Abstract Background: Acute myeloid leukemia (AML) is a group of heterogeneous hematologic malignancies correlates with poor prognosis. It is important to identify biomarkers for effective treatment of AML. Kinases participate in many regulatory pathways and biological activities in AML. Previous studies demonstrated that MAP4K1, a serine/threonine kinase, was associated with immune regulation and cancer progression. However, its role and mechanism in acute myeloid leukemia (AML) have not been explored. Methods: RNA-seq profiling was performed for Homoharringtonine (HHT)-resistant and Homoharringtonine (HHT)-sensitive cell lines. Bioinformatic tools were used for differential analysis. Cell culture and transfection, Cell proliferation, apoptosis and Cell cycle assay, Quantitative RT-PCR, and Western blotting analysis were used to explore biological phenotypes in vitro. Findings: We found that MAP4K1 was highly expressed in HHT-induced resistant AML cell lines. In addition, overexpression of MAP4K1 in AML cells induced resistance of AML cells against HHT. Not only that, the findings of this study showed that overexpression of MAP4K1 was an independent risk factor that predicts poor prognosis of AML. Further, In vitro studies showed that MAP4K1 modulated cell cycle through MAPK and DNA damage/repair pathways. Therefore, MAP4K1 is a potential target for developing therapies for AML. Interpretation: This study demonstrates that MAP4K1 not only regulates HHT resistance but also independently predicts AML prognosis. In addition, understanding the regulatory mechanism of MAP4K1 reveals novel treatment strategies for resistant and refractory AML. Fundings: This work was supported by the National Natural Science Foundation of China (NSFC) (Grant No.81800199, 81670124, 82070118) and the Natural Science Foundation of Zhejiang Province (LY20H080008). Keywords: Acute myeloid leukemia, MAP4K1, Homoharringtonine resistance, Prognosis, DNA damage, MAPK __________________________________________________________________ Research in context. Evidence before this study Acute myeloid leukemia (AML) is a group of heterogeneous hematologic malignancies with high incidence rate, high recurrence rate and high mortality. The emergence of drug resistance against conventional therapies limits the efficacy of these therapies resulting in poor patient outcomes. Therefore, it is important to identify novel therapeutic targets to improve the treatment efficacy. Previous studies demonstrated that MAP4K1, a serine/threonine kinase, is strongly implicated in immune regulation and cancer progression. However, the role of MAP4K1 in regulating AML progression and chemoresistance remains unknown. Added value of this study In this study, we developed three HHT-resistant strains for the first time and labeled them as MV4-11 R10, MV4-11 R30, MV4-11 R50. We found that the upstream MAPK gene, MAP4K1 was upregulated in all resistant strains via RNA-seq analysis. This study reveals, for the first time, the MAP4K1 regulates resistance against HHT and the prognosis of AML. In addition, MAP4K1 functions as a tumor promotor and drug mediator by modulating the DNA damage/repair system and MAPK pathway. More than that, two inhibitors, sunitinib and SP600125, were found to have good synergy with HHT, which may provide new insights into AML treatment. Implications of all the available evidence The findings of the present study suggest that MAP4K1 is an independent risk factor that influences the prognosis of AML and a regulator affects the development of drug resistance in AML. Therefore, MAP4K1 might represent a novel therapeutic target. The combination therapy proposed in this article may improve the sensitivity of AML to HHT. Alt-text: Unlabelled box 1. Introduction Acute myeloid leukemia (AML) is a group of heterogeneous hematologic malignancies characterized by numerous cytogenetic and molecular changes. AML is associated with high incidence rate, high recurrence rate and high mortality [53][1]. Nowadays, advances and development of targeted agents and modern immunotherapy have improved the responsiveness of AML patients. However, drug resistance against conventional therapies limits the efficacy of these therapies resulting in poor patient outcomes [54][2]. Therefore, it is important to identify novel therapeutic targets of AML to improve the prognosis of AML especially relapsed/refractory AML. Studies should be performed to explore the mechanisms of drug resistance to develop effective drugs against drug resistance AML. Homoharringtonine (HHT) is a cytotoxic alkaloid originally extracted from Cephalotaxus hainanensis. HHT has significant activity against various leukemia types such as acute myeloid leukemia, chronic myeloid leukemia and myelodysplastic syndrome. Our research team has been investigating the effect, mechanism and clinical application of HHT in AML for several years. A prospective, multicenter phase 3 study showed that both the complete remission (73% vs 61%, p=0.00108) and the 3-year event-free survival (35.4% vs 23.1%) in HAA treatment group were better than that in patients who received DA (doxorubicin+arac) plan [[55]3,[56]4]. Therefore, HHT could be a potential drug for the treatment of AML because of its good single effect on AML cells as well as its potential to combine with other traditional drugs. However, along with the wide use of HAA and other HHT-based plans, the resistance of HHT has become a new challenge and still lacks research. Exploring the resistant mechanism and related targets must have great practical significance for AML patients who relied on HHT-based treatment. In this study, we developed three resistant strains to HHT and labeled them as MV4-11 R10, MV4-11 R30, MV4-11 R50. Resistant strains were developed by long-term induction of increasing concentrations of HHT. Further, we used second-generation high-throughput sequencing technology to determine the expression level of genes before and after induction of resistance. The findings of this study showed that the MAPK pathway was upregulated in the three resistant strains with significantly high expression level of an upstream MAPK gene named MAP4K1. MAP4K1 knockdown increased sensitivity of AML cells to HHT whereas overexpression of MAP4K1 increased resistance of AML cells to HHT. These findings imply that MAP4K1 may play a key role in regulating HHT resistance of AML. Kinases are involved in many regulatory pathways and biological activities. Kinases play essential roles in tumor development and chemotherapy resistance thus they are desirable targets for improving survival of cancer patients and overcoming drug resistance [57][5]. Mitogen-activated protein kinases (MAPKs) belong to serine/threonine kinase family and they modulate embryogenesis, cell differentiation, proliferation, and cell death pathways [58][6]. In addition, enzymes involved in these pathways are potential targets for the development of antitumor therapy [[59]7,[60]8]. Not only that, the MAP kinase signaling pathway is closely related to hematological malignancies [61][9]. MAP4K1 was originally cloned from human hematopoietic myeloid cells and always has a typically high expression in myeloid and lymphoid lineage cells. Studies have reported that the expression of MAP4K1 is associated with the pathogenesis of several cancer types. Yang, H.S., et, al. reported that inhibition of MAP4K1 suppressed colon cancer progression [62][10]. Furthermore, Wang, J., et, al. reported that HPK1 positive expression associated with longer overall survival in patients with estrogen receptor-positive invasive ductal carcinoma [63][11]. MAP4K1 affected cell-cycle progression and mediated Vitamin D resistance in AML cell lines [64][12]. However, previous studies have not explored the mechanism and role of MAP4K1 as an independent prognostic marker for AML and its role in regulating HHT resistance. Therefore, the functional role and mechanism of MAP4K1 in AML should be explored to provide information for developing novel effective therapies. In the present study, we carried out in vitro and in vivo experiments to explore the role of MAP4K1 in regulating AML prognosis and its role in modulating drug resistance against HHT. The findings of this study show that MAP4K1 plays a key role in the pathogenesis of AML, patient survival and drug resistance. The possible regulatory mechanisms are discussed according to present data, bioinformatics and related literatures. We confirm that MAP4K1 mediates HHT resistance and is an independent risk factor for AML prognosis. These findings provide novel information for the development of AML therapies to overcome drug resistance and improve patient survival. 2. Methods 2.1. Cell culture and transfection MV4-11 cell was treated with increasing concentrations of HHT (from 1nmol/L to 50nmol/L). Cells that grew normally in complete medium containing 10 nmol / L, 30 nmol / L, and 50 nmol / L HHT were preserved. The cells were defined as MV4-11 R10, MV4-11 R30, MV4-11 R50. The MV4-11 (CVCL_0064), MV4-11 R10, MV4-11 R30, MV4-11 R50, MOLM13 (CVCL_2119) and THP-1 (CVCL_0006), KG-1 (CVCL_1824), HL-60 (CVCL_A794), Kasumi (CVCL_0589) were cultured in IMDM medium (Gibco, REF: C12440500BT) and RPMI-1640 medium (Gibco, REF: C11875500BT) containing L-glutamine (Corning) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Gibco, USA, REF: 10099-141C) at 37°C in a humidified atmosphere with 5% CO2. For MAP4K1 knockdown, three shRNA sequences were designed and the first and third sequences were selected for subsequent experiments based on qPCR and western blot results. THP-1 and MV4-11 R50 were transfected with green fluorescent protein (GFP) containing shRNA lentiviral particles directed against human MAP4K1 or with the shScramble vector. For overexpression, the coding DNA sequence of MAP4K1 was cloned into the PCDH vector and then transfected into MV4-11 and THP-1. Cells were selected in culture media containing puromycin (1μg/ml, InvivoGen, Cat: ant-pr-1) for about 1-2 weeks and used for subsequent experiments. Sequences for MAP4K1 shRNA and non-silencing control shRNA were: Sh1: 5’ - CCTGTATTCTCATAGCATCCT - 3’ Sh3: 5’ - GCCCTTCTCGTAAAGTTGTTC - 3’ shScramble vector: 5’-GCTTCGCGCCGTAGTCTTA - 3’ 2.2. Reagents and antibodies Homoharringtonine (HHT, Cat: HY-14944/CS-2872) was purchased from MCE, JNK inhibitor (SP600125, Cat: S1460) and HPK1 inhibitor (Sunitinib, Cat: S1042) were purchased from Selleck Chemicals (Houston, TX, USA). For western blotting, the following antibodies were obtained from Cell Signaling Technologies: HPK1 (4472, AB_2140826), P-JNK (4668, AB_823588), JNK (9252, AB_330894), P-C-JUN (ser63,2361, AB_490908), C-JUN (9165, AB_2130165), P-ATM (5883, AB_10835213), P-ATR (2853, AB_2290281), ATR (2790, AB_2227860), P-CHK1 (2348, AB_331212), P-CHK2 (2197, AB_2080501), CHK2 (2662, AB_2080793), P-H2AX (9718, AB_2118009), H2AX (7631, AB_10860771), P21 (2947, AB_823586), P27 (3686, AB_2077850), GAPDH (5174, AB_10622025). ATM (27156-1-AP, AB_2880780), CHK1 (25887-1-AP, AB_2880283), tubulin (66031-1-Ig, AB_11042766) were purchased from proteintech. A human CD45 antibody was purchased from MULTI SCIENCES (Cat: AH04504-100). 2.3. Cell line and antibody validation Cell lines (THP-1, MV4-11, HL-60, Kasumi, KG-1, MOLM13) used for follow-up in vitro and in vivo experiments have been validated by the institute, STR profiling of these cell lines has been listed in Supplementary Data (Supplementary materials 3) as Part1. Mycoplasma testing has been performed. Cell lines used in this article are free of mycoplasma contamination and results have been listed in Supplementary Data (Supplementary materials 3) as Part1. All antibodies are commonly used antibodies, RRID tags of all antibodies can be obtained. Information of the relevant references for antibody application has