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BACKGROUND: Rare variants in epigenes (a.k.a. chromatin modifiers), a class of genes that control epigenetic regulation, are commonly identified in both pediatric neurodevelopmental syndromes and as somatic variants in cancer. However, little is known about the extent of the shared disruption of signaling pathways by the same epigene across different diseases. To address this, we study an epigene, Additional Sex Combs-like 1 (ASXL1), where truncating heterozygous variants cause Bohring-Opitz syndrome (BOS, OMIM #605039), a germline neurodevelopmental disorder, while somatic variants are driver events in acute myeloid leukemia (AML). No BOS patients have been reported to have AML. METHODS: This study explores common pathways dysregulated by ASXL1 variants in patients with BOS and AML. We analyzed whole blood transcriptomic and DNA methylation data from patients with BOS and AML with ASXL1-variant (AML-ASXL1) and examined differential exon usage and cell proportions. RESULTS: Our analyses identified common molecular signatures between BOS and AML-ASXL1 and highlighted key biomarkers, including VANGL2, GRIK5 and GREM2, that are dysregulated across samples with ASXL1 variants, regardless of disease type. Notably, our data revealed significant de-repression of posterior homeobox A (HOXA) genes and upregulation of Wnt-signaling and hematopoietic regulator HOXB4. While we discovered many shared epigenetic and transcriptomic features, we also identified differential splice isoforms in RUNX3 where the long isoform, p46, is preferentially expressed in BOS, while the shorter p44 isoform is expressed in AML-ASXL1. CONCLUSION: Our findings highlight the strong effects of ASXL1 variants that supersede cell-type and even disease states. This is the first direct comparison of transcriptomic and methylation profiles driven by pathogenic variants in a chromatin modifier gene in distinct diseases. Similar to RASopathies, in which pathogenic variants in many genes lead to overlapping phenotypes that can be treated by inhibiting a common pathway, our data identifies common pathways for ASXL1 variants that can be targeted for both disease states. Comparative approaches of high-penetrance genetic variants across cell types and disease states can identify targetable pathways to treat multiple diseases. Finally, our work highlights the connections of epigenes, such as ASXL1, to an underlying stem-cell state in both early development and in malignancy.

Abstract Background Rare variants in epigenes (a.k.a. chromatin modifiers), a class of genes that control epigenetic regulation, are commonly identified in both pediatric neurodevelopmental syndromes and as somatic variants in cancer. However, little is known about the extent of the shared disruption of signaling pathways by the same epigene across different diseases. To address this, we study an epigene, Additional Sex Combs-like 1 (ASXL1), where truncating heterozygous variants cause Bohring-Opitz syndrome (BOS, OMIM #605039), a germline neurodevelopmental disorder, while somatic variants are driver events in acute myeloid leukemia (AML). No BOS patients have been reported to have AML. Methods This study explores common pathways dysregulated by ASXL1 variants in patients with BOS and AML. We analyzed whole blood transcriptomic and DNA methylation data from patients with BOS and AML with ASXL1-variant (AML-ASXL1) and examined differential exon usage and cell proportions. Results Our analyses identified common molecular signatures between BOS and AML-ASXL1 and highlighted key biomarkers, including VANGL2, GRIK5 and GREM2, that are dysregulated across samples with ASXL1 variants, regardless of disease type. Notably, our data revealed significant de-repression of posterior homeobox A (HOXA) genes and upregulation of Wnt-signaling and hematopoietic regulator HOXB4. While we discovered many shared epigenetic and transcriptomic features, we also identified differential splice isoforms in RUNX3 where the long isoform, p46, is preferentially expressed in BOS, while the shorter p44 isoform is expressed in AML-ASXL1. Conclusion Our findings highlight the strong effects of ASXL1 variants that supersede cell-type and even disease states. This is the first direct comparison of transcriptomic and methylation profiles driven by pathogenic variants in a chromatin modifier gene in distinct diseases. Similar to RASopathies, in which pathogenic variants in many genes lead to overlapping phenotypes that can be treated by inhibiting a common pathway, our data identifies common pathways for ASXL1 variants that can be targeted for both disease states. Comparative approaches of high-penetrance genetic variants across cell types and disease states can identify targetable pathways to treat multiple diseases. Finally, our work highlights the connections of epigenes, such as ASXL1, to an underlying stem-cell state in both early development and in malignancy.

Additional sex combs-like 1 (ASXL1) is an epigenetic modulator frequently mutated in myeloid malignancies, generally associated with poor prognosis. Current models for ASXL1-mutated diseases are mainly based on the complete deletion of Asxl1 or overexpression of C-terminal truncations in mice models. However, these models cannot fully recapitulate the pathogenesis of myeloid malignancies. Patient-derived induced pluripotent stem cells (iPSCs) provide valuable disease models that allow us to understand disease-related molecular pathways and develop novel targeted therapies. Here, we generated iPSCs from a patient with myeloproliferative neoplasm carrying a heterozygous ASXL1 mutation. The iPSCs we generated exhibited the morphology of pluripotent cells, highly expressed pluripotent markers, excellent differentiation potency in vivo, and normal karyotype. Subsequently, iPSCs with or without ASXL1 mutation were induced to differentiate into hematopoietic stem/progenitor cells, and we found that ASXL1 mutation led to myeloid-biased output and impaired erythroid differentiation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that terms related to embryonic development, myeloid differentiation, and immune- and neural-related processes were most enriched in the differentially expressed genes. Western blot demonstrated that the global level of H2AK119ub was significantly decreased when mutant ASXL1 was present. Chromatin Immunoprecipitation Sequencing showed that most genes associated with stem cell maintenance were upregulated, whereas occupancies of H2AK119ub around these genes were significantly decreased. Thus, the iPSC model carrying ASXL1 mutation could serve as a potential tool to study the pathogenesis of myeloid malignancies and to screen targeted therapy for patients. [ABSTRACT FROM AUTHOR]

Simple Summary: The expansion of mutant cells that outgrow and displace normal blood cells is called clonal hematopoiesis (CH). CH starts with the acquisition of mutations in blood stem cells that change their normal behavior and improve their fitness. Low numbers of mutant blood cells are present in many middle-aged and elderly people without noticeable effects. However, CH can progress to leukemia and contribute to diseases in other tissues, including the heart, liver, and pancreas. As these diseases progress and affect health when the number of mutant blood cells increases, preventing and/or reducing the expansion of mutant cells might delay this process. Identifying the factors driving mutant blood cell expansion is thus critical and has garnered heightened interest in the subject. Here, we review and discuss recent progress in the field that suggests that mutant blood stem cells are more resilient than normal cells and thrive in inflammatory conditions. Clonal hematopoiesis (CH), the relative expansion of mutant clones, is derived from hematopoietic stem cells (HSCs) with acquired somatic or cytogenetic alterations that improve cellular fitness. Individuals with CH have a higher risk for hematological and non-hematological diseases, such as cardiovascular disease, and have an overall higher mortality rate. Originally thought to be restricted to a small fraction of elderly people, recent advances in single-cell sequencing and bioinformatics have revealed that CH with multiple expanded mutant clones is universal in the elderly population. Just a few years ago, phylogenetic reconstruction across the human lifespan and novel sensitive sequencing techniques showed that CH can start earlier in life, decades before it was thought possible. These studies also suggest that environmental factors acting through aberrant inflammation might be a common theme promoting clonal expansion and disease progression. However, numerous aspects of this phenomenon remain to be elucidated and the precise mechanisms, context-specific drivers, and pathways of clonal expansion remain to be established. Here, we review our current understanding of the cellular mechanisms driving CH and specifically focus on how pro-inflammatory factors affect normal and mutant HSC fates to promote clonal selection. [ABSTRACT FROM AUTHOR]

Renal cell carcinoma with fibromyomatous stroma (RCC FMS), defined as an "emerging entity" in the 2016 WHO classification and recommended to be a novel entity by GUPS, is represented by tumor cells with clear to mildly eosinophilic cytoplasm displaying elongated and branching tubules and papillae. A fibromyomatous stroma could be observed in these tumors. These tumors are immunopositive for CK7 and featured by ELOC and/or TSC/mTOR gene mutations. In the 2022 WHO classification, ELOC mutated RCC is classified as a molecularly defined RCCs as an individual renal entity. However, there are limited descriptions of TSC/mTOR alterations in RCC FMS. Herein, we reported a case of 28-year-old woman with RCC FMS with intact ELOC and TSC/mTOR genes but ASXL1 mutation. The tumor cells were positive for mTOR expression. This case may indicate that altered mTOR expression, but not limited to mutated TSC/mTOR gene, that participates in the pathogenesis of RCC FMS. [ABSTRACT FROM AUTHOR]

Background: Although genetic mutations in additional sex-combs-like 1 (ASXL1) are prevalent in acute myeloid leukemia (AML), their exact impact on the AML prognosis remains uncertain. Hence, the present article was carried out to explore the prognostic importance of ASXL1 mutations in AML. Methods: We thoroughly searched electronic scientific databases to find eligible papers. Twenty-seven studies with an overall number of 8,953 participants were selected for the current systematic review. The hazard ratio (HR) and 95% confidence interval (CI) for overall survival (OS), event-free survival (EFS), and relapse-free survival (RFS) were extracted from all studies with multivariate or univariate analysis. Pooled HRs and pvalues were also calculated as a part of our work. Results: The pooled HR for OS in multivariable analysis indicated that ASXL1 significantly diminished survival in AML patients (pooled HR: 1.67; 95% CI: 1.342-2.091). Conclusions: ASXL1 mutations may confer a poor prognosis in AML. Hence, they may be regarded as potential prognostic factors. However, more detailed studies with different ASXL1 mutations are suggested to shed light on this issue. [ABSTRACT FROM AUTHOR]

Background: Although genetic mutations in additional sex-combs-like 1 (ASXL1) are prevalent in acute myeloid leukemia (AML), their exact impact on the AML prognosis remains uncertain. Hence, the present article was carried out to explore the prognostic importance of ASXL1 mutations in AML. Methods: We thoroughly searched electronic scientific databases to find eligible papers. Twenty-seven studies with an overall number of 8,953 participants were selected for the current systematic review. The hazard ratio (HR) and 95% confidence interval (CI) for overall survival (OS), event-free survival (EFS), and relapse-free survival (RFS) were extracted from all studies with multivariate or univariate analysis. Pooled HRs and p-values were also calculated as a part of our work. Results: The pooled HR for OS in multivariable analysis indicated that ASXL1 significantly diminished survival in AML patients (pooled HR: 1.67; 95% CI: 1.342-2.091). Conclusions: ASXL1 mutations may confer a poor prognosis in AML. Hence, they may be regarded as potential prognostic factors. However, more detailed studies with different ASXL1 mutations are suggested to shed light on this issue.

Objective To investigate the efficacy and influencing factors of allo-HSCT in the treatment of MDS patients with ASXL1+. Methods The second-generation sequencing technique was used to detect 22 gene mutations in 247 newly diagnosed MDS patients in our hospital. The patients were divided into chemotherapy group and transplant group according to treatment style. The differences of OS and PFS between the two groups were compared, and the influencing factors of prognosis of transplant patients were analyzed. Results ASXL1+ was detected in 75 patients (30.36%), with a median mutation ratio of 42.93 (18.10,58.39)%, 10 received supportive treatment, 43 received demethylation therapy or demethylation combined with pre-excitation therapy, and 22 received allo-HSCT. 2-year PFS rate and OS rate of transplantation group were significantly higher than that of chemotherapy group (P < 0.05). The 2-year OS rate in the low ASXL1 mutation load group (VAF ≤ 42.93%) was significantly higher than that in the high ASXL1 mutation load group (VAF > 42.93%) (P < 0.05). In the context of allo-HSCT in patients with ASXL1+, 2-year OS and PFS rates were significantly reduced in patients with RUNX1+ or ASXL1+ (P < 0.05); Multivariate analysis showed that high mutation load of ASXL1 or U2AF1+ were independent risk factors for OS in transplant patient (P < 0.05). U2AF1+ were the risk factors for PFS (P < 0.05). Conclusion allo-HSCT significantly improved the prognosis of patients with ASXL1+ MDS. High ASXL1 mutation load or U2AF1+ were independent risk factors affecting the outcome of allo-HSCT. [ABSTRACT FROM AUTHOR]

The blast phase of BCR::ABL1‐negative myeloproliferative neoplasm (MPN‐BP) represents the final stage of the disease, which is complicated by complex genomic alterations. These alterations result from sequence changes in genetic material (DNA, RNA) and can lead to either a gain or loss of function of encoded proteins, such as adaptor proteins, enzymes, components of spliceosomes, cell cycle checkpoints regulators, transcription factors, or proteins in cell signaling pathways. Interference at various levels, including transcription, translation, and post‐translational modification (such as methylation, dephosphorylation, or acetylation), can contribute to these alterations. Mutated genes such as ASXL1, EZH2, IDH1, IDH2, TET2, SRSF2, U2AF1, TP53, NRAS, KRAS, PTPN11, SH2B3/LNK, and RUNX1 play active roles at different stages of genetic material expression, modification, and protein function manipulation in MPNs. These mutations are also correlated with, and can contribute to, the progression of MPN‐BP. In this review, we summarize their common mutational profiles, functions, and associations with progression of MPN‐BP. [ABSTRACT FROM AUTHOR]

Essential thrombocythemia (ET) with double driver mutations is a rare disease. ET patients with both MPL and Type 1 CALR mutations have been reported. Here, we report the first case of an ET patient with both MPL S204P and Type 2 CALR mutations and a summary of our literature review findings. In the patient whose case is reported here, the disease progressed to an accelerated phase 3.5 months after diagnosis. CALR mutation disappeared and new mutations emerged as the disease progressed, such as ASXL1, CBL, ETV6, and PTPN11 mutations. This case highlights that screening for additional mutations using NGS should be considered in patients with ET to assess the prognosis, especially as the disease progresses. [ABSTRACT FROM AUTHOR]

Additional sex combs-like 1 (ASXL1) interacts with BRCA1-associated protein 1 (BAP1) deubiquitinase to oppose the polycomb repressive complex 1 (PRC1)-mediated histone H2A ubiquitylation. Germline BAP1 mutations are found in a spectrum of human malignancies, while ASXL1 mutations recurrently occur in myeloid neoplasm and are associated with poor prognosis. Nearly all ASXL1 mutations are heterozygous frameshift or nonsense mutations in the middle or to a less extent the C-terminal region, resulting in the production of C-terminally truncated mutant ASXL1 proteins. How ASXL1 regulates specific target genes and how the C-terminal truncation of ASXL1 promotes leukemogenesis are unclear. Here, we report that ASXL1 interacts with forkhead transcription factors FOXK1 and FOXK2 to regulate a subset of FOXK1/K2 target genes. We show that the C-terminally truncated mutant ASXL1 proteins are expressed at much higher levels than the wild-type protein in ASXL1 heterozygous leukemia cells, and lose the ability to interact with FOXK1/K2. Specific deletion of the mutant allele eliminates the expression of C-terminally truncated ASXL1 and increases the association of wild-type ASXL1 with BAP1, thereby restoring the expression of BAP1-ASXL1-FOXK1/K2 target genes, particularly those involved in glucose metabolism, oxygen sensing, and JAK-STAT3 signaling pathways. In addition to FOXK1/K2, we also identify other DNA-binding transcription regulators including transcription factors (TFs) which interact with wild-type ASXL1, but not C-terminally truncated mutant. Our results suggest that ASXL1 mutations result in neomorphic alleles that contribute to leukemogenesis at least in part through dominantly inhibiting the wild-type ASXL1 from interacting with BAP1 and thereby impairing the function of ASXL1-BAP1-TF in regulating target genes and leukemia cell growth.

Background: Acute myeloid leukemia (AML) with t(8;21)(q22;q22.1); RUNX1::RUNX1T1 has a relatively favorable prognosis with a high complete remission rate and long disease-free survival. Methods and Results: Here we describe a patient who had AML with t(8;21)(q22;q22.1); RUNX1::RUNX1T1. Cooperating mutations including KRAS and ASXL1, and with other abnormal karyotype del(17) and with a myelomonocytic differentiation. Conclusions: The patient relapsed despite achieving a morphologic complete remission (CR). [ABSTRACT FROM AUTHOR]

Background: Chromatin Modifying Disorders (CMD) have emerged as one of the most rapidly expanding genetic disorders associated with autism spectrum disorders (ASD). Motor impairments are also prevalent in CMD and may play a role in the neurodevelopmental phenotype. Evidence indicates that neurodevelopmental outcomes in CMD may be treatable postnatally; thus deep phenotyping of these conditions can improve clinical screening while improving the development of treatment targets for pharmacology and for clinical trials. Here, we present developmental phenotyping data on individuals with Bohring-Optiz Syndrome (BOS -- ASXL1) and Bainbridge-Ropers Syndrome (BRS -- ASXL3) related disorders, two CMDs highly penetrant for motor and developmental delays. Objectives: To phenotype the motor and neurodevelopmental profile of individuals with ASXL1 and ASXL3 related disorders (BOS and BRS). To provide a preliminary report on the association of motor impairments and ASD. Methods: Neurodevelopmental and motor phenotyping was conducted on eight individuals with pathogenic ASXL1 variants and seven individuals with pathogenic ASXL3 variants, including medical and developmental background intake, movement and development questionnaires, neurological examination, and quantitative gait analysis. Results: Average age of first developmental concerns was 4 months for individuals with BOS and 9 months in BRS. 100% of individuals who underwent the development questionnaire met a diagnosis of developmental coordination disorder. 71% of children with BOS and 0% of children with BRS noted movement difficulty greatly affected classroom learning. Participants with BRS and presumed diagnoses of ASD were reported to have more severe motor impairments in recreational activities compared to those without ASD. This was not the case for the individuals with BOS. Conclusion: Motor impairments are prevalent and pervasive across the ASXL disorders with and without ASD, and these impairments negatively impact engagement in school-based activities. Unique neurodevelopmental and motor findings in our data include a mixed presentation of hypo and hypertonia in individuals with BOS across a lifespan. Individuals with BRS exhibited hypotonia and greater variability in motor skills. This deep phenotyping can aid in appropriate clinical diagnosis, referral to interventions, and serve as meaningful treatment targets in clinical trials. [ABSTRACT FROM AUTHOR]

Although the majority of patients with chronic myeloid leukemia (CML) enjoy an excellent prognosis tyrosine kinase inhibitor (TKI) therapy, resistance remains a significant clinical problem. Resistance can arise from mutations in the kinase domain of ABL preventing drug binding, or due to ill-defined kinase-independent mechanisms. In this case report, we describe the case of a 27-year-old woman with a long-standing history of chronic phase (CP) CML who developed kinase-independent resistance with mutations in ASXL1 and RUNX1. As a consequence of uncontrolled disease, she progressed to a chronic myelomonocytic leukemia-like (CMML) accelerated phase (AP) disease with the acquisition of a mutation in IDH1. This disease progression was associated with the development of an inflammatory serositis, a phenomenon that has been described in CMML but not in AP-CML. This case presents key features of kinase-independent resistance with insight into potential mechanisms, highlights management challenges, and describes a novel systemic inflammatory response that occurred in this patient upon disease progression. [ABSTRACT FROM AUTHOR]

Apart from the driver mutations, high molecular risk (HMR) variants and other factors have been reported to influence the prognosis of primary myelofibrosis (PMF). The aim of our study was to investigate the impact of laboratory and molecular characteristics at the time of diagnosis (TOD) on the PMF outcome. The study group consisted of 82 patients recruited from three Polish university centers. Among the driver mutations, only CALR type 1 positively influenced the overall survival (OS). The risk of progression to accelerated or blastic disease phase (AP/BP) did not depend on the driver mutation type, but was closely associated with the presence of HMR variants (p = 0.0062). The risk of death (ROD) was higher in patients with HMR variants (OR[95%CI] = 4.33[1.52;12.34], p = 0.0044) and in patients with a platelet count at the TOD between 50–100 G/L (HR[95%CI] = 2.66[1.11;6.35]) and < 50 G/L (HR[95%CI] = 8.44[2.50;28.44]). Median survival time was 7.8, 2.2 and 1.4 years in patients with large unstained cells (LUC) count of [0.0–0.2], (0.2–0.4] and > 0.4 G/L at the TOD, respectively. We found an unexpected, hitherto undescribed, association between LUC count at the TOD and PMF prognosis. Our analysis led to the following conclusions: in PMF patients at the TOD 1) the presence of HMR variants, especially combined, is associated with an increased risk of progression to the AP and BP, and shorter OS, 2) severe thrombocytopenia confers worse prognosis than the moderate one, 3) LUC count is closely related with the disease phase, and associated with the ROD and OS. [ABSTRACT FROM AUTHOR]

Background. The development of myelofibrosis (MF) is driven by complex molecular genetic events that include driver somatic mutations responsible for the constitutive activation of the JAK/STAT signaling pathway (JAK2, CALR, and MPL), additional mutations affecting epigenetic regulators (TET2, ASXL1, IDH1/2, etc.) and RNA splicing (SRSF2, U2AF1, SF3B1, etc.), as well as genetic aberrations that contribute to genomic instability and disease progression.Aim. To analyze driver (JAK2, CALR, MPL) and prognostic (ASXL1) somatic mutations in patients with MF and evaluate their impact on survival.Materials and methods. The study included 29 patients diagnosed with MF, selected by hematologists from the City Clinical Hospital No. 7 and Regional Clinical Hospital (Krasnoyarsk).Results. 26 (89.6 %) out of 29 examined patients had some driver mutations in JAK2, CALR, MPL genes. The p.V617F mutation in the JAK2 gene was found in 20 (68.9 %) patients. Mutations in the CALR gene were detected in 4 (13.8 %) patients, mutations in the MPL gene were found in 3 patients (10.3 %). In 1 of 26 patients, 2 driver mutations were present simultaneously. 3 (10.3 %) patients were triple negative. Mutations in the ASXL1 gene were detected in 12 (41.4 %) out of 29 examined patients. Conducted targeted NGS (next generation sequencing) for 13 out of 29 patients revealed additional genetic variants that contribute to the understanding of the development mechanism and disease course. When evaluating the overall survival in the groups of patients diagnosed with MF examined by us, depending on the combination of driver (JAK2, CALR, MPL) and prognostic (ASXL1) mutations, no statistically significant differences were found (p = 0.12). This appears to be due to the small sample size. At the same time, assessment of patient survival depending on ASXL1 status showed that in the presence of mutations in the ASXL1 gene, the median survival was 45 months (range 7–120 months), while in the absence of mutations it was 48 months (range 21–359 months) (p = 0.03).Conclusion. The results obtained allow us to assume that the presence of mutations in the ASXL1 gene is an unfavorable factor in the course of the disease.

Purpose The prognostic role of TET2 and/or ASXL1 mutations which are common gene mutations in chronic myelomonocytic leukemia (CMML) remains controversial. Therefore, we conducted this meta-analysis to evaluate the prognostic efficacy of ASXL1 and TET2 mutations in CMML population.Methods PubMed, Cochrane and Embase for relevant research were employed to identify 16 studies. Overall survival rate (OS) with hazard ratios (HRs) was used for analysis, and each individual HR was applied to calculate the combined HR.Results The total HR of OS was 0.74, 95% CI = 0.61 - 0.91, P = 0.005, compared with CMML patients without TET2 mutations (TET2MT), and the total HR of OS was 1.56, 95% CI = 1.34 - 1.80, P = 0.000, compared with CMML patients without ASXL1 mutation (ASXL1WT), indicating that TET2MT and ASXL1WT were favorable for prognosis of CMML. According to whether the gene is mutated or not, the acute transformation rate of disease and mortality rate were further considered for assessment. Compared with the CMML patients with TET2MT and ASXL1WT, the HR of patients with in both TET2MT and ASXL1MT was 1.51 (95% CI = 1.14 - 1.99; P = 0.004), the HR of patients with neither TET2MT nor ASXL1MT was 1.49 (95%CI = 1.12 - 1.98; P = 0.007), and the HR of TET2WT and ASXL1MT patients was 1.88 (95%CI = 1.21 - 2.94; P = 0.005).Conclusion Presence of TET2MT and ASXL1WT genotype was the most beneficial for the survival of CMML patients.

BackgroundChromatin Modifying Disorders (CMD) have emerged as one of the most rapidly expanding genetic disorders associated with autism spectrum disorders (ASD). Motor impairments are also prevalent in CMD and may play a role in the neurodevelopmental phenotype. Evidence indicates that neurodevelopmental outcomes in CMD may be treatable postnatally; thus deep phenotyping of these conditions can improve clinical screening while improving the development of treatment targets for pharmacology and for clinical trials. Here, we present developmental phenotyping data on individuals with Bohring-Optiz Syndrome (BOS – ASXL1) and Bainbridge-Ropers Syndrome (BRS – ASXL3) related disorders, two CMDs highly penetrant for motor and developmental delays.ObjectivesTo phenotype the motor and neurodevelopmental profile of individuals with ASXL1 and ASXL3 related disorders (BOS and BRS). To provide a preliminary report on the association of motor impairments and ASD.MethodsNeurodevelopmental and motor phenotyping was conducted on eight individuals with pathogenic ASXL1 variants and seven individuals with pathogenic ASXL3 variants, including medical and developmental background intake, movement and development questionnaires, neurological examination, and quantitative gait analysis.ResultsAverage age of first developmental concerns was 4 months for individuals with BOS and 9 months in BRS. 100% of individuals who underwent the development questionnaire met a diagnosis of developmental coordination disorder. 71% of children with BOS and 0% of children with BRS noted movement difficulty greatly affected classroom learning. Participants with BRS and presumed diagnoses of ASD were reported to have more severe motor impairments in recreational activities compared to those without ASD. This was not the case for the individuals with BOS.ConclusionMotor impairments are prevalent and pervasive across the ASXL disorders with and without ASD, and these impairments negatively impact engagement in school-based activities. Unique neurodevelopmental and motor findings in our data include a mixed presentation of hypo and hypertonia in individuals with BOS across a lifespan. Individuals with BRS exhibited hypotonia and greater variability in motor skills. This deep phenotyping can aid in appropriate clinical diagnosis, referral to interventions, and serve as meaningful treatment targets in clinical trials.

Myeloid/lymphoid neoplasm with fibroblast growth factor 1 rearrangements (MLN-FGFR1) represents a rare group of hematologic neoplasms, with approximately 100 cases reported to date. A 69-year-old woman with a history of polycythemia and leukocytosis, with negative molecular testing for JAK2, CALR, and MPL, presented with diffuse adenopathy. A lymph node (LN) biopsy revealed effacement by T-lymphoblasts, consistent with T-cell acute lymphoblastic lymphoma (T-ALL). A staging bone marrow (BM) biopsy demonstrated trilineage hyperplasia, which, taken together with the patient's elevated hemoglobin and low serum erythropoietin level, fulfilled diagnostic criteria for polycythemia vera. Karyotype and fluorescence in situ hybridization on both the BM and LN demonstrated a FGFR1 rearrangement due to t(8;13), consistent with MLN-FGFR1. Whole genome sequencing on the LN additionally identified a pathogenic frameshift mutation of ASXL1 NC_000020.11:g32434646dup NM_015338.6(ASXL1):c.1934dup p.(Gly646Trpfs) predicted to result in loss of protein function, a finding also observed in 8.1% of BM reads. Both the BM and LN harbored missense variants in HDAC4 NM_001378414.1(HDAC4):c.[2763G>A]; [2763=] p.(Met921Ile) and CHEK2 NM_007194.4(CHEK2):c.[538C>T];[538=] p.(Arg180Cys), with an unknown significance. Despite initial response to Mini-CVD + venetoclax, the patient subsequently experienced rapid clinical deterioration and death. We report the second case of MLN-FGFR1 with an ASXL1 mutation and the first case with HDAC4 and CHEK2 variants. [ABSTRACT FROM AUTHOR]

Simple Summary: Therapeutic advances in acute myeloid leukemia (AML) are dependent on identifying and targeting the molecular aberrations that drive disease. The European LeukemiaNet (ELN) 2022 guidelines have improved the categorization of AML into distinct molecular subgroups. One of the most notable recent inclusions is a group of mutations highly specific for secondary AML. This review examines how each of the secondary-type mutations contributes to the genesis of leukemia while spotlighting potential therapeutic avenues. While we highlight the limitations of the ELN 2022 revision, we also emphasize current progress, recent breakthroughs, and novel therapeutic options tailored to each molecular subset. This review provides a background and foundation for rational molecular-based therapeutic approaches and combination strategies to inspire future clinical trial designs. The characterization of the molecular landscape and the advent of targeted therapies have defined a new era in the prognostication and treatment of acute myeloid leukemia. Recent revisions in the European LeukemiaNet 2022 guidelines have refined the molecular, cytogenetic, and treatment-related boundaries between myelodysplastic neoplasms (MDS) and AML. This review details the molecular mechanisms and cellular pathways of myeloid maturation aberrancies contributing to dysplasia and leukemogenesis, focusing on recent molecular categories introduced in ELN 2022. We provide insights into novel and rational therapeutic combination strategies that exploit mechanisms of leukemogenesis, highlighting the underpinnings of splicing factors, the cohesin complex, and chromatin remodeling. Areas of interest for future research are summarized, and we emphasize approaches designed to advance existing treatment strategies. [ABSTRACT FROM AUTHOR]

Purpose of Review: This article summarizes the current knowledge about clonal hematopoiesis of indeterminate potential (CHIP), its association with cardiovascular disease (CVD), and other outcomes, pathogenesis, postulated mechanisms of various pathologies, current knowledge gaps, possible targets of intervention, and therapeutic implications. Recent Findings: Recently, a common age-related hematological entity known as CHIP has been identified as the independent risk factor for CVD. CHIP is defined as the presence of clonally expanded blood cells involving leukemogenic mutations without the evidence of malignancy. CHIP is known to increase the inflammatory state which in turn is thought to be responsible for increased risk of CVD. Apart from CVD and malignancy, CHIP is also associated with pulmonary embolism, COPD, CKD, stroke, altered metabolism, obesity, liver disease, and increased all-cause mortality. At the same time surprisingly, CHIP is found to have positive outcomes in bone marrow transplant patients and similar reciprocal association with Alzheimer's disease. Summary: The risk of CVD and cancer increases with the advancing age, and these two are the leading causes of death in the USA. CHIP is an independent risk factor for CVD development. Most patients with CHIP have somatic clonal mutations in epigenetic regulators, DNA repair genes, or regulatory tyrosine kinases without evidence of overt hematological malignancy. CHIP portends increased risk for leukemia development and carries twofold increased risk of CVD including CAD, MI, and poor prognosis in heart failure. CHIP is associated with various other pathologies making CHIP an area of active research interest in recent years. Current research efforts aim to bridge many knowledge gaps in understanding of CHIP that still exist. [ABSTRACT FROM AUTHOR]

Myeloid malignancies are clonal hematopoietic disorders that are comprised of a spectrum of genetically heterogeneous disorders, including myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). Myeloid malignancies are characterized by excessive proliferation, abnormal self-renewal, and/or differentiation defects of hematopoietic stem cells (HSCs) and myeloid progenitor cells hematopoietic stem/progenitor cells (HSPCs). Myeloid malignancies can be caused by genetic and epigenetic alterations that provoke key cellular functions, such as self-renewal, proliferation, biased lineage commitment, and differentiation. Advances in next-generation sequencing led to the identification of multiple mutations in myeloid neoplasms, and many new gene mutations were identified as key factors in driving the pathogenesis of myeloid malignancies. The polycomb protein ASXL1 was identified to be frequently mutated in all forms of myeloid malignancies, with mutational frequencies of 20%, 43%, 10%, and 20% in MDS, CMML, MPN, and AML, respectively. Significantly, ASXL1 mutations are associated with a poor prognosis in all forms of myeloid malignancies. The fact that ASXL1 mutations are associated with poor prognosis in patients with CMML, MDS, and AML, points to the possibility that ASXL1 mutation is a key factor in the development of myeloid malignancies. This review summarizes the recent advances in understanding myeloid malignancies with a specific focus on ASXL1 mutations. [ABSTRACT FROM AUTHOR]

Although the majority of patients with chronic myeloid leukemia (CML) enjoy an excellent prognosis tyrosine kinase inhibitor (TKI) therapy, resistance remains a significant clinical problem. Resistance can arise from mutations in the kinase domain of ABL preventing drug binding, or due to ill-defined kinase-independent mechanisms. In this case report, we describe the case of a 27-year-old woman with a long-standing history of chronic phase (CP) CML who developed kinase-independent resistance with mutations in ASXL1 and RUNX1. As a consequence of uncontrolled disease, she progressed to a chronic myelomonocytic leukemia-like (CMML) accelerated phase (AP) disease with the acquisition of a mutation in IDH1. This disease progression was associated with the development of an inflammatory serositis, a phenomenon that has been described in CMML but not in AP-CML. This case presents key features of kinase-independent resistance with insight into potential mechanisms, highlights management challenges, and describes a novel systemic inflammatory response that occurred in this patient upon disease progression.

Background/Aim: Mutations in the ASXL transcriptional regulator 1 (ASXL1) and splicing factor 3b subunit 1(SF3B1) genes are commonly observed in myeloid neoplasms and are independent predicative factors for overall survival (OS). Only a few contradictory reports exist on the clinical significance of concurrent ASXL1 and SF3B1 mutations. Previous studies also did not exclude patients with mutations of other genes, which could be confounding factors. Materials and Methods: We identified 69 patients with mutation of only ASXL1, 89 patients with mutation of only SF3B1, and 17 patients with mutations exclusively of both ASXL1 and SF3B1 from our database of 8,285 patients and compared their clinical features and outcomes. Results: Patients with ASXL1 mutations more frequently had acute myeloid leukemia (22.47%) or clonal cytopenia of unknown significance than patients with SF3B1 mutations (1.45%) or with ASXL1/SF3B1 mutations (11.76%). Patients with SF3B1 or ASXL1/SF3B1 mutations were more frequently diagnosed with myelodysplastic syndrome (75.36% and 64.71%, respectively) than patients with ASXL1 mutations (24.72%). Patients with ASXL1/SF3B1 (23.53%) mutations more frequently had myelodysplastic/myeloid proliferative neoplasm than did patients with ASXL1 mutations (5.62%) or with SF3B1 mutations (15.94%). OS of the ASXL1 mutation-only group was worse than that of the SF3B1 mutation-only group with a hazard ratio of 5.83 (p=0.017). Finally, and most importantly, the OS of the ASXL1/SF3B1 co-mutation group was poorer than that of both singlemutation groups (p=0.005). Conclusion: ASXL1/SF3B1 comutations portend worse OS than isolated ASXL1 or SF3B1 mutations, which might be due to abnormalities in both the epigenetic-regulatory and RNA-splicing pathways or because two genes instead of one are mutated. [ABSTRACT FROM AUTHOR]

Bohring–Opitz syndrome (BOS) is a rare genetic condition caused by pathogenic variants in ASXL1, which is a gene involved in chromatin regulation. BOS is characterized by severe intellectual disabilities, distinctive facial features, hypertrichosis, facial nevus simplex, severe myopia, a typical posture in infancy, variable anomalies, and feeding issues. Wilms tumor has also been reported in two individuals. We report survey data from the largest known cohort of individuals with BOS with 34 participants from the ASXL Patient‐Driven Registry and data on five additional individuals with notable findings. Important or novel findings include hepatoblastoma (n = 1), an additional individual with Wilms tumor, two families with a parent who is mosaic including a pair of siblings, birth weights within the normal range for the majority of participants, as well as presence of craniosynostosis and hernias. Data also include characterization of communication, motor skills, and care level including hospitalization frequency and surgical interventions. No phenotype–genotype correlation could be identified. The ASXL Registry is also presented as a crucial tool for furthering ASXL research and to support the ASXL community. [ABSTRACT FROM AUTHOR]

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive lymphohematopoietic malignancy associated with poor prognosis. We aimed to improve the understanding of BPDCN, explore its prognostic significance, and identify potential therapeutic targets. Data from 14 BPDCN patients were retrospectively collected and analyzed, focusing on their clinicopathological characteristics, diagnostic features, immunophenotype, treatment regimens, and prognostic factors. Additionally, immunohistochemistry was used to detect the expression of multiple oncogenes in BPDCN. The cohort comprised 14 patients (10 males, 4 females) with a median age of 63.5 years at the time of diagnosis. Of these specimens, H3K27me3, ASXL1, BAP1, RAC1, TCF4 and AURKA were highly expressed in BPDCN, with expression rates of 71.4 % (10/14), 92.9 % (13/14), 85.7 % (12/14), 100 % (13/13), 12/14 (85.7 %) and 46.2 % (6/13), respectively. The survival of patients in this cohort ranged from 1 to 84 months, with a median overall survival (OS) of 18.5 months. The survival rates for 1, 2, 3, 4 and 5 years were 71.43 %, 53.57 %, 44.64 %, 44.64 %, and 44.64 %, respectively. In the overall BPDCN cohort, patients with positive expression of H3K27me3 exhibited significantly better overall survival compared to those with negative expression H3K27me3 (P = 0.0056). Our analysis showed that the absence of H3K27me3 expression may indicate a poor prognosis in patients with BPDCN, and H3K27me3 may be a potential prognostic indicator for BPDCN. [Display omitted] • H3K27me3, ASXL1, BAP1, RAC1, TCF4 and AURKA were highly expressed in BPDCN. • The absence of H3K27me3 expression may indicate a poor prognosis in patients with BPDCN. • H3K27me3 may play a role in BPDCN. [ABSTRACT FROM AUTHOR]

Bohring-Opitz syndrome (BOS) is a rare disease with a characteristic facial appearance and limb position. This report describes a case of BOS complicated by persistent pulmonary hypertension of the newborn (PPHN) and formation of abnormal alveoli that was confirmed by autopsy. A female neonate was born by cesarean section at 37 weeks and 2 days of gestation and found to have a nevus flammeus, exophthalmos, abnormal palate, retraction of the mandible, and a posture characteristic of BOS. The patients had severe PPHN requiring inhalation of nitric oxide. Genetic testing revealed a de novo frameshift variant in ASXL1. Autopsy revealed that the lung was at the saccular stage, equivalent to 28–34 weeks of gestation. This is the first report to present pathological evidence of immaturity of the lung that may be associated with PPHN in a patient with BOS caused by a variant in ASXL1. • The pathogenesis of PPHN complicating Bohring-Opitz syndrome is unclear. • The ASXL1 variant is associated with abnormal alveoli formation in mice. • Our Bohrng-Opitz syndrome patient with ASXL1 variant died by PPHN. • Autopsy revealed the abnormal alveoli formation associated with ASXL1 variants. [ABSTRACT FROM AUTHOR]

Background: Genetic variants causing diseases with hypertension as a secondary feature have previously been identified. Studies focussing on primary hypertension have utilised common and latterly rare genetic variants in attempts to elucidate the genetic contribution to the risk of primary hypertension. Summary: Using genome-wide association studies (GWASs), associations of hypertension with hundreds of common genetic variants have been reported, implicating thousands of genes. Individual variants have small effect sizes and cumulatively account for around 6% of genetic risk. The common variant signal is enriched for relevant tissues and physiological processes, while some variants are associated with traits expected to have secondary impacts on hypertension risk, such as fruit intake, BMI, or time watching television. Studies using rare variants obtained from exome sequence data have implicated a small number of genes for which impaired function has moderate effects on blood pressure and/or hypertension risk. Notably, genetic variants which impair elements of guanylate cyclase activation, stimulated by either natriuretic hormones or nitric oxide, increase hypertension risk. Conversely, variants impairing dopamine beta-hydroxylase or renin production are associated with lower blood pressure. Variants for which a definite effect can be designated remain cumulatively extremely rare and again make only a small contribution to overall genetic risk. Although these results are of interest, it is not clear that they provide radical new insights or identify drug targets which were not previously known. Nor does it seem that genetic testing could be useful in terms of quantifying disease risk or guiding treatment. Key Messages: Research has increased our knowledge about the relationship between naturally occurring genetic variation and risk of hypertension. Although some results serve to confirm our understanding of underlying physiology, their value in terms of potentially leading to practical advances in the management of hypertension appears questionable.

Introduction: A previous study of 200,000 exome-sequenced UK Biobank participants to test for association of rare coding variants with hypertension implicated two genes at exome-wide significance, DNMT3A and FES. A total of 42 genes had an uncorrected p value

This is a systematic review and meta-analysis evaluating the prognostic significance of epigenetic mutations on the overall survival (OS) in Acute Myeloid Leukemia (AML). We searched for studies evaluating epigenetic mutations in AML (up to November 2018) in PubMed, Trip database and Cochrane library. Hazard ratio (HR) of outcomes were extracted, and random-effects model was used to pool the results. A total of 10,002 citations were retrieved from the search strategy; 42 articles were identified for the meta-analysis (ASXL1 = 7, TET2 = 8, DNMT3A = 12, IDH =15), with fair to good-quality studies. The pooled HR was 1.88 (95% CI: 1.49−2.36) for ASXL1 mutation, 1.39 (95% CI: 1.18−1.63) for TET2 mutation, 1.35 (95% CI 1.16-1.56) for DNMT3a and 1.54 (95% CI: 1.15-2.06) for IDH mutation. However, there was a substantial heterogeneity in the DNMT3a and IDH studies. In conclusion epigenetic mutations in ASXL1, TET2, DNMT3a and IDH adversely impact OS in patients with AML albeit with considerable heterogeneity and possibly publication bias. Further studies are required to address these limitations. [ABSTRACT FROM AUTHOR]

Introduction: The additional sex combs like 1 (ASXL1) gene is frequently mutated in a number of haematological neoplasms. The c.1934dupG, known to be the most common alteration in ASXL1, is associated with poor clinical outcome. A systematic determination of ASXL1 mutational status in myeloid malignancies is therefore necessary for prognostic stratification. Methods: Because direct sequencing is not sensitive and next‐generation sequencing (NGS) is time‐consuming, expensive and sometimes does not allow the detection of the c.1934dupG, we have developed a fragment analysis assay, complementary to NGS, that allows the detection of c.1934dupG mutation in addition to other nearby insertions/deletions of ASXL1 located close to it. We called this assay the "PCR‐Fluo‐ASXL1‐FA." Results: First, we evaluated the efficiency of our approach compared to NGS and Sanger. We showed that "PCR‐Fluo‐ASXL1‐FA" could detect all insertional mutations of ASXL1 located on its area, with a high sensitivity (1.5%). Then, we have illustrated the interest of this technique by three concrete cases. Discussion: In summary, we have established a fragment analysis approach, which can detect most ASXL1 mutations, in particular the c.1934dupG, in a sensitive, fast and inexpensive manner. We therefore recommend the synchronous use of this method with NGS, to ensure complete detection of all clinically relevant ASXL1 mutations in patients suffering with myeloid neoplasms. [ABSTRACT FROM AUTHOR]

Development of secondary CML has only been casually described, with few reports attempting to analyze and explain the mechanisms behind this phenomenon. Reported cases vary with regard to presumed pathogenesis and clinical characteristics, but similarities can be observed. This report presents the case of a patient diagnosed with CALR and ASXL1-mutated primary myelofibrosis who developed CML 13 years after the initial diagnosis. In contrast with previously reported cases, this patient did not have JAK2 or ABL1 gene mutations, and also exhibited primary resistance to tyrosine kinase inhibitor (TKI) treatment. Here, we analyze the molecular evolution of CML and describe successful treatment with concomitant therapy including a TKI and JAK inhibitor. This report aims to deepen clinical experience and further broaden knowledge about chronic myeloproliferative neoplasms. [ABSTRACT FROM AUTHOR]

This is a systematic review and meta-analysis evaluating the prognostic significance of epigenetic mutations on the overall survival (OS) in Acute Myeloid Leukemia (AML). We searched for studies evaluating epigenetic mutations in AML (up to November 2018) in PubMed, Trip database and Cochrane library. Hazard ratio (HR) of outcomes were extracted, and random-effects model was used to pool the results. A total of 10,002 citations were retrieved from the search strategy; 42 articles were identified for the meta-analysis (ASXL1 = 7, TET2 = 8, DNMT3A = 12, IDH =15), with fair to good-quality studies. The pooled HR was 1.88 (95% CI: 1.49−2.36) for ASXL1 mutation, 1.39 (95% CI: 1.18−1.63) for TET2 mutation, 1.35 (95% CI 1.16-1.56) for DNMT3a and 1.54 (95% CI: 1.15-2.06) for IDH mutation. However, there was a substantial heterogeneity in the DNMT3a and IDH studies. In conclusion epigenetic mutations in ASXL1, TET2, DNMT3a and IDH adversely impact OS in patients with AML albeit with considerable heterogeneity and possibly publication bias. Further studies are required to address these limitations.

Our understanding of chronic myelomonocytic leukemia (CMML) has evolved tremendously over the past decade. Large-scale sequencing studies have led to increased insight into the genomic landscape of CMML and clinical implications of these changes. This in turn has resulted in refined and improved risk stratification models, which to date remain versatile and subject to remodeling, as new and evolving studies continue to refine our understanding of this disease. In this article, we present an up-to-date review of CMML from a hematopathology perspective, while providing a clinically practical summary that sheds light on the constant evolution of our understanding of this disease.

To evaluate the frequency and prognosis of runt‐related transcription factor 1 (RUNX1) and additional sex combs like‐1 (ASXL1) mutations in acute myeloid leukaemia (AML) patients in northeastern Iran. This cross‐sectional study was performed on 40 patients with AML (including 35 patients with denovo AML and five patients with secondary AML) from February 2018 to February 2021. All patients were followed up for 36 months. We evaluated the frequency and survival rate of RUNX1 and ASXL1 mutations in AML patients. To detect mutations, peripheral blood samples and bone marrow aspiration were taken from all participants. One male patient (2.5%) had RUNX1 mutations and four cases (10%; 3 females vs. 1 male) had ASXL1 mutations. The survival rates of AML patients after 1, 3, 6, 9, 12, 24 and 36 months were 98%, 90%, 77%, 62%, 52%, 27% and 20%, respectively. There was a significant relationship between the occurrence of ASXL1 mutations and the survival of patients with AML (p = 0.027). Also, there was a significant relationship between the incidence of death and haemoglobin levels in patients with AML (p = 0.045). Thus, with an increase of one unit in patients' haemoglobin levels, the risk of death is reduced by 16.6%. Patients with AML had a high mortality rate, poor therapy outcome and low survival rate. ASXL1 and RUNX1 mutations are associated with a worse prognosis in patients with newly diagnosed AML. Also, we witnessed that the prevalence of ASXL1 to RUNX1 mutations was higher in northeastern Iran compared with other regions. [ABSTRACT FROM AUTHOR]

Simple Summary: Systemic mastocytosis (SM) is a clonal haematopoietic stem cell disease typically characterized by the expansion and accumulation of neoplastic mast cells carrying the activating KIT D816V as a driver mutation. Multilineage involvement of haematopoiesis by this KIT mutation, particularly in a multi-mutated context, also involving other genes (e.g., SRSF2, ASXL1, DNMT3A, RUNX1, EZH2, CBL and NRAS) found to be frequently mutated in other myeloid neoplasms, have recently emerged as a genetic background associated with malignant transformation of SM. Therefore, assessment of multilineage involvement of haematopoiesis by KIT D816V and additional mutations in genes known to be associated with the prognosis of SM have become of great help to identify good vs. poor-prognosis SM patients who could benefit from a closer follow-up and, eventually, also early cytoreductive treatment. Systemic mastocytosis (SM) is a rare clonal haematopoietic stem cell disease in which activating KIT mutations (most commonly KIT D816V) are present in virtually every (>90%) adult patient at similar frequencies among non-advanced and advanced forms of SM. The KIT D816V mutation is considered the most common pathogenic driver of SM. Acquisition of this mutation early during haematopoiesis may cause multilineage involvement of haematopoiesis by KIT D816V, which has been associated with higher tumour burden and additional mutations in other genes, leading to an increased rate of transformation to advanced SM. Thus, among other mutations, alterations in around 30 genes that are also frequently mutated in other myeloid neoplasms have been reported in SM cases. From these genes, 12 (i.e., ASXL1, CBL, DNMT3A, EZH2, JAK2, KRAS, NRAS, SF3B1, RUNX1, SF3B1, SRSF2, TET2) have been recurrently reported to be mutated in SM. Because of all the above, assessment of multilineage involvement of haematopoiesis by the KIT D816V mutation, in the setting of multi-mutated haematopoiesis as revealed by a limited panel of genes (i.e., ASXL1, CBL, DNMT3A, EZH2, NRAS, RUNX1 and SRSF2) and associated with a poorer patient outcome, has become of great help to identify SM patients at higher risk of disease progression and/or poor survival who could benefit from closer follow-up and eventually also early cytoreductive treatment. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is a major public health problem on a global scale by virtue of its relatively high incidence. The transition of tumor cells from an epithelial to a mesenchymal-like phenotype, so-called epithelial-to-mesenchymal transition (EMT), is a key hallmark of human cancer metastasis, including CRC. Understanding the signaling events that initiate this phenotypic switch may provide opportunities to limit the metastasis of CRC. In this study, we aim to identify long non-coding RNA (lncRNA) mediated epigenetic regulation under the context of CRC. 54 paired samples of tumor tissues and surrounding non-tumor tissues were collected from CRC patients. Cultured human CRC cells HCT116 and LoVo were assayed for their viability and migration using CCK-8 tests and transwell migration assays. The expression of EMT-specific markers (E-cadherin, N-cadherin and vimentin) was analyzed biochemically by RT-qPCR and immunoblot analyses. Interaction among LINC00586, LSD1, and ASXL1 was determined by RNA immunoprecipitation and chromatin immunoprecipitation. In vivo analysis of LINC00586 was performed in nude mice xenografted with HCT116 cells. LINC00586 was overexpressed in CRC tissues and associated with patient survival. LINC00586 knockdown repressed HCT116 and LoVo cell viability, migration, their phenotypic switch from epithelial to a mesenchymal, and tumorigenesis in vivo. We demonstrated LINC00586 recruited the LSD1 into the ASXL1 promoter region and epigenetically silenced the ASXL1 expression. An ASXL1 gene resisting to LINC00586 attack was demonstrated in cultured HCT116 and LoVo cells and mouse xenograft models of human CRC. Overall, discovery of the LINC00586/LSD1/ASXL1 axis partially explains epigenetic mechanism regulating EMT in CRC, providing a therapeutic target to limit CRC metastasis. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases (IBDs) are a group of chronic conditions of the gastrointestinal tract in which nationwide studies have revealed a higher risk of hematological malignancies (HMs). Clonal hematopoiesis (CH) is a premalignant condition defined by the presence of an acquired somatic mutation characterized by a variant allele frequency (VAF) of ≥2%, in a gene frequently associated with HMs. A growing body of evidence suggests a correlation between inflammation and CH; its occurrence in the context of IBD has been previously demonstrated. With the aim to assess CH possible co-occurrence in patients with an IBD associated with HMs, we performed a targeted next-generation sequencing analysis in a cohort of thirteen patients who were referred to our center with IBD associated with HMs. Eleven (85%) patients showed one or more mutations in CH-associated genes; DNMT3A was the most frequently mutated gene, followed by ASXL1 and JAK2. These results may suggest that the mechanisms at the basis of the inflammatory environment could potentially select for the growth of hematopoietic clones harboring specific mutations. In this context, CH emergence may be boosted by the proinflammatory IBD environment, thus acting as a biological link between IBD and the HM onset. If these data are confirmed, IBD patients screened and positive for CH should undergo a hematologic follow-up to assess the risk of developing HM. Future study will clarify the relationship between these conditions. [ABSTRACT FROM AUTHOR]

Clonal hematopoiesis of indeterminate potential (CHIP) is an age‐associated phenomenon characterized by clonal expansion of blood cells harboring somatic mutations in hematopoietic genes, including DNMT3A, TET2, and ASXL1. Clinical evidence suggests that CHIP is highly prevalent and associated with poor prognosis in solid‐tumor patients. However, whether blood cells with CHIP mutations play a causal role in promoting the development of solid tumors remained unclear. Using conditional knock‐in mice that express CHIP‐associated mutant Asxl1 (Asxl1‐MT), we showed that expression of Asxl1‐MT in T cells, but not in myeloid cells, promoted solid‐tumor progression in syngeneic transplantation models. We also demonstrated that Asxl1‐MT–expressing blood cells accelerated the development of spontaneous mammary tumors induced by MMTV‐PyMT. Intratumor analysis of the mammary tumors revealed the reduced T‐cell infiltration at tumor sites and programmed death receptor‐1 (PD‐1) upregulation in CD8+ T cells in MMTV‐PyMT/Asxl1‐MT mice. In addition, we found that Asxl1‐MT induced T‐cell dysregulation, including aberrant intrathymic T‐cell development, decreased CD4/CD8 ratio, and naïve‐memory imbalance in peripheral T cells. These results indicate that Asxl1‐MT perturbs T‐cell development and function, which contributes to creating a protumor microenvironment for solid tumors. Thus, our findings raise the possibility that ASXL1‐mutated blood cells exacerbate solid‐tumor progression in ASXL1‐CHIP carriers. [ABSTRACT FROM AUTHOR]

Abstract Only proven pathogenic mutations associated with myeloid neoplasms are key to establish the clonal nature of the bone marrow fibrosis. In cases with genetic variants of uncertain meaning, the clinical picture may be required to rule out secondary causes.

Abstract Additional sex combs-like 1 (ASXL1) interacts with BRCA1-associated protein 1 (BAP1) deubiquitinase to oppose the polycomb repressive complex 1 (PRC1)-mediated histone H2A ubiquitylation. Germline BAP1 mutations are found in a spectrum of human malignancies, while ASXL1 mutations recurrently occur in myeloid neoplasm and are associated with poor prognosis. Nearly all ASXL1 mutations are heterozygous frameshift or nonsense mutations in the middle or to a less extent the C-terminal region, resulting in the production of C-terminally truncated mutant ASXL1 proteins. How ASXL1 regulates specific target genes and how the C-terminal truncation of ASXL1 promotes leukemogenesis are unclear. Here, we report that ASXL1 interacts with forkhead transcription factors FOXK1 and FOXK2 to regulate a subset of FOXK1/K2 target genes. We show that the C-terminally truncated mutant ASXL1 proteins are expressed at much higher levels than the wild-type protein in ASXL1 heterozygous leukemia cells, and lose the ability to interact with FOXK1/K2. Specific deletion of the mutant allele eliminates the expression of C-terminally truncated ASXL1 and increases the association of wild-type ASXL1 with BAP1, thereby restoring the expression of BAP1-ASXL1-FOXK1/K2 target genes, particularly those involved in glucose metabolism, oxygen sensing, and JAK-STAT3 signaling pathways. In addition to FOXK1/K2, we also identify other DNA-binding transcription regulators including transcription factors (TFs) which interact with wild-type ASXL1, but not C-terminally truncated mutant. Our results suggest that ASXL1 mutations result in neomorphic alleles that contribute to leukemogenesis at least in part through dominantly inhibiting the wild-type ASXL1 from interacting with BAP1 and thereby impairing the function of ASXL1-BAP1-TF in regulating target genes and leukemia cell growth.

Background. There are problems related to both quantitative assessment of an allele burden level of a mutant gene and interpretation of results in DNA samples with the burden level of the mutant allele less than 15–20 %, when using Sanger sequencing for analyzing somatic mutations. Applied Biosystems (USA) has developed new software Minor Variant Finder, which allows determining mutations with the allele burden level from 5 %.The objective: to determine the allele burden level and identification of minor variants of somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene using Minor Variant Finder software in patients with myeloproliferative neoplasms.Materials and methods. The level of mutant allele burden for 15 patients with myeloproliferative neoplasms was determined by the identified mutations using the Minor Variant Finder software, after analysis of point somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene by Sanger sequencing.Results. The allele burden level in all 5 ASXL1-positive samples and BCR-ABL-positive sample was determined as higher than 20 % using the Minor Variant Finder software. The allele burden level in 2 cases was higher than 20 % and in 7 cases lower than 20 %, when we analyzed 9 JAK2-positive samples.Conclusion. Minor Variant Finder software can be used to estimate the allele burden level and to identify minor variants of somatic mutations in the ASXL, JAK2 and BCR-ABL genes.

Abstract Acute myeloid leukemia with myelodysplasia‐related changes (AML‐MRC) are poor outcome leukemias. Its diagnosis is based on clinical, cytogenetic, and cytomorphologic criteria, last criterion being sometimes difficult to assess. A high frequency of ASXL1 mutations have been described in this leukemia. We sequenced ASXL1 gene mutations in 61 patients with AML‐MRC and 46 controls with acute myeloid leukemia without other specifications (AML‐NOS) to identify clinical, cytomorphologic, and cytogenetic characteristics associated with ASXL1 mutational status. Mutated ASXL1 (ASXL1+) was observed in 31% of patients with AML‐MRC compared to 4.3% in AML‐NOS. Its presence in AML‐MRC was associated with older age, a previous history of myelodysplastic syndrome (MDS) or myelodysplastic/myeloproliferative neoplasms (MDS/MPN), leukocytosis, presence of micromegakaryocytes in bone marrow, lower number of blasts in bone marrow, myelomonocytic/monocytic morphological features and normal karyotype. ASXL1 mutation was not observed in patients with myelodysplastic syndrome‐related cytogenetic abnormalities or TP53 mutations. Differences in terms of overall survival were found only in AML‐MRC patients without prior MDS or MDS/MPN and with intermediate‐risk karyotype, having ASXL1+ patients a worst outcome than ASXL1−. We conclude that the ASXL1 mutation frequency is high in AML‐MRC patients being its presence associated with specific characteristics including morphological signs of dysplasia. This association raises the possible role of ASXL1 as a surrogate marker in AML‐MRC, which could facilitate the diagnosis of patients within this group when the karyotype is normal, and especially when the assessment of multilineage dysplasia morphologically is difficult. This mutation could be used as a worst outcome marker in de novo AML‐MRC with intermediate‐risk karyotype.

Abstract Background Precise regulation of neural precursor cell (NPC) proliferation and differentiation is essential to ensure proper brain development and function. The HCFC1 gene encodes a transcriptional co-factor that regulates cell proliferation, and previous studies suggest that HCFC1 regulates NPC number and differentiation. However, the molecular mechanism underlying these cellular deficits has not been completely characterized. Methods Here we created a zebrafish harboring mutations in the hcfc1a gene (the hcfc1a co60/+ allele), one ortholog of HCFC1, and utilized immunohistochemistry and RNA-sequencing technology to understand the function of hcfc1a during neural development. Results The hcfc1a co60/+ allele results in an increased number of NPCs and increased expression of neuronal and glial markers. These neural developmental deficits are associated with larval hypomotility and the abnormal expression of asxl1, a polycomb transcription factor, which we identified as a downstream effector of hcfc1a. Inhibition of asxl1 activity and/or expression in larvae harboring the hcfc1a co60/+ allele completely restored the number of NPCs to normal levels. Conclusion Collectively, our data demonstrate that hcfc1a regulates NPC number, NPC proliferation, motor behavior, and brain development.

Colorectal cancer (CRC) is a major public health problem on a global scale by virtue of its relatively high incidence. The transition of tumor cells from an epithelial to a mesenchymal-like phenotype, so-called epithelial-to-mesenchymal transition (EMT), is a key hallmark of human cancer metastasis, including CRC. Understanding the signaling events that initiate this phenotypic switch may provide opportunities to limit the metastasis of CRC. In this study, we aim to identify long non-coding RNA (lncRNA) mediated epigenetic regulation under the context of CRC. 54 paired samples of tumor tissues and surrounding non-tumor tissues were collected from CRC patients. Cultured human CRC cells HCT116 and LoVo were assayed for their viability and migration using CCK-8 tests and transwell migration assays. The expression of EMT-specific markers (E-cadherin, N-cadherin and vimentin) was analyzed biochemically by RT-qPCR and immunoblot analyses. Interaction among LINC00586, LSD1, and ASXL1 was determined by RNA immunoprecipitation and chromatin immunoprecipitation. In vivo analysis of LINC00586 was performed in nude mice xenografted with HCT116 cells. LINC00586 was overexpressed in CRC tissues and associated with patient survival. LINC00586 knockdown repressed HCT116 and LoVo cell viability, migration, their phenotypic switch from epithelial to a mesenchymal, and tumorigenesis in vivo. We demonstrated LINC00586 recruited the LSD1 into the ASXL1 promoter region and epigenetically silenced the ASXL1 expression. An ASXL1 gene resisting to LINC00586 attack was demonstrated in cultured HCT116 and LoVo cells and mouse xenograft models of human CRC. Overall, discovery of the LINC00586/LSD1/ASXL1 axis partially explains epigenetic mechanism regulating EMT in CRC, providing a therapeutic target to limit CRC metastasis.

Inflammatory bowel diseases (IBDs) are a group of chronic conditions of the gastrointestinal tract in which nationwide studies have revealed a higher risk of hematological malignancies (HMs). Clonal hematopoiesis (CH) is a premalignant condition defined by the presence of an acquired somatic mutation characterized by a variant allele frequency (VAF) of ≥2%, in a gene frequently associated with HMs. A growing body of evidence suggests a correlation between inflammation and CH; its occurrence in the context of IBD has been previously demonstrated. With the aim to assess CH possible co-occurrence in patients with an IBD associated with HMs, we performed a targeted next-generation sequencing analysis in a cohort of thirteen patients who were referred to our center with IBD associated with HMs. Eleven (85%) patients showed one or more mutations in CH-associated genes; DNMT3A was the most frequently mutated gene, followed by ASXL1 and JAK2. These results may suggest that the mechanisms at the basis of the inflammatory environment could potentially select for the growth of hematopoietic clones harboring specific mutations. In this context, CH emergence may be boosted by the proinflammatory IBD environment, thus acting as a biological link between IBD and the HM onset. If these data are confirmed, IBD patients screened and positive for CH should undergo a hematologic follow-up to assess the risk of developing HM. Future study will clarify the relationship between these conditions.

Ostra białaczka szpikowa jest nowotworem złośliwym układu krwiotwórczego, w patogenezie której centralną rolę pełnią mutacje genów kontrolujących dojrzewanie i apoptozę komórek progenitorowych szpiku. Istotna rokowniczo jest mutacja w genie ASXL1, którego produkt białkowy bierze udział w regulacji ekspresji genów. Jest ona negatywnym czynnikiem prognostycznym i predykcyjnym. Szacuje się, że mutacja genu ASXL1 występuje u 14,4-19,1% pacjentów z AML i jest częstsza u mężczyzn powyżej 60 roku życia. Aberracje chromosomów 8 i 11 często współwystępują z mutacją genu ASXL1 w AML i mogą stanowić dodatkową, istotną prognostycznie informację. Leczenie chorych z AML dopasowywane jest do pacjenta w zależności od grupy ryzyka, zmian kariotypu oraz współistniejących mutacji. Duża liczba chorych nie kwalifikuje się do allo-HSCT, choć ta metoda znacznie poprawia przeżywalność pacjentów z AML z mutacją genu ASXL1. Zastosowanie w terapii tych pacjentów może mieć również gilterytynib, jednak mnogość towarzyszących mutacji zmusza do bazowania na chemioterapii opartej na niespecyficznych lekach cytotoksycznych. Celem pracy jest podsumowanie najważniejszych informacji dotyczących patogenezy, cech szczególnych, diagnostyki i terapii AML z współistniejącą mutacją ASXL1.