Endogenous DNA 3' Blocks Are Vulnerabilities for BRCA1 and BRCA2 Deficiency and Are Reversed by the APE2 Nuclease.

The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3' DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3'-blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3' blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.

Modification of the HLA-A*24:02 Peptide Binding Pocket Enhances Cognate Peptide-Binding Capacity and Antigen-Specific T Cell Activation.

The immunogenicity of a T cell Ag is correlated with the ability of its antigenic epitope to bind HLA and be stably presented to T cells. This presents a challenge for the development of effective cancer immunotherapies, as many self-derived tumor-associated epitopes elicit weak T cell responses, in part due to weak binding affinity to HLA. Traditional methods to increase peptide-HLA binding affinity involve modifying the peptide to reflect HLA allele binding preferences. Using a different approach, we sought to analyze whether the immunogenicity of wild-type peptides could be altered through modification of the HLA binding pocket. After analyzing HLA class I peptide binding pocket alignments, we identified an alanine 81 to leucine (A81L) modification within the F binding pocket of HLA-A*24:02 that was found to heighten the ability of artificial APCs to retain and present HLA-A*24:02-restricted peptides, resulting in increased T cell responses while retaining Ag specificity. This modification led to increased peptide exchange efficiencies for enhanced detection of low-avidity T cells and, when expressed on artificial APCs, resulted in greater expansion of Ag-specific T cells from melanoma-derived tumor-infiltrating lymphocytes. Our study provides an example of how modifications to the HLA binding pocket can enhance wild-type cognate peptide presentation to heighten T cell activation.

Affinity-matured HLA class II dimers for robust staining of antigen-specific CD4+ T cells.

Peptide-major histocompatibility complex (pMHC) multimers enable the detection of antigen-specific T cells in studies ranging from vaccine efficacy to cancer immunotherapy. However, this technology is unreliable when applied to pMHC class II for the detection of CD4+ T cells. Here, using a combination of molecular biological and immunological techniques, we cloned sequences encoding human leukocyte antigen (HLA)-DP, HLA-DQ and HLA-DR molecules with enhanced CD4 binding affinity (with a Kd of 8.9 ± 1.1 µM between CD4 and affinity-matured HLA-DP4) and produced affinity-matured class II dimers that stain antigen-specific T cells better than conventional multimers in both in vitro and ex vivo analyses. Using a comprehensive library of dimers for HLA-DP4, which is the most frequent HLA allele in many ancestry groups, we mapped 103 HLA-DP4-restricted epitopes derived from diverse tumor-associated antigens and cloned the cognate T-cell antigen receptor (TCR) genes from in vitro-stimulated CD4+ T cells. The availability of affinity-matured class II dimers across HLA-DP, HLA-DQ and HLA-DR alleles will aid in the investigation of human CD4+ T-cell responses.

Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy.

Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA, and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy.

A conserved CCM complex promotes apoptosis non-autonomously by regulating zinc homeostasis.

Apoptotic death of cells damaged by genotoxic stress requires regulatory input from surrounding tissues. The C. elegans scaffold protein KRI-1, ortholog of mammalian KRIT1/CCM1, permits DNA damage-induced apoptosis of cells in the germline by an unknown cell non-autonomous mechanism. We reveal that KRI-1 exists in a complex with CCM-2 in the intestine to negatively regulate the ERK-5/MAPK pathway. This allows the KLF-3 transcription factor to facilitate expression of the SLC39 zinc transporter gene zipt-2.3, which functions to sequester zinc in the intestine. Ablation of KRI-1 results in reduced zinc sequestration in the intestine, inhibition of IR-induced MPK-1/ERK1 activation, and apoptosis in the germline. Zinc localization is also perturbed in the vasculature of krit1-/- zebrafish, and SLC39 zinc transporters are mis-expressed in Cerebral Cavernous Malformations (CCM) patient tissues. This study provides new insights into the regulation of apoptosis by cross-tissue communication, and suggests a link between zinc localization and CCM disease.