In acute promyelocytic leukemia (APL), differentiation therapy with all-trans retinoic acid (ATRA) and/or arsenic trioxide can induce a differentiation syndrome (DS) with massive pulmonary infiltration of differentiating leukemic cells. Because chemokines are implicated in migration and extravasation of leukemic cells, chemokines might play a role in DS. ATRA stimulation of the APL cell line NB4 induced expression of multiple CC-chemokines (CCLs) and their receptors (> 19-fold), resulting in increased chemokine levels and chemotaxis. Induction of CCL2 and CCL24 was directly mediated by ligand-activated retinoic acid receptors. In primary leukemia cells derived from APL patients at diagnosis, ATRA induced chemokine production as well. Furthermore, in plasma of an APL patient with DS, we observed chemokine induction, suggesting that chemokines might be important in DS. Dexamethasone, which efficiently reduces pulmonary chemokine production, did not inhibit chemokine induction in APL cells. Finally, chemokine production was also induced by arsenic trioxide as single agent or in combination with ATRA. We propose that differentiation therapy may induce chemokine production in the lung and in APL cells, which both trigger migration of leukemic cells. Because dexamethasone does not efficiently reduce leukemic chemokine production, pulmonary infiltration of leukemic cells may induce an uncontrollable hyperinflammatory reaction in the lung. IntroductionAcute promyelocytic leukemia (APL) is characterized by a specific t(15;17) chromosomal translocation, which fuses the promyelocytic leukemia (PML) gene on chromosome 15 to the retinoic acid receptor-␣ (RARA) gene on chromosome 17. This translocation results in blockage of terminal granulocytic differentiation at the promyelocytic stage. 1,2 Treatment with high dosage of the RARA ligand all-trans retinoic acid (ATRA) relieves this blockage, resulting in terminal differentiation of the APL blasts. Presently, standard therapy for patients with newly diagnosed APL consists of a combination of ATRA and anthracycline-based chemotherapy, which results in the induction of a complete remission in more than 90% of the patients and a 5-year overall survival rate of more than 80%. [3][4][5][6][7][8][9][10][11] In addition, arsenic trioxide (ATO) treatment has been shown to be highly effective in relapsed APL patients and has also been used successfully as a single agent or in combination with ATRA in newly diagnosed APL patients. [12][13][14][15][16][17][18][19][20][21] Despite the high cure rates, induction mortality is a still a problem in APL. In a large series of 732 APL patients who received ATRA plus idarubicin, induction mortality was 9%. The most common causes of death were hemorrhage, infection, and the differentiation syndrome (DS), formerly known as retinoic acid syndrome. 22 DS is reported in 2.5% to 31% of the APL patients who receive induction therapy with ATRA and/or ATO. [5][6][7]10,14,16,19,20,[23][24][25][26][27][28][29][30] DS is not observed during consolidation o...

Recently, de novo heterozygous loss-of-function mutations in beta-catenin (CTNNB1) were described for the first time in four individuals with intellectual disability (ID), microcephaly, limited speech and (progressive) spasticity, and functional consequences of CTNNB1 deficiency were characterized in a mouse model. Beta-catenin is a key downstream component of the canonical Wnt signaling pathway. Somatic gain-of-function mutations have already been found in various tumor types, whereas germline loss-of-function mutations in animal models have been shown to influence neuronal development and maturation. We report on 16 additional individuals from 15 families in whom we newly identified de novo loss-of-function CTNNB1 mutations (six nonsense, five frameshift, one missense, two splice mutation, and one whole gene deletion). All patients have ID, motor delay and speech impairment (both mostly severe) and abnormal muscle tone (truncal hypotonia and distal hypertonia/spasticity). The craniofacial phenotype comprised microcephaly (typically -2 to -4 SD) in 12 of 16 and some overlapping facial features in all individuals (broad nasal tip, small alae nasi, long and/or flat philtrum, thin upper lip vermillion). With this detailed phenotypic characterization of 16 additional individuals, we expand and further establish the clinical and mutational spectrum of inactivating CTNNB1 mutations and thereby clinically delineate this new CTNNB1 haploinsufficiency syndrome.

Summary(De-)regulation of apoptosis plays an important role in normal and malignant lymphopoiesis. Apoptosis-regulating genes of the BCL-2 family and the recently identified inhibitors of apoptosis (IAP) family have been implicated in different types of non-Hodgkin lymphoma (NHL). To investigate whether expression of specific apoptosis-regulating genes correlated with different types of lymphoid malignancies, we measured the expression of five BCL-2 family genes, four IAP family genes and SMAC by real-time quantitative polymerase chain reaction in patient samples. In total, 137 samples from B-and T-cell acute lymphoblastic leukaemia (ALL), B-cell chronic lymphocytic leukaemia (CLL), six different NHL types and three control tissue types were analysed. The data were further analysed using cluster and discriminant analysis. Three specific expression patterns were identified for CLL, ALL and NHL respectively. CLL samples, as well as B-ALL and follicular lymphoma samples showed high similarity in the expression of these apoptosis-regulating genes and could be distinguished from each other and other diseases and controls. Discriminant analysis identified three members of the IAP family, C-IAP1, C-IAP2 and SURVIVIN, as the most informative genes to discriminate between these lymphoid malignancies.

Ubiquitination plays a major role in many aspects of hematopoiesis. Alterations in ubiquitination have been implicated in hematological cancer. The ubiquitin ligase Triad1 controls the proliferation of myeloid cells. Here, we show that two RING (really interesting new gene) domains in Triad1 differentially bind ubiquitin-conjugating enzymes, UbcH7 and Ubc13. UbcH7 and Ubc13 are known to catalyze the formation of different polyubiquitin chains. These chains mark proteins for proteasomal degradation or serve crucial non-proteolytic functions, respectively. In line with the dual Ubc interactions, we observed that Triad1 catalyzes the formation of both types of ubiquitin chains. The biological relevance of this finding was studied by testing Triad1 mutants in myeloid clonogenic assays. Full-length Triad1 and three mutants lacking conserved domains inhibited myeloid colony formation by over 50%. Strikingly, deletion of either RING finger completely abrogated the inhibitory effect of Triad1 in clonogenic growth. We conclude that Triad1 exhibits dual ubiquitin ligase activity and that both of its RING domains are crucial to inhibit myeloid cell proliferation. The differential interaction of the RINGs with Ubcs strongly suggests that the ubiquitination mediated through UbcH7 as well as Ubc13 plays a major role in myelopoiesis.

Growth factor independence 1 (Gfi1) is a transcriptional repressor essential for the function and development of many different hematopoietic lineages. The Gfi1 protein expression is regulated by the ubiquitin-proteasome system. In granulocytes, Gfi1 is rapidly degraded by the proteasome, while it is more stable in monocytes. How the ubiquitination and degradation of Gfi1 is regulated is unclear. Here, we show that the ubiquitin ligase Triad1 interacts with the DNA-binding domain of Gfi1. Unexpectedly, we found that Triad1 inhibited Gfi1 ubiquitination, resulting in a prolonged half-life. Down-regulation of endogenous Triad1 by siRNAs resulted in increased Gfi1 ubiquitination. In U937 cells, Triad1 caused an increase in endogenous Gfi1 protein levels and slowed cell proliferation in a similar manner when Gfi1 itself was expressed. A Triad1 mutant that lacks the Gfi1-binding domain did not affect Gfi1 levels and proliferation. Because neither proteasomeubiquitin nor Triad1 ubiquitin ligase activity was required for the inhibition of Gfi1 ubiquitination, these data suggest that Triad1 competes for Gfi1 binding with as yet to be identified E3 ubiquitin ligases that do mark Gfi1 for proteasomal degradation. The finetuning of Gfi1 protein levels regulated by Triad1 defines an unexpected role for this protein in hematopoiesis. (Blood.

Summary. Selection procedures in bone marrow transplantation (BMT) would benefit from the development of easy-to-perform cellular assays with high discriminative power. We tested a cytokine-based mixed lymphocyte culture (MLC) and compared its outcome to the routinely used MLC, helper T-lymphocyte precursor (HTLp)-f and cytotoxic Tlymphocyte precursor (CTLp)-f assays. Interferon (IFN)g was selected as a marker cytokine for (deleterious) T-helper 1 like responses and 36 (potential) BMT donor-recipient pairs were analysed. The IFNg-MLC appeared sensitive to HLA class II (subtype) differences, but not to isolated class I differences, or to mismatches other than HLA (identical siblings). The test enabled a distinction between combinations with positive MLC (proliferation) and HTLp-f, exemplified by the fact that although high IFNg levels were observed in the class II mismatched group, certain DRB3, DQB1-subtype and DRB1-subtype mismatches did not give rise to IFNg production. This might be of relevance for the detection of so-called permissible mismatches. With regard to prediction of acute graft-versus-host disease (aGVHD) in unrelated BMT, the data indicated that high levels of IFNg coincided with severe aGVHD, whereas low levels were largely associated with grades 0-I. However, in the case of isolated class I mismatches the test had no predictive value. The cellsaving IFNg-MLC provides an alternative for the assays currently in use, but should be employed along with an assay that is sensitive to class I differences to correct for false negatives. Consequently, a combination of IFNg-MLC and CTLp-f assays seems most promising for donor selection, other than identical siblings.

Selection procedures in bone marrow transplantation (BMT) would benefit from the development of easy-to-perform cellular assays with high discriminative power. We tested a cytokine-based mixed lymphocyte culture (MLC) and compared its outcome to the routinely used MLC, helper T-lymphocyte precursor (HTLp)-f and cytotoxic T-lymphocyte precursor (CTLp)-f assays. Interferon (IFN)gamma was selected as a marker cytokine for (deleterious) T-helper 1 like responses and 36 (potential) BMT donor-recipient pairs were analysed. The IFNgamma-MLC appeared sensitive to HLA class II (subtype) differences, but not to isolated class I differences, or to mismatches other than HLA (identical siblings). The test enabled a distinction between combinations with positive MLC (proliferation) and HTLp-f, exemplified by the fact that although high IFNgamma levels were observed in the class II mismatched group, certain DRB3, DQB1-subtype and DRB1-subtype mismatches did not give rise to IFNgamma production. This might be of relevance for the detection of so-called permissible mismatches. With regard to prediction of acute graft-versus-host disease (aGVHD) in unrelated BMT, the data indicated that high levels of IFNgamma coincided with severe aGVHD, whereas low levels were largely associated with grades 0-I. However, in the case of isolated class I mismatches the test had no predictive value. The cell-saving IFNgamma-MLC provides an alternative for the assays currently in use, but should be employed along with an assay that is sensitive to class I differences to correct for false negatives. Consequently, a combination of IFNgamma-MLC and CTLp-f assays seems most promising for donor selection, other than identical siblings.

The most important complication of all-trans retinoic acid (ATRA) treatment in acute promyelocytic leukemia (APL) is the retinoic acid syndrome (RAS), a life-threatening hyper-inflammatory reaction with a distinct capillary leak syndrome and multi-organ failure. Once established, the syndrome has proven very difficult to manage. Early recognition in combination with prompt corticosteroid treatment significantly reduces the mortality rate of patients with this syndrome. Nonetheless, still 15% of the induction deaths in APL is caused by RAS. The pathogenic mechanism of the hyper-inflammatory cascade in RAS is not fully understood yet. Infiltration of differentiating APL cells in the lung is important in the development of RAS. In addition, differentiation of APL cells by ATRA is associated with increased expression and release of pro-inflammatory cytokines, chemokines and adhesion molecules in vitro and it has been hypothesized that this is important for the triggering of the hyper-inflammatory cascade in RAS. We investigated the effect of ATRA on the expression and secretion of chemokines in APL cells in vitro and in vivo. Using microarray, quantitative PCR and ELISA we demonstrated significant induction (up to 16000-fold) of 8 CC-chemokines (CCLs) in the NB4 APL cell line after differentiation induction with ATRA in vitro. To demonstrate the significance of chemokine induction by ATRA in vivo, we measured plasma levels of CCLs in 3 APL patients, treated with a combination of ATRA, idarubicine and prednisone according to the AIDA-2000/P protocol. One of these patients developed an obvious retinoic acid syndrome. During therapy we observed increased plasma levels of 3 CCLs in all three patients. Induction of 5 other CCLs (CCL3, CCL4, CCL7, CCL8, CL11) was only observed during RAS, suggesting that upregulation of these 5 CC-chemokines is specific for RAS. To investigate whether the plasma levels of CC-chemokines are functionally relevant, we measured chemo-attraction of peripheral blood leukocytes towards plasma of an APL patient who developed RAS using a transwell system. Plasma from this patient during RAS showed significant more chemo-attraction than plasma from the same patient before treatment with ATRA, idarubicine and prednisone. Subsequently we investigated whether the therapeutic effect of dexamethasone in RAS can be ascribed to downregulation of chemokine expression in APL cells. Despite dexamethasone, the induction of chemokine expression in NB4 cells by ATRA sustained. We conclude that ATRA causes massive secretion of chemokines by APL cells, which might trigger the hyper-inflammatory cascade in RAS by continuous attraction of APL cells and other inflammatory cells towards tissues like the lung. Dexamethasone does not abrogate the production of CC-chemokines by APL cells, but rather seems to inhibit the hyper-inflammatory cascade at the level of the effector cells and target tissues like the lung. This might explain why dexamethasone is not able to sufficiently reverse a retinoic acid syndrome once it has been established. The application of neutralizing CC-chemokine receptor antibodies or other antagonists might be an alternative route to treat an established retinoic acid syndrome.