BRCA1 -linked cancer genomes contain abundant genome-wide ∼10 kb 'Group 1' tandem duplications (TDs) that are drivers of tumorigenesis. Group 1 TD formation is recapitulated at a chromosomal Tus/ Ter site-specific replication fork barrier in DNA end resection-defective mouse embryonic stem (mES) cells lacking Brca1 exon 11. To explore relationships between DNA end resection and Group 1 TD formation, we analyzed Brca1 coiled coil (CC) domain mutants-separation-of-function alleles that are impaired for homologous recombination but competent for DNA end resection. Notably, Brca1 CC mutants retain the ability to suppress Group 1 TDs in the Tus/ Ter system and in a mouse model of Brca1 -linked tumorigenesis. These data show that Brca1 CC domain mutant cancers follow a path of tumorigenesis distinct from that of other pathogenic Brca1 alleles. FANCM is a TD co-suppressor, the loss of which is synthetic lethal/sick in combination with Brca1 exon 11 mutation. In contrast, Fancm deletion is well-tolerated by Brca1 CC mutant mES cells. Thus, Group 1 TD formation and Fancm synthetic lethality are linked phenotypes related to defective BRCA1-mediated DNA end resection.

ABSTRACT:Innate immunity is increasingly recognized as a driver of neurodegeneration, although pathogenic mechanisms are incompletely understood. Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplastic disorder caused by activating somatic mutations in MAPK pathway genes, most commonly BRAFV600E, in myeloid precursors. A subset of patients with LCH develop progressive neurodegeneration (LCH-ND). We generated a human induced pluripotent stem cell (iPSC) model from patients with somatic hematologic mosaicism for BRAFV600E. Brain macrophages/microglia from LCH iPSCs exhibit unique disease-specific pathogenic features. Stepwise differentiation identified hematopoietic progenitors as hyperproliferative, whereas brain macrophages were apoptosis resistant. Through application of cerebral organoids and a humanized murine xenotransplantation model, we identify marked heterogeneity of differentiation potential within clonal BRAFV600E lines in vivo. This model phenocopied human-specific phenotypes, including dense basal ganglia foci of abnormal macrophages, marked neurodegeneration with astrogliosis, and progressive ataxia. This approach will allow for preclinical testing of therapeutics for LCH-ND.

Homologous recombination (HR) requires the resection of DNA breaks and RAD51 filament formation. Protein complexes that control end resection have been characterized, but regulators of RAD51 loading are not well defined. PALB2 is a mediator of BRCA2-RAD51 DNA break localization; it can also bind BRCA1 or form homodimers with DNA-binding activity via its coiled-coil (CC) domain. Here, we created a CC-mutated mouse allele (Palb2CC) that disrupts CC-mediated interactions. While Palb2CC/CC embryos were not viable, remarkably, intercrossing with mice lacking 53BP1, an inhibitor of PALB2-chromatin contacts, produced live births. However, Palb2CC/CC53bp1-/- mice were tumor prone, and cells had limited RAD51 foci. HR remained inefficient because the CC domain was required for PALB2 to bind to single-stranded (ss)DNA overhangs and subsequently promote PALB2 and RAD51 accumulation. These findings underscore the role of ssDNA binding in localizing PALB2 to DNA breaks while establishing genetic interactions that control the post-end resection steps of mammalian HR.

Mutations in the global transcriptional activator EP300/KAT3B are being reported in aggressive malignancies. However, the mechanistic contribution of EP300 dysregulation to cancer is currently unknown. While EP300 has been implicated in regulating cell cycle and DNA replication, the role of EP300 in maintaining replication fork integrity has not been studied. Here, using EP300-mutated adult T-cell leukemia/lymphoma cells and an EP300-selective degrader, we reveal that EP300 loss leads to pronounced dysregulations in DNA replication dynamics and persistent genomic instability. Aberrant DNA replication in EP300-mutated cells is characterized by elevated replication origin firing due to replisome pausing. EP300 deficiency results in a prominent defect in fork protection resulting in the accumulation of single-stranded DNA gaps. Importantly, we find that the loss of EP300 results in decreased expression of BRCA2 protein leading to sensitivity to treatments that are cytotoxic to BRCA-deficient cancers. Overall, we demonstrate that EP300-mutated cells recapitulate features of BRCA-deficient cancers.

Background: Recent research has increasingly highlighted alterations in the proto-oncogene MET, whose abnormal activation has been implicated in multiple cancers. MET encodes c-MET, a receptor tyrosine kinase critical for cellular growth, survival, and migration. Aberrant c-MET signaling, driven by mutations or gene amplification, promotes proliferation and invasion, contributing to tumorigenesis. Scope of the Review: While MET mutations are most often observed in non-small cell lung cancer (NSCLC), they also occur in other malignancies, including breast and gastric cancers. This review highlights key MET alterations, such as gene amplification, gene fusions, and exon 14 skipping deletions, and examines their prevalence across various tumor types. Major Conclusions: We discuss the clinical significance of c-MET as a therapeutic target and identify gaps in knowledge that could inform the development of alternative treatment strategies.

Microglia, the brain's resident macrophages, arise from yolk sac hematopoietic progenitor cells (HPCs) that migrate into the brain during early embryonic development and differentiate in response to microenvironment-specific signals. The resulting spatial and stage-specific programs of gene expression enable microglia to function as key modulators of diverse homeostatic processes that include synaptic pruning, myelination, and neurogenesis throughout the lifespan. Dysregulation of these core microglia functions has been linked to numerous neurodevelopmental and neurodegenerative diseases. Although normally a closed niche, studies in mice indicate that peripheral monocytes, originating from hematopoietic stem cells (HSCs), can infiltrate the brain in circumstances in which the blood brain barrier is disrupted, with context-dependent protective or detrimental consequences. A major unanswered question with significant implications for therapy of CNS diseases driven by microglia dysfunction is the extent to which human HSC-derived cells can adopt microglia-like phenotypes that would allow them to restore brain homeostasis by replacement of pathologic HPC-derived microglia. To address this question, we directly compared the differentiation potential of primary human microglia, human iPSC-derived HPCs and human HSCs in the brain utilizing a murine xenotransplantation model. HSCs and monocytes were capable of differentiating into microglia like cells in this model, they also acquired a strong interferon, phagocytic, and antigen presenting phenotype distinct from engrafted primary human microglia and HPC-derived cells. Analyses of the epigenetic landscapes of the engrafted HPC and HSC-derived cells enabled identification of the transcription factors networks underlying ontogeny-specific brain myeloid fates. Ultimately, human peripheral myeloid cells in the CNS led to astrogliosis, myelin fragmentation and synaptic loss. These findings reveal transcriptional network differences influenced by ontogeny, and together with the accompanying study by Davtvan and colleagues provide critical insights for developing human microglial or bone marrow transplant-based therapies for CNS disorders.

EHTM1 (GLP) and EHMT2 (G9a) are closely related protein lysine methyltransferases often thought to function together as a heterodimer to methylate histone H3 and non-histone substrates in diverse cellular processes including transcriptional regulation, genome methylation, and DNA repair. Here we show that EHMT1/2 inhibitors cause ATM-mediated slowdown of replication fork progression, accumulation of single-stranded replication gaps, emergence of cytosolic DNA, and increased expression of STING. EHMT1/2 inhibition strongly potentiates the efficacy of alkylating chemotherapy and anti-PD-1 immunotherapy in mouse models of tripe negative breast cancer. The effects on DNA replication and alkylating agent sensitivity are largely caused by the loss of EHMT1-mediated methylation of LIG1, whereas the elevated STING expression and remarkable response to immunotherapy appear mainly elicited by the loss of EHMT2 activity. Depletion of UHRF1, a protein known to be associated with EHMT1/2 and LIG1, also induces STING expression, and depletion of either EHMT2 or UHRF1 leads to demethylation of specific CpG sites in the STING1 promoter, suggestive of a distinct EHMT2-UHRF1 axis that regulates DNA methylation and gene transcription. These results highlight distinct functions of the two EHMT paralogs and provide enlightening paradigms and corresponding molecular basis for combination therapies involving alkylating agents and immune checkpoint inhibitors.

DSS1, essential for BRCA2-RAD51 dependent homologous recombination (HR), associates with the helical domain (HD) and OB fold 1 (OB1) of the BRCA2 DSS1/DNA-binding domain (DBD) which is frequently targeted by cancer-associated pathogenic variants. Herein, we reveal robust ss/dsDNA binding abilities in HD-OB1 subdomains and find that DSS1 shuts down HD-OB1’s DNA binding to enable ssDNA targeting of the BRCA2-RAD51 complex. We show that C-terminal helix mutations of DSS1, including the cancer-associated R57Q mutation, disrupt this DSS1 regulation and permit dsDNA binding of HD-OB1/BRCA2-DBD. Importantly, these DSS1 mutations impair BRCA2/RAD51 ssDNA loading and focus formation and cause decreased HR efficiency, destabilization of stalled forks and R-loop accumulation, and hypersensitize cells to DNA-damaging agents. We propose that DSS1 restrains the intrinsic dsDNA binding of BRCA2-DBD to ensure BRCA2/RAD51 targeting to ssDNA, thereby promoting optimal execution of HR, and potentially replication fork protection and R-loop suppression. DSS1 is crucial for BRCA2 dependent genome repair. Here, the authors reveal how DSS1 restricts the dsDNA binding ability of BRCA2 to ensure BRCA2/RAD51 loading on ssDNA. Mutations in DSS1 disrupt this regulation, leading to impaired DNA repair, replication fork protection and R-loop suppression.

Germline mutations of homologous-recombination (HR) genes are among the top contributors to medulloblastomas. A significant portion of human medulloblastomas exhibit genomic signatures of HR defects. Whether ablation of Brca2 and Palb2, and their related Brca1 and Bccip genes, in the mouse brain can differentially initiate medulloblastomas was explored here. Conditional knockout mouse models of these HR genes and a conditional knockdown of Bccip (shBccip-KD) were established. Deletion of any of these genes led to microcephaly and neurologic defects, with Brca1- and Bccip- producing the worst defects. Trp53 co-deletion significantly rescued the microcephaly with Brca1, Palb2, and Brca2 deficiency but exhibited limited impact on Bccip- mice. For the first time, inactivation of either Brca1 or Palb2 with Trp53 was found to induce medulloblastomas. Despite shBccip-CKD being highly penetrative, Bccip/Trp53 deletions failed to induce medulloblastomas. The tumors displayed diverse immunohistochemical features and chromosome copy number variation. Although there were widespread up-regulations of cell proliferative pathways, most of the tumors expressed biomarkers of the sonic hedgehog subgroup. The medulloblastomas developed from Brca1-, Palb2-, and Brca2- mice were highly sensitive to a poly (ADP-ribose) polymerase inhibitor but not the ones from shBccip-CKD mice. These models recapitulate the spontaneous medulloblastoma development with high penetrance and a narrow time window, providing ideal platforms to test therapeutic agents with the ability to differentiate HR-defective and HR-proficient tumors.

Materials and Methods for supplementary data.

Gene Expression of Capan1 olaparib resistant cells

Supplementary Figures - PDF file 1145K, These supplementary figures (Fig. S1-S5) show the results of additional analyses of Palb2-null tumors and drived cells

Effect of N-acetylcystein (NAC) on the DPP3-KEAP1 interaction.

Supplementary methods and figure legends. Figure S1 shows further characterization of Capan1 olaparib resistant cells and rucaparib resistant Capan1 cells. Figure S2 shows that drug efflux is unchanged in Capan1 olaparib resistant cells and truncated BRCA2 is overexpressed in rucaparib resistant Capan1 cells. Figure S3 shows that Capan1 olaparib resistant cells express truncated BRCA2 Figure S4 shows that Capan1 PARP inhibitor resistant cells form Rad51 and BRCA1 foci after irradiation. Figure S5 shows that BRCA2 knockdown sensitizes Capan1 olaparib resistant cells to olaparib. Figure S6 shows that BRCA2 peptide coverage and pinometostat-mediated reduction in H3K79Me in olaparib resistant Capan1 cells. Also DOT1L expression conveys poor prognosis in ovarian cancer and DOT1L knockdown reduced viability of an olaparib resistant ovarian cancer cell line.

Immunoprecipitation of BRCA2 . List of proteins identified via mass spectrometry.

Carriers of BRCA1 germline pathogenic variants are at substantially higher risk of developing breast and ovarian cancer than the general population. Accurate identification of at-risk individuals is crucial for risk stratification and the implementation of targeted preventive and therapeutic interventions. Despite significant progress in variant classification efforts, a sizable portion of reported BRCA1 variants remain as variants of uncertain clinical significance (VUSs). Variants leading to premature protein termination and loss of essential func-tional domains are typically classified as pathogenic. However, the impact of frameshift variants that result in an extended incorrect ter -minus is not clear. Using validated functional assays, we conducted a systematic functional assessment of 17 previously reported BRCA1 extended incorrect terminus variants (EITs) and concluded that 16 constitute loss-of-function variants. This suggests that most EITs are likely to be pathogenic. However, one variant, c.5578dup, displayed a protein expression level, affinity to known binding partners, and activity in transcription and homologous recombination assays comparable to the wild-type BRCA1 protein. Twenty-three additional carriers of c.5578dup were identified at a US clinical diagnostic lab and assessed using a family history likelihood model providing, in combination with the functional data, a likely benign interpretation. These results, consistent with family history data in the current study and available data from ClinVar, indicate that most, but not all, BRCA1 variants leading to an extended incorrect terminus consti-tute loss-of-function variants and underscore the need for comprehensive assessment of individual variants.

Effect of mutating individual cysteine residues of DPP3 on its interaction with KEAP1.

Quantification of mRNA amount, qRT-PCR primer sequences, and transfection and immunoprecipitation methods.