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Acute graft-versus-host disease (GvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT), a potentially curative treatment for leukemia. Endoplasmic reticulum (ER) stress occurs when the protein folding capacity of the ER is oversaturated. How ER stress modulates tissue homeostasis in the context of alloimmunity is not well understood. We show that ER stress contributes to intestinal tissue injury during GvHD and can be targeted pharmacologically. We observed high levels of ER stress upon GvHD onset in a murine allo- HCT model and in human biopsies. These levels correlated with GvHD severity, underscoring a novel therapeutic potential. Elevated ER stress resulted in increased cell death of intestinal organoids. In a conditional knockout model, deletion of the ER stress regulator transcription factor Xbp1 in intestinal epithelial cells induced a general ER stress signaling disruption and aggravated GvHD lethality. This phenotype was mediated by changes in the production of antimicrobial peptides and the microbiome composition as well as activation of pro-apoptotic signaling. Inhibition of inositol-requiring enzyme 1α (IRE1α), the most conserved signaling branch in ER stress, reduced GvHD development in mice. IRE1α blockade by the small molecule inhibitor 4m8c improved intestinal cell viability, without impairing hematopoietic regeneration and T-cell activity against tumor cells. Our findings in patient samples and mice indicate that excessive ER stress propagates tissue injury during GvHD. Reducing ER stress could improve the outcome of patients suffering from GvHD.

Acute graft-versus-host disease causes significant mortality in patients undergoing allogeneic hematopoietic cell transplantation. Immunosuppressive treatment for graft-versus-host disease can impair the beneficial graft-versus-leukemia effect and facilitate malignancy relapse. Therefore, novel approaches that protect and regenerate injured tissues without impeding the donor immune system are needed. Bile acids regulate multiple cellular processes and are in close contact with the intestinal epithelium, a major target of acute graft-versus-host disease. Here, we found that the bile acid pool is reduced following graft-versus-host disease induction in a preclinical model. We evaluated the efficacy of bile acids to protect the intestinal epithelium without reducing anti-tumor immunity. We observed that application of bile acids decreased cytokine-induced cell death in intestinal organoids and cell lines. Systemic prophylactic administration of tauroursodeoxycholic acid, the most potent compound in our in vitro studies, reduced graft-versus-host disease severity in three different murine transplantation models. This effect was mediated by decreased activity of the antigen presentation machinery and subsequent prevention of apoptosis of the intestinal epithelium. Moreover, bile acid administration did not alter the bacterial composition in the intestine suggesting that its effects are cell-specific and independent of the microbiome. Treatment of human and murine leukemic cell lines with tauroursodeoxycholic acid did not interfere with the expression of antigen presentation-related molecules. Systemic T cell expansion and especially their cytotoxic capacity against leukemic cells remained intact. This study establishes a role for bile acids in the prevention of acute graft-versus-host disease without impairing the graft-versus-leukemia effect. In particular, we provide a scientific rationale for the systematic use of tauroursodeoxycholic acid in patients undergoing allogeneic hematopoietic cell transplantation.

Background and Objectives We sought to identify new fusion genes with involvement of the platelet-derived growth factor receptor β gene ( PDGFRB ) in three patients presenting with various subtypes of chronic myeloproliferative disorders associated with chromosomal aberrations involving chromosome bands 5q31–33. Design and Methods We performed 5′ rapid amplification of cDNA ends (5′-RACE)-polymerase chain reaction (PCR) with RNA/cDNA derived from a patient (case #1) with a t(5;12)(q31–33;q24) and a second patient (case #2) with a complex rearrangement involving chromosomes 1, 5 and 11. A newly developed DNA-based ‘longdistance inverse PCR’ (LDI-PCR) was performed on a third patient (case #3) with a t(4;5;5)(q23;q31;q33). Results In cases #1 and #2, we identified mRNA fusions between GIT2 exon 12 and GPIAP1 exon 7, respectively, and PDGFRB exon 11. In case #3, LDI-PCR revealed a fusion between PRKG2 exon 5 and a truncated PDGFRB exon 12. The region encoding the catalytic domain of PDGFRβ is retained in all three cases, with the partner contributing a coiled-coil domain (GPIAP1, PRKG2) or an ankyrin protein interaction motif (GIT2) that may potentially lead to dimerization and constitutive activation of the fusion proteins. Treatment with imatinib (400 mg/day) has led to sustained complete hematologic remission in all three patients. Interpretation and Conclusions These data provide further evidence that numerous partner genes fuse to PDGFRB in BCR-ABL negative chronic myeloproliferative disorders. Although these fusion genes occur rarely, their identification is essential in order to detect patients in whom targeted treatment with tyrosine kinase inhibitors is likely to be successful.

Anagrelide is an agent with a significant platelet-lowering activity in humans. Contrasting the wealth of clinical data, bone marrow (BM) changes during therapy have been relatively rarely studied; information is particularly lacking regarding specific features of megakaryocytopoiesis.A study was performed on 15 patients with essential thrombocythemia and early stage chronic idiopathic myelofibrosis presenting with an elevated platelet count. These patients received anagrelide for 25 months resulting in a significant improvement of thrombocytosis. Evaluations were carried out by morphometry on sequential BM biopsies following enzyme- and immunohistochemical stainings that also included those for proliferative capacity and apoptosis.No significant change in proliferation or apoptosis was recognizable during anagrelide treatment. The most conspicuous alterations were those of the CD61+ megakaryocytopoiesis. Megakaryocytopoiesis revealed an increase in promegakaryoblasts together with an enhancement of proliferating cell nuclear antigen activity. This feature is in keeping with an inhibitory effect on endoreduplication implying an arrest of polyploidization and maturation into platelet-shedding large megakaryocytes. On the other hand, a significant increase in the number of megakaryocytes was not detectable. Anagrelide failed to exert a stimulating influence on the progression of myelofibrosis or on the amount of CD34+ progenitor cells. Regarding angiogenesis, there was no increase in the density of BM vessels, but distension of the vascular lumina corresponded with a vasodilatory effect.Anagrelide exerts a significant effect on endoreduplicative activity of megakaryocytes consistent with an inhibition of maturation and therefore, generates a relative predominance of precursor cells but fails to stimulate myelofibrosis.

According to the recently published WHO-classification essential thrombocythemia with ringed sideroblasts (ET/RS) remains an ambiguous category which may be considered as myelodysplastic/myeloproliferative disease, unclassifiable. Because until now only case reports or very small series of patients have been described, a more systematically performed study is warranted.A retrospective evaluation was carried out on 38 patients with the diagnosis of ET/RS and more than 15 % ringed sideroblasts on smears. Simultaneously performed bone marrow biopsies, follow-up examinations and survival data were also available.Based on cytological features and particular bone marrow findings including immunohistochemistry three patterns could be determined. These were associated with different clinical features and in particular prognosis. Group I included six patients whose diagnosis was consistent with ET, group II comprised 21 patients revealing prefibrotic and early fibrotic chronic idiopathic myelofibrosis (CIMF) and finally 11 patients (group III) displayed myelodysplastic syndromes (MDS). Follow-up studies revealed that no patient with ET showed a fiber increase but eight CIMF patients developed overt myelofibrosis and four patients of the MDS group developed secondary acute myeloid leukemia. In comparison with a control group of 39 patients with true ET, prognosis was significantly different because our cohort showed a median survival of 100 months that contrasted significantly with the 170 months in the patients with true ET.Ringed sideroblasts are not a pathognomonic feature of MDS, but may indicate a dysplasia probably associated with a primary or secondary disturbance of iron metabolism in a variety of disorders. For this reason, a more accurate classification of so-called ET/RS patients is warranted by evaluation of smears and in particular bone marrow biopsy specimens. According to our findings these patients should be classified as having either ET, CIMF or MDS and show a significantly different survival pattern.