Introduction: Multiple myeloma (MM) is a genetically complex disease with extensive clonal heterogeneity. Substantial genomic instability in MM is illustrated by extensive copy number variations (CNVs) that can be detected in almost every MM patient. The molecular basis of this genomic instability in MM is not clear. Linker histones are dynamic components of chromatin and mutations in these molecules are present in ~6% of MM patients. Two of the linker histone super-family, HIST1H1Eand HIST1H1Care the most frequently mutated members of the family in MM and their mutations mostly occur in a clonal fashion. Interestingly, it has been reported in human cell lines and in other species that linker histone loss affects DNA damage/repair pathways and leads to transcription-replication conflicts. Based on these data we hypothesized that mutation or genomic loss of linker histones affects the genome stability of MM cells.
To test this hypothesis, we developed an experimental system using CRISPR/Cas9 genome editing to generate MM linker histone-deficient cells. Low-pass whole genome sequencing (LPWGS), immunoblotting and immunofluorescent experiments were performed for genomic, molecular and functional characterization. We found that HIST1H1E,HIST1H1Cand H1FXwere the most abundantly expressed members of the linker histone family in primary myeloma cells and that myeloma cells have the highest dependency on HIST1H1Eand HIST1H1Cwhen compared to all other cancer cell lines derived from other tissues. We used OPM2 and U266 myeloma cell lines and generated knock-out variants of HIST1H1E, HIST1H1Cand H1FXlinker histones by inserting a biallelic stop codon, followed by generation of individual single-cell clones that were used as replicates. We first asked if linker histone deficient cells preserve genome stability. To address this question, we performed low pass whole genome sequencing and found more copy number abnormalities in linker histone deficient myeloma cells, when compared to wild-type cells. Moreover, linker histone deficient cells showed increased DNA damage as indicated by higher frequency of nuclear foci that were positive for damage dependent phosphorylation of the histone variant H2AX ( γH2AX). This was associated with an increased frequency of micronuclei in linker histones deficient cells, suggesting defects in mitotic fidelity and in genome stability. These micronuclei were positive for γH2AX by microscopic staining, indicative of DNA damage. We then asked if the DNA damage in micronuclei is due to defective and asynchronous DNA replication when the myeloma cells are exposed to etoposide, a topoisomerase inhibitor that induces DNA replication stress and double-strand DNA breaks (DSBs). Etoposide treatment of myeloma cells caused DNA replication stress, as measured by immunofluorescent staining of micronuclei for Replication Protein A (RPA).
Conclusions: Our results demonstrate that loss of linker histones is associated with increased copy number abnormalities, extensive DNA damage and increased frequency of micronuclei, most likely as a consequence of replication stress. These data provide a potential mechanism of how chromatin structure dynamics preserve genome stability in myeloma cells.
Disclosures
Lohr: Celgene: Research Funding; T2 Biosystems: Honoraria.
Human subtelomeric copy number gains suggest a DNA replication mechanism for formation: beyond breakage–fusion–bridge for telomere stabilization
