FUS-ERG induces late-onset azacitidine resistance in acute myeloid leukaemia cells.

FUS-ERG is a chimeric gene with a poor prognosis, found in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). It remains unclear whether DNA hypomethylating agents, including azacitidine (Aza), are effective in FUS-ERG-harbouring AML and how FUS-ERG induces chemoresistance. Stable Ba/F3 transfectants with FUS-ERG were repeatedly exposed to Aza for 7 days of treatment and at 21-day intervals to investigate Aza sensitivity. Stable FUS-ERG transfectants acquired resistance acquired resistance after three courses of Aza exposure. RNA sequencing (RNA-seq) was performed when Aza susceptibility began to change; genes with altered expression or transcript variants were identified. Molecular signatures of these genes were analysed using gene ontology. RNA-seq analyses identified 74 upregulated and 320 downregulated genes involved in cell motility, cytokine production, and kinase activity. Additionally, 1321 genes with altered transcript variants were identified, revealing their involvement in chromatin organisation. In a clinical case of AML with FUS-ERG, we compared whole-genome alterations between the initial MDS diagnosis and AML recurrence after Aza treatment. Genes with non-synonymous or near mutations in transcription regulatory areas (TRAs), additionally detected in AML recurrence, were collated with the gene list from RNA-seq to identify genes involved in acquiring Aza resistance in the presence of FUS-ERG. Whole-genome sequencing of clinical specimens identified 29 genes with non-synonymous mutations, including BCOR, and 48 genes located within 20 kb of 54 TRA mutations in AML recurrence. These genes were involved in chromatin organisation and included NCOR2 as an overlapping gene with RNA-seq data. Transcription regulators involved in mutated TRAs were skewed and included RCOR1 in AML recurrence. We tested the efficacy of BH3 mimetics, including venetoclax and S63845, in primary Aza-resistant AML cells treated with FUS-ERG. Primary FUS-ERG-harbouring AML cells acquiring Aza resistance affected the myeloid cell leukaemia-1 (MCL1) inhibitor S63845 but not while using venetoclax, despite no mutations in BCL2. FUS-ERG promoted Aza resistance after several treatments. The disturbance of chromatin organisation might induce this by co-repressors, including BCOR, NCOR2, and RCOR1. MCL1 inhibition could partially overcome Aza resistance in FUS-ERG-harbouring AML cells.

Loss of Nudt15 thiopurine detoxification increases direct DNA damage in hematopoietic stem cells.

Thiopurines, such as 6-mercaptopurine (6-MP), are widely used as cytotoxic agents and immunosuppressants for leukemia and autoimmune or inflammatory diseases. A nonsynonymous single nucleotide polymorphism (p.Arg139Cys; R139C) of the nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) gene causes the loss of thiopurine detoxification, inducing myelosuppression. To understand such hematotoxicity, we investigate the effects of NUDT15 R139C on hematopoietic stem cells (HSCs) upon thiopurine administration. Using previously established Nudt15R138C knock-in mice, which mimic myelosuppression in NUDT15R139C homozygous or heterozygous patients following thiopurine administration, we investigated the numerical changes of HSCs and hematopoietic progenitor cells following 6-MP administration using in vivo flowcytometry and ex vivo HSC expansion. Genes differentially expressed between Nudt15+/+ HSCs and Nudt15R138C/R138C HSCs were identified using RNA-sequencing before the emergence of 6-MP-induced HSC-damage. Gene Ontology (GO) and Transcriptional Regulatory Relationships Unraveled by Sentence-based Text Mining (TRRUST) analyses were performed to elucidate the molecular effects of 6-MP on HSCs. In Nudt15R138C/R138C mice, 6-MP induced exhaustion of HSCs faster than that of multipotent progenitors and as fast as that of myeloid-committed progenitors. Ex vivo-expanded Nudt15R138C/R138C HSCs were dose- and time-dependently damaged by 6-MP. GO analysis identified the DNA damage response and cell cycle process as the most strongly influenced processes in Nudt15R138C/R138C HSCs. TRRUST analysis revealed that the Trp53-regulated transcriptional regulatory network is influenced prior to HSC exhaustion in Nudt15R138C/R138C HSCs. The loss of NUDT15 thiopurine detoxification enhances thiopurine-mediated DNA damage via the Trp53 networks in HSCs. Therefore, caution is required in long-term thiopurine use in patients with NUDT15 R139C in view of its adverse effects on HSCs in the form of DNA damage.

Thiopurine pharmacogenomics and pregnancy in inflammatory bowel disease.

The thiopurine drugs azathioprine and 6-mercaptopurine are widely used for the maintenance of clinical remission in steroid-dependent inflammatory bowel disease (IBD). Thiopurines are recommended to be continued throughout pregnancy in IBD patients, but conclusive safety data in pregnant patients remain still insufficient. On the other hand, a strong association between a genetic variant of nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15 p.Arg139Cys) and thiopurine-induced myelotoxicity has been identified. Pharmacokinetic studies have revealed that thiopurine metabolism is altered in pregnant IBD patients and suggested that the fetus may be exposed to the active-thiopurine metabolite, 6-thioguaninetriphosphate, in the uterus. A recent study using knock-in mice harboring the p.Arg138Cys mutation which corresponds to human p.Arg139Cys showed that oral administration of 6-MP at clinical dose induces a severe toxic effect on the fetus harboring the homozygous or heterozygous risk allele. This suggests that NUDT15 genotyping may be required in both women with IBD who are planning pregnancy (or pregnant) and their partner to avoid adverse outcomes for their infant. The risk to the fetus due to maternal thiopurine use is minimal but there are some concerns that are yet to be clarified. In particular, a pharmacogenomic approach to the fetus is considered necessary.

LSD1-mediated repression of GFI1 super-enhancer plays an essential role in erythroleukemia.

Super-enhancers (SEs) consist of enhancer clusters with abundant binding of transcription factors (TFs) and cofactors. LSD1 is a histone modifier that eliminates SE activity. However, whether SE suppression by LSD1 is associated with leukemogenesis remains unknown. In erythro-megakaryocyte lineage leukemia cells, activation of the SE of GFI1 (GFI1-SE) is related to induction of myeloid differentiation by LSD1 inhibitors NCD38 and NCD25 and to their antileukemia effect. Although functional TF-motifs were concentrated in an evolutionally conserved area, NCD38 barely induced additional TF recruitment. Instead, the transcription cofactors including LSD1, CoREST, HDAC1, and HDAC2 were evicted from GFI1-SE. Deletion of GFI1-SE impaired induction of myeloid differentiation by NCD38 and NCD25 in erythroleukemia cells. Gene set enrichment analysis revealed that the GFI1-SE deletion impaired NCD38-induced programs related to granulocyte differentiation and the CEBPA network, but restored NCD38-suppressed programs related to erythroid development, GATA1 targets, and acute myeloid leukemia (AML) clusters including FAB subtype M6 and AML with myelodysplastic syndrome-related chromosomal abnormalities. Ontologies of genes whose expression changes by NCD38 were canceled due to the GFI1-SE deletion showed enrichment in AML and neutropenia signatures. Collectively, our data suggest that sustainable repression of GFI1-SE by LSD1 is essential for sustenance of erythroleukemia cells.

Thiopurine-mediated impairment of hematopoietic stem and leukemia cells in Nudt15R138C knock-in mice.

Thiopurines are widely used as antileukemia agents and immunosuppressants. Recent large-scale clinical studies revealed a strong association between the NUDT15 p.Arg139Cys (NUDT15R139C) polymorphism and severe thiopurine-induced leukocytopenia. We established knock-in mice harboring p.Arg138Cys (Nudt15R138C), which corresponds to the human polymorphism. A clinically relevant dose of mercaptopurine (MP) induced lethal cytopenia in Nudt15R138C-harboring mice. MP dose reduction attenuated the hematopoietic toxicity, phenocopying clinical observations and providing Nudt15 genotype-based tolerable doses of MP. High-dose MP induced acute damage to hematopoietic stem and progenitor cells (HSPCs) in Nudt15R138C/R138C mice. A competitive transplantation assay revealed that not only Nudt15R138C/R138C HSPCs, but also Nudt15+/R138C HSPCs suffered stronger damage than Nudt15+/+ HSPCs, even by lower-dose MP, after long-term administration. In a Nudt15 genotype-based posttransplantation leukemia recurrence model generated by bone marrow replacement with congenic wild-type cells and a small number of leukemia stem cells, MP prolonged the survival of mice with posttransplantation Nudt15R138C/R138C leukemia recurrence. In conclusion, our model will facilitate NUDT15 genotype-based precision medicine by providing safer estimates for MP dosing, and our findings highlighted the high susceptibility of hematopoietic stem cells to MP and suggested that exploiting thiopurine toxicity might be a novel treatment approach for leukemia in NUDT15R139C-harboring patients.

Selective dissociation between LSD1 and GFI1B by a LSD1 inhibitor NCD38 induces the activation of ERG super-enhancer in erythroleukemia cells.

Lysine-specific demethylase 1 (LSD1) is a histone modifier for transcriptional repression involved in the regulation of hematopoiesis. We previously reported that a LSD1 inhibitor NCD38 induces transdifferentiation from erythroid lineage to granulomonocytic lineage and exerts anti-leukemia effect through de-repression of the specific super-enhancers of hematopoietic regulators including ERG in a human erythroleukemia cell line, HEL. However, the mechanistic basis for this specificity of NCD38 has remained unclear. Herein, we report major partners associated with LSD1 and clarify the mechanism in HEL cells. Proteome analysis identified 54 candidate proteins associated with LSD1, including several transcription factors such as GFI1B and RUNX1 as well as BRAF-histone deacetylase complex (BHC) components such as CoREST, HDAC1, and HDAC2. NCD38 selectively disrupted the interaction of LSD1 with GFI1B but not with RUNX1, CoREST, HDAC1 and HDAC2. Erg was downregulated in murine erythroid progenitors with prominent upregulation of Gfi1b. NCD38 induced ERG and attenuated an erythroid marker CD235a in HEL while this attenuation was mimicked by the lentiviral overexpression of ERG. The ERG super-enhancer contained the conserved binding motif of GFI1B and was actually occupied by GFI1B. NCD38 dissociated LSD1 and CoREST but not GFI1B from the ERG super-enhancer. Collectively, the selective separation of LSD1 from GFI1B by NCD38 restores the ERG super-enhancer activation and consequently upregulates ERG expression, inducing the transdifferentiation linked to the anti-leukemia effect.

[Successful treatment with recombinant thrombomodulin for disseminated intravascular coagulation complicated with hemophagocytic syndrome].

Recombinant human thrombomodulin (rTM) improves the blood coagulation disorder characteristic of disseminated intravascular coagulation (DIC) as well as, or even better than, other anti-DIC drugs. On post-marketing surveillance, its effectiveness has been recognized for hematologic disorders, sepsis and solid tumor subgroups. However, the effect on hemophagocytic syndrome (HPS) complicated by DIC remains unclear. We treated three HPS patients with rTM in addition to chemotherapy for the underlying diseases including nasal NK/T cell lymphoma, angioimmunoblastic T-cell lymphoma and refractory acute myeloid leukemia post cord blood transplantation. Although being refractory to medical management was suspected in our cases, clinical status rapidly came under control including not only amelioration of the blood coagulation disorder but also inflammatory reactions, such as serum ferritin and lactic acid dehydrogenase abnormalities, which represent HPS activity. These observations suggest that rTM might exert marked synergistic effects on HPS with DIC. Given the results obtained in these three cases, administration of rTM appears to offer a promising method of treating HPS complicated by DIC.

Primary hepatic lymphoma in a patient with rheumatoid arthritis treated with methotrexate.

Primary hepatic lymphoma (PHL) has rarely been reported in patients with immunosuppression. We herein describe a case of Epstein-Barr virus- (EBV-) positive PHL in a 67-year-old Japanese woman receiving methotrexate (MTX) treatment for rheumatoid arthritis (RA). The patient, who had been receiving MTX therapy for more than 6 years, presented with low-grade fever and abdominal pain. Initial laboratory tests showed mildly elevated liver enzymes with normal levels of alpha-fetoprotein and carcinoembryonic antigen, and computed tomography scans revealed multiple hepatic tumors with no lymph-node swelling. Examination of liver specimens obtained via ultrasonography-guided needle biopsy indicated EBV-positive diffuse large B cell lymphoma; therefore, she was diagnosed with PHL. MTX was discontinued, and she was carefully monitored thereafter owing to the prolonged history of MTX administration for RA. Rapid progression of PHL was observed; therefore 10 days after the PHL diagnosis, she received 6 cycles of R-THP-COP (rituximab, cyclophosphamide, pirarubicin, vincristine, and prednisolone) therapy and achieved complete remission for more than 1 year. Although MTX-associated lymphoproliferative disorders often show remission after withdrawal of MTX, early diagnosis and treatment are essential for PHL in patients with RA treated with MTX, because of the aggressive nature of the disease.

Multiple splenic abscesses in 2 patients with myelodysplastic syndrome.

Splenic abscess is a rare clinical condition with a high mortality rate. Multiple splenic abscesses, rather than a solitary abscess, are present in immunocompromised states including hematological malignancies. As symptoms of splenic abscesses, fever and abdominal pain, are non-specific, timely and adequate use of imaging studies is crucial for early diagnosis. We report the cases of 2 patients with myelodysplastic syndrome and multiple splenic abscesses. Notwithstanding the higher mortality rate of patients with multiple splenic abscesses as compared with those with a solitary splenic abscess, we successfully treated the 2 patients by using antibiotic therapy and fine needle aspiration.