Targeting Calprotectin S100A8/A9 to Overcome AML Progression in DNMT3A-Mutant Cells

Abstract

Objective

To investigate the effects of calprotectin (S100A8/A9) on the biological activity of acute myeloid leukemia (AML) cells harboring a DNA methyltransferase 3A (DNMT3A) mutation and to explore the underlying molecular mechanisms involved.

Methods

AML monoclonal cell lines harboring the DNMT3A^R882H mutation were generated via lentiviral transduction and limiting dilution. RNA sequencing was used for differential gene expression analysis, followed by bioinformatic pathway enrichment and gene correlation analyses. The biological effects of paquinimod, a selective S100A8/A9 inhibitor, on DNMT3A^R882H AML cells were assessed via Cell Counting Kit (CCK-8) proliferation assays, Annexin V/PI staining, cell cycle analysis, cell adhesion assays, and transwell migration assays.

Results

Differential gene expression analysis revealed 442 upregulated and 535 downregulated genes in DNMT3A-mutated (DNMT3A^mut) cells compared with those in DNMT3A wild-type (DNMT3A^wt) cells, with the S100A8/A9 complex recurrently enriched in Reactome pathway analysis. Compared with healthy controls, patients with AML presented increased expression of S100A8 and S100A9 and increased expression of DNMT3A^mut cells relative to DNMT3A^wt cells, which was correlated with poor prognosis in patients with AML. There were no notable differences in proliferation among the DNMT3A^mut, DNMT3A^wt, and empty vector cells under normal or starvation conditions. However, paquinimod treatment notably inhibited the proliferation, migration, and adhesion of DNMT3A^mut AML cells in a dose-dependent manner, causing G0/G1 cell cycle arrest, whereas no significant effects on apoptosis were observed. Paquinimod also downregulated key adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1), and matrix metalloproteinase-2 (MMP-2). Additionally, S100A8 and S100A9 expression was upregulated in a dose-dependent manner in response to cytarabine treatment.

Conclusion

Elevated S100A8/A9 expression contributes to the abnormal proliferation, migration, adhesion, and chemoresistance of DNMT3A^mut AML cells. Targeting S100A8/A9 alone or in combination with other treatments represents a promising therapeutic strategy for DNMT3A^mut AML.

Keywords

Acute myeloid leukemia / DNA methyltransferase 3A / S100A8 / S100A9

References

[1] AssiSA, ImperatoMR, ColemanDJL, et al.. Subtype-specific regulatory network rewiring in acute myeloid leukemia. Nat Genet., 2019, 51(1): 151-162.

[2] DöhnerH, EsteyE, GrimwadeD, et al.. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood., 2017, 129(4): 424-447.

[3] TamamyanG, KadiaT, RavandiF, et al.. Frontline treatment of acute myeloid leukemia in adults. Crit Rev Oncol Hematol., 2017, 110: 20-34.

[4] BrunettiL, GundryMC, GoodellMA. DNMT3A in leukemia. Cold Spring Harb Perspect Med., 2017, 72a030320.

[5] VenugopalK, FengY, ShabashviliD, et al.. Alterations to DNMT3A in hematologic malignancies. Cancer Res., 2021, 81(2): 254-263.

[6] SekeresMA, GuyattG, AbelG, et al.. American Society of Hematology 2020 guidelines for treating newly diagnosed acute myeloid leukemia in older adults. Blood Adv., 2020, 4(15): 3528-3549.

[7] PatelJP, GönenM, FigueroaME, et al.. Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med., 2012, 366(12): 1079-1089.

[8] GaidzikVI, SchlenkRF, PaschkaP, et al.. Clinical impact of DNMT3A mutations in younger adult patients with acute myeloid leukemia: results of the AML Study Group (AMLSG). Blood., 2013, 121(23): 4769-4777.

[9] KadiaTM, CortesJ, RavandiF, et al.. Cladribine and low-dose cytarabine alternating with decitabine as front-line therapy for elderly patients with acute myeloid leukaemia: a phase 2 single-arm trial. Lancet Haematol., 2018, 5(9): e411-e421.

[10] YanXJ, XuJ, GuZH, et al.. Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia. Nat Genet., 2011, 43(4): 309-315.

[11] Russler-GermainDA, SpencerDH, YoungMA, et al.. The R882H DNMT3A mutation associated with AML dominantly inhibits wild-type DNMT3A by blocking its ability to form active tetramers. Cancer Cell., 2014, 25(4): 442-454.

[12] BalasubramanianSK, AlyM, NagataY, et al.. Distinct clinical and biological implications of various DNMT3A mutations in myeloid neoplasms. Leukemia., 2018, 32(2): 550-553.

[13] LiaoM, ChenR, YangY, et al.. Aging-elevated inflammation promotes DNMT3A R878H-driven clonal hematopoiesis. Acta Pharm Sin B., 2022, 12(2): 678-691.

[14] QueY, LiH, LinL, et al.. Study on the immune escape mechanism of acute myeloid leukemia with DNMT3A mutation. Front Immunol., 2021, 12. 653030

[15] KarjalainenR, LiuM, KumarA, et al.. Elevated expression of S100A8 and S100A9 correlates with resistance to the BCL-2 inhibitor venetoclax in AML. Leukemia., 2019, 33(10): 2548-2553.

[16] WangS, SongR, WangZ, et al.. S100A8/A9 in Inflammation. Front Immunol., 2018, 91298.

[17] OmettoF, FrisoL, AstorriD, et al.. Calprotectin in rheumatic diseases. Exp Biol Med (Maywood)., 2017, 242(8): 859-873.

[18] CherJZB, AkbarM, KitsonS, et al.. Alarmins in frozen shoulder: a molecular association between inflammation and pain. Am J Sports Med., 2018, 46(3): 671-678.

[19] PatelS. Danger-associated molecular patterns (DAMPs): the derivatives and triggers of inflammation. Curr Allergy Asthma Rep., 2018, 181163.

[20] ChengP, EksiogluEA, ChenX, et al.. S100A9-induced overexpression of PD-1/PD-L1 contributes to ineffective hematopoiesis in myelodysplastic syndromes. Leukemia., 2019, 33(8): 2034-2046.

[21] ZhouZ, ChenX, LiZ, et al.. Overexpression of S100A9 in tumor stroma contribute to immune evasion of NK/T cell lymphoma and predict poor response rate. Sci Rep., 2021, 11111220.

[22] GebhardtC, BreitenbachU, TuckermannJP, et al.. Calgranulins S100A8 and S100A9 are negatively regulated by glucocorticoids in a c-Fos-dependent manner and overexpressed throughout skin carcinogenesis. Oncogene., 2002, 21(27): 4266-4276.

[23] ChenY, OuyangY, LiZ, et al.. S100A8 and S100A9 in cancer. Biochim Biophys Acta Rev Cancer., 2023, 18783. 188891

[24] LiangW, YangY, GongS, et al.. Airway dysbiosis accelerates lung function decline in chronic obstructive pulmonary disease. Cell Host Microbe., 2023, 31(6): 1054-1070.e1059.

[25] FengX, WangL, ZhouR, et al.. Senescent immune cells accumulation promotes brown adipose tissue dysfunction during aging. Nat Commun., 2023, 1413208.

[26] RazmkhahF, KimS, LimS, et al.. S100A8 and S100A9 in hematologic malignancies: from development to therapy. Int J Mol Sci., 2023, 241713382.

[27] ShlushLI, ZandiS, MitchellA, et al.. Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia. Nature., 2014, 506(7488): 328-333.

[28] BelizaireR, WongWJ, RobinetteML, et al.. Clonal haematopoiesis and dysregulation of the immune system. Nat Rev Immunol., 2023, 23(9): 595-610.

[29] AsadaS, KitamuraT. Clonal hematopoiesis and associated diseases: a review of recent findings. Cancer Sci., 2021, 112(10): 3962-3971.

[30] PalamLR, RamdasB, PickerellK, et al.. Loss of Dnmt3a impairs hematopoietic homeostasis and myeloid cell skewing via the PI3Kinase pathway. JCI Insight., 2023, 89e163864.

[31] PruensterM, KurzAR, ChungKJ, et al.. Extracellular MRP8/14 is a regulator of β2 integrin-dependent neutrophil slow rolling and adhesion. Nat Commun., 2015, 66915.

[32] De LucaG, GoetteNP, LevPR, et al.. Elevated levels of damage-associated molecular patterns HMGB1 and S100A8/A9 coupled with toll-like receptor-triggered monocyte activation are associated with inflammation in patients with myelofibrosis. Front Immunol., 2024, 151365015.

[33] KwonCH, MoonHJ, ParkHJ, et al.. S100A8 and S100A9 promotes invasion and migration through p38 mitogen-activated protein kinase-dependent NF-κB activation in gastric cancer cells. Mol Cells., 2013, 35(3): 226-234.

[34] NarumiK, MiyakawaR, UedaR, et al.. Proinflammatory proteins S100A8/S100A9 activate NK cells via interaction with RAGE. J Immunol., 2015, 194(11): 5539-5548.

[35] EggersK, SikoraK, LorenzM, et al.. RAGE-dependent regulation of calcium-binding proteins S100A8 and S100A9 in human THP-1. Exp Clin Endocrinol Diabetes., 2011, 119(6): 353-357.

[36] DeguchiA, TomitaT, OhtoU, et al.. Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment. Oncogene., 2016, 35(11): 1445-1456.

[37] BresnickAR, WeberDJ, ZimmerDB. S100 proteins in cancer. Nat Rev Cancer., 2015, 15(2): 96-109.

[38] LiuK, ZhangY, ZhangC, et al.. Methylation of S100A8 is a promising diagnosis and prognostic marker in hepatocellular carcinoma. Oncotarget., 2016, 7(35): 56798-56810.

[39] ZengW, GaoY, WangQ, et al.. Preliminary clinical analysis and pathway study of S100A8 as a biomarker for the diagnosis of acute deep vein thrombosis. Sci Rep., 2024, 14113298.

[40] KimDH, GuA, LeeJS, et al.. Suppressive effects of S100A8 and S100A9 on neutrophil apoptosis by cytokine release of human bronchial epithelial cells in asthma. Int J Med Sci., 2020, 17(4): 498-509.

[41] WangS, WangY, WangS, et al.. Single-cell RNA sequencing reveals S100A8/A9(hi) neutrophils-induced endothelial cell death and lymphocyte infiltration after ischemic stroke. Biochem Biophys Res Commun., 2024, 741. 151023

[42] MondetJ, LaurinD, Lo PrestiC, et al.. Increased S100A8 expression in bone marrow plasma by monocytic cells from acute myeloid leukemia patients. Hematol Oncol., 2020, 38(1): 114-118.

[43] NicolasE, RamusC, BerthierS, et al.. Expression of S100A8 in leukemic cells predicts poor survival in de novo AML patients. Leukemia., 2011, 25(1): 57-65.

[44] StewartHJS, ChaudryS, CrichlowA, et al.. BET inhibition suppresses S100A8 and S100A9 expression in acute myeloid leukemia cells and synergises with daunorubicin in causing cell death. Bone Marrow Res., 2018, 20185742954.

[45] YangL, LiD, TangP, et al.. Curcumin increases the sensitivity of K562/DOX cells to doxorubicin by targeting S100 calcium-binding protein A8 and P-glycoprotein. Oncol Lett., 2020, 19(1): 83-92

Funding

Ministry of Education(82270177)

the China Medicine Education Association 2024 Medical Science and Technology Research Project(2024KTZ035)

RIGHTS & PERMISSIONS

The Author(s), under exclusive licence to Huazhong University of Science and Technology