Targeting STAT3-VISTA axis to suppress tumor aggression and burden in acute myeloid leukemia
Journal of Hematology & Oncology volume 16, Article number: 15 (2023)
The acute myeloid leukemia (AML) patients obtain limited benefits from current immune checkpoint blockades (ICBs), although immunotherapy have achieved encouraging success in numerous cancers. Here, we found that V-domain Ig suppressor of T cell activation (VISTA), a novel immune checkpoint, is highly expressed in primary AML cells and associated with poor prognosis of AML patients. Targeting VISTA by anti-VISTA mAb boosts T cell-mediated cytotoxicity to AML cells. Interestingly, high expression of VISTA is positively associated with hyperactive STAT3 in AML. Further evidence showed that STAT3 functions as a transcriptional regulator to modulate VISTA expression by directly binding to DNA response element of VISTA gene. We further develop a potent and selective STAT3 inhibitor W1046, which significantly suppresses AML proliferation and survival. W1046 remarkably enhances the efficacy of VISTA mAb by activating T cells via inhibition of STAT3 signaling and down-regulation of VISTA. Moreover, combination of W1046 and VISTA mAb achieves a significant anti-AML effect in vitro and in vivo. Overall, our findings confirm that VISTA is a potential target for AML therapy which transcriptionally regulated by STAT3 and provide a promising therapeutic strategy for immunotherapy of AML.
To the editor,
Despite encouraging responses elicited by immune checkpoint blockades (ICBs) have been shown in many types of cancers, the acute myeloid leukemia (AML) patients obtain little benefit from current ICBs [1, 2]. However, the mechanisms that determine immune evasion in AML are remain poorly understood. Herein, we found that V-domain immunoglobulin suppressor of T cell activation (VISTA) regulated by STAT3 is highly expressed in AML. VISTA is a novel immune checkpoint mediating immune evasion especially by impeding T cells activation [2,3,4]. Furthermore, we confirmed that combination of STAT3 inhibitor and VISTA mAb is a promising immunotherapeutic strategy for AML.
We found that VISTA was significantly higher in AML cells and was the most correlative immune checkpoint for overall prognosis of AML patients basing on TCGA database (Fig. 1A, B, Additional file 1: Fig. S1A), the same higher expression as in CD34 + AML cells and CD34- myeloid cells from bone marrow of AML patients (Fig. 1C, Additional file 1: Fig. S1B-S1C). Additionally, VISTA mAb significantly enhanced T cell-mediated cytotoxicity and prolonged survival of AML mice (Fig. 1D, E). These results together suggest that targeting VISTA might be a promising strategy for AML immunotherapy. As a member of B7 family , previous evidences suggesting that STAT3 activation is also correlated with expression of VISTA , but the molecular mechanisms remain a mystery. We found that high expression of VISTA was positive associated with STAT3 activation in AML cells (Additional file 1: Fig. S2A) and bone marrow of AML patients (Fig. 1F, G). Both genetic inhibition by specific shRNA and pharmacological inhibition by selective inhibitor W1046 of STAT3 significantly decreased the expression of VISTA at mRNA and protein level (Fig. 1H, I, Additional file 1: Fig. S2B-S2E). The same decrease of VISTA was found on AML cell membranes (Additional file 1: Fig. S2F-S2G). As a transcription factor closely associated with immune response, STAT3 regulates expression of immune genes transcriptionally [7, 8]. In this study, we found that STAT3 has two evident binding peaks in the promoter and the first intron of VISTA gene from Cistrome Data Browser database (Fig. 1J). ChIP-qPCR assay proved the bond between STAT3 and VISTA gene (Fig. 1K). Moreover, STAT3 significantly promoted transcription of VISTA, whereas the transcription activity was inhibited by loss-of-function mutation and selective STAT3 inhibitor W1046 or SH-4–54 (Fig. 1L, M, Additional file 1: Fig. S2H-S2J). The results suggest that STAT3 transcriptionally regulates VISTA by directly binding to promoter and intron region of VISTA.
Basing on above results, we assumed STAT3 inhibitor can act as an immunomodulator in AML immunotherapy. On this account, we designed a novel STAT3 inhibitor W1046 (Fig. 1N), which we first introduced the pharmacophore of boronic acid targeting SH2 domain of STAT3 in the STAT3 inhibitor design . W1046 significantly inhibited AML cells with hyperactive STAT3 but was lacked of sensitivity in MCF-7 cells with low STAT3 activation and in MOLM-13 cells with STAT3 deletion by CRISPR-Cas9 (Fig. 1O, P, Additional file 1: Fig. S3B). W1046 occupied the pivotal core of SH2 domain and elevated thermal stability of STAT3 protein (Fig. 1Q, Additional file 1: Fig. S3A, S3C). Further study showed W1046 significantly inhibited the phosphorylation and transcription of STAT3, but had no or slight effect on the phosphorylation of STAT1, STAT5, and upstream kinase JAK2 (Fig. 1R, S, Additional file 1: Fig. S3D-S3F). W1046 significantly inhibited proliferation and induced apoptosis both in AML cells lines and in primary AML patient samples (Additional file 1: Fig. S4A-S4F). Notably, W1046 remarkedly suppressed leukemia aggression, reduced leukemia burden and prolonged the survival of leukemia mice dosage-dependently (Fig. 2A–C, Additional file 1: Fig. S4G-S4H). Above results indicated that W1046 displays potent therapeutic efficacy on AML in vitro and in vivo as a novel STAT3 inhibitor.
Emerging evidences reported that combination of immunomodulator and immune checkpoint blockade generally has synergistic effect and maximizes benefits [10,11,12]. Given that the potential role of VISTA in AML immunotherapy, we next explored whether combination of STAT3 inhibitor W1046 with VISTA blockade would be a promising therapeutic strategy for AML. Firstly, we showed that combination of W1046 and VISTA mAb dramatically elevated T cell cytotoxicity in vitro (Fig. 2D, E, Additional file 1: Fig. S5A-S5B). Further study showed that combination of W1046 and VISTA mAb significantly upregulated secretion of IFN-γ and IL-2, promoted T cells proliferation, increased CD4+ T cells and CD8+ T cells (Fig. 2F, G, Additional file 1: Fig. S5C-S5J). Notably, combination of W1046 and VISTA mAb remarkedly suppressed leukemia aggression, reduced leukemia burden and prolonged the survival of leukemia mice by elevating infiltration of cytotoxic T cell in vivo (Fig. 2H–M, Additional file 1: Fig. S5K-S5L). Taken together, we testified that combination of W1046 and VISTA mAb displays significant anti-leukemia effect by decreasing VISTA and then enhancing T cells activation (Fig. 2N). The combined tactics would be a promising therapeutic strategy for AML.
Overall, our study firstly suggested that VISTA is a promising target for AML immunotherapy, regulated by STAT3 transcriptionally. A novel and selective STAT3 inhibitor W1046 was discovered and showed significant anti-tumor efficacy in AML. Combination of VISTA mAb and STAT3 inhibitor demonstrated remarkable anti-leukemia efficacy in AML by restoring T cells activation in vitro and in vivo, which shed a light on a new therapeutic strategy for AML therapy.
Availability of data and materials
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Acute myeloid leukemia
V-domain Ig suppressor of T cell activation
Immune checkpoint blockades
Signal transducer and activator of transcription 3
Ghosh A, Barba P, Perales MA. Checkpoint inhibitors in AML: are we there yet? Br J Haematol. 2020;188(1):159–67.
Dufva O, et al. Immunogenomic landscape of hematological malignancies. Cancer Cell. 2020;38(3):380–99.
Yuan L, et al. VISTA: a mediator of quiescence and a promising target in cancer immunotherapy. Trends Immunol. 2021;42(3):209–27.
ElTanbouly MA, et al. VISTA: a novel immunotherapy target for normalizing innate and adaptive immunity. Semin Immunol. 2019;42: 101308.
Xu W, et al. The structure, expression, and multifaceted role of immune-checkpoint protein VISTA as a critical regulator of anti-tumor immunity, autoimmunity, and inflammation. Cell Mol Immunol. 2018;15(5):438–46.
Wu L, et al. Expression of VISTA correlated with immunosuppression and synergized with CD8 to predict survival in human oral squamous cell carcinoma. Cancer Immunol Immunother. 2017;66(5):627–36.
Johnson DE, O’Keefe RA, Grandis JR. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat Rev Clin Oncol. 2018;15(4):234–48.
Zou S, et al. Targeting STAT3 in Cancer Immunotherapy. Mol Cancer. 2020;19(1):145.
Deng L, et al. Boronic acid: a novel pharmacophore targeting src homology 2 (SH2) domain of STAT3. J Med Chem, 2022.
Luo F, et al. Niclosamide, an antihelmintic drug, enhances efficacy of PD-1/PD-L1 immune checkpoint blockade in non-small cell lung cancer. J Immunother Cancer. 2019;7(1):245.
Stahl M, Goldberg AD. Immune checkpoint inhibitors in acute myeloid leukemia: novel combinations and therapeutic targets. Curr Oncol Rep. 2019;21(4):37.
Li H, et al. The allergy mediator histamine confers resistance to immunotherapy in cancer patients via activation of the macrophage histamine receptor H1. Cancer Cell. 2022;40(1):36–52.
This work was supported by the National Natural Science Foundation of China (81973359, 82273958, 21977128), Guangdong Basic and Applied Basic Research Foundation (2022A1515012204), Joint Foundation of Guangdong and Macau for Science and Technology Innovation (2022A0505020024), Guangzhou Basic and Applied Basic Research Foundation (202002030408, 202103000097, 202206080007). The Science and Technology Development Fund, Macau SAR (File no. 0053–2021-AGJ), Jilin Province Science and Technology Development Project (20200404105YY, 20210204055YY), National Major Special Projects for the Creation and Manufacture of New Drugs (2019ZX09301104), Key-Area Research and Development Program of Guangdong Province (2020B1111110003), Guangdong Provincial Key Laboratory of Construction Foundation (2019B030301005) are also appreciated.
Ethics approval and consent to participate
All in vivo experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (SYSU-IACUC-2022–000185) and followed the Guide for the Care and Use of Laboratory Animals. The primary leukemia patient samples were obtained under protocols reviewed and approved by the Human Ethics Committee of the Third Affiliated Hospital of Sun Yat-Sen University.
Consent for publication
Consent to publish has been obtained from all authors.
All the authors declare no competing interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Mo, J., Deng, L., Peng, K. et al. Targeting STAT3-VISTA axis to suppress tumor aggression and burden in acute myeloid leukemia. J Hematol Oncol 16, 15 (2023). https://doi.org/10.1186/s13045-023-01410-y