The multiple‐kinase inhibitor lenvatinib inhibits the proliferation of acute myeloid leukemia cells

Abstract Background Current chemotherapy for acute myeloid leukemia (AML) mainly involves cytotoxic agents such as doxorubicin (DNR), mitoxantrone (Mito) or 2‐aminopurine‐6‐thiol (6‐TG). However, because these agents are relatively ineffective, discovering other more effective drugs for AML treatment would be valuable. Methods The in vitro antitumor effect of lenvatinib on AML cells was examined using the colorimetric MTT assay for assessing cell metabolic activity. AML cells mixed with Poloxamer 407 were injected into nude mice to form subcutaneous tumors. Tumor‐bearing mice received lenvatinib by oral administration. The antitumor effect of lenvatinib was established by measuring tumor volumes and weights. Results Lenvatinib inhibited the growth of AML cells in a dose‐dependent manner. We used AML cells to establish subcutaneous tumor tissues by mixing the cell suspension with Poloxamer 407. Poloxamer 407 alone did not influence the subcutaneous growth of AML cells. Treatment of lenvatinib inhibited in vivo tumor growth of AML cells. Conclusion The multiple‐kinase inhibitor lenvatinib inhibits the in vitro proliferation of AML cells, and restricts the in vivo growth of AML tumors.


| INTRODUC TI ON
Acute myeloid leukemia (AML) is hematological malignancy that has been characterized by infiltration of the bone marrow, blood and other hematopoietic tissues. 1,2 Current chemotherapeutic options for AML include cytotoxic agents such as DNR, Mito, etoposide and 6-TG. 3,4 However, treatment of AML patients with these agents has not shown the expected promising outcomes. Due to the complex multiple causes of AML, it is hard to achieve tumor regression by simply administrating a single functional cytotoxic agent. 5 Over the last decade, molecule-targeting agents have become widely recognized as "first choice" treatments for certain hardcore tumors, and researchers have now started to focus on treating one kind of tumor using kinase inhibitors approved to treat another. 6,7 A number of studies have provided insights into the genetic variability of AML and indicated potential targets for drug development.
In addition to mutations found in genes including NPM1, CEBPA, DNMT3A, TET2, RUNX1, ASXL1, IDH2, and MLL, dysfunctional receptor tyrosine kinases (RTKs) such as Flt, VEGFR or c-Kit have been shown to be drivers of the progress of AML. 5,8 Therefore, it would be valuable to discover whether effective existing kinase inhibitors restrict AML tumors.
Lenvatinib is a newly approved multi-targeted kinase inhibitor. 9 Oral administration of lenvatinib can repress the proliferation or metastasis | 179 FENG Et al of human malignancies by inhibiting the activation of RTKs. 10,11 In this work, we found lenvatinib inhibited the growth of AML cells in vitro. By mixing AML cells with Poloxamer 407, we were able to establish subcutaneous AML tumors in nude mice. Using this tumor model, we found that treatment of lenvatinib inhibited in vivo tumor growth of AML cells.

| Subcutaneous tumor tissue shape regularity
Photographs of subcutaneous tumors were quantitatively analyzed to determine the length of the long axis of the tumor, and the area of the perfect circle (the total number of pixels) having the diameter of the long axis was calculated. At the same time, the tumor tissue was delineated by marking the edge of the tumor on the photograph and the total area of the tumor tissue (total number of pixels) was calculated. The ratio of the total area of the long-axis circle to the total area of the delineated tumor reflected the regularity of the shape of the tumor tissue: the closer the ratio to 1, the more regular the shape of the tumor tissue.

| Statistical analysis
Statistical analysis was performed using a Bonferroni correction with two-way analysis of variance and P < 0.05 were considered as statistically significant between groups. The statistical analysis was performed using SPSS software (IBM Corporation) and the IC 50 values of lenvatinib on AML cells were calculated using Origin software (OriginLab Corporation). The concentrations and the inhibition rates were analyzed with Origin software using the Sigmoidal Fit methods.

| Lenvatinib inhibits the proliferation of cultured AML cells in a dose-dependent manner
In order to study the effect of lenvatinib on AML cells, we first examined whether it affected the proliferation of the target cells. We treated in vitro cultured AML Kg-1 cells with incremental concentrations of lenvatinib for 48 hours, followed by MTT assay. We then observed whether lenvatinib inhibited the proliferation of other AML cells in a dose-dependent manner as expected ( Figure 1; Table 1). Our data show that lenvatinib inhibits the proliferation of cultured AML cells in a dose-dependent manner.

| Establishing an AML subcutaneous tumor model in nude mice
To overcome the difficulty that AML cells rarely form regular solid tumors subcutaneously and to enable measurement of tumor growth, we attempted to establish a subcutaneous growth model for these hematological malignant cells. As shown in Figure 2

| Lenvatinib inhibits the subcutaneous growth of AML cells
The in vivo antitumor effect of lenvatinib on AML cells was then examined. In our newly established tumor models, we found that oral administration of lenvatinib inhibits the subcutaneous growth of  Table 3). Thus, our results suggest lenvatinib can inhibit in vivo AML tumors.  There are many kinds of tumors of the blood system, and cells may form solid tumors in organs, eg lymph nodes. This makes the method used in the present work more relevant to blood tumor research.

| Poloxamer 407 alone does not influence the survival or growth of AML cells
Poloxamer 407 is a biodegradable pharmaceutical excipient that is safe to use on its own. 12 Results showed that Poloxamer 407 alone did not affect the subcutaneous growth of cells in nude mice. The

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
All authors made substantial contributions to the design and conception of the study and acquisition, analysis or interpretation of data. All authors took part in either drafting or revising the manuscript. All authors also read and approved the final version manuscript for publication, and agree to be accountable for all aspects of the work by ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.