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Echinomycin as a promising therapeutic agent against KSHV-related malignancies
Journal of Hematology & Oncology volume 16, Article number: 48 (2023)
Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several human cancers, including Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL), which preferentially arise in immunocompromised patients while lack of effective therapeutic options. Oncoproteins Myc and hypoxia-inducible factor-1α (HIF1α) have been found closely related to KSHV infection, replication and oncogenesis. However, the strategies of dual targeting these two oncoproteins have never been developed and tested for treatments of KSHV-related malignancies. In the current study, we report that treatment of echinomycin dramatically regresses cell growth both in vitro-cultured KSHV + tumor cells and in vivo KS or PEL xenograft mice models, through simultaneously inhibiting Myc and HIF1α expression. Echinomycin treatment also induces viral lytic gene expression whereas not increasing infectious virions production from KSHV + tumor cells. Our comparative transcriptomic analysis has identified a bunch of new Echinomycin-regulated, Myc- and HIF1α-related genes contributed to KSHV pathogenesis, including KDM4B and Tau, which are required for the survival of KSHV + tumor cells with functional validation. These data together reveal that dual targeting Myc and HIF1α such as using Echinomycin may represent a new and promising option for treatments of these virus-associated malignancies.
To the editor:
Kaposi’s Sarcoma-associated Herpesvirus (KSHV) represents a principal causative agent of several cancers arising in patients with compromised immune systems, including Kaposi's Sarcoma (KS) and Primary Effusion Lymphoma (PEL) . KSHV-induced malignancies represent a serious threat to immunosuppressed patients due to lack of effective therapies . Myc is one of the most potent and commonly activated oncoproteins, whose activation is thus considered as a hallmark of cancer initiation and maintenance . Hypoxia-inducible factor-1 (HIF1) is a master regulator mediating response to hypoxic stress in both normal tissues and tumors . Echinomycin is a bis-intercalator peptide and is biosynthesized by a unique nonribosomal peptide synthetase (NRPS), and it belongs to a family of quinoxaline antibiotics. Interestingly, Huang et al. recently reported that Echinomycin simultaneously inhibited Myc and HIF1α through proteasomal degradation . Although both Myc and HIF1α are found driven oncogenesis induced by KSHV [6, 7], dual targeting Myc and HIF1α by one agent against KSHV-related malignancies have never been reported.
Here we found that even at very low concentrations Echinomycin treatment effectively inhibited the growth of KSHV + tumor cell lines (CC50 only ~ 0.1–2 nM, Fig. 1A, B). In contrast, Echinomycin showed much less effective on the growth of normal cells such as HUVEC and peripheral blood B cells (CC50 > > 1000 nM). In addition, Echinomycin showed effective inhibition of the growth of a KSHV-infected lymphoma cell line, BJAB.219, but much less on its parental KSHV negative cell line, BJAB (Additional file 1: Fig. S1). Our further data showed a dose-dependent and time-dependent inhibition of cell growth by Echinomycin for both TIVE-LTC and BCBL-1 cell lines (Fig. 1C, D). By using soft agar assays, we found that Echinomycin treatment effectively inhibited the anchorage independent growth of KSHV + tumor cells (Fig. 1E). By using a KS-like nude mice model , we found that Echinomycin treatment significantly repressed tumor growth in mice when compared to the vehicle-treated group (Fig. 1F). At the end of experiments, the tumors isolated from Echinomycin-treated mice shrunk with much smaller size than those from vehicle-treated mice (Fig. 1G). In addition, we found that Echinomycin treatment dramatically suppressed PEL tumor progression in an established xenograft model , including reducing ascites formation and spleen enlargement over this timeframe (Fig. 1H, I).
We further found that Echinomycin treatment significantly induced both BCBL-1 and TIVE-LTC cell apoptosis as well as cell cycle arrest (Additional file 1: Fig. S2). Echinomycin treatment affected the expression of several apoptosis- or cell cycle-related proteins through repression of Myc and HIF1α expression (Fig. 1J). Since Echinomycin has been found to promote Myc and HIF1α proteasomal degradation , our results confirmed that MG132 effectively prevented the reduction of Myc and HIF1α by Echinomycin from KSHV + tumor cells (Additional file 1: Fig. S3). Echinomycin treatment significantly increased the transcription and expression of viral lytic genes, such as RTA and ORF26 (Additional file 1: Fig. S4). However, in contrast to NaB (a classical lytic inducer) leading to a pronounced increase in mature virion production, Echinomycin displayed inhibitory effects on virion production from BCBL-1 cells, instead (Additional file 1: Fig. S4).
We then compared the gene profiles between vehicle- and Echinomycin-treated KSHV + tumor cell lines, using RNA-Sequencing analyses. The volcano plots showed the scattering of genes which were significantly upregulated or downregulated (FDR < 0.05) in Echinomycin-treated BCBL-1 or TIVE-LTC (Fig. 2A). The intersection analysis identified 234 genes commonly changed in both BCBL-1 and TIVE-LTC (Fig. 2B). The top 20 commonly upregulated or downregulated genes in both BCBL-1 and TIVE-LTC were listed in a heat map (Additional file 1: Fig. S5) as well as Additional file 1: Table S1. The GO_enrichment analysis of these commonly changed genes identified several major functional categories they belong to such as extracellular structure organization, regulation of apoptotic cells, nucleic acid metabolic process and regulation of humoral immune response (Additional file 1: Fig. S5).
We selected KDM4B (lysine demethylase 4B) and MAPT (microtubule associated protein Tau) for subsequent functional validation. KDM4B is broadly defined as an oncoprotein that plays key roles in processes related to tumorigenesis . Tau is a protein that stabilizes and promotes the assembly of microtubules, which has been reported to be implicated in different types of cancer [11, 12]. We first confirmed the downregulation of these two proteins by Echinomycin in vitro and in vivo (Additional file 1: Fig. S6). Next, we demonstrated that direct knockdown of KDM4B or Tau by RNAi significantly inhibited the growth and colonies formation of KSHV + tumor cells (Fig. 2C, D, F, G), as well as downregulated the expression of both Myc and HIF1α (Fig. 2E, H). We further confirmed that direct knockdown of either Myc or HIF1α was able to downregulate both KDM4B and Tau expression from KSHV + tumor cells (Additional file 1: Fig. S7). By using immunofluorescence assay (IFA), knockdown of Tau severely impaired the structure and assembly of microtubules in TIVE-LTC (Fig. 2I). In addition, similar effects were observed within Echinomycin-treated TIVE-LTC in a dose-dependent manner (Additional file 1: Fig. S8). For clinical relevance, our results showed that the expression of KDM4B and Tau was upregulated in AIDS-KS tissues from two cancer patients when compared to normal skin tissues (Fig. 2J).
Taken together, our data reveal that dual targeting Myc and HIF1α by Echinomycin may represent a new and promising option for treatments of these virus-associated malignancies.
Availability of data and materials
All the data shown in this paper are available from the corresponding authors upon reasonable request.
Kaposi’s Sarcoma-associated Herpesvirus
Multicentric Castleman Disease
Primary Effusion Lymphoma
Master regulator of cell cycle entry and proliferative metabolism
Lysine demethylase 4B
The histone lysine demethylases
Combination antiretroviral therapy
Latency-associated nuclear antigen
Replication and transcription activator
Viral G protein-coupled receptor
Next-generation sequencing analysis
Quantitative reverse transcription PCR
Human umbilical vein endothelial cells
KSHV long-term-infected telomerase-immortalized HUVEC cells
- CC50 :
The 50% cytotoxicity concentration
Talaporfin sodium-photodynamic therapy
Migration inhibitory factor
Reactive oxygen species
Lurain K, Yarchoan R, Ramaswami R. Immunotherapy for KSHV-associated diseases. Curr Opin Virol. 2022;55:101249.
Naimo E, Zischke J, Schulz TF. Recent advances in developing treatments of Kaposi’s Sarcoma Herpesvirus-related diseases. Viruses. 2021;13:1797.
Gabay M, Li Y, Felsher DW. MYC activation is a hallmark of cancer initiation and maintenance. Cold Spring Harbor Perspect Med. 2014;4:a014241.
Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A. 1995;92:5510–4.
Huang X, Liu Y, Wang Y, Bailey C, Zheng P, Liu Y. Dual targeting oncoproteins MYC and HIF1α regresses tumor growth of lung cancer and lymphoma. Cancers. 2021;13:694.
Sin SH, Kim Y, Eason A, Dittmer DP. KSHV latency locus cooperates with myc to drive lymphoma in mice. PLoS Pathog. 2015;11:e1005135.
Shrestha P, Davis DA, Veeranna RP, Carey RF, Viollet C, Yarchoan R. Hypoxia-inducible factor-1 alpha as a therapeutic target for primary effusion lymphoma. PLoS Pathog. 2017;13:e1006628.
Dai L, Del Valle L, Miley W, Whitby D, Ochoa AC, Flemington EK, et al. Transactivation of human endogenous retrovirus K (HERV-K) by KSHV promotes Kaposi’s sarcoma development. Oncogene. 2018;37:4534–45.
Dai L, Trillo-Tinoco J, Cao Y, Bonstaff K, Doyle L, Del Valle L, et al. Targeting HGF/c-MET induces cell cycle arrest, DNA damage, and apoptosis for primary effusion lymphoma. Blood. 2015;126:2821–31.
Wang Z, Cai H, Zhao E, Cui H. The diverse roles of histone demethylase KDM4B in normal and cancer development and progression. Front Cell Dev Biol. 2021;9:790129.
Souter S, Lee G. Microtubule-associated protein tau in human prostate cancer cells: isoforms, phosphorylation, and interactions. J Cell Biochem. 2009;108:555–64.
Miyazono M, Iwaki T, Kitamoto T, Shin RW, Fukui M, Tateishi J. Widespread distribution of tau in the astrocytic elements of glial tumors. Acta Neuropathol. 1993;86:236–41.
This work was supported by NIH R01CA228166 (to SR.P.), R01HL146713 (to S.M.), R03DE031978 (to L.D.), the Arkansas Bioscience Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000 (to Z.Q.). This work was also supported by NIH COBRE grants P20GM121288, a U.S.-Japan Cooperative Medical Sciences Program Collaborative Award from the NIAID and CRDF Global (Grant Number DAA3-19-65602-1), a Ladies Leukemia League Research Grant and a Carol Lavin Bernick faculty grant to Z.L. Funding sources had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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The study was approved by the Institutional Review Board for Human Research (approval no. 8079) at LSUHSC. All subjects have been provided with written informed consent.
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Chen, J., Lin, Z., Song, J. et al. Echinomycin as a promising therapeutic agent against KSHV-related malignancies. J Hematol Oncol 16, 48 (2023). https://doi.org/10.1186/s13045-023-01441-5