Monocyte-Driven Aberrant Inflammation in Myeloproliferative Neoplasms Is Regulated By Galectin-1

JDP and STO co-corresponding authors

Previous studies by our group and others have elucidated a key role for monocytes in driving hyperinflammation in myeloproliferative neoplasms (MPNs). To further interrogate the cellular landscape of aberrant inflammation in MPNs, we subjected MPN patient samples to single cell RNA-seq (scRNA-seq) analysis and found significant enrichment of inflammation-related genes, including galectins, in MPN patient monocytes. Cell-cell communication networks inferred from expression of ligands and receptors predicted monocytes as a pivotal mediator of cell interactions and galectin signaling as one of the most robust input/output pathways for monocytes. Galectins are a class of proteins that bind to glycosylated proteins and mediate broad biological functions, including cell proliferation, apoptosis, adhesion, and inflammation. Dysregulation of galectins and global protein glycosylation has been reported in various cancers, but its role in MPN has remained incompletely understood.

To validate our scRNA-seq findings, we performed flow cytometry analysis which demonstrated elevated expression of galectin-1 (Gal-1) in MPN patient CD14+ monocytes. Increased plasma levels of Gal-1 were also identified in MPN patients compared to healthy individuals. We also observed enrichment of Gal-1 in mouse CD11b+ myeloid cells, with further elevation in JAK2^V617F knock-in mice compared to wild-type controls. Additionally, Gal-1 expression was induced by MPL^W515L and JAK2^V617F and inhibited by ruxolitinib, a JAK inhibitor, in Ba/F3 cells. Thus, our results confirm enrichment of Gal-1 in MPN monocytes, with evidence of direct contribution from specific MPN driver mutations.

To comprehensively characterize functional effects of Gal-1, we performed mass cytometry (CyTOF) analysis of MPN samples which demonstrated that Gal-1 stimulation induced multiple inflammatory cytokines, including TNF and IL-6, in monocytes specifically without affecting other types of cells. We further incubated CD14+ monocytes from MPN patients with recombinant Gal-1 (rGal-1) and observed markedly stimulated transcription and secretion of inflammatory cytokines, such as IL-1α, IL-1β, IL-6, IL-8 and TNF. Pharmacologic inhibition of Gal-1 by OTX008 suppressed the expression and secretion of inflammatory cytokines in MPN monocytes and monocytic cell lines. Furthermore, we observed crosstalk between LPS-TLR4 and Gal-1 signaling pathways. Using protein 3D structure prediction and co-immunoprecipitation, we confirmed interaction of Gal-1 with TLR4. Notably, targeting TLR4 via both neutralizing antibody and pharmacologic inhibition (TAK-242) abrogated the proinflammatory effects of Gal-1 on monocytes. Taken together, these findings uncover a monocyte-specific pro-inflammatory effect of Gal-1 mediated by TLR4 in MPN.

To explore molecular mechanisms underlying its proinflammatory functions, we performed gene co-expression analysis for Gal-1 in monocytes and identified OXPHOS and PI3K-AKT-mTOR signaling pathways as top candidates. Both genetic and pharmacologic inhibition of Gal-1 reduced cellular ATP levels and oxygen consumption rate, suggesting a metabolic reprogramming by Gal-1 in MPN. Incubation of monocytic cell lines and MPN monocytes with rGal-1 stimulated PI3K-AKT-mTOR signaling, represented by increased levels of phosphorylated mTOR, AKT and S6. Consistently, genetic and pharmacologic inhibition of Gal-1 inhibited activation of the PI3K-AKT-mTOR pathway.

Across in vivo models, both Gal-1 knockout and OTX008 ameliorated key MPN disease features, including leukocytosis and splenomegaly, driven by MPL^W515L and JAK2^V617F. Gal-1 inhibition also suppressed carrageenan-induced thrombosis and inflammation in mice. We further evaluated therapeutic effects of targeting global glycosylation in MPN via 2-Deoxy-D-glucose (2-DG), a global glycosylation inhibitor, which decreased splenomegaly and reduced elevated platelets and hematocrit in JAK2^V617F knock-in mice.

In summary, we identify enrichment of Gal-1 in MPN monocytes, potentially due to activation of JAK2-driven signaling. We further demonstrate that Gal-1 fuels monocyte inflammation by interacting with TLR4 and activation of OXPHOS and PI3K-AKT-mTOR signaling pathways. Our results uncover a novel therapeutic avenue for targeting aberrant Gal-1 and global glycosylation in MPNs.