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1. Blazar, B.R., Murphy, W.J. & Abedi, M. Advances in graft-versus-host disease biology and therapy. Nat Rev Immunol 12, 443-458 (2012).‘

2. Tawara, I. et al. Interleukin-6 modulates graft-versus-host responses after experimental allogeneic bone marrow transplantation. Clin Cancer

Res 17, 77-88 (2011). 3. Chen, X. et al. Blockade of interleukin-6 signaling augments regulatory T-cell reconstitution and attenuates the severity of

graft-versus-host disease. Blood 114, 891-900 (2009). 4. Sun, K. et al. Inhibition of acute graft-versus-host disease with retention of graft-versus-tumor effects by the proteasome inhibitor bortezomib. Proc Natl Acad Sci U S A 101, 8120-8125 (2004). 5. Sun, K. et al. Differential effects of proteasome inhibition by bortezomib on murine acute graft-versus-host disease (GVHD): delayed administration of bortezomib results in increased GVHD-dependent gastrointestinal toxicity. Blood 106, 3293-3299 (2005).

Abstract

Treatment of Chronic Graft-versus-Host Disease with the Proteasome Inhibitor Bortezomib Chien-Chun Pai1, Erik Ames, PhD2, Mingyi Chen, MD, PhD3, Lam Khuat1, Annie Mirsoian1, Anthony E Zamora1, Arta Monjazeb, MD, PhD4, Julian Perks, PhD4, Shuaib Juma4,

Mehrdad Abedi, MD5 and William J Murphy, PhD1

1Department of Dermatology, School of Medicine, University of California, Davis, Sacramento, CA; 2School of Medicine, University of California, Davis, Sacramento, CA; 3Department of Pathology and Laboratory Medicine, UC Davis Medical Center, Sacramento, CA; 4Department of Radiation Oncology, School of Medicine, University of California, Davis, Sacramento, CA; 5Internal Medicine, School of Medicine, University of California, Davis, Sacramento, CA

Materials and Methods

Irradiation 800 cGy B10D2 Mice (H2d)

Balb/c Mice (H2d)

I.V. injection

BMCs Spleen

cGvHD

Results

We have shown the organ-specific protection of low-dose bortezomib in both acute and chronic MHC-matched, miHAg-mismatched GvHD murine models. The specific skin protection was associated with decreased IL-6 levels.

Bortezomib administration resulted in a significant reduction in the total

numbers of donor-derived B cells in both the spleen and skin, despite increased overall donor chimerism.

Lymphoma-bearing mice receiving allogeneic HSCT with bortezomib

administration ameliorate skin GvHD while preserving graft versus tumor (GvT) effects.

Based on these animal studies, a clinical trial, with an intra patient

bortezomib dose escalation design, in patients with steroid intolerant, dependent, or resistant extensive cGVHD, was initiated with encouraging results.

Conclusions

References

Acknowledgements

We thank Monja Metcalf and Weihong Ma for technical help and useful advice. This work has been supported by National Cancer Institute (NCI) grant 5R01CA102282-08.

Fig 1. Dose response of bortezomib on cutaneous lesions. Irradiated (800 cGy) recipient Balb/c mice underwent BMT as described in the Methods. Different doses of bortezomib (from 0.1 mg/kg to 0.4 mg/kg) or vehicle were treated intraperitoneally from D20 and every other five days (A) Skin clinical scores were evaluated twice a week during cGvHD pathogenesis. (B) Skin samples were harvested at Day 57 and evaluated by pathological scores in a blinded fashion. All the data were conducted from two to three independent experiments with at least 8 mice per groups. The data are shown as mean ± SEM and analyzed in one or two-way ANOVA with post- Tukey test to compare between individual groups. P<0.05 (*), P< 0.01 (**) and P<0.001(***) were considered as significant

Chronic GvHD model

Fig 3. BM BM+SC BM+SC+Bort

BM BM+SC+Vehicle

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Fig 5. Bortezomib ameliorates cGVHD skin lesions while maintaining GvT effects. Irradiated Balb/c mice were transplanted with bone marrow cells with or without spleen cells at Day 0. Six hours later, A20-leciferase tumor cell lines (1 x 106) were injected via tail veins into different groups. (A) Bioluminescent images displaying tumor growth and metastasis pattern as measured by IVIS Spectrum. (B) Survival curves from different experimental treated groups. (C) Clinical skin scores were evaluated twice a week. Data are collected from one experiment with 8 mice per groups. Survival data were plotted by the Kaplan-Meier method and analyzed by the log-rank test. Skin clinical score were analyzed by two-way ANOVA with post- Tukey test.

Fig 5.

B.

A.

Fig 2. B. A.

C. D.

E. F.

Fig 2. Serum cytokine levels post allogeneic HSCT with bortezomib treatment. Serum samples were collected from bone marrow only groups, therapeutic bortezomib treatment groups (0.1 mg/kg; I.P. injected from Day 20) and GvHD vehicle control groups at Day 55. Skin tissues and GI tract tissue samples were also collected for RNA extraction. (A-D) Serum cytokine levels (IL-6, IL-17,IFN-Υ and TNF-α ) were detected by cytometric beads array and shown as mean ± SEM (N=4). (E and F) RT-PCR samples from skin or GI tract were analyzed for gene expression levels (N=4). The data are shown as mean ± SEM and analyzed in one-way ANOVA with post-Tukey test to compare between individual groups. P< 0.01 (**) and P<0.0001(****) were considered as significant.

A.

B. C.

BM BM+SC BM+SC+Bort

Fig 3. Decrease in B cells post allogeneic HSCT with bortezomib treatment. Irradiated Balb/c mice transplanted with bone marrow cells with or without spleen cells were injected with either bortezomib (0.1 mg/kg) or vehicle control starting at day 20. (A) Spleen cells were collected at Day 55 and gated on CD45+ CD19+ populations. (B and C) Data showing total numbers of B cells populations in the spleen and skin. (D) Skin samples were collected at Day 55 and RT-PCR was performed to detect BAFF gene expression levels. All the data are shown as mean ± SEM and was analyzed by Student’s t test. P<0.05 (*), P< 0.01 (**) and P<0.001(***) were considered as s ign i f i cant . Dat e w er e co l lec ted f ro m 8 mi c e pe r group f ro m two independent exp er im ents .

D.

Fig 3.

Figure 6. Treatment effects of bortezomib on clinical cGVHD human patients. A single institution pilot study of bortezomib was initiated in patients with steroid-dependent, -intolerant, or -refractory cGVHD. (A) Patient 4 showed extensive grade III skin sclerodermatous GVHD covering >50% of the body. The abdominal region before and after bortezomib treatments are shown. (B) Representative images of the pretreatment skin biopsies taken from the patient shown in A. (C) Immunohistochemical staining for CD3 and CD20 in pretreatment skin biopsy samples from patient 4. (D) CBC and biochemistry data from patient 5 were collected through the trial period. (E) Total numbers of peripheral blood B cells (CD45+CD19+) from 3 patients were analyzed by flow cytometry before and after bortezomib treatment. (F-G) Treg cell populations (CD4+CD25+Foxp3+) were analyzed by flow cytometry and shown as total numbers and percentage. All the data were collected from individual cGVHD patients that underwent bortezomib treatment. The data are shown as mean 6 SEM and analyzed by Student t test to compare pre- and post-bortezomib treatments. *P<0.05 was considered significant.

Fig 4.Engraftment of donor T cells post BMT To evaluate the effects of bortezomib on T cells, irradiated Balb/c mice were transplanted with bone marrow cells with or without spleen cells and treated with bortezomib (0.1 mg/kg) or vehicle control. (A) Spleen cells were isolated at Day 58 and analyzed for donor derived cells (Ly9.1-) by flow cytometry. (B and C) Percentage of CD4+CD25+Foxp3+ population in the spleen and total numbers. All the data are shown as mean ± SEM and analyzed by one-way ANOVA with post-Tukey test to compare between individual groups. P<0.05 (*) were considered as significant. Data were collected from two independent experiments with 8 mice per group.

Allogeneic hematopoietic stem cell transfer (HSCT) can act as a powerful immunotherapy and is a quintessential treatment for many malignant hematologic diseases. However, development of graft versus host disease (GvHD) remains as a major complication after HSCT, and affects numerous organs. Furthermore, chronic GvHD is emerging as a predominant cause of morbidity. Chronic GvHD has a distinctive pathology and pathogenesis and can develop in the form of scleroderma manifested with cutaneous sclerosis, loss of hair follicles, epidermal atrophy and replacement of peri-eccrine fat. We and others have previously demonstrated that bortezomib, a proteasome inhibitor, can prevent acute GvHD if given immediately after HSCT but that continuous treatment of mice resulted in accelerated GvHD-induced gut pathology due to CD4+ T cells. We therefore wanted to assess the effects of bortezomib on chronic GvHD or in particular acute GvHD models where CD8+ T cells were responsible for the disease. Treatment of ongoing GvHD resulted in organ-specific protection on skin GvHD pathology. Using a chronic GvHD model, treatment of ongoing GvHD with bortezomib also resulted in skin, but not intestine pathology protection using the H2d strain combination (B10D2 donors into Balb/c recipients; minor MHC mismatch). The contrasting organ specific effects of bortezomib are further contingent on the timing of administration. In marked contrast to the effects observed with early bortezomib administration in acute GvHD prevention, in cGvHD models, early administration after HSCT worsened the scleroderma pathogenesis, while the later administration of bortezomib during active cGvHD ameliorated the cutaneous lesions. The divergent dose-dependent characteristic of bortezomib also suggests a narrow therapeutic window for the treatment of sclerodermatous chronic GvHD. Continuous administration of bortezomib led to down-regulation of serum IL-6 and which was also correlated with skin pathology. Total numbers of donor-derived spleen B cells were significantly reduced and treatment also correlated with lower BAFF gene expression levels in the peripheral skin tissues. Importantly, later bortezomib administration also preserved graft versus tumor (GvT) effects when challenged with a B cell lymphoma tumor model. Thus bortezomib can produce skin specific protection effects in both acute and chronic GvHD responses. The organ specificity and time sensitivity of bortezomib treatment on sclerodermatous GvHD can provide valuable insights for future clinical trials.

Fig 6.

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