RUNX1::ETO and CBFß::MYH11 converge on aberrant activation of BCAT1 to confer a therapeutic vulnerability in core-binding factor-acute myeloid leukaemia.

Effectively targeting transcription factors in therapeutic interventions remains challenging, especially in core-binding factor-acute myeloid leukaemia (CBF-AML) characterized by RUNX1::ETO and CBFß::MYH11 fusions. However, recent studies have drawn attention towards aberrant amino acid metabolisms as actionable therapeutic targets. Here, by integrating the expression profile and genetic makeup in AML cohort, we found higher BCAT1 expression in CBF-AML patients compared with other subtypes. Metabolic profiling revealed that high BCAT1 expression led to reprogrammed branch amino acid metabolism in CBF-AML and was associated with sphingolipid pathway relating to the fitness of leukaemia cells, supported by transcriptomic profiling. Mechanistically, we demonstrated in cell lines and primary patient samples that BCAT1 was directly activated by RUNX1::ETO and CBFß::MYH11 fusion proteins similarly in a RUNX1-dependent manner through rewiring chromatin conformation at the BCAT1 gene locus. Furthermore, BCAT1 inhibition resulted in blunted cell cycle, enhanced apoptosis and myeloid differentiation of CBF-AML cells in vitro, and alleviated leukaemia burden and prolonged survival in vivo. Importantly, pharmacological inhibition of BCAT1 using the specific inhibitor Gabapentin demonstrated therapeutic effects, as evidenced by delayed leukaemia progression and improved survival in vivo. In conclusion, our study uncovers BCAT1 as a genetic vulnerability and a promising targeted therapeutic opportunity for CBF-AML.

Recurrent noncoding somatic and germline WT1 variants converge to disrupt MYB binding in acute promyelocytic leukemia.

Genetic alternations can occur at noncoding regions, but how they contribute to cancer pathogenesis is poorly understood. Here, we established a mutational landscape of cis-regulatory regions (CREs) in acute promyelocytic leukemia (APL) based on whole-genome sequencing analysis of paired tumor and germline samples from 24 patients and epigenetic profiling of 16 patients. Mutations occurring in CREs occur preferentially in active enhancers bound by the complex of master transcription factors in APL. Among significantly enriched mutated CREs, we found a recurrently mutated region located within the third intron of WT1, an essential regulator of normal and malignant hematopoiesis. Focusing on noncoding mutations within this WT1 intron, an analysis on 169 APL patients revealed that somatic mutations were clustered into a focal hotspot region, including one site identified as a germline polymorphism contributing to APL risk. Significantly decreased WT1 expression was observed in APL patients bearing somatic and/or germline noncoding WT1 variants. Furthermore, biallelic WT1 inactivation was recurrently found in APL patients with noncoding WT1 variants, which resulted in the complete loss of WT1. The high incidence of biallelic inactivation suggested the tumor suppressor activity of WT1 in APL. Mechanistically, noncoding WT1 variants disrupted MYB binding on chromatin and suppressed the enhancer activity and WT1 expression through destroying the chromatin looping formation. Our study highlights the important role of noncoding variants in the leukemogenesis of APL.

IRF4-BLOC1S5: the first rearrangement gene identified in TEMPI syndrome.

Not available.

Combination of eriocalyxin B and homoharringtonine exerts synergistic anti-tumor effects against t(8;21) AML.

Understanding the molecular pathogenesis of acute myeloid leukemia (AML) with well-defined genomic abnormalities has facilitated the development of targeted therapeutics. Patients with t(8;21) AML frequently harbor a fusion gene RUNX1-RUNX1T1 and KIT mutations as "secondary hit", making the disease one of the ideal models for exploring targeted treatment options in AML. In this study we investigated the combination therapy of agents targeting RUNX1-RUNX1T1 and KIT in the treatment of t(8;21) AML with KIT mutations. We showed that the combination of eriocalyxin B (EriB) and homoharringtonine (HHT) exerted synergistic therapeutic effects by dual inhibition of RUNX1-RUNX1T1 and KIT proteins in Kasumi-1 and SKNO-1 cells in vitro. In Kasumi-1 cells, the combination of EriB and HHT could perturb the RUNX1-RUNX1T1-responsible transcriptional network by destabilizing RUNX1-RUNX1T1 transcription factor complex (AETFC), forcing RUNX1-RUNX1T1 leaving from the chromatin, triggering cell cycle arrest and apoptosis. Meanwhile, EriB combined with HHT activated JNK signaling, resulting in the eventual degradation of RUNX1-RUNX1T1 by caspase-3. In addition, HHT and EriB inhibited NF-κB pathway through blocking p65 nuclear translocation in two different manners, to synergistically interfere with the transcription of KIT. In mice co-expressing RUNX1-RUNX1T1 and KITN822K, co-administration of EriB and HHT significantly prolonged survival of the mice by targeting CD34+CD38- leukemic cells. The synergistic effects of the two drugs were also observed in bone marrow mononuclear cells (BMMCs) of t(8;21) AML patients. Collectively, this study reveals the synergistic mechanism of the combination regimen of EriB and HHT in t(8;21) AML, providing new insight into optimizing targeted treatment of AML.

Arsenic trioxide replacing or reducing chemotherapy in consolidation therapy for acute promyelocytic leukemia (APL2012 trial).

As all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are widely accepted in treating acute promyelocytic leukemia (APL), deescalating toxicity becomes a research hotspot. Here, we evaluated whether chemotherapy could be replaced or reduced by ATO in APL patients at different risks. After achieving complete remission with ATRA-ATO-based induction therapy, patients were randomized (1:1) into ATO and non-ATO groups for consolidation: ATRA-ATO versus ATRA-anthracycline for low-/intermediate-risk patients, or ATRA-ATO-anthracycline versus ATRA-anthracycline-cytarabine for high-risk patients. The primary end point was to assess disease-free survival (DFS) at 3 y by a noninferiority margin of -5%; 855 patients were enrolled with a median follow-up of 54.9 mo, and 658 of 755 patients could be evaluated at 3 y. In the ATO group, 96.1% (319/332) achieved 3-y DFS, compared to 92.6% (302/326) in the non-ATO group. The difference was 3.45% (95% CI -0.07 to 6.97), confirming noninferiority (P < 0.001). Using the Kaplan-Meier method, the estimated 7-y DFS was 95.7% (95% CI 93.6 to 97.9) in ATO and 92.6% (95% CI 89.8 to 95.4) in non-ATO groups (P = 0.066). Concerning secondary end points, the 7-y cumulative incidence of relapse (CIR) was significantly lower in ATO (2.2% [95% CI 1.1 to 4.2]) than in non-ATO group (6.1% [95% CI 3.9 to 9.5], P = 0.011). In addition, grade 3 to 4 hematological toxicities were significantly reduced in the ATO group during consolidation. Hence, ATRA-ATO in both chemotherapy-replacing and -reducing settings in consolidation is not inferior to ATRA-chemotherapy (https://www.clinicaltrials.gov/, NCT01987297).

A PML/RARα direct target atlas redefines transcriptional deregulation in acute promyelocytic leukemia.

Transcriptional deregulation initiated by oncogenic fusion proteins plays a vital role in leukemia. The prevailing view is that the oncogenic fusion protein promyelocytic leukemia/retinoic acid receptor-α (PML/RARα), generated by the chromosome translocation t(15;17), functions as a transcriptional repressor in acute promyelocytic leukemia (APL). Here, we provide rich evidence of how PML/RARα drives oncogenesis through both repressive and activating functions, particularly the importance of the newly identified activation role for the leukemogenesis of APL. The activating function of PML/RARα is achieved by recruiting both abundant P300 and HDAC1 and by the formation of super-enhancers. All-trans retinoic acid and arsenic trioxide, 2 widely used drugs in APL therapy, exert synergistic effects on controlling super-enhancer-associated PML/RARα-regulated targets in APL cells. We use a series of in vitro and in vivo experiments to demonstrate that PML/RARα-activated target gene GFI1 is necessary for the maintenance of APL cells and that PML/RARα, likely oligomerized, transactivates GFI1 through chromatin conformation at the super-enhancer region. Finally, we profile GFI1 targets and reveal the interplay between GFI1 and PML/RARα on chromatin in coregulating target genes. Our study provides genomic insight into the dual role of fusion transcription factors in transcriptional deregulation to drive leukemia development, highlighting the importance of globally dissecting regulatory circuits.

Transcriptomic profiling of pemphigus lesion infiltrating mononuclear cells reveals a distinct local immune microenvironment and novel lncRNA regulators.

Pemphigus is an autoimmune skin disease. Ectopic lymphoid-like structures (ELSs) were found to be commonly present in the pemphigus lesions, presumably supporting in situ desmoglein (Dsg)-specific antibody production. Yet functional phenotypes and the regulators of Lymphoid aggregates in pemphigus lesions remain largely unknown. Herein, we used microarray technology to profile the gene expression in skin lesion infiltrating mononuclear cells (SIMC) from pemphigus patients. On top of that, we compared SIMC dataset to peripheral blood mononuclear cells (PBMC) dataset to characterize the unique role of SIMC. Functional enrichment results showed that mononuclear cells in skin lesions and peripheral blood both had over-represented IL-17 signaling pathways while neither was characterized by an activation of type I Interferon signaling pathways. Cell-type identification with relative subsets of known RNA transcripts (CIBERSORT) results showed that naïve natural killer cells (NK cells) were significantly more abundant in pemphigus lesions, and their relative abundance positively correlated with B cells abundance. Meanwhile, plasma cells population highly correlated with type 1 macrophages (M1) abundance. In addition, we also identified a lncRNA LINC01588 which might epigenetically regulate T helper 17 cells (Th17)/regulatory T cells (Treg) balance via the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Here, we provide the first transcriptomic characterization of lesion infiltrating immune cells which illustrates a distinct interplay network between adaptive and innate immune cells. It helps discover new regulators of local immune response, which potentially will provide a novel path forward to further uncover pemphigus pathological mechanisms and develop targeted therapy.

Interplay between hypertriglyceridemia and acute promyelocytic leukemia mediated by the cooperation of peroxisome proliferator-activated receptor-α with the PML/RAR α fusion protein on super-enhancers.

Patients with newly diagnosed acute promyelocytic leukemia (APL) are often obese or overweight, accompanied by metabolic disorders, such as dyslipidemia. However, the link between dyslipidemia and leukemia is obscure. Here, we conducted a retrospective study containing 1,412 cases (319 newly diagnosed APL patients, 393 newly diagnosed non-APL acute myeloid leukemia patients, and 700 non-tumor controls) and found that APL patients had higher triglyceride levels than non- APL and control groups. Using clinical data, we revealed that hypertriglyceridemia served as a risk factor for early death in APL patients, and there was a positive correlation between triglyceride levels and leukocyte counts. RNA sequencing analysis of APL patients having high or normal triglyceride levels highlighted the contribution of peroxisome proliferatoractivated receptor-α (PPARα), a crucial regulator of cell metabolism and a transcription factor involved in cancer development. The genome-wide chromatin occupancy of PPARα revealed that PPARα co-existed with PML/RARα within the super-enhancer regions to promote cell proliferation. PPARα knockdown affected the expression of target genes responsible for APL proliferation, including FLT3, and functionally inhibited the proliferation of APL cells. Moreover, in vivo results in mice having high fat diet-induced high triglyceride levels supported the connection between high triglyceride levels and the leukemic burden, as well as the involvement of PPARα-mediated-FLT3 activation in the proliferation of APL cells. Our findings shed light on the association between APL proliferation and high triglyceride levels and provide a genetic link to PPARα-mediated hyperlipidemia in APL.

Homoharringtonine deregulates MYC transcriptional expression by directly binding NF-κB repressing factor.

Homoharringtonine (HHT), a known protein synthesis inhibitor, has an anti-myeloid leukemia effect and potentiates the therapeutic efficacy of anthracycline/cytarabine induction regimens for acute myelogenous leukemia (AML) with favorable and intermediate prognoses, especially in the t(8;21) subtype. Here we provide evidence showing that HHT inhibits the activity of leukemia-initiating cells (Lin-/Sca-1-/c-kit+; LICs) in a t(8;21) murine leukemia model and exerts a down-regulating effect on MYC pathway genes in human t(8;21) leukemia cells (Kasumi-1). We discovered that NF-κB repressing factor (NKRF) is bound directly by HHT via the second double-strand RNA-binding motif (DSRM2) domain, which is the nuclear localization signal of NKRF. A series of deletion and mutagenesis experiments mapped HHT direct binding sites to K479 and C480 amino acids in the DSRM2 domain. HHT treatment shifts NKRF from the nucleus (including nucleoli) to the cytoplasm by occupying the DSRM2 domain, strengthens the p65-NKRF interaction, and interferes with p65-p50 complex formation, thereby attenuating the transactivation activity of p65 on the MYC gene. Moreover, HHT significantly decreases the expression of KIT, a frequently mutated and/or highly expressed gene in t(8;21) AML, in concert with MYC down-regulation. Our work thus identifies a mechanism of action of HHT that is different from, but acts in concert with, the known mode of action of this compound. These results justify further clinical testing of HHT in AML.

Genetically modified pigs are protected from classical swine fever virus.

Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is one of the most detrimental diseases, and leads to significant economic losses in the swine industry. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread. Here, antiviral small hairpin RNAs (shRNAs) were selected and then inserted at the porcine Rosa26 (pRosa26) locus via a CRISPR/Cas9-mediated knock-in strategy. Finally, anti-CSFV transgenic (TG) pigs were produced by somatic nuclear transfer (SCNT). Notably, in vitro and in vivo viral challenge assays further demonstrated that these TG pigs could effectively limit the replication of CSFV and reduce CSFV-associated clinical signs and mortality, and disease resistance could be stably transmitted to the F1-generation. Altogether, our work demonstrated that RNA interference (RNAi) technology combining CRISPR/Cas9 technology offered the possibility to produce TG animal with improved resistance to viral infection. The use of these TG pigs can reduce CSF-related economic losses and this antiviral strategy may be useful for future antiviral research.

Three-Field versus Two-Field Lymphadenectomy for Esophageal Squamous Cell Carcinoma: A Meta-analysis.

BACKGROUND: Most surgeons now accept lymphadenectomy as an essential feature of the operative treatment of esophageal squamous cell carcinoma. Three-field and two-field lymphadenectomy are two of the most popular excision scopes among surgeons. Over recent years, researchers have performed a range of comparative studies regarding these techniques, although the conclusions remain inconsistent. METHOD: We systematically retrieved the records of PubMed, Embase, The Cochrane Library, and ClinicalTrials.gov until October 2019 and performed preliminary and full-text screening of the articles. We used the NOS scale to evaluate the quality of the enrolled studies, with only medium- and high-quality studies included. Review Manager 5.3 and Stata15 were used for the meta-analysis. RESULTS: A total of eight studies involving 1676 patients were included in the meta-analysis. The results showed that for esophageal squamous cell carcinoma using with two-field and three-field lymphadenectomy, although three-field lymphadenectomy led to the gaining of a higher number of lymph nodes, there were no significant differences between the two in terms of the number of positive lymph nodes and overall survival. Three-field lymphadenectomy also caused higher levels of intraoperative blood loss and higher morbidity of the anastomotic fistula. No significant differences in operation time, recurrent laryngeal nerve injury, pneumonia, chylothorax, anastomotic stenosis, ileus, cervical nodal recurrence and hospital mortality were observed. CONCLUSIONS: According to our meta-analysis, two-field lymphadenectomy is recommended as a first-choice surgical treatment for esophageal squamous cell carcinoma. However, since the results showed a risk of bias, they should be treated with caution.

Assembly and Regulation of CRL Ubiquitin Ligases.

Cullin-RING ubiquitin ligases (CRLs) determine the substrate specificity of ubiquitination reactions, and substrates are recruited to the cullin core through binding to their cognate substrate receptor modules. Because a family of substrate receptors compete for the same cullin core, the assembly and activity of CRLs are dynamically regulated to fulfill the needs of the cell to adapt to the changing pool of proteins demanding ubiquitination. Cullins are modified by NEDD8, a ubiquitin-like protein. This process, referred to as neddylation, promotes the E3 activity of CRLs by inducing conformational rearrangement in the Cullin-RING catalytic core. Cand1 is a cullin-associated protein whose binding is excluded by cullin neddylation. Although early biochemical studies suggested that Cand1 inhibits CRL activity, genetic studies revealed its positive role in ubiquitination. Emerging evidence from kinetic and quantitative proteomic studies demonstrated that Cand1 stimulates assembly of new Skp1-Cul1-F-box protein (SCF) complexes by exchanging the Skp1-F-box protein substrate receptor modules. Furthermore, aided by refined experimental design as well as computational simulation, an attractive model has been developed in which substrate, neddylation cycle and Cand1-mediated "adaptive exchange" collaborate to maintain the dynamics of the cellular SCF repertoire. Here, we review and discuss recent advances that have deepened our understanding of CRL regulation.

The Possible Role of Complete Loss of Myostatin in Limiting Excessive Proliferation of Muscle Cells (C2C12) via Activation of MicroRNAs.

Myostatin (MSTN) is a member of the TGF-ß superfamily that negatively regulates skeletal muscle growth and differentiation. However, the mechanism by which complete MSTN deletion limits excessive proliferation of muscle cells remains unclear. In this study, we knocked out MSTN in mouse myoblast lines using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system and sequenced the mRNA and miRNA transcriptomes. The results show that complete loss of MSTN upregulates seven miRNAs targeting an interaction network composed of 28 downregulated genes, including TGFB1, FOS and RB1. These genes are closely associated with tumorigenesis and cell proliferation. Our study suggests that complete loss of MSTN may limit excessive cell proliferation via activation of miRNAs. These data will contribute to the treatment of rhabdomyosarcoma (RMS).

PML-RARα interferes with erythropoiesis by repressing LMO2 in acute promyelocytic leukaemia.

The PML-RARα fusion gene, generated by the t(15;17) chromosome translocation, is regarded as the initiating factor of acute promyelocytic leukaemia (APL). In addition to the well-known effects on blocking myeloid differentiation at the promyelocytic stage, promyelocytic leukaemia-retinoic acid receptor α (PML-RARα) has also been reported to interfere with multiple differentiation processes, including erythroid differentiation. However, the detailed molecular mechanism by which PML-RARα impairs erythropoiesis has not yet been fully addressed. By chromatin immunoprecipitation-PCR assay, we found that PML-RARα bound to the distal promoter region of LMO2 (LIM-only protein 2), a critical erythroid-specific transcription factor. Luciferase reporter assays and qRT-PCR results demonstrated that PML-RARα down-regulated the expression of the LMO2 distal transcript through transrepressing its promoter activity. Analysis of gene expression profiling data from large cohorts of acute myeloid leukaemia (AML) patients confirmed that LMO2 expressed at a markedly lower level in APL patients in comparison to non-APL AML patients. Further flow cytometry analysis demonstrated that PML-RARα inhibited erythropoietin-induced erythroid differentiation by down-regulating LMO2 expression. Our findings reveal a previously unidentified mechanism, by which PML-RARα interferes with erythropoiesis through directly targeting and transrepressing LMO2 expression in the development of APL.

Genome-wide studies identify a novel interplay between AML1 and AML1/ETO in t(8;21) acute myeloid leukemia.

The AML1/ETO fusion protein is essential to the development of t(8;21) acute myeloid leukemia (AML) and is well recognized for its dominant-negative effect on the coexisting wild-type protein AML1. However, the genome-wide interplay between AML1/ETO and wild-type AML1 remains elusive in the leukemogenesis of t(8;21) AML. Through chromatin immunoprecipitation sequencing and computational analysis, followed by a series of experimental validations, we report here that wild-type AML1 is able to orchestrate the expression of AML1/ETO targets regardless of being activated or repressed; this is achieved via forming a complex with AML1/ETO and via recruiting the cofactor AP-1 on chromatin. On chromatin occupancy, AML1/ETO and wild-type AML1 largely overlap and preferentially bind to adjacent and distinct short and long AML1 motifs on the colocalized regions, respectively. On physical interaction, AML1/ETO can form a complex with wild-type AML1 on chromatin, and the runt homology domain of both proteins are responsible for their interactions. More importantly, the relative binding signals of AML1 and AML1/ETO on chromatin determine which genes are repressed or activated by AML1/ETO. Further analysis of coregulators indicates that AML1/ETO transactivates gene expression through recruiting AP-1 to the AML1/ETO-AML1 complex. These findings enrich our knowledge of understanding the significance of the interplay between the wild-type protein and the oncogenic fusion protein in the development of leukemia.

Arsenic trioxide at conventional dosage does not aggravate hemorrhage in the first-line treatment of adult acute promyelocytic leukemia.

OBJECTIVES: The arsenic trioxide (ATO) plus all-trans retinoic acid (ATRA) therapy has demonstrated a tremendous success in the first-line treatment of acute promyelocytic leukemia (APL). Actually, early death (ED) is currently thought as a major challenge in APL. ATO has been reported to inhibit platelet function in vitro, and whether it increases the ED rate by exacerbating the hemorrhagic symptoms remains to be investigated. METHODS: Effects of ATO on platelet aggregation and adhesion were evaluated in vitro and in thirty-two complete remission (CR) and four newly diagnosed APL patients. Furthermore, concentrations of plasma total arsenic were monitored in APL patients via ICP-MS. RESULTS: The inhibition of platelet function, either aggregation or adhesion, did occur in vitro when the concentration of ATO reached 2 µmol/L. However, in CR APL patients receiving ATO with normal platelet count, the platelets responded normally when being activated and so did those in the newly diagnosed patients with thrombocytopenia. Our data further showed that the conventional dosage of ATO reached a plasma concentration substantially below the required concentration to inhibit platelets. CONCLUSIONS: In the first-line treatment of APL, the use of ATO is safe and effective and does not compromise the hemostatic potential that may eventually increase ED rate.

CRISPR/Cas9-mediated knockout of myostatin in Chinese indigenous Erhualian pigs.

CRISPR/Cas9 has emerged as one of the most popular genome editing tools due to its simple design and high efficiency in multiple species. Myostatin (MSTN) negatively regulates skeletal muscle growth and mutations in myostatin cause double-muscled phenotype in various animals. Here, we generated myostatin mutation in Erhualian pigs using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. The protein level of myostatin precursor decreased dramatically in mutant cloned piglets. Unlike myostatin knockout Landrace, which often encountered health issues and died shortly after birth, Erhualian pigs harboring homozygous mutations were viable. Moreover, myostatin knockout Erhualian pigs exhibited partial double-muscled phenotype such as prominent muscular protrusion, wider back and hip compared with wild-type piglets. Genome editing in Chinese indigenous pig breeds thus holds great promise not only for improving growth performance, but also for protecting endangered genetic resources.

DNMT3A Arg882 mutation drives chronic myelomonocytic leukemia through disturbing gene expression/DNA methylation in hematopoietic cells.

The gene encoding DNA methyltransferase 3A (DNMT3A) is mutated in ∼20% of acute myeloid leukemia cases, with Arg882 (R882) as the hotspot. Here, we addressed the transformation ability of the DNMT3A-Arg882His (R882H) mutant by using a retroviral transduction and bone marrow transplantation (BMT) approach and found that the mutant gene can induce aberrant proliferation of hematopoietic stem/progenitor cells. At 12 mo post-BMT, all mice developed chronic myelomonocytic leukemia with thrombocytosis. RNA microarray analysis revealed abnormal expressions of some hematopoiesis-related genes, and the DNA methylation assay identified corresponding changes in methylation patterns in gene body regions. Moreover, DNMT3A-R882H increased the CDK1 protein level and enhanced cell-cycle activity, thereby contributing to leukemogenesis.