Non-Canonical Gli1 Activation Mediates Profibrotic Hematopoiesis-Stroma Crosstalk in MPN

Recent work demonstrated that Gli1⁺ cells are fibrosis-driving cells in myeloproliferative neoplasms (MPNs). Genetic ablation of Gli1⁺ cells or pharmacologic targeting of Hedgehog (Hh)-Gli signaling ameliorated fibrosis in mouse models of myelofibrosis (MF). Although preclinical and clinical data suggest that Hh pathway inhibitors have therapeutic activity in MF, the role of canonical vs. non-canonical Hh-signaling in MF was never systematically analysed.

We first asked if Gli1, as a readout for Hh signaling, is expressed at higher levels in myeloid cells from MPN patients compared to controls. PBMCs from patients with varying MF grades were isolated and intracellular Gli1 levels determined for different cell populations via flow cytometry. In particular, CD66b⁺ granulocytes showed significantly increased levels of Gli1 in MPN patients in correlation with MF grade. We hypothesized that inhibition of Hh/Gli signaling in hematopoietic cells may ameliorate bone marrow fibrosis and potentially interrupt crosstalk with fibrosis-driving stromal cells.

To systematically interrogate the role of the Hh pathway in hematopoietic cells, we transplanted ckit⁺ hematopoietic stem and progenitor cells (HSPCs) isolated from 1) Gli1, 2) Gli2 or 3) Smoothened (Smo) knockout mice which were transduced with a thrombopoietin (ThPO) overexpression or JAK2V617F (JAK2VF) plasmid, and respective controls. These models robustly induce MPN and MF. The knockout of Gli1, but not Gli2 or Smo, in HSPCs significantly reduced measures of progressed MF in vivo such as splenomegaly and anemia and, importantly, also reduced fibrosis. These data suggest that Smo independent, non-canonical Hh signaling results in Gli1 activation in HSPCs.

To understand how inhibition of Gli1 in HSPCs in vivo can ameliorate the activation of fibrosis-driving stromal cells, we performed single cell RNA sequencing on HSPCs and stromal cells in the presence or absence of hematopoietic Gli1, comparing the JAK2VF-induced MPN model with the control. In normal HSPCs inhibition of Gli1 resulted in downregulation of EGFR, PI3K and MAPK pathways, which are all associated with non-canonical Hh/Gli activation. In JAK2VF induced MPN, HSPCs showed upregulation of VEGF and WNT signaling. These were downregulated in the absence of Gli1, in line with the observed reduced disease severity.

Stromal cells were significantly affected by JAK2VF mutant HSPCs and upregulate key fibrosis pathways such as VEGF, p53, hypoxia and TGFb. Without Gli1 in JAK2VF HSPCs these pathways were instead downregulated in stromal cells, corroborating a profibrotic, Gli1-mediated crosstalk between HSPCs and stroma.

We next focused on analysis of receptor-ligand pairs which revealed changes in cell-cell interactions associated with the absence of Gli1 in HSPCs. Inhibition of Gli1 signaling in HSPCs resulted in increased stroma-stroma interactions both under normal conditions and in the context of MPN. Particularly in the MPN setting, HSPC-stroma interactions were decreased, potentially resulting in dysregulation of hematopoiesis and stromal autonomy. Inhibition of Gli1 in HSPCs in MPN restored this crosstalk. One affected interaction was between macrophage migration inhibitory factor (MIF) on HSPCs and CD74 on stromal cells. CD74 and MIF have been discussed as potential therapeutic targets in solid organ fibrosis.

To determine the clinical relevance of MIF we measured levels in MPN patient serum. MIF levels were significantly increased in MPN compared to controls and were significantly correlated with platelet counts. To determine the role of hematopoietic MIF in fibrosis, we transplanted HSPCs from MIF knockout mice transduced with ThPO or control. Whilst there was no significant reduction in fibrosis grade, there were significant reductions in cell populations associated with more advanced disease such as monocytes and macrophages.

Here we demonstrate that Gli1 in the MPN clone can be activated in a non-canonical fashion and may represent a therapeutic target, in particular since Hh signaling is dispensable for normal hematopoiesis. We demonstrate that hematopoietic Gli1 has a significant effect on stromal cells, partially mediated through a MIF/CD74 axis. These data highlight the complex interplay between alterations in the MPN clone, immune cell subsets such as monocytes and macrophages, and activation of stromal cells.

Koschmieder:Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Bristol Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; CTI Biopharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; PharmaEssentia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Alexion: Other: Travel support; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Karthos: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; RWTH Aachen University: Patents & Royalties: BET Inhibitor; AOP Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Geron: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding. Berres:Bayer: Consultancy; Roche Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel and Accommodation support; Pierre Fabre Pharm: Honoraria; Lilly: Honoraria; MSD Sharp&Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees. Kramann:Pfizer: Consultancy; Gruenenthal: Consultancy; Chugai: Research Funding; Bayer: Consultancy; Travere Therapeutics: Research Funding; Galapagos: Research Funding; Novo Nordisk: Consultancy, Research Funding.