Novel Anti-Acute Myeloid Leukemia Targeted Short Peptide ETP-3904 And Notable Inhibition Of KIR2DL1-HLA Pathway In The Cytotoxicity Of Allo-NK Cell Against KG1a Cell

Acute leukemia (AL) is one of the most common form of cancers. It is characterized by symptoms of short duration, many immature cell forms in the bone marrow and/or peripheral blood. Acute myeloid leukemia (AML) is the most common leukemia subtype in adults. Initial disease complete remission (CR) can be achieved in 60-80% of AML patients after standard induction chemotherapy regimens. Despite this success, only 20-30% of patients have long-term, disease-free survival after achieving complete remission with more than 50% of patients experiencing disease of relapse. Only about 40% of younger patients and 10% of older patients with this disease are alive at 5 years. Refractory and relapsed disease remains a major challenge in all patients. Besides, hematopoietic stem cell transplantation (HSCT) from matched or partially matched unrelated donors as well as mismatched related donors has become an established procedure for the treatment of AML patients. Natural killer (NK) cell alloreactivity is found to be associated with a reduced risk of relapse in AML after human leukocyte antigen (HLA) haplotype mismatched HSCT, but the undefined influence of killer cell immunoglobulin-like receptors (KIR)2DL1 expression and its correlation with KIR2DL1-HLA pathway is still not clear.The epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinase (RTK), plays a critical role in tumorigenesis and tumor progression which makes it an attractive candidate for anticancer therapies. However, problems of intrinsic and acquired resistance to EGFR inhibitors arise in parallel. Recently, role of the juxtamembrane domain (JMD) has been highlighted in the activation and regulation of EGFR activity, especially in regulating allosteric tyrosine kinase activation. Researchers found that the phosphorylation of all tyrosine residues of EGFR was abolished when JMD was replaced by unstructured sequence. Albeit the JMDs of other RTKs generally participate in auto-inhibitory interactions, the activating role of the JMD of EGFR seems unique, which serves as a new approach to EGFR-targeting therapies. Besides, the JMD was found to be responsible for stabilizing the asymmetric dimmer of the tyrosine kinase, which was involved in the activation process of EGFR. Boran, A.D. et al and Bennasroune, A. et al designed peptide inhibitors derived from JMD of EGFR, which were found to down-regulate downstream signaling of EGFR.According to the latest data from 2015 America Society of Clinical Oncology (ASCO), novel protein sites such as HER2, PD-1, HER3 and EGFR represent the main targets of novel anti-cancer drugs in recent years, and EGFR plays vital roles in cancer cell malignant proliferation. EGFR consists of three main parts, the extracellular domain (ECD 1-620), transmembrane domain (TCD 621-644) and intracellular domain (ICD 645-1186). ICD includes JMD (645-682), kinase domain (KD,683-954) and C-terminal tail (955-1186). EGFR inhibitors are roughly divided into three generations. The first generation---Gefitinib and Erlotinib won a great success in clinical applcation, but most patients produced drug resistance in 1-2 years, mainly (-60%) because of EGFR 790Tâ†'M. The second generation--Afatinib and Dacomitinib failed to overcome these above drug resistance in clinical trials. The third generation--- ADZ9291 and Rociletinib received significant treatment effect, and showed initial clinical efficacy to EGFR 790Tâ†'M mutation patients received EGFR inhibitor treatment.Over the past decade, based on understanding of protein spatial structure and physiological function, drugs treating leukemia have become more and more targeted. The introduction of computer technology has provided technical support for cancer targeting design. Several drug aided design softwares, such as Sybyl, AutoDock have become increasingly mature and widely used. A growing understanding of the underlying molecular mechanisms of AML has led to development of novel agents, most of which selectively target leukemic cells, such as monoclonal antibodies, tyrosine kinase inhibitors, farnesyl transferase inhibitors, hypomethylating agents, histone deacetylase inhibitors, etc. Given the rising incidence of AL and the identification of molecular markers in AML has greatly improved our understanding of disease pathogenesis and facilitated the discrimination of biologically and clinically distinct subgroups, novel mechanism and target-based approaches are needed to control this malignancy.Epidermal growth factor receptor pathway substrate No.8 (EPS8) represents a vital substrate of EGFR kinase, specifically binding to the JMD of EGFR[15-18]. It is a typical signaling protein of 821 amino acid long, containing a phosphotyrosine binding protein (PTB) domain, a Src homology 3 (SH3) domain and a sterile alpha-pointed (SAM-PNT) domain. There exist two additional functional regions, the C terminal effector region (aa:641-821) which interacts with Sos-1 and subsequently actives Rac specific GEF activity and the EGFR binding region (aa: 296-362) which provides a binding surface for the JMD of EGFR. Elevated level of EPS8 expression was found in various solid tumors and several hematological malignancies. Studies have shown that EPS8 is critical to tumorigenesis, proliferation, migration and metastasis, and is a biomarker for poor prognosis of cancer patients. However, the role of EPS8 in hematopoietic malignancies especially in AML remains unclear. And our previous study has provided a comprehensive picture of the role of EPS8 in different tumor biological behaviors and proposed that EPS 8 may become a potential target for tumor prevention and therapy.On the other hand, NK cell is the major component of human naturized immunity and take charge of transmitting inhibitorory signals in killing target cells via KIR-HLA pathway. The main KIRs involved are KIR2DL1 interacted with HLA-C2 and KIR2DL2/3 paired with HLA-C1, and these two KIR ligands are distinguished primarily by the presence of asparagine or lysine at position 80 in the protein sequence. Niu et al. proved that the lysis of KG1a cells by NK cells may be dominated by correlation of KIR-HLA mismatch. Therefore, the aim of this research is to prove this undefined influence of KIR2DL1 expression, as well as to confirm whether the KIR2DL1-HLA signaling pathway exerted the notable inhibition during the lysis of KG1a cells by NK cells.Taken together, in this study, our work consisted of three major parts:first, we found that PI3K/AKT pathway may play a critical role in EPS8 related AML cell proliferation, apoptosis and drug sensitivity. Second, we attempted to establish a new peptide inhibitor as a novel anti-AML agent based on the structure of the EGFR binding region of EPS8. Our peptide inhibitor displayed significant activities in inhibiting proliferation, facilitating apoptosis and enhancing drug sensitivity of AML cell lines with high expression levels of EPS8. Third, we confirmed whether the existence of KIR2DL1, a forceful inhibitory receptor binding with C2 epitope of KGla cell lines, will deliver strong inhibition which prevents NK cells from killing KG la cell, and whether the KIR2DL1-HLA signaling pathway exerted the notable inhibition during the lysis of KG1a cells by NK cells.Objects:1. Introduction of computer aided drug design (CADD) and establishment of EPS8 stable overexpression cell lines.2. Design and synthesize anti-AML short peptide ETP-3904 by CADD.3. Detect anti-AML activity of ETP-3904 and downstream pathway.4. Verify KIR2DL1-HLA pathway and its inhibitory role in the cytotoxicity of allo-NK cell against KG1a cell.Methods:1. EPS8 specifically interacts with JMD of EGFR through its EGFR binding region (aa:298-362) and the crystal structure of EGFR JMD has been previously defined, which makes it possible for drug design by CADD. In this work, a peptide named ETP-3904 containing 11 amino acids with Tat-conjunction was designed.2. Cellular entry kinetics studies were conducted to observe the transmembrane activation of ETP-3904. MTT assays were conducted to detect the cytotoxicity of ETP-3904 against leukemia cell lines and normal PBMCs. Flow cytometry assays were utilized to detect influence of ETP-3904 on AML cell apoptosis and cell cycle. Drug combination assays between ETP-3904 with therapeutic agents (DNR, Ara-c, Perifosine) were conducted to detect influence on drug sensitivity.3. PI3K/AKT and ERK pathways were examined by western blot assay in AML cell lines (KGla, HL-60 and THP-1). Akt, Erk and Stat3 phosphorylation were detected in these three cells treated with ETP-3904.4. NK cells were isolated by magnetic activated cell sorting (MACS) from peripheral blood of 12 healthy unrelated male donors. Flow cytometry was used to evaluate the purity of NK cells and phenotypic KIR2DL1 expression on them. The lysis of KG1a by NK cells was analyzed in the lactate dehydrogenase (LDH) assay to investigate whether KIR2DL1 expression on NK cells would affect the cytotoxicity.Results:1. Molecular docking and dynamics simulation results showed that the peptide inhibitor ETP-3904 could successfully bind to JMB segment of EGFR JMD.2. Cellular entry kinetics studies confirmed the transmembrane activation of ETP-3904. MTT assay results suggested the specific cytotoxicity of ETP-3904 against 6 AML cell lines and 1 CML cell lines, while the cytotoxic effect was not observed in normal PBMCs. Flow cytometry assay results suggested that ETP-3904 could induce apoptosis and result in a robust accumulation in S and G2-M phases of the cell cycle against 6 AML cell lines. Combining ETP-3904 with therapeutic agents (DNR, Ara-c, Perifosine) induced significant cytotoxic effect against AML cells. MTT analysis and combination index (CI) analysis showed that CI values of ETP-3904 and therapeutic agents were below 1, which suggested the synergism effect.3. Akt, Erk and Stat3 phosphorylation were inhibited in three AML cells (KG1a, HL-60 and THP-1) treated with ETP-3904.4. Significant differences in KIR2DL1 frequency were noted among the donors (range,27.14%-92.49%). NK cells with lower KIR2DL1 expression exerted higher cytolytic activity against KG1a cells, whereas those with higher KIR2DL1 expression exerted significantly lower lysis against KG1a cells (R2=0.8169, p< 0.05).Conclusions:1. Successfully designed and synthesized a peptide inhibitor ETP-3904.2. ETP-3904 showed remarkable activities of cellular entry, anti-proliferation, pro-apoptosis, cell cycle arrest against AML cells and enhanced drug sensitivity to chemotherapeutic drugs3. Western blot assay showed that the anti-AML effects of ETP-3904 might result from the down-regulation of PI3K/AKT pathway and ERX pathway.4. KIR2DL1 expression frequency was negatively correlated with the cytotoxicity of NK cells against KG1a cells.