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DNA double-strand breaks are repaired by multiple pathways, including non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ). The balance of these pathways is dependent on the local chromatin context, but the underlying mechanisms are poorly understood. By combining knockout screening with a dual MMEJ:NHEJ reporter inserted in 19 different chromatin environments, we identified dozens of DNA repair proteins that modulate pathway balance dependent on the local chromatin state. Proteins that favor NHEJ mostly synergize with euchromatin, while proteins that favor MMEJ generally synergize with distinct types of heterochromatin. Examples of the former are BRCA2 and POLL, and of the latter the FANC complex and ATM. Moreover, in a diversity of human cancer types, loss of several of these proteins alters the distribution of pathway-specific mutations between heterochromatin and euchromatin. Together, these results uncover a complex network of proteins that regulate MMEJ:NHEJ balance in a chromatin context-dependent manner. DNA double-strand breaks are repaired by multiple pathways. The balance of these pathways depends on the local chromatin context, but the underlying mechanisms are poorly understood. Here the authors uncover a network of proteins that regulate pathway balance in a chromatin context-dependent manner.

Faithful and timely repair of DNA double‐strand breaks (DSBs) is fundamental for the maintenance of genomic integrity. Here, we demonstrate that the meiotic recombination co‐factor MND1 facilitates the repair of DSBs in somatic cells. We show that MND1 localizes to DSBs, where it stimulates DNA repair through homologous recombination (HR). Importantly, MND1 is not involved in the response to replication‐associated DSBs, implying that it is dispensable for HR‐mediated repair of one‐ended DSBs. Instead, we find that MND1 specifically plays a role in the response to two‐ended DSBs that are induced by irradiation (IR) or various chemotherapeutic drugs. Surprisingly, we find that MND1 is specifically active in G2 phase, whereas it only marginally affects repair during S phase. MND1 localization to DSBs is dependent on resection of the DNA ends and seemingly occurs through direct binding of MND1 to RAD51‐coated ssDNA. Importantly, the lack of MND1‐driven HR repair directly potentiates the toxicity of IR‐induced damage, which could open new possibilities for therapeutic intervention, specifically in HR‐proficient tumors. Hypothetical model of the specific action of MND1/HOP2 complex on the repair of DSBs. We observe that the meiotic recombination co‐factor MND1 facilitates the repair of DSBs in somatic cells. Surprisingly, we find that MND1 is specifically active in G2 phase, whereas it only marginally affects repair during S phase.

Hydrolethalus Syndrome (HLS) is a lethal, autosomal recessive ciliopathy caused by the mutation of the conserved centriole protein HYLS1. However, how HYLS1 facilitates the centriole-based templating of cilia is poorly understood. Here, we show that mice harboring the HYLS1 disease mutation die shortly after birth and exhibit developmental defects that recapitulate several manifestations of the human disease. These phenotypes arise from tissue-specific defects in cilia assembly and function caused by a loss of centriole integrity. We show that HYLS1 is recruited to the centriole by CEP120 and functions to recruit centriole inner scaffold proteins that stabilize the centriolar microtubule wall. The HLS mutation disrupts the interaction of HYLS1 with CEP120 leading to HYLS1 displacement and degeneration of the centriole distal end. We propose that tissue-specific defects in centriole integrity caused by the HYLS1 mutation prevent ciliogenesis and drive HLS phenotypes.

Faithful and timely repair of DNA double-strand breaks (DSBs) is fundamental for the maintenance of genomic integrity. Here, we demonstrate that the meiotic recombination co-factor MND1 facilitates the repair of DSBs in somatic cells. We show that MND1 localizes to DSBs, where it stimulates DNA repair through homologous recombination (HR). Importantly, MND1 is not involved in the response to replication-associated DSBs, implying that it is dispensable for HR-mediated repair of one-ended DSBs. Instead, we find that MND1 specifically plays a role in the response to two-ended DSBs that are induced by IR or various chemotherapeutic drugs, specifically in G2. MND1 localization to DSBs is dependent on resection of the DNA ends, and seemingly occurs through direct binding of MND1 to RAD51-coated ssDNA. Importantly, the lack of MND1-driven HR repair directly potentiates the toxicity of IR-induced damage, which could open new possibilities for therapeutic intervention, specifically in HR-proficient tumors.

Abstract Acquired drug resistance is a major problem in the treatment of cancer. hTERT-immortalized, untransformed RPE-1 (RPE) cells can acquire resistance to taxol by derepressing the ABCB1 gene, encoding for the multidrug transporter P-gP. Here we have investigated how the ABCB1 gene is derepressed. We show that activation of the ABCB1 gene is associated with reduced DNA methylation, reduced H3K9 trimethylation and increased H3K27 acetylation at the ABCB1 promoter. In addition, we find that the ABCB1 locus has moved away from the nuclear lamina in the taxol-resistant cells. This raises the question which of these alterations were causal to derepression. Directly modifying DNA methylation or H3K27 methylation had neither significant effect on ABCB1 expression, nor did it promote drug resistance. In contrast, the disruption of Lamin B Receptor (LBR), a component of the nuclear lamina involved in genome organization, did promote the acquisition of a taxol-resistant phenotype in a subset of cells. Using CRISPRa-mediated gene activation, we could further substantiate a model in which disruption of lamina association renders the ABCB1 gene permissive to derepression. Based on these data we propose a model in which nuclear lamina dissociation of a repressed gene allows for its activation, implying that deregulation of the 3D genome topology could play an important role in tumor evolution and the acquisition of drug resistance. Competing Interest Statement This work was supported by NWO Zwaartekracht (58588) (to R.H.M.) and by NIH Common Fund 4D Nucleome Program grant U54DK107965 (to B.v.S.). The Oncode Institute is partly supported by KWF Dutch Cancer Society. E.d.W. is a co-founder of Cergentis B.V.

Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress-response programs that counteract the inherent toxicity of such aberrant signaling. While inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of Protein Phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor suppressive resistance.Competing Interest StatementR.B., J.K. and M.H.D are listed as inventors of a patent describing the drug combinations discovered here. R.B. is a member of the board of directors of Lixte Biotechnology. R.B and M.H.D. are shareholders of Lixte Biotechnology. J.K. is employee of Lixte and shareholder in the company. This work was supported in part by a research grant for Lixte Biotechnology. A.V. and A.Vil. are co-founders of Xenopat S.L.