dThe histone variant H2AX is a principal component of chromatin involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). H2AX is thought to operate primarily through its C-terminal S139 phosphorylation, which mediates the recruitment of DNA damage response (DDR) factors to chromatin at DSB sites. Here, we describe a comprehensive screen of 67 residues in H2AX to determine their contributions to H2AX functions. Our analysis revealed that H2AX is both sumoylated and ubiquitylated. Individual residues defective for sumoylation, ubiquitylation, and S139 phosphorylation in untreated and damaged cells were identified. Specifically, we identified an acidic triad region in both H2A and H2AX that is required in cis for their ubiquitylation. We also report the characterization of a human H2AX knockout cell line, which exhibits DDR defects, including p53 activation, following DNA damage. Collectively, this work constitutes the first genetic complementation system for a histone in human cells. Finally, our data reveal new roles for several residues in H2AX and define distinct functions for H2AX in human cells. N uclear DNA is bound by histones within nucleosomes to form chromatin (1). Core nucleosomes consist of two copies each of four canonical histones (H2A, H2B, H3, and H4) in an octamer that contains ϳ146 bp of DNA wrapped around the histone protein core. In mammalian genomes, several histone variants resembling core histones exist, such as the histone variant H2AX, which is nearly identical to H2A except for a divergent and extended C terminus. Histones can be modified on specific amino acid residues by various posttranslational chemical modifications (PTMs), including methylation, acetylation, and phosphorylation (2-4). In addition, lysine residues can be modified by the covalent attachment of small polypeptides such as ubiquitin (Ub) and SUMO (small ubiquitin-like modifier) (5). These various PTMs are catalyzed by "writer" enzymes and are removed by additional enzymes that act to "erase" these marks (3). Together, these enzymes and chromatin binding proteins dynamically regulate the structure and functions of chromatin, which in turn regulates fundamental nuclear processes, such as chromosome replication and segregation, transcription, and DNA repair.The protection of our genetic material is paramount for averting various human diseases, and chromatin plays an important role in coordinating the repair of nuclear DNA (6, 7). Cells have evolved a complex network of diverse cellular pathways, termed the DNA damage response (DDR), which detects damaged DNA, signals its presence, and promotes DNA repair (6, 7). DNA double-strand breaks (DSBs) represent a particularly challenging and cytotoxic form of DNA damage. DSBs create discontinuities in chromosomal DNA that, if not repaired or repaired incorrectly, result in mutations, chromosome loss, and/or ongoing genome instability. DSBs are predominantly repaired by either homologous recombination (HR) or nonhomologous end joining (NHEJ) (8). DSB rep...

Background:Methotrexate targets dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS) to prevent cancer growth, and increases DHFR expression when applied. Results: TYMS resistant to methotrexate inhibits the methotrexate induced increase in DHFR expression. Conclusion: Thymidine synthesis regulates post-transcriptional expression of DHFR and TYMS. Significance: Methotrexate-resistant DHFR and TYMS can be used to regulate cis and trans transgene in primary T cells.

T cell receptor (TCR)–based immunotherapy has emerged as a promising therapeutic approach for the treatment of patients with solid cancers. Identifying peptide–human leukocyte antigen (pHLA) complexes highly presented on tumors and rarely expressed on healthy tissue in combination with high-affinity TCRs that when introduced into T cells can redirect T cells to eliminate tumor but not healthy tissue is a key requirement for safe and efficacious TCR-based therapies. To discover promising shared tumor antigens that could be targeted via TCR-based adoptive T cell therapy, we employed population-scale immunopeptidomics using quantitative mass spectrometry across ~1500 tumor and normal tissue samples. We identified an HLA-A*02:01-restricted pan-cancer epitope within the collagen type VI α-3 ( COL6A3 ) gene that is highly presented on tumor stroma across multiple solid cancers due to a tumor-specific alternative splicing event that rarely occurs outside the tumor microenvironment. T cells expressing natural COL6A3-specific TCRs demonstrated only modest activity against cells presenting high copy numbers of COL6A3 pHLAs. One of these TCRs was affinity-enhanced, enabling transduced T cells to specifically eliminate tumors in vivo that expressed similar copy numbers of pHLAs as primary tumor specimens. The enhanced TCR variants exhibited a favorable safety profile with no detectable off-target reactivity, paving the way to initiate clinical trials using COL6A3-specific TCRs to target an array of solid tumors.

Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αβ T-cell receptors (TCRαβ) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαβs obtained from single T cells. Our approach is based on the rapid expression of cloned TCRαβ genes as Sleeping Beauty transposons and the determination of the introduced TCRαβs' antigen specificity and avidity using a reporter cell line. The platform enables the very rapid identification of tumor-reactive TCRs for the bioengineering of T cells with redirected specificity.immunotherapy is that ex vivo culture and numeric expansion typically leads to the clonal and/ or oligoclonal expansion of terminally differentiated T cells. Together, these clinical data suggest that the administration of "young" T cells that are sourced from peripheral blood and genetically modified to be neoantigen-specific offers an advantage over TIL-based immunotherapy.The bioengineering of neoantigen-specific T cells requires identifying individual TCRαβs and determining their antigen specificity. Next-generation sequencing (NGS) was used to identify non-synonymous tumor-specific mutations and single-cell RNA sequencing (scRNAseq) to identify paired full-length TCRαβ sequences [18]. This enabled us to reconstruct tumor-specific TCRs and evaluate their antigen specificity to engineer clinical-grade T cells. This was undertaken by very rapidly constructing a library of TCRαβ genes expressed in DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system and then inducing the expression of cloned TCRαβs in a reporter cell line to determine their antigen specificity and avidity. These reporter cells were co-cultured with genetically edited HLA null HEK293 cells and genetically modified with monoallelic HLA and the putative neoantigen as a minigene construct to serve as artificial antigen-presenting cells. This suite of technologies could be used to determine the antigen specificity of TCRs retrieved from primary tumors. In summary, this platform serves as a resource for the very rapid, robust, and high-throughput identification of immunogenic neoantigens and their cognate antigen-specific TCRs. Materials and methods Ethical statementPeripheral blood mononuclear cells (PBMCs) were obtained from patients who had provided written informed consent in accordance with a protocol established and approved by MD Anderson's Institutional Review Board (#LAB07-0296, Acquisition of Peripheral Blood from Healthy Donors). The identities of all patients were kept private. Animals were handled in accordance with strict guidelines established by MD Anderson's Institutional Animal Care and Use Committee (IACUC), which specifically approved this study (#00001131-RN02, Adoptive Immunotherapy w...

Antithymidylates (AThy) constitute a class of drugs used in the treatment of cancers such as lung, colon, breast and pancreas. These drugs inhibit DNA synthesis by targeting the enzymes dihydrofolate reductase (DHFR) and/or thymidylate synthase (TYMS). AThys effectively inhibit cancer cells, and also inhibit T cells, preventing anticancer immunity, which might otherwise develop from AThy-induced cancer destruction. We establish that T cells expressing mutant DHFR--DHFR L22F, F31S (DHFR(FS))--and/or mutant TYMS--TYMS T51S, G52S (TYMS(SS))-effectively survive in toxic concentrations of AThys methotrexate, pemetrexed and 5-fluorouracil. Furthermore, we show that DHFR(FS) permitted rapid selection of an inducible suicide transgene in T cells. These findings demonstrate that AThy resistances prevent AThy cytotoxicity to T cells while permitting selection of important transgenes. This technological development could enhance in vitro and in vivo survival and selection of T-cell therapeutics being designed for a broad range of cancers.