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Corticosteroids (CS) represent first-line treatment for gastrointestinal graft vs host disease (GI GVHD), and CS failure is associated with severe morbidity and mortality. While the immune system is the intended target of CS treatment, the glucocorticoid receptor (GR) is widely expressed, and there is limited understanding of the direct effects of CS on intestinal epithelium following immune-mediated damage. We thus investigated how CS treatment could impact intestinal homeostasis and regeneration following experimental bone marrow transplantation (BMT). In healthy C57BL/6 (B6) mice, in vivo administration of clinically relevant CS doses reduced Ki67 + epithelial proliferation in the ileum (p We next investigated CS effects on epithelial cells during immune-mediated damage. Pre-treatment of mice with 2 mg/kg MP x 7 days in vivo prior to crypt harvest and organoid culture increased organoid sensitivity to T-cell-mediating killing ex vivo (p Despite potential harmful side effects, CS are frequently necessary for treatment of clinical GVHD. We hypothesized that CS-mediated epithelial suppression could be mitigated by concurrent administration of agents capable of inducing tissue regeneration. Interleukin-(IL)-22 has been shown to promote epithelial proliferation and recovery following GI damage. We thus investigated whether IL-22 treatment could counterbalance CS-induced impairment of epithelial recovery in GVHD. Indeed, addition of IL-22 to MP-treated organoids promoted organoid growth without inducing toxicity/organoid loss in both murine and human SI organoid cultures (p In summary, these findings indicate that CS treatment can suppress epithelial proliferation in the intestines and exacerbate GI damage if it fails to control the pathologic immune response. However, deleterious CS side effects can be counterbalanced by promotion of epithelial regeneration, providing rationale for combining immunosuppression with tissue-supporting therapeutics such as IL-22 to optimize intestinal recovery in GVHD. Figure 1 Figure 1. Disclosures Blazar: Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rheos Medicines: Research Funding; Equilibre Pharmaceuticals Corp: Research Funding; Carisma Therapeutics, Inc: Research Funding; Tmunity Therapeutics: Other: Co-founder. Hanash: Evive Biotech: Ended employment in the past 24 months.

Glucocorticoids (GC) are a standard therapy of immune-mediated gastrointestinal (GI) damage. However, there is little understanding of the effects that GC use may have on intestinal epithelium and its regenerative function. Using ex vivo organoid models of immune-mediated intestinal injury, we sought to investigate direct effects of GC on intestinal epithelium and test the impact of GC on cytokine- and T-cell-mediated effects. GC suppressed proliferation of small intestine (SI) organoids, reducing organoid size and decreasing cell cycle progression of Lgr5+ intestinal stem cells. Consistently, GC treatment of C57BL/6J mice suppressed epithelial proliferation, decreasing crypt height and reducing the percentage of Ki67+ crypt cells in vivo. We next investigated the role of GC-mediated epithelial suppression in the setting of tissue damage, modeling immune-mediated GI pathology by culturing organoids in the presence of IFNγ and by co-culturing organoids with allogeneic T-cells. Culture with GC in addition to IFNγ significantly increased the toxicity of IFNγ leading to reduced efficiency of organoid formation. Consistently, GC pretreated organoids were more susceptible to allogeneic T-cells with reduced formation efficiency. Finally, we studied cytokine-mediated epithelial regeneration in the context of GC treatment. Culturing organoids with both GC and IL-22 indicated that IL-22 could promote regeneration of intestinal epithelium in the presence of GC. Taken together, our findings indicate that GC, which are necessary clinically for suppressing pathologic inflammatory immune responses, may have direct effects on the epithelium potentially impairing protective regeneration responses and augmenting toxicity.

Innate lymphoid cells (ILCs) are associated with protective mucosal responses after allogeneic bone marrow transplantation (allo-BMT). However, the specific in vivo roles of ILCs after BMT remain unclear. We thus sought to characterize the kinetics and significance of ILC reconstitution after allo-BMT. In a sex-mismatched acute graft-vs-host-disease (GVHD) model, depletion of recipient ILCs induced significantly worse systemic signs of GVHD, as well as increased thymic injury and intestinal pathology, indicating a contributory role for ILCs in tissue protection post-BMT. Analysis of ILCs in distinct GVHD target organs after T-cell-depleted BMT demonstrated a high frequency of NK cells and ILC1s in the liver, a higher proportion of ILC2s in lungs, and a higher proportion of ILC3s in the small intestine post-transplant in the absence of GVHD. For all tissues analyzed, the majority of ILCs were host-derived. In contrast, in the presence of GVHD, all ILC subsets were significantly reduced in all tissues analyzed. Furthermore, ILC precursors were significantly reduced in the bone marrow of mice with GVHD, suggesting that alloreactive immunity can impair ILC reconstitution by targeting both mature ILCs and their developmental niche. To examine the clinical relevance of these findings, human ILCs were analyzed by FACS of lamina propria lymphocytes isolated from patient duodenal biopsy specimens obtained to evaluate symptoms of acute GI GVHD. Consistent with experimental findings, duodenal ILC frequencies were significantly lower in patients with histologic evidence of GVHD. In conclusion, tissue-resident ILCs contribute to tissue protection after allo-BMT, but GVHD leads to ILC elimination and impairs their reconstitution.

The successful use of SMO inhibitors in tumors with activating mutations in hedgehog signaling raised interests in their exploitation against other malignancies. The role of hedgehog signaling in pediatric malignancies remains unclear. We investigated the hedgehog signaling and its inhibition in a panel of 18 tumor cell lines derived from six of the most common and highly aggressive pediatric tumor types. None of the cell lines was known to stem from tumors with activating hedgehog mutations. Tetrazolium-based assays (MTT and MTS) and BrdU assays were used to analyze cell viability and proliferation after exposure to SANT1 and GANT61. Expression analysis of hedgehog signaling members and cyclins was performed by quantitative real-time PCR and Western blot. Key members of hedgehog signaling (SHH, PTCH1, SMO, GLI1, GLI2 and SUFU) were expressed in all cell lines. In 50 % of the cell lines viability was significantly increased by SHH exposure. Stimulation was not restricted to distinct tumor types, but related to cell lines with higher mRNA levels of PTCH1, SMO, GLI1 and GLI2. SMO inhibition by SANT1 moderately decreased cell viability with GI50s between 28 and 93 µmol/l. Sensitivity to SANT1 was not related to distinct tumor types. The GLI inhibitor GANT61 inhibited cell viability and proliferation more effectively than SANT1. Our preclinical data provide evidence that hedgehog signaling is active and can be stimulated by PTCH1 ligands in various pediatric tumors. We suggest further evaluation of GLI inhibitors as inhibitors of hedgehog signaling for the treatment of the investigated tumor types.

Fewer than 50% of patients with high-risk neuroblastoma survive five years after diagnosis with current treatment protocols. Molecular targeted therapies are expected to improve survival. Although MDM2 has been validated as a promising target in preclinical models, no MDM2 inhibitors have yet entered clinical trials for neuroblastoma patients. Toxic side effects, poor bioavailability and low efficacy of the available MDM2 inhibitors that have entered phase I/II trials drive the development of novel MDM2 inhibitors with an improved risk-benefit profile. We investigated the effect of the novel MDM2 small molecular inhibitor, DS-3032b, on viability, proliferation, senescence, migration, cell cycle arrest and apoptosis in a panel of six neuroblastoma cell lines with different TP53 and MYCN genetic backgrounds, and assessed efficacy in a murine subcutaneous model for high-risk neuroblastoma. Re-analysis of existing expression data from 476 primary neuroblastomas showed that high-level MDM2 expression correlated with poor patient survival. DS-3032b treatment enhanced TP53 target gene expression and induced G1 cell cycle arrest, senescence and apoptosis. CRISPR-mediated MDM2 knockout in neuroblastoma cells mimicked DS-3032b treatment. TP53 signaling was selectively activated by DS-3032b in neuroblastoma cells with wildtype TP53, regardless of the presence of MYCN amplification, but was significantly reduced by TP53 mutations or expression of a dominant-negative TP53 mutant. Oral DS-3032b administration inhibited xenograft tumor growth and prolonged mouse survival. Our in vitro and in vivo data demonstrate that DS-3032b reactivates TP53 signaling even in the presence of MYCN amplification in neuroblastoma cells, to reduce proliferative capacity and cause cytotoxicity.

A potential role for endogenous innate lymphoid cells (ILCs) in prevention of gastrointestinal (GI) GVHD has recently been described in murine and clinical models, and experimental approaches for adoptive cellular therapy with ILCs have recently been described as well. However, the specific in vivo roles of tissue-resident ILCs and their capacity for reconstitution post-transplant remain unclear. We thus sought to characterize the significance and tissue distribution of ILCs after allogeneic bone marrow transplant (allo-BMT) in mouse models and in patients. In order to specifically assess the functional role of ILCs after allo-BMT, we established a clinically relevant acute GVHD model based on sex mismatched H-Y antigens. Lethally irradiated Rag2-/-male mice (Thy1.2, B6) were transplanted with T cell-depleted (TCD) BM and purified T cells obtained from wild-type female mice (Thy1.1, B6). To specifically deplete host-derived ILCs (and not donor cells), recipient mice were treated with an anti-Thy1.2 antibody or an anti-isotype antibody prior to BMT. Pre-transplant depletion of ILCs induced significantly worse systemic signs of GVHD, as well as increased thymic injury and intestinal GVHD pathology, indicating an important role for endogenous ILCs in tissue protection after allo-BMT in this context. To better understand the roles of tissue-resident ILCs in reducing pathology post-BMT, we evaluated ILC subsets in different GVHD target organs after allo-BMT. Two weeks after MHC-mismatched (C57BL6 into BALB/c) TCD-BMT, both donor- and host-derived ILCs could be detected in the liver, lungs, and intestines. Liver was characterized by a high number of NK cells and ILC1s, while lungs contained a high proportion of ILC2s. The small intestine contained a relatively greater proportion of ILC3s. For all tissues analyzed, the majority of ILCs were host-derived. During T cell replete BMT resulting in GVHD, both donor and host-derived ILCs were significantly reduced in all three tissues compared to TCD-BMT mice, and this was true for all ILC lineages. To better understand the loss of donor-derived ILCs in GVHD, we investigated ILC precursors in recipient marrow post-BMT, hypothesizing that an impairment of ILC precursors could explain the loss of all ILC lineages in the various tissues analyzed. Indeed, we found a significant loss of CLPs, αLPs, CHILPs, and ILC2Ps in mice with GVHD, suggesting that bone marrow involvement with GVHD induced a loss of ILC precursors impairing their reconstitution. We next sought to evaluate if the loss of tissue-resident ILCs in GVHD target organs after experimental BMT was relevant for clinical transplantation. To evaluate ILC frequencies in patient tissues post-transplant, duodenal biopsies were collected from patients undergoing clinically indicated endoscopy to work up symptoms of acute upper GI (UGI) GVHD. In total, duodenal samples were collected from ten patients post-transplant and 6/10 had histologic evidence of GVHD on biopsy (Table 1). While frequencies were low, ILCs could be identified by FACS in digested lamina propria of duodenal biopsy specimens. Notably, ILC frequencies were significantly lower in patients with biopsy-proven UGI GVHD, and duodenal ILCs could be identified in only 1/6 patients with histologic evidence of GVHD (Fig 1). Among the ILCs that could be identified, ILC1s represented the greatest proportion of ILC populations within the duodenal lamina propria. Accordingly, the frequency of ILC1s was significantly reduced in transplant patients with biopsy-proven UGI GVHD. Additionally, we were able to evaluate paired peripheral blood and duodenal biopsy samples in a small subset of patients. Although the numbers were limiting, this cohort suggested that ILC2s may represent a greater proportion of ILCs in peripheral blood than in the duodenum post-transplant. Overall, clinical findings were consistent with experimental models demonstrating a reduction of ILCs in GVHD. In conclusion, tissue-resident ILCs contribute to tissue protection after allo-BMT, but GVHD leads to elimination of pre-existing ILCs and loss of ILC precursors in the marrow, impairing their reconstitution. Most duodenal ILCs in clinical biopsy samples post-transplant were ILC1s in this patient cohort, and patients with biopsy-proven UGI GVHD demonstrated significant loss of the few lamina propria ILCs that could be identified in clinical duodenal biopsies. Disclosures Hanash: Nexus Global Group LLC: Consultancy.

Atypical teratoid/rhabdoid tumor (AT/RT) is an aggressive embryonal tumor of the central nervous system with a generally dismal prognosis, especially in patients younger than 12 months. We here describe the unusual case of an infant with AT/RT with long-term survival despite low-cumulative dose chemotherapy after subtotal resection. Due to a poor neurological situation and an unfavorable oncological prognosis, therapy was halted after two partial surgical resections and four of the nine chemotherapy courses recommended by the European Rhabdoid Registry, without the patient receiving either radiotherapy or high-dose chemotherapy. The patient is alive without evidence of disease 52 months after diagnosis. This case report highlights that independent prognostic factors are urgently needed for optimizing treatment stratification and preventing overtreatment.