DNA damage induces p53-independent apoptosis through ribosome stalling.

In response to excessive DNA damage, human cells can activate p53 to induce apoptosis. Cells lacking p53 can still undergo apoptosis upon DNA damage, yet the responsible pathways are unknown. We observed that p53-independent apoptosis in response to DNA damage coincided with translation inhibition, which was characterized by ribosome stalling on rare leucine-encoding UUA codons and globally curtailed translation initiation. A genetic screen identified the transfer RNAse SLFN11 and the kinase GCN2 as factors required for UUA stalling and global translation inhibition, respectively. Stalled ribosomes activated a ribotoxic stress signal conveyed by the ribosome sensor ZAKα to the apoptosis machinery. These results provide an explanation for the frequent inactivation of SLFN11 in chemotherapy-unresponsive tumors and highlight ribosome stalling as a signaling event affecting cell fate in response to DNA damage.

Discovery of tumor-reactive T cell receptors by massively parallel library synthesis and screening.

T cell receptor (TCR) gene therapy is a potent form of cellular immunotherapy in which patient T cells are genetically engineered to express TCRs with defined tumor reactivity. However, the isolation of therapeutic TCRs is complicated by both the general scarcity of tumor-specific T cells among patient T cell repertoires and the patient-specific nature of T cell epitopes expressed on tumors. Here we describe a high-throughput, personalized TCR discovery pipeline that enables the assembly of complex synthetic TCR libraries in a one-pot reaction, followed by pooled expression in reporter T cells and functional genetic screening against patient-derived tumor or antigen-presenting cells. We applied the method to screen thousands of tumor-infiltrating lymphocyte (TIL)-derived TCRs from multiple patients and identified dozens of CD4+ and CD8+ T-cell-derived TCRs with potent tumor reactivity, including TCRs that recognized patient-specific neoantigens.

Identification of patient-specific CD4+ and CD8+ T cell neoantigens through HLA-unbiased genetic screens.

Cancer neoantigens that arise from tumor mutations are drivers of tumor-specific T cell responses, but identification of T cell-recognized neoantigens in individual patients is challenging. Previous methods have restricted antigen discovery to selected HLA alleles, thereby limiting the breadth of neoantigen repertoires that can be uncovered. Here, we develop a genetic neoantigen screening system that allows sensitive identification of CD4+ and CD8+ T cell-recognized neoantigens across patients' complete HLA genotypes.

Bone marrow-derived myeloid progenitors as driver mutation carriers in high- and low-risk Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a myeloid neoplasia, driven by sporadic activating mutations in the MAPK pathway. The misguided myeloid dendritic cell (DC) model proposes that high-risk, multisystem, risk-organ-positive (MS-RO+) LCH results from driver mutation in a bone marrow (BM)-resident multipotent hematopoietic progenitor, while low-risk, MS-RO- and single-system LCH would result from driver mutation in a circulating or tissue-resident, DC-committed precursor. We have examined the CD34+c-Kit+Flt3+ myeloid progenitor population as potential mutation carrier in all LCH disease manifestations. This population contains oligopotent progenitors of monocytes (Mo's)/macrophages (MΦs), osteoclasts (OCs), and DCs. CD34+c-Kit+Flt3+ cells from BM of MS-RO+ LCH patients produced Langerhans cell (LC)-like cells in vitro. Both LC-like and DC offspring from this progenitor carried the BRAF mutation, confirming their common origin. In both high- and low-risk LCH patients, CD34+c-Kit+Flt3+ progenitor frequency in blood was higher than in healthy donors. In one MS-RO+ LCH patient, CD34+c-Kit+Flt3+ cell frequency in blood and its BRAF-mutated offspring reported response to chemotherapy. CD34+c-Kit+Flt3+ progenitors from blood of both high- and low-risk LCH patients gave rise to DCs and LC-like cells in vitro, but the driver mutation was not easily detectable, likely due to low frequency of mutated progenitors. Mutant BRAF alleles were found in Mo's /MΦs, DCs, LC-like cells, and/or OC-like cells in lesions and/or Mo and DCs in blood of multiple low-risk patients. We therefore hypothesize that in both high- and low-risk LCH, the driver mutation is present in a BM-resident myeloid progenitor that can be mobilized to the blood.