targeting syk activated b-cells in murine and human

1

Targeting Syk Activated B-cells in murine and human chronic graft-versus-host disease

Ryan Flynn1, Jessica L. Allen2, Leo Luznik3, Kelli P. MacDonald4, Katelyn Paz1,

Kylie A. Alexander4, Ante Vulic3, Jing Du1, Angela Panoskaltsis-Mortari1, Patricia A. Taylor1, Jonathan C. Poe2, Jonathan S. Serody5, William J. Murphy6, Geoffrey R. Hill4, Ivan Maillard7, John Koreth8, Corey S. Cutler8, Robert J. Soiffer8, Joseph

H. Antin8, Jerome Ritz8, Nelson J. Chao2, Raphael A. Clynes*9, Stefanie Sarantopoulos*2 and Bruce R. Blazar*1.

1Division of Blood and Marrow Transplantation, University of MN, Masonic Cancer Center and Department of Pediatrics, Minneapolis, MN; 2Division of Hematological Malignancies and Cellular Therapy and Duke Cancer Institute, Duke University Department of Medicine and Duke Cancer Institute, Durham, NC; 3Sidney Kimmel Comprehensive Cancer Center and Department of Oncology, The Johns Hopkins University, Baltimore, MD; 4Department of Immunology, Queensland Institute of Medical Research, Brisbane, Australia; 5Lineberger Comprehensive Cancer Center Division of Hematology/Oncology, University of North Carolina, Chapel Hill, NC; 6Department of Dermatology, School of Medicine, University of California, Davis, Sacramento, CA; 7Life Sciences Institute and Department of Internal Medicine, Division of Hematology-Oncology, University of Michigan, Ann Arbor, MI; 8Dana Farber Cancer Institute, Boston, MA; 9Columbia University Medical Center, Columbia University. *Equal contributors

Short title: Chronic GVHD suppression by Syk inhibition

Correspondence: Bruce R. Blazar, M.D.; MMC 109, University of Minnesota 420 Delaware St. SE; Minneapolis, MN 55455 USA; E-mail: [email protected] Tel: 612-626-1926 Fax: 612-624-3913 Word No: Abstract: 200; Text: 3889; Figures: 7; Supplemental Figures 1; Supplemental Table 1; References: 37; Category: TRANSPLANTATION

Blood First Edition Paper, prepublished online April 7, 2015; DOI 10.1182/blood-2014-08-595470

Copyright © 2015 American Society of Hematology

For personal use only.on June 11, 2015. by guest www.bloodjournal.orgFrom

http://www.bloodjournal.org/

http://www.bloodjournal.org/site/subscriptions/ToS.xhtml

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Key Points

1) Syk is required for increased B cell activation and cGVHD generation and

maintenance.

2) The Syk inhibitor Fostamatinib can treat murine cGVHD and increase

human cGVHD B cell death.




3

Abstract

Novel therapies for chronic graft-versus-host disease (cGVHD) are needed.

Aberrant B cell activation has been demonstrated in mice and humans with

cGVHD. Having previously found that human cGVHD B cells are activated and

primed for survival, we sought to further evaluate the role of the spleen tyrosine

kinase (Syk) in cGVHD in multiple murine models and human peripheral blood

cells. In a murine model of multi-organ system, non-sclerodermatous disease

with bronchiolitis obliterans where cGVHD is dependent on antibody and

germinal center (GC) B cells, we found that activation of Syk was necessary in

donor B cells but not T cells for disease progression. BM-specific Syk deletion in

vivo was effective in treating established cGVHD as was a small molecule

inhibitor of Syk, fostamatinib which normalized GC formation and decreased

activated CD80/86+ dendritic cells. In multiple distinct models of sclerodermatous

cGVHD, clinical and pathological disease manifestations were not eliminated

when mice were therapeutically treated with fostamatinib, though both clinical

and immunological effects could be observed in one of these scleroderma

models. We further demonstrated that Syk inhibition was effective at inducing

apoptosis of human cGVHD B cells. Together these data demonstrate a

therapeutic potential of targeting B-cell Syk signaling in cGVHD.




4

Introduction

The development of cGVHD is a major complication following allogeneic

hematopoietic stem cell transplantation. Discovery of new therapies has been

limited by the absence of murine models that closely represent the clinical human

disease and pathogenesis1, 2. Herein we use multiple mouse models to assess

the effectiveness of therapies during cGVHD. We utilize a mouse model of

antibody-dependent, multi-organ system cGVHD, previously demonstrated to

mimic several aspects of human cGVHD pathology, with the exception of

sclerodermatous cGVHD3, 4and three models of sclerodermatous cGVHD with

skin manifestations5, 6.

While the exact mechanisms of cGVHD remain unknown, recent studies

have elucidated a role for antibody production by B-cells. The increase of B-cells

in the germinal center (GC) has been shown to be necessary for the

development of an antibody-facilitated multi-organ system cGVHD model that

includes bronchiolitis obliterans (BO)3 Spleen tyrosine kinase (Syk) is activated

by B-cell receptor (BCR) engagement. After antigen-BCR engagement, Syk is

phosphorylated at Y348, allowing for B-cell survival and proliferation. Increased

proximal BCR signaling was recently described in cGVHD patient B cells.7

Syk also affects myeloid cell [macrophages; neutrophils and dendritic cells

(DCs)] including phagocytosis, signal transduction via activating FcRs, and

antigen-uptake, internalization and upregulation of costimulatory molecules in

DCs.8 Syk also has been demonstrated to have an impact on cell-migration of

monocytes.9 Given the function of Syk in myeloid cells and the critical role of




5

macrophages and DCs in cGVHD5,10, Syk inhibition may have additional

advantages of treating cGVHD via macrophage and DC effects.

We sought to determine whether B-cell Syk activation was a critical

component in cGVHD pathophysiology. First, we demonstrated that Syk was

hyper-responsive in B-cells during cGVHD and that Syk was necessary in B- but

not T-cells for murine cGVHD progression. Next, we demonstrated that the

deletion of Syk in donor BM cells in vivo in mice with established cGVHD with BO

was able to reverse disease, a finding that was phenocopied by the in vivo

molecular inhibition of Syk with fostamatinib. Inhibition of Syk decreased the

frequency of GCs and expression of the activation costimulatory molecules CD80

and CD86 on CD11c+ cells in vivo. In multiple distinct models of sclerodermatous

cGVHD, clinical and pathological disease manifestations were not eliminated

when mice were therapeutically treated with fostamatinib, though both clinical

and immunological effects could be observed in one of the sclerodermatous

cGVHD models. Human cGVHD B cells had increased death when treated with

fostamatinib, demonstrating BCR-activated B cells can be preferentially targeted.

These data together suggest that Syk could be a novel therapeutic for cGVHD

patients.




6

Materials and Methods

Mice

C57Bl/6 (B6; H2b) mice were purchased from the National Cancer Institute.

B10.BR (H2k) mice were purchased from Jackson Laboratories. Female C57Bl/6

(B6) (H-2b, CD45.2), and B6D2F1 (H-2b/d, CD45.2) mice were purchased from

the Animal Resources Center. BALB/c, B10.D2-Thy1.2 and B10-.D2-Thy1.1 mice

were propagated in the animal facility at the Johns Hopkins University Cancer

Research Building I. The Syk fl/fl x ERT2-cre mice were provided by RAC from

Columbia University. Deletion of Syk (Syk KO) occurred with administration of

tamoxifen (Sigma) PO for 5 days and confirmed by Western Blot. Mice were

housed in a specific pathogen-free facility and used with the approval of each

institution’s animal care committee.

BMT

For model 1 (systemic cGVHD with BO, MHC disparate), B10.BR recipients were

conditioned with cyclophosphamide and total body irradiation (8.3 Gy).

Recipients received 10x106 T cell depleted (TCD) bone marrow (BM) cells from

B6 wildtype (WT) or BM cells from donors that were induced to delete Syk by

tamoxifen administration. Cohorts received no supplemental T cells or purified

splenic T-cells (0.1x106) from WT vs KO donors3. In one experiment, as

indicated, mice were given donor BM from Syk fl/fl x ERT2-cre or WT mice with

or without purified T cells; on D28 post-BMT, mice were given tamoxifen to delete

Syk from donor BM cells. For model 2 (sclerodermatous cGVHD, semi-allogeneic




7

recipients), on day -1, B6D2F1 recipient mice received total body irradiation (11

Gy; 137Cs source), split into 2 doses separated by 3 hours to minimize

gastrointestinal toxicity5. On day 0, recipients underwent transplantation with 5 x

106 BM with or without 1 x 106 purified T cells from B6 mice. Non-GVHD control

groups were injected with TCD BM grafts. For model 3 (sclerodermatous cGVHD

with G-CSF mobilized splenocytes, MHC disparate), on day –1, mice received

total-body irradiation (1Gy; 137 Cs source), delivered as in model 2. On day 0, B6

mice each received 25 × 106 donor splenocytes from G-GSF–mobilized BALB/c

donors. Spleen were depleted of T cells as previously described for non-GVHD

control groups11. Transplanted mice were monitored daily, and those with GVHD

clinical scores of ≥610, 11 were sacrificed, and the date of death registered as the

next day in accordance with institutional animal ethics committee guidelines. For

model 4 (sclerodermatous cGVHD, minor histocompatibility antigen disparate), a

single lethal irradiation dose of 7.75 Gy was administered using a 137Cs irradiator.

Animals were reconstituted with 107 B10.D2-Thy1.2 TCD BM cells (107) alone or

supplemented 1.8 × 106 CD4+ and 0.9 × 106 CD8+ Thy1.1+ T cells, reflecting T

cells found in 1.2 × 107 B10.D2 donor splenocytes, a dose that reproducibly

induces GVHD6. Purified populations of donor T cells were obtained using T cell

isolation kits (Dynabeads, Invitrogen, Carlsbad, CA).

Where indicated, cGVHD recipients were given R788 (30

mg/Kg/animal/twice PO daily) or 0.1% carboxymethylcellulose vehicle from d28-

56 (BO model 1), D14-28 (B6 into B6D2F1, scleroderma, model 2), days 7-21

(G-CSF mobilized Balb/c splenocytes into B6 recipients, scleroderma model 3),




8

or B10.D2 into Balb/c (experiment 1, D21-35; experiment 2, D14-28). Both pro-

drug, R788, and its active metabolite, R406 (both known as fostamatinib) were

kindly provided by Rigel Pharmaceuticals. Lung function was assessed as

previously described.12

Patient Samples

Samples were obtained from patients following written informed consent in

accordance with the Declaration of Helsinki. Viably frozen peripheral blood

samples from patient with either active clinical manifestations of cGVHD or no

active cGVHD were randomly selected from cell banks for study (patient

characteristic details are shown in (Supplemental Table 1). The Institutional

Review Boards at the University of North Carolina Chapel Hill, Duke University

Medical Center, and the Dana-Farber Cancer Institute approved all studies.

Analysis of Syk Activation and In Vitro Effects of Syk Inhibition

For analysis of Syk phosphorylation in murine cGVHD, B-cells were purified from

murine cGVHD spleens or BM only controls on D60 in the cGVHD/BO model and

activated by 5 μg/mL anti-IgM Fab (Jackson Research), fixed with BD Cytofix

Fixation Buffer and permeabilized with BD Perm Buffer III. Cells were

subsequently stained with anti-Syk Y348 PE (eBioscience) as previously

described13.

For analysis of the effects of in vitro Syk inhibition, cryopreserved PBMCs

from cGVHD or non-cGVHD control patients were thawed and allowed to rest

overnight. The Syk inhibitor, R406, (0.01 or 0.1 μM) or DMSO control (0.1%) for




9

48 hours at 37°C. Apoptosis was measured by gating on CD19+ or CD19- cells

and co-staining with Annexin V and 7-AAD.

Flow Cytometry

GC B cells and CD11c+ cells were labeled with anti-CD19 (Clone: eBio1D3), anti-

CD11c (Clone: N418), anti-CD80 (Clone:16-10A1), anti-CD86 (Clone: GL1), anti-

MHCII (Clone: M5/114.15.2), anti-GL7 (Clone: GL-7) (eBioscience), anti-CD4

(clone: RM4-5) anti-CD95 (Clone: Jo2) (BD Biosciences), anti-F4/80 (Clone:

BM8), anti-CXCR4 (Clone: L276F12), anti-Ly6G (Clone: 1A8), anti-CD3

(Clone:145-2C11), anti-Ly6C (Clone: HK1.4), anti-CD8 (Clone: 53-6.7), and anti-

CD11b (Clone: M1/70), (biolegend) were analyzed on BD LSRFortessa. Ficolled

human PBMC were stimulated and stained for Annexin V as previously

described.14, 15

Frozen tissue preparation, pathology and immunofluorescence Organs

harvested were embedded in Optimal Cutting Temperature compound, snap

frozen, and stored at -80°. GC detection and pathologic scoring were

accomplished as previously described4. At various times post transplantation,

GVHD target tissues were harvested, fixed in 10% formalin for 24hrs, embedded

in paraffin, and processed to generate 5µm-thick sections. H&E sections of skin

were examined in a blinded fashion using a semi quantitative scoring system for

GVHD as previously published.4, 16 Samples were scored from 0 to 4 for




10

epidermal and dermal inflammation, dermal fibrosis and subcutaneous fibrosis,

epidermal apoptosis, and loss of subcutaneous fat.

Statistics

Group comparisons of pathology, pulmonary function tests, cell counts and flow

cytometry data were analyzed by Student t-test or one-way ANOVA.




11

Results

Syk phosphorylation is increased during murine cGVHD and is necessary

in donor BM derived cells for the development and maintenance of cGVHD.

To determine if Syk activation is important in murine cGVHD, purified B

cells (Figure 1) from day 60 cGVHD spleens were assayed for Syk

phosphorylation. Purified B cells from cGVHD mice were activated with anti-IgM

and the amount of phosphorylated Syk at Y348 was measured. There was an

increase in the percentage pSyk in both BCR stimulated B-cells and unstimulated

B-cells in cGVHD (Figure 1). These data extend a recent report that human B-

cells have increased pSyk during cGVHD7.

To test whether Syk is necessary for cGVHD progression, mice were

transplanted with WT or induced Syk KO BM with WT T-cells and analyzed for

disease on day 60. Mice receiving Syk KO BM and WT T-cells did not develop

cGVHD pulmonary dysfunction compared to those receiving WT BM and T-cells

(Figure 2 A). Since Syk is necessary for the proliferation of B-cells following

antigen stimulation17, we analyzed the spleens of transplanted mice to determine

if there was a defect in the maintenance of Syk KO BM-derived B-cells. We found

that total B-cell frequency in mice receiving Syk KO BM was decreased 8-fold

(Figure 2B). These data are consistent with the dependency of activated B-cells

on Syk for proliferation and survival, and a requirement for activated donor-

derived BM B-cells in cGVHD pathogenesis3, 4.

In contrast to Syk dependence in BM derived cells, the addition of Syk KO

donor T-cells did not attenuate the functional manifestations of BO (with the




12

exception of resistance) compared to Syk WT T cell transplanted mice (Figure 2

C). In addition cGVHD pathology in the lungs and liver were similar in recipients

of Syk WT vs KO T cells (Figure 2D). While T cell effects after Syk inhibition were

previously described in acute18 and sclerodermatous cGVHD19, in this systemic

cGVHD model with BO and without scleroderma, donor T-cell Syk was not fully

required for cGVHD development.

To determine whether Syk function in BM-derived B cells was essential for

sustaining cGVHD in mice with established disease, recipients were given donor

BM from Syk fl/fl x ERT2-cre or WT mice with or without purified T cells. On D28

post-BMT, mice were given tamoxifen. Induced deletion of Syk in donor BM cells

significantly improved all pulmonary function parameters to levels of non-cGVHD

controls (Figure 3).

Therapeutic pharmacological inhibition of Syk decreases pathology in a

murine BO model of cGVHD but had little clinical therapeutic effect in

scleroderma models of cGVHD.

Fostamatinib is a potent small molecule inhibitor of Syk. Studies in

rheumatoid arthritis have demonstrated efficacy with fostamatinib in randomized

phase II clinical trials.20, 21 Fostamatinib was notably safe in patients treated for

non-Hodgkin’s lymphoma, including some who had received prior autologous

HCT.22. Additionally, Leonhardt et. al.18 demonstrated that Syk inhibition

decreased costimulatory molecules on antigen-presenting cells and increased

survival of mice during acute GVHD while preserving anti-tumor and anti-viral




13

immunity. In addition, other studies have demonstrated the importance of BCR

signaling for the development of murine cGVHD.23

Having demonstrated the requirement for Syk in donor BM cells in mice

with established cGVHD and BO, we next sought to determine whether drug

treatment with fostamatinib in mice with established cGVHD would be similarly

efficacious as Syk deletion in donor BM cells.3, 4 Mice receiving fostamatinib

beginning on D28 had restoration of pulmonary function, similar to the healthy

transplanted BM only controls (Figure 4 A). Improvement in pulmonary function

correlated with a reduction in cGVHD pathology in the lung (Figure 4B). The

number of GC reactions in the spleen was decreased in fostamatinib versus

vehicle treated cGVHD mice (Figure 4C). This was highlighted by a decrease in

frequency of splenic GC B-cells in fostamatinib treated cGVHD mice, matching

BM only controls (Figure 4D). These data demonstrate the importance of Syk in

B cell signaling during active disease.

Previous reports demonstrated a decrease in activation of CD11c+ cells18

in mice with acute GVHD treated with fostamatinib. In the BO cGVHD model we

found increased expression of costimulatory molecules CD80 and CD86 on

CD11c+ spleen cells compared to BM only transplanted mice. When these

cGVHD mice are treated with fostamatinib beginning D28, we found a significant

decrease in the frequency of CD11c+ cells expressing CD80 and CD86 though

without affecting MHC class II expression (Figure 4 E-G). These data highlight a

potentially beneficial alternative effect of Syk inhibition CD11c+ myeloid cells

during cGVHD.




14

In order to determine whether the effects of Syk inhibition attenuate

murine sclerodermatous cGVHD in our hands, we utilized three different cGVHD

scleroderma models involving lethally irradiated recipients: B6 into B6D2F1

model (model 2); G-CSF mobilized BALB/c splenocytes into B6 (model 3), and a

minor-mismatch model of B10.D2 into Balb/c (model 4). In the B6 into F1 model

(fostatinib, D14-28) and in the G-CSF-mobilized Balb/c into B6 models (fostatinib,

D7-21), the frequency of splenic CD19+ B cells in vehicle and fostatinib treated

cGVHD were >10- and 5-fold fold lower than non-cGVHD, BM only controls in

both the B6 into B6D2F1 and G-CSF Balb/c into B6 models, respectively (Figure

5 A and Supplemental Figure 1A). Neither a significant beneficial clinical

therapeutic nor pathological effect as reflected by total skin inflammation score

was seen in either model (Figure 5 B and Supplemental Figure 1B). However, we

found a significant decrease in peripheral blood CD11b+F4/80+ and Ly6Chi

monocytes numbers in the B6 into B6D2F1 model after treatment with

fostamatinib in the B6 into B6D2F1 model (Figure 5 C-E), which was also seen in

the G-CSF mobilized Balb/c into B6 model but did not reach statistical

significance (Supplemental Figure 1 C-E).

In the B10.D2 into Balb/c model of scleroderma, fostamatinib treatment

from D.21-35 did not impact skin disease or clinical score (not shown). However,

beginning fostatinib treatment 1 week earlier on D14, as used by Huu et. al.19, did

significantly improve these parameters on D19 and there was a reduction in the

both mean skin score from 3 to 1 and the GVHD clinical score from 6 to 4 in the

fostatinib vs vehicle treated controls on D35, the end of the study (Figure 6 A and




15

B). In the latter experiment, there also was a decrease in total numbers of viable

cervical and mesenteric lymph node (but not splenic) B cells and frequency of

CD11b+ cervical lymph node cells. CXCR4+ T cells in the spleen and cervical

lymph node were also decreased in fostamatinib vs vehicle treated cGVHD mice

(Figure 6 C-G), suggesting an impairment to the overall immune response and

migration of T cells and myeloid cells to tissues. Taken together, these data

suggest that early post-transplant treatment of sclerodermatous cGVHD may

attenuate disease progression.

Fostamatinib increases apoptosis in B cells purified from cGVHD patients.

To determine if human cGVHD B-cells are more susceptible to Syk blockade,

human peripheral blood (Supplementary Table 1) were treated in vitro with R406,

the active form of R788. B cells from patients with active cGVHD had increased

apoptotic and total cell death compared to patients with inactive or no cGVHD

after R406 (Figure 7). These data, consistent with work by Allen et. al,7 reveal

that R406 preferentially kills cGVHD B cells via apoptosis. Global targeting of B

cells with rituximab has been met with mixed success possibly due to altered B

cell homeostasis perpetuated in some patients.24, 25 We now demonstrate that

constitutively activated B cells can be selectively targeted in cGVHD by Syk

inhibition (Figure 7B). Consistent with the lack of a requirement for Syk in donor

T cells in the cGVHD BO model, fostamatinib did not induce apoptosis in the

non-B cell population (Figure 7C). In 3 of samples from each patient group, T-cell

specific anti-CD3 antibody was included to stimulate T-cell proliferation in the




16

presence of R406; no increase in T-cell death was observed under these

conditions. Together, these data point to the therapeutic potential of fostatinib

and pSyk targeting in cGVHD patients.

Discussion

Here, we demonstrated that pSyk was upregulated in murine cGVHD B

cells in a multi-organ system model of cGVHD with BO that is dependent upon

donor T cell support of antibody isotype switching and immunoglobulin

deposition. Such cGVHD B cells were hyper-responsive to B cell activation

signals in vitro. Syk function in donor BM-derived B cells but not T cells was

required for cGVHD generation and Syk deletion in donor BM cells in mice with

established cGVHD on D28 post-BMT reversed disease. A small molecule

inhibitor of pSyk, fostamatinib that has been tested in the clinic for autoimmune

diseases, was able to treat established cGVHD when therapy was initiated on

D28. In cGVHD patients, previous studies have shown that cGVHD B cells have

high levels of pSyk and in the current study, we now show that B cells from

patients with active cGVHD but not B cells from patients without active cGVHD

was induced into apoptosis in vitro by the active metabolite of fostamatinib.

Whereas fostamatinib was highly effective in reversing cGVHD in the BO model,

clinical and immunological effects in sclerodermatous cGVHD appeared to be

more modest under the conditions tested.

It is unclear if B cells are intrinsically or extrinsically affected during

cGVHD. B cells might increase their activation state based on pathogen

activating molecular patterns released during conditioning treatment and




17

activating toll-like receptors on B cells. In contrast there is potential that

increased activating factors such as B cell activating factor (BAFF) are causing B

cells to increase response from factors such as Syk since BAFF receptor may be

reliant on Syk for its signaling.26 The requirement of Syk in BM derived B cells for

cGVHD development in the BO model also allowed us to elucidate a potential

mechanism of disease. BCR stimulation with antigen leads to phosphorylation of

proximally located Syk to initiate down-stream signaling for B cell survival and

proliferation. Syk signaling is necessary for survival of B cells during the GC

reaction. The decrease in B cells after Syk-deficient BM transplant in the BO

model suggests a mechanistic link between BCR-antigen engagement that

requires Syk for B cell promotion and perpetuation. The absence or relatively

modest effects of fostamatinib in sclerodermatous cGVHD is consistent with the

findings that there were few splenic B cells present in our studies in fostamatinib

or vehicle treated sclerodermatous cGVHD mice when analyzed 4-5 weeks post-

transplant. These B cell data contrast to the significantly increased splenic GC B

cell frequency seen in cGVHD mice with BO.3, 4

Studies of cGVHD patient peripheral blood have demonstrated an

important role of B cells in development of disease. The production of auto- and

allo-antibodies has previously been associated with the development of cGVHD

in patients.27-30 The dysregulation of B cells could be due to the significant

increase in BAFF present during cGVHD.15, 31 B cells in patients with cGVHD

were more sensitive to Syk inhibition compared to B cells from patients without

active cGVHD, consistent with our previous finding that these cells have




18

increased activation of Syk and the proximal BCR signalosome7. We

demonstrated that these cells had an increase in cell death rates compared to

controls. Like BCR-activated B cell lymphomas32, only those cGVHD B cells

activated through the BCR pathway are presumably susceptible to killing at low

drug concentration. Together these data demonstrate the relative selective ability

of fostamatinib to target the activated B cells in cGVHD patients.

The role of Syk in donor T cells was demonstrated to be of little

importance in the development of murine cGVHD in the BO model. However in

the minor antigen-disparate sclerodermatous cGVHD model, we did find a

decrease in the expression of CXCR4 on T cells when mice with established

clinical cGVHD were treated with R788, consistent with Huu et al.19 who also

demonstrated increased pSyk in T cells on day 14 post-transplant also showed a

reduction in proliferation of T cells from cGVHD mice when exposed in vitro to

the active fostamatinib metabolite, R406 and mice (4-6 per group) treated with

fostamatinib in vivo exhibited ~1 week shift in cGVHD onset and progression

when treatment was initiated on day 14 post-transplant, resulting in significantly

lower average skin scores and body weight between days 21-42 post-

transplant.19 The decrease in migration of T cells to effector tissues could have a

beneficial effect on the development of disease. Thus, while treatment did not

fully restore health in our study in the B10.D2 into Balb/c mode, there was a

decrease in the progression of disease associated with a lowered frequency of

CXCR4+ T cells that may contribute to disease reduction.




19

Despite the apparent lack of Syk dependency of donor T cells in cGVHD

with BO, we demonstrate that the Syk inhibitor fostamatinib is capable of

blocking the cGVHD GC response that is dependent upon T follicular helper cells

and GC B cells.3 These data point to a T cell independent effect of fostamatinib

in abrogating the cGVHD GC response, which is essential for cGVHD generation

and progression in the BO model.

In addition to targeting B cells during cGVHD, there is evidence that

antigen-presenting cells are susceptible to fostamatinib treatment. For example,

in an acute GVHD model, Leonhardt et. al.18 showed that fostamatinib reduced

costimulatory molecule expression and disrupted cytoskeletal organization, along

with blocking T cell proliferation and migration, resulting in acute GVHD

amelioration with preservation of graf-versus-leukemia and anti-viral immunity.

Consistent with other reports that inhibition of Syk can decrease costimulatory

molecules expression.19, 33-35 we demonstrated in vivo decrease in the frequency

of activated (CD80+, CD86+) DCs in mice with cGVHD and BO.

Although we could not discern effects of fostamatinib on the total skin

inflammatory scores in either MHC-disparate models of sclerodermatous

cGVHD, in the B6 into B6D2F1 model, fostamatinib significantly decreased the

absolute number of CD11b+F4/80+ monocytes and the inflammatory Ly6Chi but

not the Ly6Clo non-inflammatory subset in peripheral blood. These data are

interesting in light of the role of Syk in macrophages and monocytes and our

recent study implicating monocytes and macrophages in cGVHD generation and

maintenance in the two scleroderma cGVHD models as well as in the BO model5.




20

Notably, there was a significant decrease in CD11b+ cells in the cervical lymph

node of fostamatinib treated mice in the minor antigen disparate B10.D2 into

Balb/c sclerodermous cGVHD model. Monocyte and macrophages may

contribute to cGVHD in these models by antigen-presentation capacity or the

elaboration of pro-fibrogenic proteins such as TGFb5. Thus, the impact of

fostamatinib in cGVHD models may depend in part as to the stage of disease

progression as well as the dominant underlying mechanisms for disease

pathogenesis.

In summary, our data clearly indicate by biochemical and genetic and

pharmacological approaches a role of Syk in donor BM-derived B cells in a multi-

organ system model of cGVHD with BO. Further we have translated these

findings into a therapeutic using a pharmacological small molecule Syk inhibitor,

fostamatinib, that has progressed through phase II clinical trials20, 21 Fostamatinib

ameliorated disease in mice with cGVHD and BO and has biological effects in

some models of scleroderma. These preclinical murine and human data point to

consideration of clinical trials of fostamatinib or other Syk inhibitor alone or in

combination with other similar agents23 for the treatment of cGVHD.




21

Acknowledgements. We thank Rigel Pharmaceuticals for providing

Fostamatinib (R788/R406). Supported in part by National Institutes of Health

National Cancer Institute grants P01 CA142106-06A1 and 5P01-CA047741-20,

National Institutes of Allergy and Infectious Diseases grants P01 AI 056299 and

T32 AI 007313 and K08HL107756 and Leukemia and Lymphoma Society

Translational Research Grants 6458-15 and 6462-15.

Author contributions

RF designed experiments, performed experiments, and wrote the paper. JLA, LL,

KPM, KP, JP, KAA, JD, and JCP designed and performed experiments. AP-M

performed histological analyses, discussed experimental design, and edited the

paper. PAT performed experiments and edited the paper. JSS, WJM, GRH,

KPM, LL, IM, JK, CSC, RJS, and JR designed experiments and edited the paper;

NJC and RAC provided reagents, discussed experiments and edited the paper,

SS and BRB designed experiments and edited the paper.

Conflict of interest

No conflicts of interest are known.




22

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Figure Legends

Figure 1: Syk activation during cGVHD. B cells were purified from the spleens

of healthy control (BM Only) or cGVHD (BM + T) mice and stimulated with anti-

IgM at 5 μg/mL. Splenocytes from B10.BR mice transplanted with B6 BM and low

numbers of T cells were analyzed for phosphorylated Syk at Y348 after ex vivo

stimulation by 5 μg/mL of anti-IgM* p < 0.5; Error bars represent SEM, n=8

representative data from 2 experiments.

Figure 2: Presence of Syk in BM derived B cells but not donor T cells is

necessary for development of cGVHD pathology in BO model. A) Day 60

pulmonary function tests of mice transplanted with taxomifen induced Syk KO

BM and WT T cells. B) Frequency of B cells in transplanted mice on D60 after

transplant. C) Pulmonary function tests from mice transplanted with WT BM and

either WT T cells or Syk KO T cells. D) Pathology scores from lung and liver of

mice transplanted with Syk KO T cells. * p < 0.5; ** p < 0.01. Error Bars

represent SEM, Representative data from 3 independent experiments with n=8

per group.

Figure 3: Genetic deletion of Syk during active disease prevents

pathogenic pulmonary function. Mice were transplanted with either WT or Syk

fl/fl x ERT2-Cre BM. On D8 mice were treated with Tamoxifen for 5 days to

delete Syk in donor BM derived cells. A) Pulmonary function tests of mice on day

60. * p < 0.5; ** p < 0.01. Error Bars represent SEM, n=10 per group.




28

Figure 4: Inhibition of Syk by R788 decreases cGVHD in murine BO model.

A) Day 56 pulmonary function tests on mice treated with 30 mg/Kg of R788 twice

daily from D28-56. B) GVHD pathology scores from the lungs of mice on D60. C)

Number of GCs present in situ in spleens of mice. D) Frequency of GC B cells

(gated on CD19+GL7+CD95hi) present in the spleens of transplanted mice on day

60. E-G) Frequency of CD11c cells expressing CD80, CD86 and MHC Class II in

the spleens of transplanted mice on day 60. * p < 0.5; ** p < 0.01; *** p< 0.001.

Error Bars represent SEM, Representative data from 3 independent experiments

with n=8 per group.

Figure 5: Fostamatinib does not alter skin inflammation in B6 into B6D2F1

model of sclerodermatous GVHD but decreases peripheral blood Ly6Chi

monocytes. A) Frequency of CD19+ B cells in spleens of mice on day 21 in

vehicle treated (grey bars), R788 treated (black bars) and non-GVHD controls

(white bars). B) Total inflammation score. C) Absolute number of CD11b+ F4/80+

peripherial blood monocytes. Peripherial monocytes were examines for D) Ly6C

high or E) Ly6C low expression. * p < 0.5; ** p < 0.01; Error bars represent SEM,

n=6 per group.

Figure 6: Fostamatinib has little clinical therapeutic benefit in the B10.D2

into Balb/c sclerodermatous cGVHD model but decreases expression of

CXCR4 on CD11b+ and CD4+ cells. A) Clinical GVHD scores or b) Skin GVHD




29

Scores in vehicle treated (grey squares) or R788 treated (black circles) in B10.D2

into Balb/c model. C) Frequency of viable CD11b+ cells and D) expression of

CXCR4 on CD11b+ cells. E) Absolute number CD19+ B cells and F) expression

of CXCR4 on B cells G) expression of CXCR4 on CD4+ cells. * p < 0.5; Error

bars represent SEM. n=5 per group.

Figure 7: Increased apoptosis in human B cells when treated with R788. (A)

Representative flow plots of human PBMC with or without cGVHD. Consistent

with previous work36 unmanipulated, untreated cGVHD B cells had superior

survival compared to B cells from patients without disease. Peripheral blood

mononuclear cells from patients without cGVHD (n = 6, open circles) and with

cGVHD (n = 6, filled squares) treated with R406 (0, 0.01, and 0.1 μM) as

indicated for 48 h. Apoptotic B cells were defined as CD19+ Annexin V+ 7AAD-

cells (B) or as CD19 negative non-B cells (C). Fold increase in apoptosis by

R406 divided by PBS is depicted. Data are median +/- range pooled from 2

independent experiments. * p < 0.5; ** p < 0.01; *** p < 0.001.




doi:10.1182/blood-2014-08-595470Prepublished online April 7, 2015;

H. Antin, Jerome Ritz, Nelson J. Chao, Raphael A. Clynes, Stefanie Sarantopoulos and Bruce R. BlazarWilliam J. Murphy, Geoffrey R. Hill, Ivan Maillard, John Koreth, Corey S. Cutler, Robert J. Soiffer, JosephVulic, Jing Du, Angela Panoskaltsis-Mortari, Patricia A. Taylor, Jonathan C. Poe, Jonathan S. Serody, Ryan Flynn, Jessica L. Allen, Leo Luznik, Kelli P. MacDonald, Katelyn Paz, Kylie A. Alexander, Ante graft-versus-host diseaseTargeting Syk Activated B-cells in murine and human chronic

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