ALBERT

Beschreibung: Additional sex combs-like 1 (ASXL1), an epigenetic modulator, is frequently mutated in myeloid neoplasms. Recent analyses of mutant ASXL1 conditional knock-in (ASXL1-MT-KI) mice suggested that ASXL1-MT alone is insufficient for myeloid transformation. In our previous study, we utilized retrovirus-mediated insertional mutagenesis, which exhibited susceptibility of ASXL1-MT-KI hematopoietic cells to transform into myeloid leukemia cells. In this screening, we identified Hematopoietically expressed homeobox (HHEX) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis. Expression of HHEX enhanced proliferation of ASXL1-MT expressing HSPCs by inhibiting apoptosis and blocking differentiation, whereas it showed only modest effect in normal HSPCs. Moreover, ASXL1-MT and HHEX accelerated the development of RUNX1-ETO9a and FLT3-ITD leukemia. Conversely, HHEX depletion profoundly attenuated the colony-forming activity and leukemogenicity of ASXL1-MT-expressing leukemia cells. Mechanistically, we identified MYB and ETV5 as downstream targets for ASXL1-MT and HHEX by using transcriptome and ChIP-seq analyses. Moreover, we found that expression of ASXL1-MT enhanced the binding of HHEX to the promoter loci of MYB or ETV5 via reducing H2AK119ub. Depletion of MYB or ETV5 induced apoptosis or differentiation in ASXL1-MT-expressing leukemia cells, respectively. In addition, ectopic expression of MYB or ETV5 reversed the reduced colony-forming activity of HHEX-depleted ASXL1-MT-expressing leukemia cells. These findings indicated that the HHEX-MYB/ETV5 axis promotes myeloid transformation in ASXL1-mutated preleukemia cells.

Beschreibung: The pseudokinase Trib1 functions as a myeloid oncogene that recruits the E3 ubiquitin ligase COP1 to C/EBPa and interacts with MEK1 to enhance ERK phosphorylation. Close genetic effect of Trib1 on Hoxa9 has been observed in myeloid leukemogenesis where Trib1 overexpression significantly accelerates Hoxa9-induced leukemia onset. However, the mechanism underlying how Trib1 functionally modulates Hoxa9 transcription activity is unclear. Herein, we provide evidence that Trib1 modulates Hoxa9-associated super-enhancers. ChIP-seq analysis identified increased histone H3K27Ac signals at super-enhancers of the Erg, Spns2, Rgl1, and Pik3cd loci, as well as increased mRNA expression of these genes. Modification of super-enhancer activity was mostly achieved via the degradation of C/EBPa p42 by Trib1, with a slight contribution from the MEK/ERK pathway. Silencing of Erg abrogated the growth advantage acquired by Trib1 overexpression, indicating that Erg is a critical downstream target of the Trib1/Hoxa9 axis. Moreover, treatment of acute myeloid leukemia (AML) cells with the BRD4 inhibitor JQ1 showed growth inhibition in a Trib1/Erg-dependent manner both in vitro and in vivo. Upregulation of ERG by TRIB1 was also observed in human AML cell lines, suggesting that Trib1 is a potential therapeutic target of Hoxa9-associated AML. Taken together, our study demonstrates a novel mechanism by which Trib1 modulates chromatin and Hoxa9-driven transcription in myeloid leukemogenesis.

Beschreibung: N-glycanase 1 (NGLY1) deficiency, an autosomal recessive disease caused by mutations in the NGLY1 gene, is characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, movement disorders and other neurological phenotypes. Because of few animal models that recapitulate these clinical signatures, the mechanisms of the onset of the disease and its progression are poorly understood, and the development of therapies is hindered. In this study, we generated the systemic Ngly1-deficient rodent model, Ngly1−/− rats, which showed developmental delay, movement disorder, somatosensory impairment and scoliosis. These phenotypes in Ngly1−/− rats are consistent with symptoms in human patients. In accordance with the pivotal role played by NGLY1 in endoplasmic reticulum-associated degradation processes, cleaving N-glycans from misfolded glycoproteins in the cytosol before they can be degraded by the proteasome, loss of Ngly1 led to accumulation of cytoplasmic ubiquitinated proteins, a marker of misfolded proteins in the neurons of the central nervous system of Ngly1−/− rats. Histological analysis identified prominent pathological abnormalities, including necrotic lesions, mineralization, intra- and extracellular eosinophilic bodies, astrogliosis, microgliosis and significant loss of mature neurons in the thalamic lateral and the medial parts of the ventral posterior nucleus and ventral lateral nucleus of Ngly1−/− rats. Axonal degradation in the sciatic nerves was also observed, as in human subjects. Ngly1−/− rats, which mimic the symptoms of human patients, will be a useful animal model for preclinical testing of therapeutic options and understanding the detailed mechanisms of NGLY1 deficiency.

Beschreibung: Olivella minuta is an abundant neogastropod on sandy beaches from Texas (USA) to southern Brazil. This study aimed to characterize and compare the feeding activity and diet of a Brazilian population of O. minuta in different tidal zones (intertidal and subtidal), and different tidal levels (high and low tides), with three combinations of tidal zone and level being studied (intertidal during low tide, intertidal during high tide and subtidal). The results showed that diet composition was generally similar among tidal conditions, with O. minuta being a generalist, feeding on 45 different food items. Feeding activity, however, was higher in the intertidal during low tide, whereas the richness and diversity of food items were higher in the intertidal during high tide. The higher feeding activity of O. minuta during low tide may be linked to a lower risk of predation; at low tide organisms may be able to feed for a longer time, and this may be particularly true on beaches with fine sand, where water retention is higher than that on beaches with coarse sand. The higher diversity of food items consumed in the intertidal during high tide is likely related to the increased prevalence of planktonic food during high tide. Our results indicate that tidal zone and level may strongly influence the feeding activity of coastal soft-bottom species and that species may show higher feeding activity during low tide. Given the current loss of intertidal habitats due to anthropogenic activity and climate-change associated factors, our study has important implications, highlighting the importance of intertidal areas for the ecology and conservation of sandy beach species.

Beschreibung: Myelodysplastic syndromes (MDS) is a clonal disorder of hematopoietic stem cells (HSCs) characterized by clonal hematopoietic stem cells (HSCs) with cytopenia, morphological abnormalities, genetic alteration, ineffective normal hematopoiesis, and frequent progression to AML. It has long remained unresolved how MDS cells, which are less proliferative, inhibit normal hematopoiesis and eventually come to dominate the bone marrow space. Despite several studies of mesenchymal stem cells (MSCs), one of the principal components of HSC niche supporting normal hematopoiesis, the molecular mechanisms underlying this process remain unclear. In this study, we examined the mechanism by which less-proliferative MDS cells outcompete normal hematopoiesis through the effects on MSCs using serially transplantable Abcg2-induced MDS/AML model we recently generated. The recipient-derived normal BM cells displayed a considerably lower colony output with markedly decreased numbers of the hematopoietic stem progenitor cells (HSPCs) . However, there were no direct effects on the colony-forming ability of the recipient HSPCs co-cultured with MDS/AML cells, indicating that MDS/AML cells inhibited hematopoiesis through alteration of bone marrow microenvironment, such as MSCs, rather than direct interaction between normal and malignant HSCs. We next analyzed histological features of BM specimens. Interestingly, bone sections from the MDS/AML mice showed a reduced trabecular bone and narrowed growth plates. Moreover, micro computed tomography (micro-CT) analysis of the femora showed a significant reduction of the trabecular bone volume in the recipient mice transplanted with the MDS/AML BM cells. We detected decreased bone formation based on the calcein double labeling, but unchanged numbers of the TRAP-positive mononuclear or multinucleated (osteoclastic) cells in the MDS/AML samples, suggesting that the reduced bone volume was caused by suppressed bone formation. The impaired bone formation was also observed in the human MDS patients in terms of lower bone volume and decreased expression of BGLAP, one of osteogenic markers. In line with the above findings, single cell qRT-PCR analyses of mouse MSCs displayed downregulation of a line of osteolineage markers, indicating that MDS/AML cells suppress bone formation through inhibiting osteolineage differentiation of MSCs. Based on the findings, we next examined if re-induction of osteolineage differentiation of the MDS/AML-derived MSCs could rescue the potential of MSCs to support normal hematopoiesis. Importantly, the number of colony-forming cells (CFCs) was significantly restored by inducing differentiation of MDS/AML-derived MSCs toward osteogenic lineage both in vitro and in vivo. These results indicate that the impairment of osteolineage differentiation is the principal cause for an impaired normal hematopoiesis in MDS/AML, and that restoring the supportive niche will be a potential therapeutic option. Since extracellular vesicles (EVs) derived from MDS/AML cells are critical mediators of intercellular communication, we examined the molecular mechanism underlying the dysfunction of MSCs via EVs. As expected, EVs from MDS/AML cells were incorporated into the normal MSCs where osteolineage marker genes were clearly downregulated, and the number of CFCs significantly decreased in the HSPCs co-cultured with MSCs treated by the MDS/AML-derived EVs. Moreover, by comprehensively analyzing microRNAs (miRNAs) enriched in EVs derived from MDS/AML cells, we identified several miRNAs that impaired the differentiation of normal MSCs. These results suggested that miRNAs in EVs derived from MDS/AML cells disrupted the hematopoietic supporting niche through suppressing an osteolineage differentiation of MSCs. Here we uncover a heretofore unrecognized mechanism of bone marrow failure in MDS via the impairment of osteolineage differentiation in MSCs. EVs from MDS cells will be an attractive therapeutic target to restore the supportive niches, such as MSCs, for the remaining normal HSCs. Figure Disclosures No relevant conflicts of interest to declare.

Beschreibung: Introduction. Hypomethylating agents azacitidine (AZA) and decitabine are standard of care for myelodysplastic syndromes (MDS). Considering the result of the secondary analysis of the AZA-001 trial showing 91% of first responses occurred within 6 cycles, the concept is widely accepted that AZA should be performed at least 6 cycles even if early response is not achieved (Silverman LR, Cancer. 2011). However, 40-50% of cases finally experienced primary resistance. These facts mean almost half of the patients receive fruitless treatment for up to six months. Therefore, it is necessary to identify those patients who do not response to AZA from the early cycles, so that they can receive alternative treatment as early as possible. The Wilms' tumor 1 gene (WT1) is a tumor marker of leukemic blasts. High expression of WT1-mRNA is correlated with disease progression, efficacy of AZA and prognosis in the patients with MDS (Tamaki et al, Leukemia. 1999). In this study, we explored whether WT1-mRNA levels in peripheral blood (PB) samples at baseline and during the early cycles of AZA could be useful in predicting outcome to this therapy. Methods. We retrospectively evaluated transplant-ineligible patients with high-risk MDS, diagnosed according to the 2008 World Health Organization classification, who received more than three cycles of AZA as a 1st line therapy between March 2012 and January 2020 at Aiiku Hospital and Sapporo Hokuyu Hospital. Response to treatment was classified as complete remission (CR), partial remission (PR), hematologic improvement (HI), stable disease, and failure based on the International Working Group 2006 criteria. Patients who were not evaluated WT1-mRNA levels in PB samples at baseline and within three cycles of AZA were excluded from this study. WT1-mRNA was measured using a WT1-mRNA Assay Kit (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan). The study procedures were in accordance with the Helsinki Declaration and institutional ethical guidelines, and were approved by the institutional review boards. Results. Seventy-five patients (age 61-88, median 77; 76% male) were included in this study. The median follow-up time from AZA initiation was 11.4 months (range, 4.2-48.8 months). Median overall survival (OS) time from AZA initiation was 16.4 months (95% CI, 11.6-24.1 months). Forty-one patients (54.7%) achieved any response (CR + PR + HI). WT1-mRNA expression at the baseline varied from

Beschreibung: Introduction: Myeloid/Natural killer (NK) cell precursor acute leukemia (MNKPL) is a rare hematologic malignancy prevalent in East Asia. MNKPL is characterized by extramedullary involvement, immature lymphoblastoid morphology without myeloperoxidase (MPO) reactivity, the CD7+/CD33+/CD34+/CD56+/HLA−DR+ phenotype. MNKPL is classified as mixed phenotype acute leukemia, and not otherwise specified rare types (MPAL NOS rare types) in WHO classification. However, its characteristic clinical feature and undetermined genetic feature suggests that MNPKL leaves open the possibility of a new independent disease concept. Here, we report clinical features and genetic alterations in patients with MNKPL. Methods: The Leukemia and Lymphoma Committee of the Japanese Society of Pediatric Hematology and Oncology (JSPHO) sent out questionnaires to 110 JSPHO affiliated hospitals and collected cases of MNPKL diagnosed during the period 2000-2013. Besides, the cases published as literature were recruited. The data of clinical features, cell surface antigen profiling, overall survival (OS), and event-free survival (EFS) defined as relapse or death were also collected as a secondary survey. The protocol of this retrospective study was approved by the review boards of JSPHO and Ehime Prefectural Central Hospital. Comprehensive genetic analysis including 13 whole-exome sequences (WES), 2 target sequence, 6 RNA sequence (RNA-seq), and 8 DNA methylation analysis was performed. We also performed single-cell RNA-seq using 1 sample of MNKPL patients and a normal bone marrow sample as the reference. The research protocol was approved by the review board of TMDU. Results: Sixteen children or young adults (〈 39 years old) and 2 older adults with MNKPL were identified. The median age of MNKPL patients was 11 (0.5-75) years old. There are 12 males and 6 females. The extramedullary involvement was observed in 7 patients. Complete remission after induction therapy was achieved in 8/14 (57%) patients treated with acute myeloid leukemia (AML) type chemotherapy and 2/4 (50%) patients treated with acute lymphoblastic leukemia (ALL)/non-Hodgkin lymphoma type chemotherapy, respectively. Fifteen patients underwent hematopoietic cell transplantation (HCT). The median follow-up period was 3.8 (0.1-16.0) years. 5-year OS and 5-year EFS was 49.5% and 40.7%, respectively. In genetic analysis, median 388 somatic mutations in MNKPL were identified by WES. The recurrent mutations were observed in NOTCH1 (n=5), MAML3 (n=4), NRAS, MAP3K4, RECQL4, CREBBP, ASXL2, and KMT2D (n=3, respectively), and MAML2, MAP3K1, FLT3, CARD11, MSH4, FANCI, WT1, ZNF384, and ERG (n=2, respectively). The distinct expression pattern, higher expression of RUNX3 and NOTCH1, and lower expression of BCL11B were identified in MNKPL samples which were compared to MPAL, AML, and T cell ALL in RNA-seq. The distinct methylation profile, hypomethylation of RUNX3 regulatory region, and hypermethylation of BCL11B regulatory region were identified in DNA methylation analysis. Single-cell RNA-seq analysis also showed distinct 4 subsets of MNKPL. Discussion and Conclusions: NK cells are the founding member of a family of innate lymphoid cells (ILC). Genetic abnormality of NOTCH1 pathway is a hallmark of MNPKL. RUNX3 is required for NK cell survival and proliferation in response to IL-15 signaling. RUNX3 high expression and hypomethylation of RUNX3 regulatory region also characterize MNKPL. Currently, MNKPL is classified as MPAL NOS, our genetic analysis revealed that MNKPL is a distinct group from MPAL. The prognosis of MNKPL was not satisfactory even though HCT was performed. The development of new therapeutic approaches based on these genetic analyses is highly expected. Disclosures Saito: Toshiba Corporation: Research Funding. Nakazawa:Toshiba Corporation: Research Funding.