: Zhang Weiguo , Konopleva Marina , Shi Yue-Xi , Harris David , Small Donald , Ling Xiaoyang , Estrov Zeev , Cortes Jorge , Andreeff Michael 
Published: October 2019

Fms-like tyrosine kinase 3 (FLT3) gene mutations, including internal tandem duplication (ITD) and missense point mutations, are frequent molecular abnormalities in acute myeloid leukemia (AML), which are associated with poor prognosis. The Raf-MEK-Erk pathway is one of the downstream targets activated by FLT3 mutations. Our previous studies have demonstrated that Erk is constitutively phosphorylated in the majority of primary AML samples and is associated with poor prognosis in AML, even in the absence of Flt3 abnormalities (Ricciardi et al., 2005; Kornblau et al., 2001 and 2006). Sorafenib (BAY 43-9006), a small molecule inhibitor of c-Raf, has been shown to be a potent inhibitor of wild-type FLT3 (Wilhelm et al., 2004) in addition to targeting Raf and other kinases. We demonstrated that sorafenib selectively induces growth arrest and apoptosis in FLT3-mutant human and murine cell lines at a 1,000-fold lower concentration (IC50 1–3nM) compared to the cells with FLT3 wild-type. Sorafenib also induced a much more pronounced inhibition of colony formation in FLT3-ITD-harboring primary AML cells (IC50=10 ± 2 nM) compared to FLT3 wild-type AML samples (IC50= 2,680 ± 1.8 μM). In a murine leukemia model bearing GFP-transduced Ba/F3-ITD cells, oral administration of sorafenib, at 10 mg/kg/day for 15 days, prolonged the median survival of the mice to 36.5 days compared to 16 days of vehicle group (p=0.002). Histopathology showed significant hepato-splenomegaly in control mice compared to sorafenib-treated mice, and significant reduction of GFP positive leukemia cells in spleen, liver and bone marrow. In an ongoing Phase I clinical trial, 3 patients (pts) with FLT3-ITD have been treated: the 1st had a dramatic decrease of circulating blasts from 92% to 2% in 7 days after oral administration of sorafenib 200 mg/BID and a decrease in BM blasts (95% to 50%), associated with significant down-regulation of phospho-FLT3. The 2nd pt had clearance of peripheral blood blasts from baseline of 97% to undetectable after 7 days and bone marrow blasts decreased from 96% to 25%. The 3rd pt has had a significant decrease in peripheral blasts from 82% to 45% (absolute blasts 10.57 to 0.81 ×109/L). Mechanistically, sorafenib directly inhibited autophosphorylation on tyrosine residues in ITD mutant, but not in FLT3 wild-type protein in the in vitro kinase assays, and blocked downstream targets of mutant FLT3-ITD such as phospho-Akt, phospho-Stat5, phospho-ERK and Pim-1. Further, sorafenib inhibited expression of the anti-apoptotic Mcl-1 and Bcl-XL proteins and induced pro-apoptotic Bim levels in Ba/F3-ITD but not in Ba/F3-FLT3 cells. These results imply that sorafenib directly targets mutant FLT3-ITD protein and its downstream signaling, which may account for the over 1000-fold increased sensitivity to sorafenib in ITD-mutant-harboring AML cells, the significant abrogation of colony formation in ITD-mutant-harboring primary AML cells, and the dramatic drop in circulating blasts in ITD-harboring AML patient. In conclusion, the identification of FLT3-ITD mutant as a main therapeutic target of sorafenib may provide therapeutic guidance in the ongoing Phase I clinical trials in AML and offer therapeutic benefit to AML patients that are expected to respond poorly to chemotherapy.