Justin L. Engel

Abstract Complex genome rearrangements can be generated by the catastrophic pulverization of missegregated chromosomes trapped within micronuclei through a process known as chromothripsis 1–5 . As each chromosome contains a single centromere, it remains unclear how acentric fragments derived from shattered chromosomes are inherited between daughter cells during mitosis 6 . Here we tracked micronucleated chromosomes with live-cell imaging and show that acentric fragments cluster in close spatial proximity throughout mitosis for asymmetric inheritance by a single daughter cell. Mechanistically, the CIP2A–TOPBP1 complex prematurely associates with DNA lesions within ruptured micronuclei during interphase, which poises pulverized chromosomes for clustering upon mitotic entry. Inactivation of CIP2A–TOPBP1 caused acentric fragments to disperse throughout the mitotic cytoplasm, stochastically partition into the nucleus of both daughter cells and aberrantly misaccumulate as cytoplasmic DNA. Mitotic clustering facilitates the reassembly of acentric fragments into rearranged chromosomes lacking the extensive DNA copy-number losses that are characteristic of canonical chromothripsis. Comprehensive analysis of pan-cancer genomes revealed clusters of DNA copy-number-neutral rearrangements—termed balanced chromothripsis—across diverse tumour types resulting in the acquisition of known cancer driver events. Thus, distinct patterns of chromothripsis can be explained by the spatial clustering of pulverized chromosomes from micronuclei.

CRISPR Cas9-based screening is a powerful approach for identifying and characterizing novel drug targets. Here, we elucidate the synthetic lethal mechanism of deubiquitinating enzyme USP1 in cancers with underlying DNA damage vulnerabilities, specifically BRCA1/2 mutant tumors and a subset of BRCA1/2 wild-type (WT) tumors. In sensitive cells, pharmacologic inhibition of USP1 leads to decreased DNA synthesis concomitant with S-phase-specific DNA damage. Genome-wide CRISPR-Cas9 screens identify RAD18 and UBE2K, which promote PCNA mono- and polyubiquitination respectively, as mediators of USP1 dependency. The accumulation of mono- and polyubiquitinated PCNA following USP1 inhibition is associated with reduced PCNA protein levels. Ectopic expression of WT or ubiquitin-dead K164R PCNA reverses USP1 inhibitor sensitivity. Our results show, for the first time, that USP1 dependency hinges on the aberrant processing of mono- and polyubiquitinated PCNA. Moreover, this mechanism of USP1 dependency extends beyond BRCA1/2 mutant tumors to selected BRCA1/2 WT cancer cell lines enriched in ovarian and lung lineages. We further show PARP and USP1 inhibition are strongly synergistic in BRCA1/2 mutant tumors. We postulate USP1 dependency unveils a previously uncharacterized vulnerability linked to posttranslational modifications of PCNA. Taken together, USP1 inhibition may represent a novel therapeutic strategy for BRCA1/2 mutant tumors and a subset of BRCA1/2 WT tumors.

Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic-lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2-M arrest and DNA damage. The VRK1-VRK2 synthetic-lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line-derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM. SIGNIFICANCE: A paralog synthetic-lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.