Mechanism of FLT3 Ligand Dependent Resistance to FLT3 Inhibitors
Authors: Chen Fangli , Ishikawa Yuichi , Kiyoi Hitoshi , Naoe Tomoki 
Published: October 2019

Abstract
Background: FLT3mutation, which is found in about 30% of acute myeloid leukemia (AML) patients, is involved in the signaling pathway of autonomous proliferation and differentiation block in leukemia cells. Since FLT3 mutation is associated with a poor prognosis in AML patients, mutated FLT3 serves as an important molecular target in the treatment of leukemia. To date, several FLT3 inhibitors are undergoing investigation, but their clinical efficacies were lower than expected. The inhibitory effects of FLT3 inhibitors were mainly evaluated using the sole mutant FLT3-expressing cells in pre-clinical studies, while clinicallymost AML cells harboring FLT3 mutation co-express wild-type (Wt) FLT3, suggesting that FLT3 ligand (FL)-dependent Wt-FLT3 signal might cause the lower clinical efficacies of FLT3 inhibitors. Recently, it was reported that administration of FLT3 inhibitors induced increased concentration of FL in plasma. Thus, here we analyzed how FL-dependent signal affects the inhibitory effect of FLT3 inhibitors and proliferation on Wt- and ITD-FLT3-coexpressing cells.
Methods: 5 kinds of FLT3-expressing 32D cells were established: Wt-FLT3, FLT3-ITD, extra cellular domain-lacking FLT3-ITD (cyFLT3-ITD), Wt- and FLT3-ITD co-expressing and Wt- and cyFLT3-ITD co-expressing 32D cells. The growth inhibitory effects of 6 FLT3 inhibitors (AC220, CEP701, FI-700, KW2449, PKC412 and Sorafenib) with and without FL stimulation were evaluated by MTT assay. Furthermore, cell cycle analysis was performed to evaluate cell proliferation, and inhibitory effects on FLT3 kinase and its downstream molecules were also evaluated by western-blot. In vivo, cells were inoculated into C3H/Hej mice intravenously to follow survival rate and NOD/SCID mice subcutaneously to compare tumor volume between sole ITD-FLT3-expressing 32D cells and Wt and ITD-FLT3-coexpressing 32D cells.
Results: The FL-stimulation reduced growth inhibitory effects by FLT3 inhibitors on Wt- and ITD-FLT3-co-expressing 32D cells, while those reducing effects were little on the sole extracellular domain lacked ITD-FLT3 (cyITD-FLT3)-expressing 32D cells. Of note, FL-stimulation induced cell cycle arrest dose-dependently, resulting reduced proliferation in Wt- and ITD-FLT3-co-expressing cells. In vivo, all syngeneic C3H mice inoculated with sole cyITD-FLT3-expressing 32D cells and sole ITD-FLT3-expressing 32D cells developed leukemia within 16 days and 72 days respectively, but mice inoculated with Wt- and ITD-FLT3-co-expressing cells survived more than 100 days (P<0.0001). Furthermore, the growth of tumors driven by sole ITD- and cyITD- FLT3-expressing 32D cells was significantly faster than tumors driven by Wt- and ITD-FLT3-co-expressing cells in NOD/SCID subcutaneous model. Western blot shows AKT and ERK are activated in Wt- and ITD-FLT3-co-expressing cells by FL stimulation. STAT3 is highly phosphorylated in Wt- and ITD-FLT3-co-expressing cells and can be further activated by FL stimulation, while the phosphorylation is weak in sole ITD expressing cells. Immunopricipitation demonstrated that FLT3 ligand activated only Wt-FLT3 but not ITD-FLT3 in co-expressing cells. Furthermore, p21 (CIP1/WAF1) can be up-regulated by FL and induce cell cycle arrest in Wt- and ITD-FLT3-co-expressing cells. FL impeded the inhibitory effect of FLT3 inhibitors by persistent activation of ERK and AKT through Wt-FLT3. Also, down-regulation of p21 and Mcl-1induced by FLT3 inhibitor can be suppressed by FL.
Conclusions: These results suggested that FLT3 Ligand dependent resistance to FLT3 inhibitors were associated with reduced proliferation ability caused by up-regulation of P21 and persistent ERK and AKT activation through Wt-FLT3 signal.

Disclosures
Kiyoi: Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Zenyaku Kogyo: Research Funding; FUJIFILM Corporation: Research Funding. Naoe:Otsuka Pharmaceutical Co. LTD: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis Pharma: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Kyowa Hakko Kirin Co. LTD: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Zenyaku Kogyo: Research Funding; FUJIFILM Corporation: Research Funding.