Mutational Analysis of the Genome Stability Factor BOD1L1 in Myeloproliferative Neoplasm
Authors: Higgs Martin R , Browne Sofia , Collinson Ryan , Fuller Kathy , Erber Wendy N. , Kleppe Maria , Sirnosian Ben , Natsoulis Georges , Guo Belinda , Rienhoff Jr. Hugh Young 
Published: December 2023

The myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders that include essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF) which arise from somatic mutations in JAK2, MPL or CALR, but which can evolve to acute myeloid leukemia (AML) by the acquisition of additional somatic mutations.
To investigate the contribution that mutations might make to the pathogenesis or clinical course of MPNs, 261 genes recurrently mutated in myeloid malignancies were sequenced in DNA from germline and blood samples from patients with AML, ET and MF enrolled in studies of the LSD1 inhibitor bomedemstat. Earlier work identified somatic mutations inthegene BOD1L1 in isolated megakaryocytes in 6 of 12 patients with MPN with mutations suggestive of loss-of-function (Guo et al., 2017). BOD1L1 plays a key role in multiple pathways of cellular DNA repair in conjunction with the histone methyltransferase SETD1A, which regulates transcription and DNA repair (Higgs et al. 2018; Hoshii et al. 2018. We previously demonstrated that BOD1L1 prevents uncontrolled resection of damaged DNA forks under conditions of replication stress, and that its loss leads to severe genome instability (Higgs et al., 2015, 2018). In addition, BOD1L1 promotes repair of DNA double-strand breaks after ionizing radiation through its interaction with the DNA repair protein RIF1 (Bayley et al., 2022).
This sequencing-based analysis identified specific germline mutations in BOD1L1 that were 3-6 times more prevalent in MPN patients compared to a control population (gnomAD). This enrichment of BOD1L1 mutations was confirmed in whole-exome sequencing from an independent data set (EGA) of 183 patients with MPNs. We predicted these highly deleterious mutations, or at least a subset, may compromise the DNA repair functions of BOD1L1 and potentially influence the development and/or progression of MPN.
To investigate this prediction, we disrupted BOD1L1 using a doxycycline-inducible CRISPR-Cas9 system in HeLa cells and complemented them with gRNA-resistant wild-type BOD1L1,or one of six mutants distributed throughout the protein. None of the mutants affected BOD1L1 protein expression; however, two of the six variants were defective for RIF1 binding by co-immunoprecipitation/proximity ligation, failed to recruit this key DNA repair factor to radiation-induced double-strand breaks, and compromised repair of these breaks. Moreover, single molecule DNA fiber analyses revealed that these same two mutants were unable to protect stalled replication forks against resection, leading to elevated levels of genomic damage.
To explore the functional consequences of BOD1L1 loss-of-function on megakaryocytes, the gene was silenced using shRNA in human megakaryoblastic JAK2 V617F SET-2 cells, differentiated with thrombopoietin, and analyzed by flow cytometry. These cells were CD41-positive, consistent with megakaryocytic differentiation, and showed increased DNA damage (γH2AX) compared to controls. Depletion of BOD1L1 also lead to a greater number of cells with 2n nuclear content, and fewer with ≥8n, compared with controls, suggesting that the loss of BOD1L1 led to impaired polyploidization likely due to unrepaired DNA damage and subsequent cell death.
In summary, we have shown that MPN patients have a high prevalence of mutations in the key genome instability factor BOD1L1 and that a subset of these mutations compromised its DNA repair functions and increased genome instability. The presence of BOD1L1 somatic mutations in megakaryocytes in MF patients may deregulate polyploidization, altering megakaryocyte function that may contribute to the development of bone marrow fibrosis. That no patients with BOD1L1 germline mutations progressed to AML in these clinical studies, coupled with the fact that these variants were not enriched in patients with AML, invites the possibility that BOD1L1 mutations may be synthetically lethal with other somatic mutations that result in genome instability and, hence, protect against progression to AML.