Histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect

Abstract

Acute graft-versus-host disease (GVHD) and leukemic relapse are the two major obstacles to successful outcomes after allogeneic bone marrow transplantation (BMT), an effective therapy for hematological malignancies. Several studies have demonstrated that the dysregulation of proinflammatory cytokines and the loss of gastrointestinal tract integrity contribute to GVHD, whereas the donor cytotoxic responses are critical for graft-versus-leukemia (GVL) preservation. Suberoylanilide hydroxamic acid (SAHA) is currently in clinical trials as an antitumor agent; it inhibits the activity of histone deacetylases and at low doses exhibits antiinflammatory effects by reducing the production of proinflammatory cytokines. Using two well characterized mouse models of BMT, we have studied the effects of SAHA on GVHD severity and GVL activity. Administration of SAHA from day +3 to day +7 after BMT reduced serum levels of the proinflammatory cytokines and decreased intestinal histopathology, clinical severity, and mortality from acute GVHD compared with vehicle-treated animals. However, SAHA had no effect on donor T cell proliferative and cytotoxic responses to host antigens in vivo or in vitro. When mice received lethal doses of tumor cells at the time of BMT, administration of SAHA did not impair GVL activity and resulted in significantly improved leukemia-free survival by using two different tumor and donor/recipient combinations. These findings reveal a critical role for histone deacetylase inhibition in the proinflammatory events contributing to GVHD and suggest that this class of pharmacologic agents may provide a strategy to reduce GVHD while preserving cytotoxic T cell responses to host antigens and maintaining beneficial GVL effects.

Fig. 1.

Injection of SAHA inhibits in vivo proinflammatory cytokine production after BMT. B6D2F1 mice were given 1,300 cGy of total body irradiation and received transplants of 5 × 10⁶ T cell-depleted BM cells and 2 × 10⁶ T cells from either allogeneic (B6) or syngeneic (B6D2F1) donors as in Materials and Methods. Each F₁ recipient of the allogeneic cells were injected i.p. with 35 mg·kg^-1·day^-1 SAHA or the diluent control from day +3 to day +7 after transplantation. Sera from the recipient animals (n = 3 per group) were obtained by retroorbital venous puncture on day 7 after BMT and analyzed, as described in Materials and Methods. (a) Serum TNF-α levels are reduced in the recipients treated with SAHA. Shown are syngeneic (□) and allogeneic controls (▪) vs. SAHA allogeneic (allo) recipients (); ^*, P < 0.05. Results from one of two similar experiments are shown. (b) Serum IL1-β levels are reduced. Shown are syngeneic (□), allo controls (▪) vs. SAHA-treated allo recipients (); ^**, P < 0.02. Data are from one of two similar experiments. (c) Serum IFN-γ levels are reduced. Shown are syngeneic (□), allo controls (▪) vs. SAHA-treated allo recipients (); ^**, P < 0.02. Data from one of three similar experiments are shown.

Fig. 2.

SAHA increases the acetylation of histone H3 and down-regulates the transcription of TNF-α and IFN-γ after allogeneic (allo) BMT. B6D2F1 animals were irradiated and transplanted with cells from either syngeneic (syn) or allo donors as in Fig. 1. Allo recipients were injected with either SAHA or the control diluent for 5 days, from day +3 to day +7 after BMT. (a) On day +7 after BMT the recipient splenocytes were harvested (n = 3 per group), and histones were isolated by acid extraction, as described in Materials and Methods. Immunoblot analysis was performed to detect acetylation of histones by using antiacetylated H3 Ab. (b) Total RNA was extracted from the splenocytes harvested and pooled together on day +7 (n = 3 per group; □, syngeneic; ▪, allo controls, SAHA allo recipients) after BMT as above. The expression of TNF-α and IFN-γ mRNA levels were analyzed by RPA, as described in Materials and Methods. The TNF-α and the IFN-γ mRNA transcript levels were reduced in the recipients treated with SAHA. Shown are controls (▪) vs. SAHA allo recipients (); ^*, P < 0.05. Results are from one of two similar experiments.

Fig. 3.

Treatment with SAHA results in decreased intestinal histopathology after allogeneic (allo) BMT. B6D2F1 animals received syngeneic BMT or allo BMT from C57BL/6 donors as in Fig. 1. Allo recipients were injected with either SAHA or the control diluent for 5 days, from day +3 to day +7 after BMT. On days +7 to +8, small-bowel samples from BMT recipient mice (n = 4 per group) were obtained and analyzed microscopically, as described in Materials and Methods. (a) Syngeneic BMT recipients demonstrate reestablishment of intestinal architecture with villi of near-normal length and without significant cellular infiltration into the lamina propria. Regenerative change is evident by focally increased nuclear staining and increased nuclear/cytoplasmic ratios. (b) Recipients of allo BMT show severe intestinal toxicity, including surface erosion, villous blunting, epithelial attenuation, and an intense cellular infiltration in the lamina propria. (c) Animals treated with SAHA exhibit partial restoration of small intestinal villous architecture, regenerative change, and little inflammatory infiltration. (Original magnification, ×200.) (d) Coded slides were scored semiquantitatively for pathological damage, as described in Materials and Methods. BMT recipients of diluent (▪, n = 4) or SAHA (, n = 4) from allo donors and of syngeneic donors (□, n = 4) are shown. Total GVHD score: mean ± SE of the sum of scores for small bowel and colon from individual animals in each group. ^*, P < 0.05, control (▪) vs. the SAHA () allo group.

Fig. 4.

Treatment of allogeneic (allo) recipients with SAHA attenuates acute GVHD mortality and morbidity. B6D2F1 mice were given 1,300 cGy of total body irradiation and transplants of 5 × 10⁶ T cell-depleted BM cells and 2 × 10⁶ T cells from allo B6 donors as in Materials and Methods. Syngeneic recipients (▪, n = 5) received transplants similarly with cells from F₁ donors. Each allo recipient was injected i.p. with either 35 mg·kg^-1·day^-1 SAHA (•, n = 10) or the control vehicle (▴, n = 10) for 5 days from day +3 to day +7. Transplanted animals were monitored daily for survival and assessed weekly for clinical severity of acute GVHD, as described in Materials and Methods. Data from one of two similar experiments are shown. (a) Percent survival after BMT. • vs. ▴, ^*, P < 0.002 by Wilcoxon rank test. (b) Animals scored for clinical GVHD, as described in Materials and Methods. Data are expressed as mean ± SEM. • vs. ▴, ^**, P < 0.05 by Mann-Whitney U test from day 7 to day 35.

Fig. 5.

Effect of SAHA administration on donor T cell functions after BMT. B6 Ly5.2 (CD45.1+) donor cells were transplanted into irradiated syngeneic (B6 Ly 5.1, CD 45.2) or allogeneic (allo) (B6D2F1) recipients injected with SAHA or the vehicle as in Materials and Methods. Splenocytes were harvested from the recipients on days +7 (a) and +14 (b) after BMT (n = 4 per group) and labeled with anti-CD3 phycoerythrin and anti-CD45.1 FITC. The number of donor T cells (CD45.1⁺ CD3⁺) were determined [allo plus control () vs. allo plus SAHA (▪), P = NS]. Recipients of syngeneic BMT (□) demonstrated lower numbers of donor T cells at both time points. Data from one of three similar experiments are shown. (c) Harvested splenocytes normalized for donor T cells (CD45.1⁺ and CD3⁺) were restimulated in quadruplicate with irradiated naive host (syngeneic B6 Ly5.2 and allo F₁) splenocytes in mixed lymphocyte reaction cultures for 48 h. Proliferation was determined by incubation of cells with [³H]thymidine (1 μCi per well) for an additional 24 h. T cells from SAHA-treated animals showed equivalent proliferation ( vs.▪, P = NS). Results from one of three similar experiments are shown. (d) SAHA treatment preserves cytotoxic T lymphocyte function after BMT. Splenocytes harvested from allo animals on day +14 after BMT were pooled (n = 3 per group), normalized for donor CD8⁺ cells, and used in a ⁵¹Cr-release assay. Cytotoxic T lymphocyte activity against allo targets (P815) in control () and SAHA (▪) groups was similar, whereas no significant lysis of syngeneic targets (EL-4) by both groups (▴ and ▾) occurred. Data from one of two similar experiments are shown.

Fig. 6.

Treatment with SAHA preserves GVL activity and improves leukemia-free survival. (a) B6D2F1 mice receiving transplants of T cells from syngeneic donors and injected with either SAHA (□, n = 10) or control vehicle (▪, n = 10) or from allogeneic (allo) B6Ly5.2 donors and injected with SAHA (▵, n = 10) or vehicle (▴, n = 10) as described in Fig. 1. P815 cells (2,000) were added to the BM inoculum at day 0, as described in Materials and Methods. Animals were monitored daily for survival, and necropsy was performed on all dying animals to determine whether death was caused by leukemia or GVHD. ^**, P < 0.05 for allo plus SAHA (▵) vs. allo plus vehicle (▴) and P < 0.02 for allo plus SAHA (▵) vs. syngeneic plus SAHA (□). Data shown are from one of two similar experiments. (b) Total-body-irradiated B6 Ly5.2 mice received transplants of 5 × 10⁶ TCD BM and 2 × 10⁶ splenic T cells from allo BALB/c donors and were treated with SAHA (▵, n = 11) or control (▴, n = 10) or from syngeneic B6 donors and were injected with control (▪, n = 10) or SAHA (□, n = 11) as in Materials and Methods. All the recipient mice were injected i.v. with 2,000 EL4 tumor cells on day 0. ^**, P < 0.05 for ▵ vs. ▴ and P < 0.01 for ▵ vs. □.