microRNA, immunophenotypic, and somatic alterations in hematologic malignancies

Clinical Center

Investigators

Linked publications & trials

Abstract

A. miRNA profiles of GATA2 deficient cells. This is an ongoing project that was initially completed in 2020, but reactivated in 2021 due to additional experiments requested by reviewers for publication. GATA2 deficiency is caused by germline mutations in GATA2, leading to loss of function or haploinsufficiency of the GATA2 transcription factor. GATA2 deficiency is manifested by severe cytopenias of multiple cell lineages, including monocytes, B- and NK-cells, susceptibility to opportunistic infections and strong propensity to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia. Exact mechanisms of progressive cytopenias remain unclear. MicroRNA (miRNA) represents a unique mechanism of post-transcriptional gene regulation. In this study, miRNA profiles were evaluated and eight miRNAs were found to be differentially expressed ( 2-fold, p 0.05) in patient derived cell lines in comparison to controls. miR-9, miR-181a-2-3p, miR-181c, miR-181c-3p, miR-486-3p, and miR-582 showed increased expression, while miR-223 and miR-424-3p showed decreased expression. Cell death assays indicated that miR-181c potently induces cell death. TargetScan predicted that miR-181c targets MCL1. Transfection assays confirmed that miR-181c targets MCL1 3UTR, resulting in significantly reduced MCL1 mRNA and decreased live cell numbers. Real time RT-PCR experiments demonstrated that MCL1 levels were decreased in GATA2 deficiency cell lines and in GATA2 patient bone marrow mononuclear cells. Additional archived clinical bone marrow flow cytometry and morphologic data was analyzed on the same patient samples that were assayed for MCL1 demonstrating significant loss of monocytes, B-cells, NK-cells and dendritic cells (in comparison to healthy control marrow) which correlated with decreased MCL1. In transfection experiments, GATA2 expression led to significant repression of miR-181c expression. Conversely, knock-down of GATA2 by RNA interference led to increased miR-181c expression. These findings indicate that miR-181c expression is increased and MCL1 levels decreased in GATA2 deficiency cells, and that GATA2 represses miR-181c transcription. Increased miR-181c may contribute to elevated cell death by regulating MCL1 in GATA2 deficient cells, and may thereby contribute to cellular loss and bone marrow failure. A manuscript detailing these studies was published in 2021 in the Journal of Leukocyte Biology, Wang W. et. al. PMID 34270823. B. Immunophenotypic and morphologic analyses of rare diseases with bone marrow and peripheral blood pathology. 1. During 2020 I collaborated with David Beck, Peter Grayson and Daniel Kastner to evaluate patients with severe inflammation, rheumatologic disease and myelodysplasia. All of the patients had underlying mutations in UBA1. I discovered abnormal vacuolization of myeloid and erythroid precursors in the bone marrow of all patients. A significant subset of patients developed MDS or Myeloma/MGUS. UBA1 represents one of the first genes identified with somatic mutations that link severe autoinflammation by clonal myeloid cells and predisposition to hematologic malignancies. The first paper was published in December 2020 in the New England Journal of Medicine, and the disease was called VEXAS for Vacuoles, E1 enzyme (encoded by UBA1), X-linked, Autoinflammation, and Somatic. After the original VEXAS paper was published, my team worked with Neal Young's team in NHLBI to characterize in detail the benign and malignant hematologic manifestations of VEXAS. We combined pathologic, molecular, and clinical studies to generate a informative manuscript that was published in Blood Advances in August 2021. In brief, bone marrow evaluation was performed in 16 VEXAS patients who underwent marrow biopsy for cytopenias to rule out MDS. All subjects were male and had a history of severe autoinflammatory/rheumatologic manifestations and a somatic UBA1 mutation involving p.Met41. The median age was 57 years (range 45-77) at disease onset. Multi-organ systemic inflammatory symptoms were present in all with the commonest being: fatigue (100%), recurrent fevers (88%), pulmonary infiltrates (87%), skin lesions (88%), ear and nose chondritis (73% and 47%), and vasculitis (67%). Ten patients had hematological disorders: MDS (6/16), multiple myeloma (2/16), monoclonal gammopathy of undetermined significance (2/16), and monoclonal B cell lymphocytosis (2/16) with a few having two co-existing clonal processes. While macrocytic anemia (100%) and lymphopenia (80%) were prevalent in nearly all patients with VEXAS, thrombocytopenia and neutropenia were more common in patients with progression to MDS. All marrows in VEXAS had prominent cytoplasmic vacuoles in myeloid and erythroid precursors. None of the nine patients tested had copper deficiency which is also associated with vacuolization of marrow precursors. In addition, most marrows were hypercellular with myeloid hyperplasia, erythroid hypoplasia, and had varying degrees of dysplasia. All diagnosed MDS cases were lower-risk (low blast count, very good-intermediate cytogenetics) as per standard prognostic scoring with no known progression to leukemia. Additionally, 10/16 patients had thrombotic events including venous thromboembolism and arterial stroke. While VEXAS presents symptomatically as a rheumatologic autoinflammatory disease, morbidity and mortality are associated with progression to hematologic disease. Given the increased risk of developing MDS and multiple myeloma, we believe that surveillance for disease progression is critical. MDS and somatic mutation analysis in VEXAS. Of the 6 patients that developed MDS, 4 were diagnosed with MDS-MLD and 2 with MDS-SLD. Half of MDS cases had a normal karyotype. Three patients had cytogenetic abnormalities involving t(3;12)(q21;q13) in one patient; del (5q) and del (13) in another patient and del(20)(q11.2;q13.3) in the remaining patient. Somatic mutation analysis was performed on 4 available marrow specimens and showed that two cases had DNMT3A mutations with variant allele frequencies of 42% and 44%. One patient had no mutations detected and another had very low frequency mutations in GNA11 (3.3%) and CSF1R (3.1%) of uncertain significance. Only one of the patients without MDS had a mutation identified, involving EZH2 with a VAF of 21%. 2. GATA2 deficiency. In an ongoing collaborative study of 106 patients with germline GATA2 mutations we combined the results of NGS myeloid panel sequencing of bone marrow performed in my laboratory, with whole exome sequencing (WES) performed in Dennis Hicksteins laboratory. Correlative studies have been performed on this large dataset to characterize the molecular landscape of GATA2-deciency associated bone marrow disorder (G2BMID) characterized by hypocellular marrows without overt dysplasia, and cases with overt myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), or chronic myelomonocytic leukemia (CMML). 3. Bone marrow failure, immunodeficiency, and hematologic malignancies. I continued collaborative support of multiple clinical and translational research protocols in areas of bone marrow failure, immunodeficiency, hematologic malignancies and other rare diseases resulting in unexplained cytopenia or hematologic manifestations.