Notch—mediated Fibrogenic Activation Is a Spatial Determinant of RA Treatment Response

Kartik Bhamidipati¹, Ce Gao¹, Ksenia Anufrieva², Shideh Kazerounian³, Roopa Madhu⁴, Miles Tran¹, Vikram Khedgikar¹, Sonia Presti¹, Anna Helena Jonsson⁵, ilya Korsunsky¹ and Kevin Wei⁶, ¹Brigham and Women's Hospital, Boston, MA, ²BWH, Cambridge, MA, ³Brigham & Women's Hospital, Boston, MA, ⁴Brigham and Women's Hospital, Brookline, MA, ⁵University of Colorado School of Medicine, Aurora, CO, ⁶Brigham and Women's Hospital at Harvard Medical School, Boston, MA

Meeting: ACR Convergence 2024

Keywords: Fibroblasts, Synovial, Fibrosing syndromes, rheumatoid arthritis, Transforming Growth Factor (TGF)

Session Information

Date: Sunday, November 17, 2024

Title: Abstracts: RA – Etiology and Pathogenesis I

Session Type: Abstract Session

Session Time: 1:00PM-2:30PM

Background/Purpose: In RA, poor treatment response has been linked to a fibroid synovial tissue phenotype, characterized by lack of immune cell infiltration and an expansion of synovial fibroblasts. Recent transcriptomic analysis of synovial tissue from treatment non-responders reveals strong enrichment of a fibrogenic extracellular matrix (ECM) gene signature. Here, we leveraged spatial transcriptomics and in vitro studies to spatially map the treatment non-responder signature in RA synovium and identify key drivers of fibrogenic signaling.

Methods: Synovial FFPE tissue sections (4 RA) were subject to the Xenium in situ workflow. Samples were probed with a custom panel of 50 genes designed to identify the treatment non-responder fibrogenic signature. Trichrome and immunofluorescent staining was performed on additional RA synovial samples. For in vitro experiments, synovial fibroblasts were stimulated with plate-bound Notch ligand DLL4 or co-cultured with endothelial cells (HUVECs). Fibroblasts were subsequently lysed for qPCR or western blot. Media from cultured cells were collected for analysis of ECM production by ELISA.

Results: Through transcriptomic analysis of synovial biopsies of treatment naïve RA patients classified by TNFi response status¹, we identified a fibrogenic signature that was strongly enriched in TNFi non-responders. Interestingly, the fibrogenic signature enriched in non-responders mapped to a novel POSTN+ sublining fibroblast subset, recently described in the single-cell RA atlas². The non-responder signatures are found in the perivascular niches in RA, and given the localization of the signature, we hypothesized that Notch signaling was linked to fibrogenic activation. In organoid co-cultures of fibroblasts and HUVECs, we observed an expansion of an ECM expressing fibroblast population that was absent with inhibition of Notch signaling³. DLL4 stimulation of fibroblasts in vitro induced significant TGFβ isoform expression, driving autocrine POSTN production that was diminished by TGFβRII silencing. In co-culture, TGFβRII was downregulated by endothelial-derived Notch in a time-dependent manner, leading to fibroblast tolerization and stabilization of ECM production. Analysis of RA synovium revealed evidence of exuberant fibrogenic activation (pSMAD2/3 and POSTN) in regions with diminishing Notch activation compared to regions with sustained Notch activity. Disruption of endothelial—fibroblast crosstalk with DLL4 blockade in co-cultures led to further induction of fibrogenic ECM production via re-sensitization of fibroblasts to TGFβ.

Conclusion: Here, we defined and spatially mapped a fibrogenic fibroblast cell state associated with treatment resistance. We found that Notch signaling controls fibrogenic activation by modulating both TGFβ isoform and receptor expression. Loss of sustained Notch signaling drives a treatment non-response fibrogenic program. Targeting Notch signaling presents an alternate therapeutic axis for treatment refractory patients.

1. Wang, J. et al. Arthritis Rheumatol. 2022. doi: 10.1002/art.42295

2. Zhang, F et al. Nature. 2023. doi: 10.1038/s41586-023-06708-y

3. Wei, K et al. Nature. 2020. doi: 10.1038/s41586-020-2222-z

Figure 1: Spatial mapping of fibrogenic niches in rheumatoid arthritis. (A) Differential gene expression analysis of treatment naïve synovial biopsies classified by TNFi response. Genes enriched in non-responders are highlighted. (B) Non-responder signature score applied to fibroblast cell states as defined by the AMP RA single-cell atlas. (C) Spatial mapping of fibroblasts defined as high and low expressors of the fibrogenic non-responder signature. (D) Post-Xenium H&E of RA synovium annotated as ECM high and low corresponding to the density of pink staining. (E) Visualization of fibrogenic transcript density localization. (F-H) Trichrome and Collagen Type I staining of an additional RA synovial section

Figure 2: Notch signaling controls fibroblast TGFβ expression and responsiveness. (A) Merged UMAP representing single-cells from organoid cultures across three different conditions. DAPT (10 uM) was used to inhibit Notch signaling. (B) Expression of COL1A1 visualized in UMAP space. (C) Violin plot representing fibrogenic signature score across the three conditions. (D) qPCR of fibroblasts stimulated with or without Notch ligand DLL4 for 72 hours. (E) Fibroblasts were treated with NTC or TGFBR2 siRNA at t = 0. Supernatant was collected during a 24-hour window between day 5 and day 6 of stimulation and assayed for POSTN concentration. (F) Fibroblasts and HUVECs were cultured at a 1:1 ratio, fixed and stained for TGFβRII at various timepoints for analysis by flow. MFI of fibroblast TGFBR2 expression is reported as a function of time. (G) Fibroblasts and endothelial cells were co-cultured at a 1:1 ratio with collection of replacement of media every 24 hours. The media was subsequently assayed for POSTN concentration and plotted as a function of time.

Figure 3: Loss of sustained Notch signaling induces fibrogenic activation. (A) IF staining of RA synovium for NOTCH3, pSMAD3 and POSTN. Representative regions showing areas with high Notch activation corresponding to low fibrogenic signaling and conversely low Notch activation corresponding to high fibrogenic signaling. (B) Fibroblasts and HUVECs were cultured 1:1 for 72 hours and then treated with an antibody blocking DLL4 (Enoticumab, Regeneron) at a concentration of 25 ug/mL to disrupt endothelial-fibroblast Notch crosstalk. Media was collected and replaced every 24 hours and was assayed for POSTN concentration. (C) Schematic representing the polarization of fibroblasts from a NOTCH3+ to a myofibroblast-like state upon disruption of endothelial-derived Notch signaling.

Disclosures: K. Bhamidipati: None; C. Gao: None; K. Anufrieva: None; S. Kazerounian: None; R. Madhu: None; M. Tran: None; V. Khedgikar: None; S. Presti: None; A. Jonsson: Pfizer, 6; i. Korsunsky: Mestag Therapeutics LTD, 2; K. Wei: 10X Genomics, 5, Gilead sciences, 2, Merck/MSD, 5, Mestag, 2, santa ana bio, 2.

To cite this abstract in AMA style:

Bhamidipati K, Gao C, Anufrieva K, Kazerounian S, Madhu R, Tran M, Khedgikar V, Presti S, Jonsson A, Korsunsky i, Wei K. Notch—mediated Fibrogenic Activation Is a Spatial Determinant of RA Treatment Response [abstract]. Arthritis Rheumatol. 2024; 76 (suppl 9). https://acrabstracts.org/abstract/notch-mediated-fibrogenic-activation-is-a-spatial-determinant-of-ra-treatment-response/. Accessed .

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