Enhancing natural killer (NK) cell cytotoxicity by blocking inhibitory signaling could lead to improved NK-based cancer immunotherapy. Thus, we have developed a highly efficient method for editing the genome of human NK cells using CRISPR/Cas9 to knock out inhibitory signaling molecules. Our method efficiently edits up to 90% of primary peripheral blood NK cells. As a proof-of-principle we demonstrate highly efficient knockout of ADAM17 and PDCD1, genes that have a functional impact on NK cells, and demonstrate that these gene-edited NK cells have significantly improved activity, cytokine production, and cancer cell cytotoxicity. Furthermore, we were able to expand cells to clinically relevant numbers, without loss of activity. We also demonstrate that our CRISPR/Cas9 method can be used for efficient knockin of genes by delivering homologous recombination template DNA using recombinant adeno-associated virus serotype 6 (rAAV6). Our platform represents a feasible method for generating engineered primary NK cells as a universal therapeutic for cancer immunotherapy.

Nonhomologous end joining (NHEJ), a major pathway of DNA double-strand break (DSB) repair, is required during lymphocyte development to resolve the programmed DSBs generated during Variable, Diverse, and Joining [V(D)J] recombination. XRCC4-like factor (XLF) (also called Cernunnos or NHEJ1) is a unique component of the NHEJ pathway. Although germ-line mutations of other NHEJ factors abrogate lymphocyte development and lead to severe combined immunodeficiency (SCID), XLF mutations cause a progressive lymphocytopenia that is generally less severe than SCID. Accordingly, XLF-deficient murine lymphocytes show no measurable defects in V(D)J recombination. We reported earlier that ATM kinase and its substrate histone H2AX are both essential for V(D)J recombination in XLF-deficient lymphocytes, despite moderate role in V(D)J recombination in WT cells. p53-binding protein 1 (53BP1) is another substrate of ATM. 53BP1 deficiency led to small reduction of peripheral lymphocyte number by compromising both synapse and end-joining at modest level during V(D)J recombination. Here, we report that 53BP1/XLF double deficiency blocks lymphocyte development at early progenitor stages, owing to severe defects in end joining during chromosomal V(D)J recombination. The unrepaired DNA ends are rapidly degraded in 53BP1 −/− XLF −/− cells, as reported for H2AX −/− XLF −/− cells, revealing an end protection role for 53BP1 reminiscent of H2AX. In contrast to the early embryonic lethality of H2AX −/− XLF −/− mice, 53BP1 −/− XLF −/− mice are born alive and develop thymic lymphomas with translocations involving the T-cell receptor loci. Together, our findings identify a unique function for 53BP1 in end-joining and tumor suppression.Ataxia-Telangiectasia-Mutated | XRCC4-like factor | classical nonhomologous end joining L ymphocyte development requires the ordered assembly of variable region of antigen receptor genes from individual Variable, Diverse, and Joining gene segments through V(D)J recombination (1). V(D)J recombination is initiated by the RAG endonuclease (RAG), which introduces DNA double-strand breaks (DSBs) between the conserved recombination signal sequence (RSS) and the participating germ-line V, D, or J gene segments (1). RAG cleavage generates two types of DNA ends, the 5′-phosphorylated blunt signal ends (SEs) and the hairpin sealed coding ends (CEs) (1). In the next step, ubiquitously expressed classical nonhomologous end-joining (C-NHEJ) factors directly join the two SEs to form a signal join (SJ), and process (via hairpin opening, de novo synthesis, and loss of nucleotides) and join the two CEs to form a coding join (CJ) (1).There are seven C-NHEJ factors in mammalian cells. Ku70 and Ku80 (or KU86 in human) form heterodimers (KU) that bind DSB ends and, among other functions, recruit and activate other NHEJ factors (2). DNA-bound KU interacts with the DNA-PK catalytic subunit (DNA-PKcs), and together they activate the endonuclease function of Artemis for end processing, including opening hairpin-sealed CEs during V(D)J reco...

Expression of a kinase-deficient ATM protein leads to severe genomic instability and embryonic lethality.

Defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding escape from antitumor immunity and optimizing T cell-related therapeutic strategies. Here, we engineered nanotextured elastic platforms to study and enhance T cell migration through complex microenvironments and define how the balance between contractility localization-dependent T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues. Using these platforms, we characterize a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and force generation. In 3D environments and live tumors, we demonstrate that microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration whereas clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. We show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering, results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. Thus, engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics.

Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (AtmKD/-) is more oncogenic than loss of ATM (Atm-/-) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate AtmKD/-, but not Atm-proficientor Atm-/- leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.DOI: http://dx.doi.org/10.7554/eLife.14709.001

Defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding sanctuaries from antitumor immunity and optimizing T cellrelated therapeutic strategies. To enhance T cell migration through complex microenvironments, we engineered nanotextured platforms that allowed us to define how the balance between T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues and characterize a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and force generation. In 3D environments and live tumors, we demonstrate that microtubules instability, leading to increased Rho pathway-dependent cell contractility, promotes migration while clinically used microtubule-targeting chemotherapies profoundly decrease effective migration. Indeed, we show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering, results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. This suggests that engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics.

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