Loss of Lamin B1 in Myeloid Neoplasms with 5q Deletion Causes Myeloid-Biased Hematopoiesis and Pelger-Huet Nuclear Anomaly
Abstract Hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations and cytogenetic abnormalities leading to myeloid neoplasms (MDS/AML). A subgroup of 10-20% MDS and AML cases undergo complex chromosomal rearrangements associated with TP53 mutations and poor prognosis. One of the most common chromosomal events in high-risk myeloid neoplasms is deletion of chromosome 5q (del(5q)). In contrast to 5q- syndrome, characterized by a distal commonly deleted region (CDR) and good prognosis, the critical CDR in high-risk MDS/AML centers on 5q31. However, the key genes in the high-risk 5q-deleted region that contribute to dysregulation of hematopoiesis, chromosomal instability, and disease progression remain poorly characterized. Progression to high-risk MDS/AML is often accompanied by morphologic dysplasia in the form of Pelger-Huët cells, abnormal neutrophils with bilobed or unilobed nuclei and coarse clumping of the nuclear chromatin. The inherited form of Pelger-Huët anomaly (PHA) is caused by mutations in LBR, which encodes lamin B receptor. Acquired PHA or pseudo-PHA is commonly seen in myeloid malignancies, often in high risk MDS and AML with TP53 mutations. While LBR is not mutated in MDS/AML, its main interaction partner lamin B1/LMNB1 is encoded in the high-risk 5q-deleted region. Lamins organize chromatin into perinuclear compartments and regulate diverse biological processes, including gene expression and DNA repair. The frequent deletion of LMNB1 in 5q-deleted MDS and AML led us to hypothesize that LMNB1 loss drives both nuclear dysmorphology and functional HSC defects in malignant transformation. LMNB1 expression was broadly decreased in MDS and AML, particularly in del(5q) cases, 83% of which had deletions of the LMNB1 locus. We show that in a clinical cohort of MDS patients, the Pelger-Huët nuclear phenotype is strongly associated with del5q. To understand the role of LMNB1, we performed lentiviral shRNA knockdown in normal umbilical cord blood (CB) CD34 + HSPCs followed by transplantation into immunodeficient NSG mice. To study the role of lamin B1 in 5q-deleted MDS, we established an in vitro patient-derived induced pluripotent stem cell (iPSC) model by reprograming an MDS patient with TP53 mutation and complex karyotype, and deriving MDS HSPCs with TP53+/R209fs alone or TP53+/R209fs with an isolated del5q spanning 5q22-5q31.1 encoding LMNB1 (TP53;del5q). Using these model systems we show that LMNB1 loss is both necessary and sufficient to cause Pelger-Huët nuclear morphology in normal and MDS neutrophils. Additionally, we find that LMNB1-deficient human HSCs display increased self-renewal and profound myeloid lineage bias in vitro and in vivo. Single cell RNA expression data from LMNB1-deficient cells in vivo shows mis-expression of lineage-specifying transcription factors in single multipotent progenitors. LMNB1 deficiency also results in poor marking and propagation of unrepaired DNA breaks, leading to genome instability. The role of LMNB1 in maintaining chromatin organization prompted us to use chromosome conformation capture (Hi-C) to visualize the 3D chromatin organization of LMNB1-deficient HSPCs. We show that lamin B1 regulates enhancer-promoter loops suggesting that lamin B1 loss dysregulates HSC function by altering enhancer-promoter interactions at lineage-specifying transcription factor loci. Together, we identify lamin B1 as a novel 5q gene that contributes to malignant transformation by driving enhanced self-renewal, myeloid-bias, and genome instability. Furthermore, lamin B1 deletion is the genetic cause of acquired PHA in myeloid malignancies, providing a potential biomarker for high risk neoplasms. Disclosures Becker: Glycomimetics: Research Funding; CVS Caremark: Consultancy; Abbie: Research Funding; BMS: Research Funding; Pfizer: Research Funding; Cardiff Oncology: Research Funding; SecuraBio: Research Funding.
