Inhibition of RIP1 improves immune reconstitution and reduces GVHD mortality while preserving graft-versus-leukemia effects

Abstract

Graft-versus-host disease (GVHD) remains the major cause of morbidity and nonrelapse mortality (NRM) after hematopoietic cell transplantation (HCT). Inflammatory cytokines mediate damage to key GVHD targets such as intestinal stem cells (ISCs) and also activate receptor interacting protein kinase 1 (RIP1; RIPK1), a critical regulator of apoptosis and necroptosis. We therefore investigated the role of RIP1 in acute GVHD using samples from HCT patients, modeling GVHD damage in vitro with both human and mouse gastrointestinal (GI) organoids, and blocking RIP1 activation in vivo using several well-characterized mouse HCT models. Increased phospho-RIP1 expression in GI biopsies from patients with acute GVHD correlated with tissue damage and predicted NRM. Both the genetic inactivation of RIP1 and the RIP1 inhibitor GNE684 prevented GVHD-induced apoptosis of ISCs in vivo and in vitro. Daily administration of GNE684 for 14 days reduced inflammatory infiltrates in three GVHD target organs (intestine, liver, and spleen) in mice. Unexpectedly, GNE684 administration also reversed the marked loss of regulatory T cells in the intestines and liver during GVHD and reduced splenic T cell exhaustion, thus improving immune reconstitution. Pharmacological and genetic inhibition of RIP1 improved long-term survival without compromising the graft-versus-leukemia (GVL) effect in lymphocytic and myeloid leukemia mouse models. Thus, RIP1inhibition may represent a nonimmunosuppressive treatment for GVHD.

Fig. 1.. RIP1 phosphorylation in gastrointestinal biopsies correlates with histologic damage and long-term outcomes.

(A) Immunohistochemistry of pRIP1 (Ser166) levels in 24 GI biopsies taken from patients with GI symptoms after HCT. Histologic severity was determined by the Lerner score. Scale bar, 100 μm. (B) Violin plot of pRIP1 abundance in GI biopsies that were classified as none/mild (0/1) or moderate/marked (2/3) and correlated the severity of damage as assessed by the Lerner score. (C) Receiving operator characteristic (ROC) curve of pRIP1 abundance and 12-month NRM after biopsy. (D) Cumulative incidence of NRM according to pRIP1 abundance. **P = 0.01, unpaired two-tailed t test; *P = 0.027, Log-rank (Mantel-Cox) test.

Fig. 2.. Inactivation of RIP1 prevents apoptosis in human organoids.

(A) Concentrations of IFN-γ and TNFα in serum from patients with GVHD. (B to E) Organoids were established from healthy humans and cultured in media with the additives indicated as described in Materials and Methods. (B) Viability of ileal organoids 72 hours after the addition of serum from individual patients (P) either with no evidence of gastrointestinal GVHD (GVHD−) or with gastrointestinal GVHD symptoms (GVHD+). GNE684 was added to some cultures as described in Materials and Methods. (C) Viability of colonic organoids treated as in (B). (D and E) Viability of ileal and colonic organoids cultured with the indicated additives. (F) Organoids from human colons cultured in media with the additives as indicated and evaluated for pRIP1 by IHC as described in Materials and Methods. *P < 0.05, **P < 0.01, and ***P < 0.001, unpaired two-tailed t test.

Fig. 3.. Inactivation of RIP1 prevents apoptosis in murine organoids.

Organoids were established from mice as described in Materials and Methods and cultured in media with indicated treatments. (A) Sera from either syngeneic (open bars) or allogeneic (solid bars) transplanted mice were added to ileal mouse organoids, and viability was quantified as in Materials and Methods. (B) Viability in response to increasing concentrations of IFN-γ and TNFα. (C) Ileal organoids from Lgr5-eGFP mouse were established and treated as indicated; Lgr5⁺ ISCs were quantified by flow cytometry. (D) Crypts from Lgr5-eGFP mice were dissociated into single cells and sorted to obtain Lgr5⁺ ISCs; after sorting, ISCs were cultured with the additives indicated, and viability was assessed. (E) Ileal organoids from B6 WT mice (left) or B6-RIP1 KD mice (right) were cultured with the additives indicated, and viability was assessed as described in Materials and Methods. (F) Colonic organoids from B6 WT mice (left) or B6-RIP1 KD mice (right) were cultured as described in (E), and viability was assessed. *P < 0.05, **P < 0.01, and ***P < 0.001, unpaired two-tailed t test.

Fig. 4.. Genetic inactivation of RIP1 in HCT recipient mice prevents GI GVHD and reduces mortality.

B6 WT and B6-RIP1 KD mice received HCT from syngeneic (open red and purple bars, respectively) or allogeneic C3H.SW donors (solid red and solid purple bars, respectively). Small intestines were harvested and analyzed on day 9 after HCT and Western blots, and flow cytometry of lamina propria lymphocytes was performed as described in Materials and Methods. (A) Western blots of ileum for RIP1, RIP3, pRIP3, MLKL, pMLKL, caspase 8, caspase 3, and actin from individual mice as indicated. (B) The ratio of cleaved caspase 3 to full-length (FL) caspase 3 as measured by densitometry. IFN-γ⁺ and TNFα⁺ were quantified for (C) CD4⁺ and (D) CD8⁺ populations. (E) Intestinal lengths were measured on day 9. (F) Survival of B6 WT recipients (solid red squares, n = 16) and B6-RIP1KD recipients (solid purple squares, n = 16) transplanted from C3H.SW donors and syngeneic B6 WT donors (open squares, n = 7 per group). (G) Survival of B6 WT HCT recipients (solid red squares, n = 20) and B6-RIP1KD HCT recipients (solid purple squares, n = 20) transplanted from allogeneic Balb/c donors and from syngeneic B6 WT donors (open red and purple squares, n = 7 per group). **P < 0.01 and ***P < 0.001 [B to E: Unpaired two-tailed t test; F and G: Log-rank (Mantel-Cox) test].

Fig. 5.. Pharmacological inhibition of RIP1 by GNE684 prevents GI GVHD and improves survival in mice.

(A to D) B6 WT mice received HCT as in Fig. 4 and daily intraperitoneal injections of either GNE684 75 mg/kg (blue bars) or vehicle (red bars) from day 0 after HCT. (A) Intestines were processed on day 9 after HCT, and the numbers of leukocyte subpopulations in the lamina propria were analyzed and calculated by flow cytometry after syngeneic (open bars) or allogeneic (closed bars) HCT. Cellular subpopulations included the following: T lymphocytes (CD3⁺), effector CD4⁺ T cells (CD4⁺IFN-γ⁺ and CD4⁺TNFα⁺), effector CD8⁺ T cells (CD8⁺IFN-γ⁺ and CD8⁺TNFα⁺), T_regs (CD4⁺Foxp3⁺), conventional T cells (CD4⁺Foxp3⁻), regulatory/conventional CD4+ T cell ratios (Foxp3⁺/Foxp3⁻), macrophages (F4/80⁺), natural killer cells (NK1.1⁺), and neutrophils (Ly6G⁺). (B) ISCs of Lgr5-eGFP recipient mice as quantified by flow cytometry using the gating strategy described in Materials and Methods. (C) Small intestines were removed and measured from the proximal jejunum to the cecum. (D) Organoids from intestinal crypts of transplanted mice were cultured with additives as indicated, and viability was measured after 5 days. (E) Survival of B6 WT mice that received HCT from syngeneic B6 WT donors (open squares, n = 6 per group) or allogeneic C3H.SW donors (solid squares) that received 14 daily injections of GNE684 beginning on day 0 at the following doses (all in milligrams per kilogram): 0 (red, n = 26); 5 (light blue, n = 16); 25 (medium blue, n = 16); and 75 (navy blue, n = 10). (F) Survival of HCT recipients from syngeneic B6 WT donors (open circles, n = 6 per group) or from allogeneic Balb/c donors that received 14 daily injections from day 0 of either vehicle (red solid circles, n = 10) or GNE684 (75 mg/kg; blue solid circles, n = 10). (G) Survival of HCT recipients transplanted from syngeneic B6 WT donor (open circles, n = 6 per group) or from allogeneic C3H.SW donors (solid circles) that received 14 daily injections beginning on day +7 of either vehicle (red circles, n = 10) or GNE684 (75 mg/kg; blue circles, n = 10). Tx: daily injections, ns: nonsignificant, *P < 0.05, **P < 0.01, and ***P < 0.001 [A to D: Unpaired two-tailed t test, E to G: Log-rank (Mantel-Cox) test].

Fig. 6.. Pharmacological inhibition of RIP1 by GNE684 prevents accumulation of inflammatory cellular infiltrates in mouse liver.

Mice were transplanted and treated as in Fig. 4. Livers were harvested on day 9 and processed, and the number of inflammatory cellular subpopulations was analyzed and calculated by flow cytometry as described in Materials and Methods. Cellular subpopulations included the following: T lymphocytes (CD3⁺), effector CD4⁺ T cells (CD4⁺IFN-γ⁺ and CD4⁺TNFα⁺), effector CD8⁺ T cells (CD8⁺IFN-γ⁺ and CD8⁺TNFα⁺), T_regs (CD4⁺Foxp3⁺), conventional T cells (CD4⁺Foxp3⁻), regulatory/conventional CD4+ T cell ratios (Foxp3⁺/Foxp3⁻), macrophages (F4/80⁺), natural killer cells (NK1.1⁺), and neutrophils (Ly6G⁺). ns, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001, unpaired two-tailed t test.

Fig. 7.. Pharmacological inhibition of RIP1 by GNE684 does not suppress lymphocyte function and improves immune reconstitution during GVHD in mice.

(A) T cells isolated from naïve B6 WT mice were stained with CFSE and stimulated in vitro with antiCD3/CD28 beads with or without GNE684 for 5 days (left) and then stimulated with phorbol myristate acetate (PMA) for 4 hours to detect intracellular TNFα and IFN-γ (orange bars) or TNFα alone (green bars) (right). (B and C) B6 WT mice received HCT from syngeneic donors and were treated with vehicle or GNE684 75 mg/kg daily for 14 days (open red and blue bars, respectively), and organs were harvested on day 14. (B) Absolute numbers of CD4⁺, CD8⁺, CD4⁺CD8⁺, and CD4⁻CD8⁻ were quantified in the thymus by flow cytometry. (C) CD3⁺ T cells were isolated and quantified from the spleen (left), stained with CFSE, and stimulated with antiCD3/CD28 beads in vitro for 5 days (middle). Cells were then harvested and stimulated with PMA for 4 hours to detect intracellular IFN-γ and TNFα by flow cytometry (right). (D) B6 WT mice received HCT from syngeneic (open bars) or allogeneic C3H.SW donors (solid bars) and were treated with vehicle or GNE684 75 mg/kg daily (red and blue bars, respectively). Splenocytes were isolated on day 14, and the cell surface expression of PD-1 and TIM-3 was analyzed for both CD4⁺ and CD8⁺ T cell subsets by flow cytometry (left and middle). CD3⁺ T cells isolated from the splenocytes were stained with CFSE, stimulated in vitro with Balb/c DCs for 5 days, and then analyzed for CD4⁺ and CD8⁺ cell surface expression (right). ns, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001 (A to D: Unpaired two-tailed t test).

Fig. 8.. RIP1 inhibition preserves GVL effects in mice.

Mice received HCT as in Fig. 4. A total of 5 × 10⁵ C1498 (A and C) or 1 × 10⁶ EL-4 (B and D) leukemia cells were added to the donor inoculum of each recipient. (A and B) Death by leukemia (left) and survival (right) of B6 WT recipients (red squares) and B6-RIP1 KD HCT recipients (purple squares) transplanted from syngeneic donors (open squares, n = 15 per group) or from allogeneic C3H.SW donors (solid squares, n = 15 per group). (C and D) Death by leukemia (left) and survival (right) of mice injected for 14 days intraperitoneally with vehicle (red solid circles, n = 12) or GNE684 (75 mg/kg; blue solid circles, n = 17) that received HCT from syngeneic B6 WT donors (open circles, n = 15 per group) and from allogeneic C3H.SW donors (solid circles). Mice that died from GVHD are shown as ticks along the x axis in the left panels. Tx: daily injections, **P < 0.02 [Log-rank (Mantel-Cox) test].