LOX1 expression identifies a population of polymorphonuclear myeloid-derived suppressor cells that are highly enriched in bone marrow samples of treatment-Naïve and BTKi-Relapsed/Refractory WM patients and show constitutive activation of BTK

Abstract

Abstract Introduction: Immune microenvironment dysregulation is a hallmark of early/smoldering Waldenstrom's Macroglobulinemia (WM) that underlies disease progression (Kaushal et al 2021, Sklavenitis-Pistofidis et al 2025). The mechanisms orchestrating immune dysregulation at WM diagnosis and relapse remain poorly understood. Myeloid-derived suppressor cells (MDSC) are pathologically activated myeloid cells with potent immunosuppressive activity, able to induce T cell tolerance in cancer. Bone marrow (BM) expansion of polymorphonuclear-MDSC (PMN-MDSC) has been described in symptomatic WM (Bhardwaj et al 2024). LOX1 is a selective marker for suppressive PMN-MDSC, whose role has not been defined in WM. We showed that MDSC-related inflammatory mediators, including the S100A8 and S100A9 alarmins, are highly expressed in early/smoldering and downregulated in late/symptomatic WM, where a different set of effectors, including IL-6 and CXCL13, becomes predominant [Hunter et al, Blood 2023; 142 (Suppl. 1):756]. In this study, we sought to investigate the interplay between tumor and MDSC in BM and peripheral blood (PB) from treatment-naïve and relapsed/refractory WM patients. Methods: Spectral flow cytometry on BM (BMMC) and/or PB mononuclear cells (PBMC) from 5 WM patients (2 untreated and 3 relapsed/refractory to BTKi-containing therapeutic regimens) was performed to phenotypically profile monocytic-MDSC (M-MDSC), PMN-MDSC and the expression of the S100A8 and S100A9 receptors CD33, CD69, RAGE and TLR4. Paired BMMC/PBMC were analyzed in 2 patients; 2 healthy donor (HD) PBMC were run as controls. Intracellular staining for p-BTK, p-HCK and p-ERK1/2 was carried out in 6 samples. Data acquisition was performed on a Sony ID7000 cytometer using the Sony ID7000 software. We studied the transcriptional regulation of 16 WM-related inflammatory genes via qRT-PCR in endogenously MYD88MUT WM (BCWM.1, MWCL-1) and MYD88WT lymphoma (OCI-Ly7, Ramos) cell lines overexpressing either MYD88WT or MYD88MUT following lentiviral transduction. Transient MYD88WT or MYD88MUT transfection was performed in 293T cells as a non-B cell control. Results: By scatter gating, untreated WM BM samples showed greater enrichment of granulocytic vs monocytic populations. We characterized PMN-MDSC as CD14-CD15+CD11b+LOX1+. LOX1+ PMN-MDSC were the predominant subtype vs LOX1- granulocytes in WM untreated and BTKi-relapsed/refractory BM samples (MFI 5,988 vs 1,110; p=0.021). LOX1+ PMN-MDSC showed constitutive p-BTK activation across the analyzed HD and WM samples. This finding is particularly compelling in the context of WM, given its dependency on the BTK signaling and the critical therapeutic role of BTKi. Within the monocyte gate, CD14+CD11b+CD15-HLA-DR+ monocytes were enriched vs CD14+CD11b+CD15-HLA-DRlow/-(M-MDSC) in patient BM and PB. In BM, M-MDSC upregulated CD69 vs HLA-DR+ monocytes. Multidimensional analysis of paired samples from a BTKi relapsed/refractory patient showed the distinct expansion of RAGEhigh monocytes in PB vs BM. Furthermore, we studied the transcriptional regulation of key WM inflammatory mediators to investigate tumor-related mechanisms promoting early recruitment and activation of MDSC. MYD88WT and MYD88MUT overexpression predominantly induced gene upregulation. The MYD88MUT-driven upregulation of CXCL12, CXCL13 and IL-6 was specific to WM cell lines. Unlike BCWM.1, IL-6 was also upregulated in MWCL-1 following MYD88WT overexpression. Upregulation of the CXCL1, CXCL2 and CXCL10 chemokines was driven by both MYD88WT and MYD88MUTin WM and 293T lines; a stronger effect was driven by MYD88MUTin WM and by MYD88WT in 293T lines. Moreover, upregulation of S100A8 and S100A9 was stronger in MYD88WT vs MYD88MUT BCWM.1. S100A8 upregulation followed exclusively MYD88MUT overexpression in 293T and OCI-Ly7 lines. Unlike the WM lines, OCI-Ly7 and Ramos upregulated IL-10, especially following MYD88MUT overexpression.Conclusions: Our study identified the BM expansion of LOX1+ PMN-MDSC which showed constitutive BTK activation in untreated and BTKi relapsed/refractory WM patients, thus opening a new avenue of investigation of BTK-targeting drugs for WM-directed cellular therapies. We showed that MYD88MUT induced the transcriptional upregulation of key inflammatory mediators that were highly expressed in WM patients, thus providing a framework for the investigation of their biological role in recruiting and activating immunosuppressive MDSCs.