CRISPR-Cas9-mediated deletion enhancer of MECOM play a tumor suppressor role in ovarian cancer.

MDS1 and EVI1 complex locus (MECOM), a transcription factor encoding several variants, has been implicated in progression of ovarian cancer. The function of regulatory regions in regulating MECOM expression in ovarian cancer is not fully understood. In this study, MECOM expression was evaluated in ovarian cancer cell lines treated with bromodomain and extraterminal (BET) inhibitor JQ-1. Oncogenic phenotypes were assayed using assays of CCK-8, colony formation, wound-healing and transwell. Oncogenic phenotypes were estimated in stable sgRNA-transfected OVCAR3 cell lines. Xenograft mouse model was assayed via subcutaneous injection of enhancer-deleted OVCAR3 cell lines. The results displayed that expression of MECOM is downregulated in cell lines treated with JQ-1. Data from published ChIP-sequencing (H3K27Ac) in 3 ovarian cancer cell lines displayed a potential enhancer around the first exon. mRNA and protein expression were downregulated in OVCAR3 cells after deletion of the MECOM enhancer. Similarly, oncogenic phenotypes both in cells and in the xenograft mouse model were significantly attenuated. This study demonstrates that JQ-1 can inhibit the expression of MECOM and tumorigenesis. Deletion of the enhancer activity of MECOM has an indispensable role in inhibiting ovarian cancer progress, which sheds light on a promising opportunity for ovarian cancer treatment through the application of this non-coding DNA deletion.

Orthogonal proteogenomic analysis identifies the druggable PA2G4-MYC axis in 3q26 AML.

The overexpression of the ecotropic viral integration site-1 gene (EVI1/MECOM) marks the most lethal acute myeloid leukemia (AML) subgroup carrying chromosome 3q26 abnormalities. By taking advantage of the intersectionality of high-throughput cell-based and gene expression screens selective and pan-histone deacetylase inhibitors (HDACis) emerge as potent repressors of EVI1. To understand the mechanism driving on-target anti-leukemia activity of this compound class, here we dissect the expression dynamics of the bone marrow leukemia cells of patients treated with HDACi and reconstitute the EVI1 chromatin-associated co-transcriptional complex merging on the role of proliferation-associated 2G4 (PA2G4) protein. PA2G4 overexpression rescues AML cells from the inhibitory effects of HDACis, while genetic and small molecule inhibition of PA2G4 abrogates EVI1 in 3q26 AML cells, including in patient-derived leukemia xenografts. This study positions PA2G4 at the crosstalk of the EVI1 leukemogenic signal for developing new therapeutics and urges the use of HDACis-based combination therapies in patients with 3q26 AML.

Evaluation of European LeukemiaNet 2022 risk classification in patients undergoing allogeneic haematopoietic stem cell transplantation for acute myeloid leukaemia: Identification of a very poor prognosis genetic group.

European LeukemiaNet refined their risk classification of acute myeloid leukaemia (AML) in 2022 (ELN 2022) according to the two new myeloid classifications published the same year. We have retrospectively assessed the prognostic value of the ELN 2022 in 120 AML patients undergoing allogeneic haematopoietic cell transplantation (allo-HCT), including 99 in first complete response (CR1) from 2011 to 2021 in our centre. Adverse risk patients (Adv) presented inferior outcome in terms of overall survival (OS) and leukaemia-free survival (LFS) (OS [p = 0.003], LFS [p = 0.02]), confirmed in multivariate analysis (hazard ratio [HR] for OS = 2.00, p = 0.037). These results were also seen in patients allografted in CR1. Further analysis identified a subgroup named adverse-plus (AdvP), including complex karyotype, MECOM(EVI1) rearrangements and TP53 mutations, with worse outcomes than the rest of groups of patients, including the Adv (HR for OS: 3.14, p < 0.001, HR for LFS: 3.36, p < 0.001), with higher 2-year cumulative incidence of relapse (p < 0.001). Notably, within this analysis, the outcome of Adv and intermediate patients were similar. These findings highlight the prognostic value of ELN 2022 in patients undergoing allo-HCT, which can be improved by the recognition of a poor genetic subset (AdvP) within the Adv risk group.

Oncogene EVI1 drives acute myeloid leukemia via a targetable interaction with CTBP2.

Acute myeloid leukemia (AML) driven by the activation of EVI1 due to chromosome 3q26/MECOM rearrangements is incurable. Because transcription factors such as EVI1 are notoriously hard to target, insight into the mechanism by which EVI1 drives myeloid transformation could provide alternative avenues for therapy. Applying protein folding predictions combined with proteomics technologies, we demonstrate that interaction of EVI1 with CTBP1 and CTBP2 via a single PLDLS motif is indispensable for leukemic transformation. A 4× PLDLS repeat construct outcompetes binding of EVI1 to CTBP1 and CTBP2 and inhibits proliferation of 3q26/MECOM rearranged AML in vitro and in xenotransplant models. This proof-of-concept study opens the possibility to target one of the most incurable forms of AML with specific EVI1-CTBP inhibitors. This has important implications for other tumor types with aberrant expression of EVI1 and for cancers transformed by different CTBP-dependent oncogenic transcription factors.

Aberrant ecotropic viral integration site-1 (EVI-1) and myocyte enhancer factor 2 C gene (MEF2C) in adult acute myeloid leukemia are associated with adverse t (9:22) & 11q23 rearrangements.

Acute myeloid leukemia (AML) shows multiple chromosomal translocations & point mutations which can be used to refine risk-adapted therapy in AML patients. Ecotropic viral integration site-1 (EVI-1) & myocyte enhancer factor 2 C gene (MEF2C) are key regulatory transcription factors in hematopoiesis and leukemogenesis & both drive immune escape. This prospective study involved 80 adult de novo AML patients recruited from Oncology Center, Mansoura University, between March 2019 and July 2021. The MEF2C and EVI1 expression were measured using a Taqman probe-based qPCR assay. The results revealed that EVI1 and MEF2C expression were significantly elevated in AML patients as compared to control subjects (p = 0.001. 0.007 respectively). Aberrant expressions of EVI1 and MEF2C showed a significant negative correlation with hemoglobin levels (p = 0.034, 0.025 respectively), & bone marrow blasts (p = 0.007, 0.002 respectively). 11q23 translocation was significantly associated with EVI1 and MEF2C (p = 0.004 and 0.02 respectively). Also, t (9;22) was significantly associated with EVI1 and MEF2C (p = 0.01 and 0.03 respectively), higher expression of EVI1 and MEF2C were significantly associated with inferior outcome after induction therapy (p = 0.001 and 0.018 respectively) and shorter overall survival (p = 0.001, 0.014 respectively). In conclusion, EVI1 & MEF2C were significantly expressed in AML cases. EVI1 & MEF2C overexpression were significantly associated with 11q23 rearrangements and t (9;22) and were indicators for poor outcome in adult AML patients; These results could be a step towards personalized therapy in those patients.

Promoter-centred chromatin interactions associated with EVI1 expression in EVI1+3q- myeloid leukaemia cells.

EVI1 expression is associated with poor prognosis in myeloid leukaemia, which can result from Chr.3q alterations that juxtapose enhancers to induce EVI1 expression via long-range chromatin interactions. More often, however, EVI1 expression occurs unrelated to 3q alterations, and it remained unclear if, in these cases, EVI1 expression is similarly caused by aberrant enhancer activation. Here, we report that, in EVI1+3q- myeloid leukaemia cells, the EVI1 promoter interacts via long-range chromatin interactions with promoters of distally located, active genes, rather than with enhancer elements. Unlike in 3q+ cells, EVI1 expression and long-range interactions appear to not depend on CTCF/cohesin, though EVI1+3q- cells utilise an EVI1 promoter-proximal site to enhance its expression that is also involved in CTCF-mediated looping in 3q+ cells. Long-range interactions in 3q- cells connect EVI1 to promoters of multiple genes, whose transcription correlates with EVI1 in EVI1+3q- cell lines, suggesting a shared mechanism of transcriptional regulation. In line with this, CRISPR interference-induced silencing of two of these sites minimally, but consistently reduced EVI1 expression. Together, we provide novel evidence of features associated with EVI1 expression in 3q- leukaemia and consolidate the view that EVI1 in 3q- leukaemia is largely promoter-driven, potentially involving long-distance promoter clustering.

A novel missense mutation in the MECOM gene in a Chinese boy with radioulnar synostosis with amegakaryocytic thrombocytopenia.

Radioulnar synostosis with amegakaryocytic thrombocytopenia (RUSAT) type 2, caused by MDS1 and EVI1 complex locus (MECOM) gene mutations, is a rare inherited bone marrow failure syndrome (IBMFS) with skeletal anomalies, characterized by varying presentation of congenital thrombocytopenia (progressing to pancytopenia), bilateral proximal radioulnar synostosis, and other skeletal abnormalities. Due to limited knowledge and heterogenous manifestations, clinical diagnosis of the disease is challenging. Here we reported a novel MECOM mutation in a Chinese boy with typical clinical features for RUSAT-2. Trio-based whole exome sequencing of buccal swab revealed a novel heterozygous missense mutation in exon 11 of the MECOM gene (chr3:168818673; NM_001105078.3:c.2285G > A). The results strongly suggest that the variant was a germline mutation and disease-causing mutation. The patient received matched unrelated donor hematopoetic stem cell transplantation (HSCT). This finding was not only expanded the pathogenic mutation spectrum of MECOM gene, but also provided key information for clinical diagnosis and treatment of RUSAT-2.

Preclinical efficacy of targeting epigenetic mechanisms in AML with 3q26 lesions and EVI1 overexpression.

AML with chromosomal alterations involving 3q26 overexpresses the transcription factor (TF) EVI1, associated with therapy refractoriness and inferior overall survival in AML. Consistent with a CRISPR screen highlighting BRD4 dependency, treatment with BET inhibitor (BETi) repressed EVI1, LEF1, c-Myc, c-Myb, CDK4/6, and MCL1, and induced apoptosis of AML cells with 3q26 lesions. Tegavivint (TV, BC-2059), known to disrupt the binding of nuclear ß-catenin and TCF7L2/LEF1 with TBL1, also inhibited co-localization of EVI1 with TBL1 and dose-dependently induced apoptosis in AML cell lines and patient-derived (PD) AML cells with 3q26.2 lesions. TV treatment repressed EVI1, attenuated enhancer activity at ERG, TCF7L2, GATA2 and MECOM loci, abolished interactions between MYC enhancers, repressing AML stemness while upregulating mRNA gene-sets of interferon/inflammatory response, TGF-ß signaling and apoptosis-regulation. Co-treatment with TV and BETi or venetoclax induced synergistic in vitro lethality and reduced AML burden, improving survival of NSG mice harboring xenografts of AML with 3q26.2 lesions.

[The role of SF3B1 mutations in EVI1-rearranged myeloid neoplasms].

In acute myeloid leukemia (AML), EVI1 rearrangement represented by inv(3)(q21q26) or t(3;3)(q21;q26) causes EVI1 overexpression via structural rearrangement of an enhancer, and confers poor prognosis. My colleagues and I performed a mutational analysis of EVI1-rearranged myeloid neoplasms and identified SF3B1, a core RNA splicing factor, as the most commonly co-mutated gene. Indeed, latent leukemia development in transgenic mice bearing the humanized inv(3)(q21q26) allele was significantly accelerated by co-occurrence of Sf3b1 mutation. Intriguingly, we found that this SF3B1 mutant induced mis-splicing of EVI1 itself, which generated an aberrant EVI1 isoform with in-frame insertion of 6 amino acids near the DNA-binding domain of EVI1. This aberrant EVI1 isoform exhibited DNA-binding activity different from wild-type EVI1 and significantly enhanced the self-renewal capacity of murine hematopoietic stem cells. We also identified the cryptic branch point and exonic splicing enhancer required for this EVI1 mis-splicing induced by the SF3B1 mutant. These data provide a basis for further elucidation of the molecular mechanism and potential therapeutic candidates for EVI1-rearranged AML.

A novel mutation in MECOM affects MPL regulation in vitro and results in thrombocytopenia and bone marrow failure.

MECOM-associated syndrome (MECOM-AS) is a rare disease characterized by amegakaryocytic thrombocytopenia, progressive bone marrow failure, pancytopenia and radioulnar synostosis with high penetrance. The clinical phenotype may also include finger malformations, cardiac and renal alterations, hearing loss, B-cell deficiency and predisposition to infections. The syndrome, usually diagnosed in the neonatal period because of severe thrombocytopenia, is caused by mutations in the MECOM gene, encoding for the transcription factor EVI1. The mechanism linking the alteration of EVI1 function and thrombocytopenia is poorly understood. In a paediatric patient affected by severe thrombocytopenia, we identified a novel variant of the MECOM gene (p.P634L), whose effect was tested on pAP-1 enhancer element and promoters of targeted genes showing that the mutation impairs the repressive activity of the transcription factor. Moreover, we demonstrated that EVI1 controls the transcriptional regulation of MPL, a gene whose mutations are responsible for congenital amegakaryocytic thrombocytopenia (CAMT), potentially explaining the partial overlap between MECOM-AS and CAMT.

MECOM Deficiency: from Bone Marrow Failure to Impaired B-Cell Development.

MECOM deficiency is a recently identified inborn error of immunity and inherited bone marrow failure syndrome caused by haploinsufficiency of the hematopoietic transcription factor MECOM. It is unique among inherited bone marrow failure syndromes, many of which present during later childhood or adolescence, because of the early age of onset and severity of the pancytopenia, emphasizing the importance and gene dose dependency of MECOM during hematopoiesis. B-cell lymphopenia and hypogammaglobulinemia have been described in a subset of patients with MECOM deficiency. While the mechanisms underlying the B-cell deficiency are currently unknown, recent work has provided mechanistic insights into the function of MECOM in hematopoietic stem cell (HSC) maintenance. MECOM binds to regulatory enhancers that control the expression of a network of genes essential for HSC maintenance and self-renewal. Heterozygous mutations, as seen in MECOM-deficient bone marrow failure, lead to dysregulated MECOM network expression. Extra-hematopoietic manifestations of MECOM deficiency, including renal and cardiac anomalies, radioulnar synostosis, clinodactyly, and hearing loss, have been reported. Individuals with specific genotypes have some of the systemic manifestations with isolated mild thrombocytopenia or without hematologic abnormalities, highlighting the tissue specificity of mutations in some MECOM domains. Those infants with MECOM-associated bone marrow failure require HSC transplantation for survival. Here, we review the expanding cohort of patient phenotypes and accompanying genotypes resulting in MECOM deficiency, and the proposed mechanisms underlying MECOM regulation of human HSC maintenance and B-cell development.

High EVI1 and PARP1 expression as favourable prognostic markers in high-grade serous ovarian carcinoma.

BACKGROUND: Mechanisms of development and progression of high-grade serous ovarian cancer (HGSOC) are poorly understood. EVI1 and PARP1, part of TGF-ß pathway, are upregulated in cancers with DNA repair deficiencies with DNA repair deficiencies and may influce disease progression and survival. Therefore we questioned the prognostic significance of protein expression of EVI1 alone and in combination with PARP1 and analyzed them in a cohort of patients with HGSOC. METHODS: For 562 HGSOC patients, we evaluated EVI1 and PARP1 expression by immunohistochemical staining on tissue microarrays with QuPath digital semi-automatic positive cell detection. RESULTS: High EVI1 expressing (> 30% positive tumor cells) HGSOC were associated with improved progression-free survival (PFS) (HR = 0.66, 95% CI: 0.504-0.852, p = 0.002) and overall survival (OS) (HR = 0.45, 95% CI: 0.352-0.563, p < 0.001), including multivariate analysis. Most interestingly, mutual high expression of both proteins identifies a group with particularly good prognosis. Our findings were proven technically and clinically using bioinformatical data sets for single-cell sequencing, copy number variation and gene as well as protein expression. CONCLUSIONS: EVI1 and PARP1 are robust prognostic biomarkers for favorable prognosis in HGSOC and imply further research with respect to their reciprocity.

Epigenetic landscape reveals MECOM as an endothelial lineage regulator.

A comprehensive understanding of endothelial cell lineage specification will advance cardiovascular regenerative medicine. Recent studies found that unique epigenetic signatures preferentially regulate cell identity genes. We thus systematically investigate the epigenetic landscape of endothelial cell lineage and identify MECOM to be the leading candidate as an endothelial cell lineage regulator. Single-cell RNA-Seq analysis verifies that MECOM-positive cells are exclusively enriched in the cell cluster of bona fide endothelial cells derived from induced pluripotent stem cells. Our experiments demonstrate that MECOM depletion impairs human endothelial cell differentiation, functions, and Zebrafish angiogenesis. Through integrative analysis of Hi-C, DNase-Seq, ChIP-Seq, and RNA-Seq data, we find MECOM binds enhancers that form chromatin loops to regulate endothelial cell identity genes. Further, we identify and verify the VEGF signaling pathway to be a key target of MECOM. Our work provides important insights into epigenetic regulation of cell identity and uncovered MECOM as an endothelial cell lineage regulator.

Mecom mutation related to radioulnar synostosis with amegakaryocytic thrombocytopenia reduces HSPCs in mice.

Radioulnar synostosis with amegakaryocytic thrombocytopenia (RUSAT) is an inherited bone marrow failure syndrome characterized by the congenital fusion of the forearm bones. RUSAT is largely caused by missense mutations that are clustered in a specific region of the MDS1 and EVI1 complex locus (MECOM). EVI1, a transcript variant encoded by MECOM, is a zinc finger transcription factor involved in hematopoietic stem cell maintenance that induce leukemic transformation when overexpressed. Mice with exonic deletions in Mecom show reduced hematopoietic stem and progenitor cells (HSPCs). However, the pathogenic roles of RUSAT-associated MECOM mutations in vivo have not yet been elucidated. To investigate the impact of the RUSAT-associated MECOM mutation on the phenotype, we generated knockin mice harboring a point mutation (translated into EVI1 p.H752R and MDS1-EVI1 p.H942R), which corresponds to an EVI1 p.H751R and MDS1-EVI1 p.H939R mutation identified in a patient with RUSAT. Homozygous mutant mice died at embryonic day 10.5 to 11.5. Heterozygous mutant mice (Evi1KI/+ mice) grew normally without radioulnar synostosis. Male Evi1KI/+ mice, aged between 5 and 15 weeks, exhibited lower body weight, and those aged ≥16 weeks showed low platelet counts. Flow cytometric analysis of bone marrow cells revealed a decrease in HSPCs in Evi1KI/+ mice between 8 and 12 weeks. Moreover, Evi1KI/+ mice showed delayed leukocyte and platelet recovery after 5-fluorouracil-induced myelosuppression. These findings suggest that Evi1KI/+ mice recapitulate the bone marrow dysfunction in RUSAT, similar to that caused by loss-of-function Mecom alleles.

Expanded phenotypic and hematologic abnormalities beyond bone marrow failure in MECOM-associated syndromes.

The MECOM gene encodes multiple protein isoforms that are essential for hematopoietic stem cell self-renewal and maintenance. Germline MECOM variants have been associated with congenital thrombocytopenia, radioulnar synostosis and bone marrow failure; however, the phenotypic spectrum of MECOM-associated syndromes continues to expand and novel pathogenic variants continue to be identified. We describe eight unrelated patients who add to the previously known phenotypes and genetic defects of MECOM-associated syndromes. As each subject presented with unique MECOM variants, the series failed to demonstrate clear genotype-to-phenotype correlation but may suggest a role for additional modifiers that affect gene expression and subsequent phenotype. Recognition of the expanded hematologic and non-hematologic clinical features allows for rapid molecular diagnosis, early identification of life-threatening complications, and improved genetic counseling for families. A centralized international publicly accessible database to share annotated MECOM variants would advance their clinical interpretation and provide a foundation to perform functional MECOM studies.

The adverse impact of ecotropic viral integration site-1 (EVI1) overexpression on the prognosis of acute myeloid leukemia with KMT2A gene rearrangement in different risk stratification subtypes.

INTRODUCTION: AML patients with KMT2A-MLLT3 and other 11q23 abnormalities belong to the intermediate and high-risk level groups, respectively. Whether the poor prognostic value of Ecotropic Viral Integration site-1 (EVI1) overexpression suits either the subtypes of KMT2A-MLLT3 or Non-KMT2A-MLLT3 AML patients (intermediate and high risk group) needs to be further investigated. METHODS: We retrospectively analyzed the clinical characteristics of 166 KMT2A-r and KMT2A-PTD AML patients. RESULTS: For the Non-KMT2A-MLLT3 group, patients in the EVI1-high subgroup had shorter OS and DFS and higher CIR than those in the EVI1-low subgroup (p = .027, p = .018, and p = .020, respectively). Additionally, both KMT2A-MLLT3 and Non-KMT2A-MLLT3 patients who received chemotherapy alone had poorer prognosis than patients who also received allogeneic hematopoietic stem cell transplant (allo-HSCT) regardless of their EVI1 expression level (all p < .001). For transplanted patients with KMT2A-MLLT3 or Non-KMT2A-MLLT3 rearrangement, the EVI1-high subgroup had worse prognosis than the EVI1-low subgroup (all p < .05). The 2-year CIR of the KMT2A-MLLT3 and Non-KMT2A-MLLT3 groups with high EVI1 expression was high (52% and 49.6%, respectively). However, for patients with low EVI1 expression, the 2-year CIR of transplanted patients with KMT2A-MLLT3 and Non-KMT2A-MLLT3 was relatively low. CONCLUSIONS: Our study showed that for the Non-KMT2A-MLLT3 group, the EVI1-high group had shorter OS and DFS than the EVI1-low group. High EVI1 expression showed an adverse effect in AML with KMT2A rearrangement in different risk stratification subtypes. For the EVI1-high patients with non-KMT2A-MLLT3 rearrangement, other novel regimens towards relapse should be taken into consideration.

EVI1 exerts distinct roles in AML via ERG and cyclin D1 promoting a chemoresistant and immune-suppressive environment.

Aberrant expression of ecotropic viral integration site-1 (EVI1+) is associated with very poor outcomes in acute myeloid leukemia (AML), mechanisms of which are only partially understood. Using the green fluorescent protein reporter system to monitor EVI1 promoter activity, we demonstrated that Evi1high KMT2A-MLLT1-transformed AML cells possess distinct features from Evi1low cells: the potential for aggressive disease independent of stem cell activity and resistance to cytotoxic chemotherapy, along with the consistent gene expression profiles. RNA sequencing and chromatin immunoprecipitation sequencing in EVI1-transformed AML cells and normal hematopoietic cells combined with functional screening by cell proliferation-related short hairpin RNAs revealed that the erythroblast transformation-specific transcription factor ERG (E26 transformation-specific [ETS]-related gene) and cyclin D1 were downstream targets and therapeutic vulnerabilities of EVI1+ AML. Silencing Erg in murine EVI1+ AML models severely impaired cell proliferation, chemoresistance, and leukemogenic capacity. Cyclin D1 is also requisite for efficient EVI1-AML development, associated with gene expression profiles related to chemokine production and interferon signature, and T- and natural killer-cell exhaustion phenotype, depending on the interferon gamma (IFN-γ)/STAT1 pathway but not on CDK4/CDK6. Inhibiting the IFN-γ/STAT1 pathway alleviated immune exhaustion and impaired EVI1-AML development. Overexpression of EVI1 and cyclin D1 was associated with IFN-γ signature and increased expression of chemokines, with increased exhaustion molecules in T cells also in human AML data sets. These data collectively suggest that ERG and cyclin D1 play pivotal roles in the biology of EVI1+ AML, where ERG contributes to aggressive disease nature and chemoresistance, and cyclin D1 leads to IFN-γ signature and exhausted T-cell phenotypes, which could potentially be targeted.

EVI1 drives leukemogenesis through aberrant ERG activation.

Chromosomal rearrangements involving the MDS1 and EVI1 complex locus (MECOM) on chromosome 3q26 define an aggressive subtype of acute myeloid leukemia (AML) that is associated with chemotherapy resistance and dismal prognosis. Established treatment regimens commonly fail in these patients, therefore, there is an urgent need for new therapeutic concepts that will require a better understanding of the molecular and cellular functions of the ecotropic viral integration site 1 (EVI1) oncogene. To characterize gene regulatory functions of EVI1 and associated dependencies in AML, we developed experimentally tractable human and murine disease models, investigated the transcriptional consequences of EVI1 withdrawal in vitro and in vivo, and performed the first genome-wide CRISPR screens in EVI1-dependent AML. By integrating conserved transcriptional targets with genetic dependency data, we identified and characterized the ETS transcription factor ERG as a direct transcriptional target of EVI1 that is aberrantly expressed and selectively required in both human and murine EVI1-driven AML. EVI1 controls the expression of ERG and occupies a conserved intragenic enhancer region in AML cell lines and samples from patients with primary AML. Suppression of ERG induces terminal differentiation of EVI1-driven AML cells, whereas ectopic expression of ERG abrogates their dependence on EVI1, indicating that the major oncogenic functions of EVI1 are mediated through aberrant transcriptional activation of ERG. Interfering with this regulatory axis may provide entry points for the development of rational targeted therapies.

Clinical features and prognostic implications of ecotropic viral integration site 1 (EVI1) in childhood acute lymphoblastic leukemia.

In contrast to the extensive knowledge on EVI1 in myeloid malignancies, few data are available on the EVI1 transcript in pediatric ALL. The purpose of this study was to examine the clinical and biological significance of EVI1 and validate its prognostic significance in pediatric patients with ALL. Here, we examined the clinical and biological significance of EVI1 expression, as measured by real-time polymerase chain reaction (PCR) in 837 children with newly diagnosed ALL treated on the National Protocol of Childhood Leukemia in China (NPCLC)-ALL-2008 protocol, and aimed to explore their prognostic significance in pediatric ALL patients. The EVI1 expression was detected in 27 of 837 (3.2%) patients. No statistically significant differences in prednisone response, complete remission (CR) rates and relapse rates were found between EVI1 overexpression (EVI1+) group and EVI1- group. Moreover, we found no significant difference in event-free survival (EFS) and overall survival (OS) between these two groups, also multivariate analysis did not identify EVI1+ as an independent prognostic factor. In the subgroup analysis, there was no difference in clinical outcome between EVI1+ and EVI1- patients in standard­risk (SR), intermediate-risk (IR) and high-risk (HR) groups. In the minimal residual disease (MRD)<10-4 group, EVI1+ patients have significantly lower EFS and OS rates compared to EVI1- patients. Further large­scale and well­designed prospective studies are required to confirm the results in the future.

Case report: A rare case of coexisting Waldenstrom Macroglobulinemia and B-cell acute lymphoblastic leukemia with KMT2D and MECOM mutations.

Background: Waldenstrom Macroglobulinemia (WM) is a rare and indolent lymphoma of B-cell origin characterized by elevated monoclonal IgM, with MYD88L265P mutation and CXCR4 mutation as common molecular alterations. B-cell Acute Lymphoblastic Leukemia (B-ALL) is clinically heterogeneous, characterized by abnormal proliferation and aggregation of immature lymphocytes in the bone marrow and lymphoid tissue. WM and ALL are hematologic malignancies of B-cell origin with completely different clinical manifestations and biological features. KMT2D and MECOM mutations are very rare in ALL and usually indicate poor disease prognosis. The coexistence of WM and ALL with KMT2D and MECOM mutations have not been reported. Case presentation: A 74-year-old female patient was diagnosed with WM in July 2018 and received four cycles of chemotherapy of bortezomib and dexamethasone. In November 2018, she received immunomodulator thalidomide as maintenance therapy. In November 2020, Bruton's Tyrosine Kinase inhibitors (BTKi) has been introduced into the Chinese market and she took zanubrutinib orally at a dose of 80 mg per day. The disease remained in remission. In December 2021, she presented with multiple enlarged lymph nodes throughout the body. Bone marrow and next-generation sequencing (NGS) suggested the coexistence of WM and B-ALL with KMT2D and MECOM mutations. The patient was treated with zanubrutinib in combination with vincristine and dexamethasone, after which she developed severe myelosuppression and septicemia. The patient finally got remission. Due to the patient's age and poor status, she refused intravenous chemotherapy and is currently treated with zanubrutinib. Conclusions: The coexistence of WM and B-ALL is very rare and has not been reported. The presence of both KMT2D and MECOM mutations predicts a poor prognosis and the possibility of insensitivity to conventional treatment options. BTKi achieves its anti-tumor effects by inhibiting BTK activation and blocking a series of malignant transformations in B-cell tumors. In addition, it also acts on T-cell immunity and tumor microenvironment. Combination therapy based on BTKi may improve the prognosis of this patient.