Translational Research From the Tropical Forest: Silvestrol, a Natural Product From the Plant Aglaia Foveolata inhibits the Expression of Tyrosine Kinases and Shows a Significant In Vivo Activity in Acute Myeloid Leukemia (AML)
Authors: Alachkar Houda , Santhanam Ramasamy , Lucas David M. , Schwind Sebastian , Hickey Christopher , Pang Jiuxia , Perrotti Danilo , Pan Li , Kinghorn A Douglas , Marcucci Guido , Grever Michael R 
Published: October 2019

Abstract
Abstract 2616
Despite the progress made in the diagnosis, classification, prognostication and treatment of AML, the majority of patients still fail to achieve long-term survival and die of their disease. Several cytogenetic and molecular markers including gene mutations and aberrant expression have been shown to impact the outcome of AML patients and some of these are also potential therapeutic targets. Receptor tyrosine kinases (RTKs) regulate cell survival, proliferation and differentiation in normal hematopoiesis. Gain-of-function mutations of genes encoding RTKs, such as internal tandem duplication of FLT3 (FLT3-ITD) and mutations of KIT, result in constitutive tyrosine kinase activity and have been associated with poor outcome in distinct cytogenetic (i.e., normal karyotype and core binding factor, respectively) subsets of AML. In addition, overexpression of FLT3 and wild type (wt) KIT was also found to negatively impact outcome. Tyrosine kinase (TK) inhibitors (Is) (e.g., imatinib, dasatinib or PKC412) have been shown to suppress the aberrant activity of these mutants, but their activities as single agents have been limited by their relatively elevated IC50 values for myeloid blasts and early onset of resistance. Indeed, even in combination with chemotherapy, TKIs have not yet been shown to impact favorably on AML patient outcome. This supports the need for development of novel strategies that effectively interfere with aberrant TK activity in leukemia blasts. Silvestrol, a cyclopenta[b]benzofuran derivative isolated from Aglaia foveolata, a plant that grows in the tropical rain forests of Southeast Asia, has been shown to exhibit potent inhibitory activity against a panel of human cancer cell lines and shows in vitro and in vivo efficacy in B-cell malignancies. The mechanism of action has been thought to involve inhibition of translation initiation, which in turn results in selective depletion of actively translated, short half-lives proteins. We hypothesized that silvestrol-mediated inhibition of the translation of mutated and/or overexpressed RTKs results in a significant antileukemic activity in AML cells that rely on the aberrant TK activity of these proteins. Silvestrol showed antiproliferative and proapoptotic activities in representative AML cell lines harboring activated TK proteins: FLT3-ITD-positive MV4-11, FLT3-overexpressing THP-1 KIT-mutated Kasumi-1 and also BCR-ABL-positive K562, an erythroblastic cell line notoriously resistant to chemotherapy. Importantly, silvestrol treatment reduced the expression levels of FLT3 in MV4-11 (92 %) and THP-1 (84 %), KIT in Kasumi-1 (46 %), and BCR-ABL in K562 (64 %), compared to the respective vehicle-treated controls. The silvestrol IC50 at 48 hr was less than 10nM for all cell lines (MTS assay). Silvestrol also showed antiproliferative activity in FLT3-ITD-positive AML patient blasts (IC50 24 nM at 48 hr). To evaluate in vivo activity, NOD-SCID Gamma (NSG) mice were engrafted with FLT3-ITD-positive MV4-11 cells. Twenty-one days post-engraftment, mice were treated intraperitoneally with vehicle or silvestrol (1.5 mg/kg every 48 hr for 3 weeks). After 3 doses (day 6 of treatment), spleens were obtained from three mice from each group. Spleens from silvestrol treated mice were 60% decreased in size (P=0.016) and showed 80% FLT3 protein downregulation (P=0.002) compared to vehicle-treated controls. Silvestrol treated leukemic mice (n=10) survived significantly longer than vehicle treated control (n=6) (median survival: 55.5 days vs. 29 days from engraftment day respectively; P<0.0001). All vehicle-treated mice died by day 31 from engraftment (day 11 from treatment start), while 50% of silvestrol-treated mice are still alive on day 60 from engraftment (day 40 from treatment start; updated survival curves will be provided). We conclude that silvestrol has a potent in vitro and in vivo antileukemic activity in AML through downregulation of the expression and activity of FLT3 and other TKs driving leukemia. Given the excellent antileukemic activity of silvestrol via a novel mechanism of TK inhibition that is potentially complementary to that of TKIs (i.e., silvestrol-induced suppression of TK protein expression vs. TKI-induced protein enzymatic inhibition), a rapid translation of this natural product to the clinic as a single agent or in combination with other TKIs ± chemotherapy seems warranted.

Disclosures:
No relevant conflicts of interest to declare.