S100a9 deficiency accelerates MDS-associated tumor escape via PD-1/PD-L1 overexpression

**Background:** Myelodysplastic Syndromes (MDS) are characterized by ineffective hematopoiesis and progressive bone marrow failure, with the bone marrow microenvironment playing a key pathogenic role. S100a8/a9 proteins have demonstrated anti-tumor effects in human cancer cell lines, but their function in hematological malignancies is poorly understood. Previous work by the authors showed S100a9 is highly expressed in lower-risk MDS bone marrow mononuclear cells (BM-MNCs) and expressed at low levels in higher-risk MDS BM-MNCs. Programmed cell death protein 1 (PD-1) is expressed on exhausted T cells and represents a major mechanism of immune escape in malignancies. This study aimed to explore the role of S100a9 in tumor escape and identify its potential mechanisms.

**Methods:** Fourteen MDS patients and three healthy controls were included. Patients were classified as lower-risk (IPSS-R≤3) and higher-risk (IPSS-R≥4.5). Human MDS cell lines SKM-1 and K562 were used. rhS100a9 was administered at 20 μg/mL for SKM-1 cells and 40 μg/mL for K562 cells. Cytotoxicity was measured by CCK8 assay. Cell apoptosis was determined using PI and Annexin-V staining with flow cytometry. PI3K/AKT/mTOR protein levels were measured by western blot analysis. PD-1/PD-L1 expression was measured by flow cytometry. For animal studies, NHD13 transgenic mice, Setd2fl/fl mice, and Lep-CreSetd2fl/fl mice were used. Bone marrow transplantation was performed by transplanting NHD13-expressing WT or Setd2fl/fl BM cells into lethally irradiated recipient mice. Complete blood counts, erythroid differentiation stages (using Ter119 and CD71 staining), c-kit+ cell frequencies, and PD-L1 expression were analyzed by flow cytometry. Bone marrow and spleen pathology was examined using Wright's stain and hematoxylin and eosin staining. Immunohistochemistry for CD41 and CD61 was performed on spleen sections.

**Key Results:** S100a9 induced cytotoxicity in a concentration- and time-dependent manner, with higher cytotoxicity in SKM-1 cells than K562 cells. Early and late apoptosis levels were significantly increased after rhS100a9 treatment. Lymphocyte cytotoxicity was higher in lower-risk MDS-lymphocytes than in high-risk MDS-lymphocytes, and S100a9 partially rescued the exhausted cytotoxicity in lymphocytes. PI3K, AKT, and mTOR protein levels were significantly increased after rhS100a9 treatment. PD-1/PD-L1 levels were markedly decreased in SKM-1 and K562 cells after S100a9 treatment. Anti-PD-1 blocking antibody (5 μg/mL) treatment increased PI3K, AKT, and mTOR levels. In mouse models, the (NHD13)Setd2fl/fl and (NHD13)Lep-creSetd2fl/fl groups showed trends toward lower WBC and hemoglobin compared with WT group (not statistically significant). Altered erythroid differentiation with decreased frequencies of mature erythroid cells was observed in transgenic mice. Both transgenic groups had significantly higher PD-L1 expression than the WT group (P<0.05). (NHD13)Lep-creSetd2fl/fl mice showed increased bone marrow hematopoiesis aggressiveness (annular nuclei, neutrophils lacking granules) and promoted megakaryocyte proliferation in the spleen, confirmed by CD41 and CD61 immunohistochemistry.

**Clinical Implications:** This study demonstrates that S100a9 induces apoptosis in MDS clone cells and rescues exhausted CD8+ T cells via PI3K/AKT/mTOR pathway activation. S100a9 acts as an endogenous inhibitor of PD-1/PD-L1, and its deficiency in high-risk MDS may accelerate tumor escape through PD-1/PD-L1 overexpression. The findings suggest that high-concentration S100a9 treatment could partially rescue exhausted lymphocyte cytotoxicity in higher-risk MDS. The results are consistent with clinical trials showing potential benefit of anti-PD-1 agents in MDS treatment. The authors propose that combination therapy with AZA and nivolumab may have better efficacy than AZA monotherapy, and that anti-PD-1 agents could provide mutation-specific supplementary therapy for high-risk MDS patients with mutations such as TP53, N-RAS, and other complex mutations that cannot be overcome by allogeneic stem cell transplantation.