br Funding This work was supported

Funding This work was supported by Department of Neurosurgery, the University of Texas Health Science Center at Houston, Memorial Hermann Foundation-Staman Ogilvie Fund, the Bentsen Stroke Center, the TIRR Foundation through Mission Connect (No. 014-115), Craig H. Neilsen Foundation (No. 338617), NIH (R01 NS061975), NSF (CBET1134449). We also thank the IMM FACS core facility and the Cancer Prevention and Research Institute of Texas (CPRIT) for the supported use of flow cytometry.
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Introduction Chemotherapy resistant leukemic stem cells (LSCs) are the major cause for relapse of leukemia, since the chemotherapeutic drugs eradicate differentiated cells while LSCs remain unaffected. Characterizing and targeting LSCs using specific surface markers is a rational therapeutic approach to prevent disease relapse. Various surface markers like CD34, CD90, CD123, CD25, CD32, CD96 and CD47 have been identified in LSCs that could be used for therapeutic targeting. CD25 was reported to be a potential target for LSC specific therapy since it is highly expressed in LSCs and not in normal hematopoietic stem cells (HSCs). The safety of targeting CD25 was also demonstrated by the long term multi-lineage hematopoietic reconstitution capacity of normal HSCs depleted of CD25-positive cells. CD25-positive LSCs were quiescent, chemotherapy-resistant and could initiate AML in vivo (Saito et al., 2010). Therefore, we developed an immunotoxin using CD25 ligand IL2α to target LSCs. Recently, we reported the potential of CD25 specific targeting in leukemia using immunotoxin conjugated with recombinant human TNF-related apoptosis inducing ligand (TRAIL) (Madhumathi et al., 2016). Recombinant TRAIL protein, TRAIL derivatives or monoclonal droperidol targeting TRAIL receptors have been successfully used as anti-tumor agents (Mérino et al., 2007, Cretney et al., 2007). Interestingly, drug resistant side population (SP) showed higher expression of TRAIL receptors compared to non-SP cells. Chemoresistant SP cells of colon cancer, displayed higher sensitivity to TRAIL compared to non-SP cells, suggesting the possible role of TRAIL in targeting drug resistant CSCs (Sussman et al., 2007). Upregulation of TRAIL receptors in CSCs have been reported in other studies (Signore et al., 2013, He et al., 2014). Hence, targeting TRAIL receptors in CSCs is an attractive strategy to induce selective apoptosis. However, using recombinant TRAIL protein have been reported to result in TRAIL resistance due to binding of TRAIL to decoy receptors that lack intracellular death domain and does not induce apoptosis. Targeting TRAIL R2/DR5 receptor specifically using anti-DR5 mab or DR5 specific TRAIL peptide mimetic (Pavet et al., 2010) could overcome TRAIL resistance by inhibition of non-specific binding to antagonistic decoy receptors (van der Sloot et al., 2006, Mérino et al., 2007). We hypothesized that IL2 conjugated with TRAIL peptide mimetic could effectively bind and destroy LSCs specifically since LSCs over express both CD25 and TRAIL receptors. Here, we report for first time a novel immunotoxin conjugate IL2-TRAIL peptide to target leukemic stem cells isolated from drug resistant cell lines and relapsed leukemic patient samples.
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Discussion Mutation in a stem cell or a progenitor cell may give rise to LSCs that exhibits stem cell properties. Although the nature of LSC varies depending upon the stage or cell type at which the mutations appear, one common characteristic of all LSCs is drug resistance. The vast majority of therapeutic agents used for treating leukemia are not effective against LSCs, leading to rapid disease recurrence. The conventional leukemic drugs either do not target or lack specificity in targeting LSCs (Jordan, 2007). Novel therapeutic strategies targeting LSCs specifically are required to eradicate leukemia completely. Consequently, various therapeutic targets for drug resistant LSCs were identified (Saito et al., 2010).