Germ line DDX41 mutations define a unique subtype of myeloid neoplasms.

Germ line DDX41 variants have been implicated in late-onset myeloid neoplasms (MNs). Despite an increasing number of publications, many important features of DDX41-mutated MNs remain to be elucidated. Here we performed a comprehensive characterization of DDX41-mutated MNs, enrolling a total of 346 patients with DDX41 pathogenic/likely-pathogenic (P/LP) germ line variants and/or somatic mutations from 9082 MN patients, together with 525 first-degree relatives of DDX41-mutated and wild-type (WT) patients. P/LP DDX41 germ line variants explained ∼80% of known germ line predisposition to MNs in adults. These risk variants were 10-fold more enriched in Japanese MN cases (n = 4461) compared with the general population of Japan (n = 20 238). This enrichment of DDX41 risk alleles was much more prominent in male than female (20.7 vs 5.0). P/LP DDX41 variants conferred a large risk of developing MNs, which was negligible until 40 years of age but rapidly increased to 49% by 90 years of age. Patients with myelodysplastic syndromes (MDS) along with a DDX41-mutation rapidly progressed to acute myeloid leukemia (AML), which was however, confined to those having truncating variants. Comutation patterns at diagnosis and at progression to AML were substantially different between DDX41-mutated and WT cases, in which none of the comutations affected clinical outcomes. Even TP53 mutations made no exceptions and their dismal effect, including multihit allelic status, on survival was almost completely mitigated by the presence of DDX41 mutations. Finally, outcomes were not affected by the conventional risk stratifications including the revised/molecular International Prognostic Scoring System. Our findings establish that MDS with DDX41-mutation defines a unique subtype of MNs that is distinct from other MNs.

Application of prime editing system to introduce TP53 R248Q hotspot mutation in acute lymphoblastic leukemia cell line.

In childhood acute lymphoblastic leukemia (ALL), TP53 gene mutation is associated with chemoresistance in a certain population of relapsed cases. To directly verify the association of TP53 gene mutation with chemoresistance of relapsed childhood ALL cases and improve their prognosis, the development of appropriate human leukemia models having TP53 mutation in the intrinsic gene is required. Here, we sought to introduce R248Q hotspot mutation into the intrinsic TP53 gene in an ALL cell line, 697, by applying a prime editing (PE) system, which is a versatile genome editing technology. The PE2 system uses an artificial fusion of nickase Cas9 and reverse-transcriptase to directly place new genetic information into a target site through a reverse transcriptase template in the prime editing guide RNA (pegRNA). Moreover, in the advanced PE3b system, single guide RNA (sgRNA) matching the edited sequence is also introduced to improve editing efficiency. The initially obtained MDM2 inhibitor-resistant PE3b-transfected subline revealed disrupted p53 transactivation activity, reduced p53 target gene expression, and acquired resistance to chemotherapeutic agents and irradiation. Although the majority of the subline acquired the designed R248Q and adjacent silent mutations, the insertion of the palindromic sequence in the scaffold hairpin structure of pegRNA and the overlap of the original genomic DNA sequence were frequently observed. Targeted next-generation sequencing reconfirmed frequent edit errors in both PE2 and PE3b-transfected 697 cells, and it revealed frequent successful edits in HEK293T cells. These observations suggest a requirement for further modification of the PE2 and PE3b systems for accurate editing in leukemic cells.

Two novel high-risk adult B-cell acute lymphoblastic leukemia subtypes with high expression of CDX2 and IDH1/2 mutations.

The genetic basis of leukemogenesis in adults with B-cell acute lymphoblastic leukemia (B-ALL) is largely unclear, and its clinical outcome remains unsatisfactory. This study aimed to advance the understanding of biological characteristics, improve disease stratification, and identify molecular targets of adult B-ALL. Adolescents and young adults (AYA) (15 to 39 years old, n = 193) and adults (40 to 64 years old, n = 161) with Philadelphia chromosome-negative (Ph-) B-ALL were included in this study. Integrated transcriptomic and genetic analyses were used to classify the cohort into defined subtypes. Of the 323 cases included in the RNA sequencing analysis, 278 (86.1%) were classified into 18 subtypes. The ZNF384 subtype (22.6%) was the most prevalent, with 2 novel subtypes (CDX2-high and IDH1/2-mut) identified among cases not assigned to the established subtypes. The CDX2-high subtype (3.4%) was characterized by high expression of CDX2 and recurrent gain of chromosome 1q. The IDH1/2-mut subtype (1.9%) was defined by IDH1 R132C or IDH2 R140Q mutations with specific transcriptional and high-methylation profiles. Both subtypes showed poor prognosis and were considered inferior prognostic factors independent of clinical parameters. Comparison with a previously reported pediatric B-ALL cohort (n = 1003) showed that the frequencies of these subtypes were significantly higher in AYA/adults than in children. We delineated the genetic and transcriptomic landscape of adult B-ALL and identified 2 novel subtypes that predict poor disease outcomes. Our findings highlight the age-dependent distribution of subtypes, which partially accounts for the prognostic differences between adult and pediatric B-ALL.

High IL2RA/CD25 expression is a prognostic stem cell biomarker for pediatric acute myeloid leukemia without a core-binding factor.

CD25 is an aberrant marker expressed on the leukemic stem cell (LSC) surface and an immunotherapy target in acute myeloid leukemia (AML). However, the clinical prevalence and significance of CD25 expression in pediatric AML are unknown. High IL2RA/CD25 expression in pediatric AML showed a stem cell-like phenotype, and elevated CD25 expression was associated with lower overall survival (p < .001) and event-free survival (p < .001) in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 study. This finding was reproduced in AML without a core-binding factor in the Children's Oncology Group study cohort. High CD25 expression has prognostic significance in pediatric AML.

Gemtuzumab Ozogamicin Followed by Unrelated Cord Blood Transplantation With Reduced-intensity Conditioning for a Child With Refractory Acute Promyelocytic Leukemia.

There is no established treatment for patients with acute promyelocytic leukemia (APL) refractory to targeted therapies with all-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO). We report here a case of an 8-month-old girl with APL who failed standard ATRA-combined chemotherapy. Although molecular remission was achieved after introducing ATRA/ATO combination therapy, molecular relapse occurred during the ATO consolidation courses. Subsequent molecular remission was rapidly achieved after administering 2 doses of gemtuzumab ozogamicin. She was successfully treated with unrelated cord blood transplantation using reduced-intensity conditioning. Gemtuzumab ozogamicin might be a preferable choice for patients with APL refractory to standard therapy.

Uracil-auxotrophic marker recycling system for multiple gene disruption in Pseudozyma antarctica.

The basidiomycetous yeast Pseudozyma antarctica, which has multiple auxotrophic markers, was constructed, without inserting a foreign gene, as the host strain for the introduction of multiple useful genes. P. antarctica was more resistant to ultraviolet (UV) irradiation than the model yeast Saccharomyces cerevisiae, and a Paura3 mutant (C867T) was obtained after 3 min of UV exposure. A uracil-auxotrophic marker (URA3) recycling system developed in ascomycetous yeasts and fungi was applied to the P. antarctica Paura3 strain. The PaLYS12 and PaADE2 loci were disrupted via site-directed homologous recombination of PaURA3 (pop-in), followed by the removal of PaURA3 (pop-out). In the obtained double auxotrophic strain (Palys12Δ, Paura3), PaADE2 was further disrupted, and PaURA3 was removed to obtain the triple auxotrophic strain PGB800 (Paura3, Palys12Δ, Paade2Δ). The whole-genome sequence of the PGB800 strain did not contain foreign genes used for genetic manipulation and disrupted PaADE2 and PaLYS12, and removed PaURA3, as planned.

Cutinase-like biodegradable plastic-degrading enzymes from phylloplane yeasts have cutinase activity.

Phylloplane yeast genera Pseudozyma and Cryptococcus secrete biodegradable plastic (BP)-degrading enzymes, termed cutinase-like enzymes (CLEs). Although CLEs contain highly conserved catalytic sites, the whole protein exhibits ≤30% amino acid sequence homology with cutinase. In this study, we analyzed whether CLEs exhibit cutinase activity. Seventeen Cryptococcus magnus strains, which degrade BP at 15 °C, were isolated from leaves and identified the DNA sequence of the CLE in one of the strains. Cutin was prepared from tomato leaves and treated with CLEs from 3 Cryptococcus species (C. magnus, Cryptococcus flavus, and Cryptococcus laurentii) and Pseudozyma antarctia (PaE). A typical cutin monomer, 10,16-dihydroxyhexadecanoic acid, was detected in extracts of the reaction solution via gas chromatography-mass spectrometry, showing that cutin was indeed degraded by CLEs. In addition to the aforementioned monomer, separation analysis via thin-layer chromatography detected high-molecular-weight products resulting from the breakdown of cutin by PaE, indicating that PaE acts as an endo-type enzyme.

Luseogliflozin, a SGLT2 Inhibitor, Does Not Affect Glucose Uptake Kinetics in Renal Proximal Tubules of Live Mice.

Proximal tubules (PTs) take up most of the glucose in the glomerular filtrate and return it to peritubular capillary blood. Sodium-glucose cotransporter 2 (SGLT2) at the apical membrane takes up glucose into the cell. Glucose then flows across the cells and is transported to the interstitium via glucose transporter 2 (GLUT2) at the basolateral membrane. However, glucose transport under SGLT2 inhibition remains poorly understood. In this study, we evaluated the dynamics of a fluorescent glucose analog, 2-NBDG, in the PTs of live mice treated with or without the SGLT2 inhibitor, luseogliflozin. We employed real-time multiphoton microscopy, in which insulin enhanced 2-NBDG uptake in skeletal muscle. Influx and efflux of 2-NBDG in PT cells were compared under hypo-, normo-, and hyperglycemic conditions. Luseogliflozin did not exert significant effects on glucose influx parameters under any level of blood glucose. Our results suggest that blood glucose level per se does not alter glucose influx or efflux kinetics in PTs. In conclusion, neither SGLT2 inhibition nor blood glucose level affect glucose uptake kinetics in PTs. The former was because of glucose influx through basolateral GLUT2, which is an established bidirectional transporter.

Fungal community dynamics during degradation of poly(butylene succinate-co-adipate) film in two cultivated soils in Japan.

Fungi play an important role in the degradation of biodegradable plastics (BPs) in soil. However, little is known about their dynamics in the soil during the degradation of BPs. We studied the community dynamics of BP-degrading fungi during poly(butylene succinate-co-adipate) (PBSA) film degradation in two different types of soils using culture-dependent and culture-independent methods. The Fluvisol and the Andosol soils degrade embedded PBSA films at high and low speeds, respectively. The number of PBSA emulsion-degrading fungi that increased in the Fluvisol soil was higher than that in the Andosol soil after embedding with PBSA films. We succeeded in detecting internal transcribed spacer 1 (ITS1) regions those matched that of the fungi by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) in both soils. Our results suggest that fungal community analyses using PCR-DGGE in combination with BP degraders isolation techniques enables the monitoring of BP films-degrading fungi.

Genetic abnormalities in myelodysplasia and secondary acute myeloid leukemia: impact on outcome of stem cell transplantation.

Genetic alterations, including mutations and copy-number alterations, are central to the pathogenesis of myelodysplastic syndromes and related diseases (myelodysplasia), but their roles in allogeneic stem cell transplantation have not fully been studied in a large cohort of patients. We enrolled 797 patients who had been diagnosed with myelodysplasia at initial presentation and received transplantation via the Japan Marrow Donor Program. Targeted-capture sequencing was performed to identify mutations in 69 genes, together with copy-number alterations, whose effects on transplantation outcomes were investigated. We identified 1776 mutations and 927 abnormal copy segments among 617 patients (77.4%). In multivariate modeling using Cox proportional-hazards regression, genetic factors explained 30% of the total hazards for overall survival; clinical characteristics accounted for 70% of risk. TP53 and RAS-pathway mutations, together with complex karyotype (CK) as detected by conventional cytogenetics and/or sequencing-based analysis, negatively affected posttransplant survival independently of clinical factors. Regardless of disease subtype, TP53-mutated patients with CK were characterized by unique genetic features and associated with an extremely poor survival with frequent early relapse, whereas outcomes were substantially better in TP53-mutated patients without CK. By contrast, the effects of RAS-pathway mutations depended on disease subtype and were confined to myelodysplastic/myeloproliferative neoplasms (MDS/MPNs). Our results suggest that TP53 and RAS-pathway mutations predicted a dismal prognosis, when associated with CK and MDS/MPNs, respectively. However, for patients with mutated TP53 or CK alone, long-term survival could be obtained with transplantation. Clinical sequencing provides vital information for accurate prognostication in transplantation.