FY23 Artesunate as Treatment for Childhood Acute Myeloid Leukemia

Specific Aims Acute myeloid leukemia (AML) accounts for ~15 percent of childhood leukemia. Standard treatment of AML involves multiple rounds of intensive chemotherapy and may include a bone marrow transplant. While most patients with AML are able to achieve a complete response with this treatment, ~30% will relapse and these children have a very poor prognosis. Recent evidence suggests that mutations in Ras/MAPK/MEK pathways are present in 90% of patients and this pathway is predominantly altered in relapsed pediatric AML.(Farrar et al., 2016) Therefore an effective and tolerable oral therapy that targets these pathways has the potential to prevent AML relapse and substantially improve outcomes for children with AML. The plant Artemisia annua (Aa), also known as sweet wormwood, has been used for centuries as infusions or powders to treat malaria. Artemisinin is extracted from Aa and artesunate is then semi-synthetically derived from artemisinin.1 Artesunate is a Nobel Prize winning, highly effective and well tolerated treatment for malaria in both adults and children.2 In addition to its antimalarial activity, the existing literature and our preliminary data demonstrate intriguing anti-cancer activity in many types of cancer including AML.2,3,4 Further preliminary data from our lab demonstrates artesunate may inhibit Src. In the context of mutated Ras, activation of Src is a driver of both tumor progression and metastasis in AML.(Shields et al., 2011) Anticancer activity specifically targeting the predominant pathway in relapsed AML, without significant adverse effects makes artesunate and Aa extracts attractive candidates for further development. Aim 1. Fractionate and assess the anticancer activity of the constituents of Artemisia annua extracts. As described in our preliminary data, we have developed an extraction method to obtain artemisinin and constituents from the leaf of the Kentucky grown Aa plant and that Aa extracts were more active than artemisinin alone. Working hypothesis. We hypothesize that a specific fraction is responsible for the anticancer activity of Aa. We will fractionate and quantitate the differential components of the Aa varieties from currently propagated varieties, CNAP8001 and Apollon with methanol, hexane or supercritical fluid extraction system (SFE) and test their anticancer activity in our AML cell models with proliferation assays. This aim will identify the most active combination and concentration of Aa extracts for further testing in xenograft models. Aim 2. Validate Src as an artesunate target. Based on our preliminary data, which identified Src as a likely target of artesunate, that Keap1 is required for artesunate sensitivity and in the absence of Keap1, inhibition of Nrf2 overcomes artesunate resistance, we will interrogate this pathway to determine critical determinants of artesunate activity. Working hypothesis: Artesunate is a Src inhibitor and dual Src inhibition will enhance artesunate sensitivity. We will assess the synergy of artesunate and Src inhibition with both siRNA and small molecules (dasatinib) using A549 and H1299 and patient derived cell lines. We will probe artesunate-Src binding interactions though immunoprecipitation affinity based arrays. Ability of artesunate to decrease Src will be assessed via Western and mRNA. This aim will identify critical proteins for artesunate activity and allow rational development of synergistic combinations for further testing. Aim 3. Assess the ability of artesunate to modulate downstream biomarkers in children who have completed therapy for AML. The objective of this aim is to determine the safety of orally administered artesunate in children after AML treatment and to assess the ability of artesunate to modulate key downstream biomarkers. As demonstrated in our preliminary data, Keap-1 is required for artesunate activity. Since Keap-1 is a negative regulator of the transcription factor Nrf2, the central regulator of cell response to oxidative stress, we anticipate that artesunate treatment prevents Nrf2 from activating antioxidant response genes like Nqo1. Working hypothesis: Artesunate administration will reduce the gene and protein expression of Nqo1. We will conduct a pilot study in ten children with AML who have completed therapy and assess adverse effects and ability of artesunate to modify biomarkers. This aim lays the foundation for a subsequent efficacy clinical trial to assess the ability of artesunate to reduce relapse after treatment for AML. The Kentucky Tobacco Research and Development Center (KTRDC), working with Kentucky farmers, is actively growing Aa in Kentucky, and the plant has the potential to replace tobacco as a cash crop for Kentucky farmers. Significant investment by venture capital and the state government resulted in planting a second crop in 2019, and a processing facility, which will isolate artesunate from Aa, is under construction in northern Kentucky. By developing Aa extracts as anticancer agents, we are uniquely positioned to substantially reduce the burden of childhood AML and economic impact of tobacco in Kentucky. ,