BCR Hyper-Responsiveness In B Cells From Patients With Chronic Gvhd Is Blocked With The Syk Inhibitor R406
Authors: Allen Jessica L. , Tata Prasanthi V. , Wooten Jenna , Fore Matthew S. , Deal Allison M. , Sharf Andrew , Hoffert Todd , Roehrs Philip A. , Armistead Paul M. , Coghill James , Wood William A. , Shea Thomas C. , Serody Jonathan , Richards Kristy L. , Jagasia Madan , Lee Stephanie J. , Rowe Krista , Rizzieri David , Horwitz Mitchell E. , Chao Nelson J. , Sarantopoulos Stefanie 
Published: October 2019

Abstract
Our previous findings indicate that the excess BAFF present in patients with chronic GVHD (cGVHD) induces increased metabolic activity and B-cell survival ex vivo (Allen et al., Blood. 2012. 120:2529). Mechanistic work in murine B cells shows that BAFF treatment increases proliferation in response to B-cell receptor (BCR) stimulation (Patke et al., J Exp Med. 2006. 203: 2551). Taken together, these data suggested that B cells in patients with cGVHD might respond more readily to the allo- and neo-autoantigens present post-transplant. We aimed to determine whether B cells from cGVHD patients were hyper-responsive to BCR stimulation. B cells from 13 allo-HSCT patients who were >12 months post-transplant and not receiving high dose steroid were purified. Proliferation was determined by CFSE incorporation. B cells from patients with cGVHD (n = 6) had a significantly increased proliferative response to BCR stimulation with anti-IgM, even with limiting amounts of ligand, compared to patients without cGVHD (n = 7; p = 0.01). This B-cell hyper-proliferation was specific to BCR signaling as proliferation in response to anti-CD40 plus IL-4 was similar between patient phenotypes.
To determine potential mechanisms underlying the increased BCR-driven proliferation we performed pathway-focused mRNA expression profiling of 84 genes in highly purified CD27- and antigen-experienced CD27+ B cells from patients with cGVHD. One gene integral to BCR-signaling, BLNK, was increased >5-fold in both cGVHD B cell subsets compared to healthy donors. BLNK is a central adapter protein in B-cell activation. It couples antigen-BCR engagement with SYK activation and is required for BCR-driven proliferation in mice. We examined baseline protein levels of BLNK and SYK in un-manipulated B cells from 10 patients by mean fluorescence intensity (MFI). The MFI of BLNK and SYK were significantly increased in B cells from patients with cGVHD (n = 4) compared to B cells from patients without cGVHD (n = 6; BLNK: p = 0.009. SYK: p = 0.009). We next determined that such elevated baseline levels of BLNK and SYK in B cells from patients with cGVHD might amplify BCR signaling. Specifically, stimulation with anti-IgM resulted in increased phosphorylation of BLNK and SYK in cGVHD B cell subsets. Antigen-experienced CD27+ B cells from patients with cGVHD (n = 5) had significantly increased BCR-driven phosphorylation of BLNK (pY84: p < 0.05) and SYK (pY348: p < 0.005) compared to those B cells from patients without cGVHD (n = 6). CD27- B cells from patients with cGVHD (n = 6) also had significantly increased pBLNK compared to non-cGVHD B cells (n = 7; p < 0.0005). Of note, BCR (IgM, IgD and IgG) surface expression on peripheral B cells was similar between cGVHD phenotypes suggesting that our results were not due to altered receptor expression.
BAFF and BCR signaling operate together to convey proliferation, activation and survival signals. To determine if the increased activation of BLNK and SYK were contributing to the B-cell proliferation and survival in cGVHD B cells, we studied the effects of the SYK-inhibitor R406 (the active metabolite of Fostamatinib, kindly provided by Rigel Pharmaceuticals). B cells from patients with and without cGVHD were stimulated with anti-IgM and treated with R406. As expected, B cells from patients with cGVHD hyper-proliferated in response to anti-IgM and SYK inhibition prevented BCR-driven proliferation in vitro in both patient phenotypes. Strikingly, BCR stimulation ex vivo induced a significant increase in surviving B cell number from patients with cGVHD (n = 4) compared to patients without cGVHD (n = 4) (Figure 1). Importantly, inhibition of SYK with low-dose R406 (0.01 μM) abrogated the survival advantage of B cells from patients with cGVHD (Figure 1). These data suggest that B cell hyper-responsiveness in patients with cGVHD is due to elevated levels and activation of proximal BCR signaling components. Our findings suggest novel therapeutic targets in patients with cGVHD.Figure 1SYK inhibition with R406 abrogates BCR-driven proliferation and survival of B cells from patients with cGVHD. Peripheral B cells from patients without cGVHD (grey bars) and with cGVHD (white bars) treated as indicated for 6 days. [Percent change = (V2 – V1) / V1 *100]. Data are median +/- range, pooled from 3 independent experiments. n = 4 / phenotype. One-Way ANOVA, p = 0.0002. Tukey's Post Hoc, * p < 0.05, ** p < 0.005, *** p <0.0005.Figure 1. SYK inhibition with R406 abrogates BCR-driven proliferation and survival of B cells from patients with cGVHD. Peripheral B cells from patients without cGVHD (grey bars) and with cGVHD (white bars) treated as indicated for 6 days. [Percent change = (V2 – V1) / V1 *100]. Data are median +/- range, pooled from 3 independent experiments. n = 4 / phenotype. One-Way ANOVA, p = 0.0002. Tukey's Post Hoc, * p < 0.05, ** p < 0.005, *** p <0.0005.

Disclosures:
Rizzieri: Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau.