BackgroundDot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined.Methodology/Principal FindingsUsing the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1L−/− and wild type cells are equally resistant to ionising radiation, whereas 53Bp1−/−/Dot1L−/− cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the mRNA levels of over 1200 genes involved in diverse biological functions. These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression. In 53Bp1−/− cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed. To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line.Conclusions/SignificanceTaken together, our results uncover a negative role for Dot1L and H3K79 methylation in the DNA damage response in the absence of 53Bp1. They also enlighten the roles of Dot1L and 53Bp1 in gene expression and the control of DNA double-strand repair pathways in the context of chromatin.

Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy. KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers. Here we show the identification and development of an affinity-enhanced T cell receptor (TCR) that recognizes a peptide derived from the most common KRAS mutant, KRASG12D, presented in the context of HLA-A*11:01. The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG12D over KRASWT. While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG12D, thermodynamic analysis and molecular dynamics simulations reveal that TCR specificity is driven by differences in indirect electrostatic interactions. The affinity enhanced TCR, fused to a humanized anti-CD3 scFv, enables selective killing of cancer cells expressing KRASG12D. Our work thus reveals a molecular mechanism that drives TCR selectivity and describes a soluble bispecific molecule with therapeutic potential against cancers harboring a common shared neoantigen.

Loss of p53, a transcription factor activated by cellular stress, is a frequent event in cancer. The role of p53 in tumour suppression is largely attributed to cell fate decisions. Here, we provide evidence supporting a novel role for p53 in the regulation of DNA double-strand break (DSB) repair pathway choice. 53BP1, another tumour suppressor, was initially identified as p53 Binding Protein 1, and has been shown to inhibit DNA end resection, thereby stimulating non-homologous end joining (NHEJ). Yet another tumour suppressor, BRCA1, reciprocally promotes end resection and homologous recombination (HR). Here, we show that in both human and mouse cells, the absence of p53 results in impaired 53BP1 focal recruitment to sites of DNA damage induced by ionizing radiation. This effect is largely independent of cell cycle phase and the extent of DNA damage. In p53-deficient cells, diminished localization of 53BP1 is accompanied by a reciprocal increase in BRCA1 recruitment to DSBs. Consistent with these findings, we demonstrate that DSB repair via NHEJ is abrogated, while repair via homology-directed repair (HDR) is stimulated. Overall, we propose that in addition to its role as an ‘effector’ protein in the DNA damage response, p53 plays a role in the regulation of DSB repair pathway choice.

Background: Bispecific immunotherapies have been validated for the treatment of hematologic tumors but none have yet been approved for solid tumor indications, including high prevalent tumors such as non-small-cell lung carcinoma (NSCLC). Immunocore is developing ImmTAC® molecules, a new class of TCR/anti-CD3 bispecific fusion protein, that target intracellularly derived peptides presented at the tumor cell surface in complex with human leukocyte antigen (HLA). The ImmTAC IMC-F106C is in development for the treatment of advanced cancers that are positive for Preferentially Expressed Antigen in Melanoma (PRAME). PRAME is a cancer-testis antigen (CTA) that is highly expressed in normal testis and a range of solid and hematologic malignancies. The aim of this study was to characterise the expression of PRAME in a variety of human malignancies (mRNA and protein) and demonstrate that IMC-F106C can potently redirect T cells to eliminate indication-relevant tumor cells in vitro. Method: FFPE tumor samples were analysed by RT-qPCR and IHC for a number of tumor indications, to determine levels of PRAME mRNA and protein expression in patient samples for each indication. The activity of IMC-F106C was investigated in cellular assays using healthy donor PBMCs as effectors, targeting a variety of indication-relevant tumor cell lines expressing a PRAME-specific peptide complexed with HLA-A*02:01, including NSCLC non-small-cell lung carcinoma, ovarian carcinoma, and acute myeloid leukemia cell lines. T cell activation was assessed by cytokine release and T cell-mediated target-cell killing was evaluated by measurement of cell death (xCELLigence). Results: PRAME mRNA and protein expression was highly prevalent in samples of NSCLC, including both the adenocarcinoma and squamous cell carcinoma subtypes, SCLC, melanoma, ovarian, endometrial carcinoma samples, and in triple negative breast cancer (TNBC). Over 60% of samples demonstrated some level of PRAME expression by IHC and RT-qPCR in these 6 cancer indications. In the PRAME positive HLA-A*02:01 positive cell lines, IMC-F106C redirected donor effector cells to release IFNγ and GrB and kill tumor cells in a dose-dependent manner, with activity demonstrated as low as < 1 pM. By contrast, cell lines negative for PRAME or HLA-A*02:01 expression failed to induce responses < 1 nM of IMC-F106C. Conclusion: These data indicate that PRAME is expressed in a number of solid tumors, and is highly prevalent in lung tumours, irrespective of EGFR status, as well as female-oriented cancers. In conjunction, IMC-F106C efficiently redirects T cell activity against tumor cell lines that express PRAME across a range of tumor indications. Taken together, IMC-F106C could prove to be a highly effective immunotherapy option for HLA*02:01 positive patients with PRAME positive tumors. Citation Format: Sylvie Moureau, Alessio Vantellini, Florence Schlosser, Jacob Robinson, Jane Harper, Athiva Shankar, Greg Dobrynin, Gabrielle Le Provost, Amanda Williams, David Berman, Laure Humbert. IMC-F106C, a novel and potent immunotherapy approach to treat PRAME expressing solid and hematologic tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5572.

BackgroundKRAS is the most frequently mutated oncogene, yet mutant KRAS has historically been a challenging target for conventional small molecule drug development. Tumour specific neoantigen peptides derived from KRAS are presented by cell surface human leucocyte antigens (HLA) and form a class of shared, tumour-specific antigens that are attractive targets for immunotherapy.MethodsA T cell clone that specifically recognizes the most common KRAS G12D mutant presented as a peptide in the context of HLA-A*11:01 was isolated from healthy donor PBMCs. The affinity of the respective T cell receptor (TCR) was enhanced by phage display and the x-ray crystal structures of the affinity-enhanced TCR bound to HLA presenting mutant KRAS G12D and wildtype (KRAS WT) peptides were solved. We used structural, biochemical, and computational approaches to investigate the molecular interactions underlying TCR selectivity for mutant KRAS G12D. Finally, the high affinity TCR was engineered into a soluble T cell engaging ImmTAC (Immune mobilizing monoclonal TCR Against Cancer) molecule, IMC-KRAS-G12D, and in vitro cell-based assays were performed to evaluate its potency and selectivity.ResultsThe affinity of the engineered TCR was enhanced by a million-fold and demonstrated remarkable ability to distinguish between KRAS G12D and KRAS WT peptide presented by HLA-A*11:01. X-ray crystal structures demonstrate that TCR binding is almost identical between KRAS G12D and KRAS WT despite a binding affinity difference of >4000 fold. The mutant residue G12D is buried into the HLA peptide binding groove and acts as a secondary anchor, making it inaccessible to the TCR. Thermodynamic analysis of TCR-HLA interaction combined with molecular dynamics simulations indicates a novel mechanism of peptide selectivity, mediated by an indirect energetic mechanism driven by an induced fit in the peptide upon TCR binding. In functional assays, this molecular differentiation translated into biological specificity with IMC-KRAS-G12D mediating T cell activation in response to cells pulsed with or expressing KRAS G12D but not KRAS WT. Furthermore, IMC-KRAS-G12D was able to redirect T cell cytotoxicity towards target KRAS G12D presenting colon cancer cells, while sparing normal colon epithelial cellsConclusionsWe developed a high affinity TCR bispecific with exquisite specificity towards a common shared neoantigen, KRAS G12D, that is a relevant therapeutic target in a wide range of cancers. These findings reveal a novel molecular mechanism for TCR selectivity for a neoantigen that differs from self-antigen by only a single amino acid, with attendant implications for therapeutic development.