CRL4DCAF1/VprBP E3 ubiquitin ligase controls ribosome biogenesis, cell proliferation, and development

Evolutionarily conserved DCAF1 is a major substrate receptor for the DDB1-CUL4-ROC1 E3 ubiquitin ligase (CRL4) and controls cell proliferation and development. The molecular basis for these functions is unclear. We show here that DCAF1 loss in multiple tissues and organs selectively eliminates proliferating cells and causes perinatal lethality, thymic atrophy, and bone marrow defect. Inducible DCAF1 loss eliminates proliferating, but not quiescent, T cells and MEFs. We identify the ribosome assembly factor PWP1 as a substrate of the CRL4DCAF1 ligase. DCAF1 loss results in PWP1 accumulation, impairing rRNA processing and ribosome biogenesis. Knockdown or overexpression of PWP1 can rescue defects or cause similar defects as DCAF1 loss, respectively, in ribosome biogenesis. DCAF1 loss increases free RPL11, resulting in L11-MDM2 association and p53 activation. Cumulatively, these results reveal a critical function for DCAF1 in ribosome biogenesis and define a molecular basis of DCAF1 function in cell proliferation and development.

Simple, 1-Day Manufacturing of Quiescent Chimeric Antigen Receptor T Cells for Adoptive Immunotherapy

The recent success of immunotherapy using chimeric antigen receptor modified T cells (CAR T) in B-cell malignancies highlights the potential of these cytotoxic "drugs" for cancer therapy. CAR T therapies generally rely upon manufacturing approaches that include prior T cell activation through engagement of the TCR and costimulatory receptors followed by ex vivo expansion of patient-derived T cells over days to weeks. We previously reported that CD3/CD28 stimulation prior to transduction promotes progressive T cell effector differentiation over time in culture with loss of CAR T cell potency (Ghassemi et al. 2018 PMID: 30030295). Since cell division is not a prerequisite for lentiviral vector-mediated gene delivery, we hypothesized that lentiviral transduction of quiescent T cells without prior activation will enhance engraftment and persistence of CART cells that is associated with long-term leukemia control. Here, we show that functional CD19-specific CAR T cells (CART19) can be generated in as little as 24 hours using lentiviral vectors without the need for prior T cell activation. We showed using a non-optimized process that a mean of 6.5% (range 2%-10%) of freshly isolated quiescent T cells can be transduced using an infrared red fluorescent protein (iRFP)-expressing lentiviral vector with slower kinetics of expression compared with activated T cell transduction (peak at 96 hrs vs. 48 hrs for quiescent and activated T cells, respectively). Although substantially less efficient compared to activated T cells, transduction was detected across all T cells subsets with central memory T cells showing the greatest transduction efficiency with a mean of 4-fold greater transduction compared with naïve T cells. Somewhat unexpectedly, CART19 cells generated from quiescent T cells using a CD19-specific CAR vector showed a 3-5 fold greater transgene expression compared with iRFP vectors transduced at similar MOI. However, we show that CAR expression can occur in quiescent T cells even without reverse transcription or integrase function, so called "pseudotransduction". Importantly, we show that this CAR expression produces T cells with cytolytic activity and effector cytokine production in response to antigen that is similar to activated and transduced CAR T cells. Using the well-characterized Nalm6 model of acute lymphoblastic leukemia, we show that CART19 cells generated by transduction of quiescent T cells for 16 hours followed by washing to remove vector exhibit dose-dependent anti-leukemic activity that is durable with injection of as little as 2x105 total T cells. We estimate the latter to contain ~2x104 T cells with integrated lentiviral vector based upon transduction efficiency determined in studies using non-tumor bearing mice. (Fig 1). In summary, our results support the need for further investigation of CAR T cells that are generated using engineering of quiescent T cells. Taking advantage of the ability of lentiviral vectors to transfer genes to quiescent T cells, the highly abbreviated and simplified manufacturing approach described here has the potential to enhance therapeutic potency while also substantially reducing the materials and labor costs associated with current manufacturing approaches that use activated and expanded T cells. In addition, the rapid nature of this manufacturing has the potential to extend the population of patients that may be treated with these therapies by shortening the interval between aphaeresis collection and re-infusion of CAR T cells, which prevents the treatment of some patients with rapidly progressive disease. Disclosures Ghassemi: Novartis: Patents & Royalties. Milone:Novartis: Patents & Royalties, Research Funding.

Metabolic control of regulatory T cell (Treg) survival and function by Lkb1

The metabolic programs of functionally distinct T cell subsets are tailored to their immunologic activities. While quiescent T cells use oxidative phosphorylation (OXPHOS) for energy production, and effector T cells (Teffs) rely on glycolysis for proliferation, the distinct metabolic features of regulatory T cells (Tregs) are less well established. Here we show that the metabolic sensor LKB1 is critical to maintain cellular metabolism and energy homeostasis in Tregs. Treg-specific deletion of Lkb1 in mice causes loss of Treg number and function, leading to a fatal, early-onset autoimmune disorder. Tregs lacking Lkb1 have defective mitochondria, compromised OXPHOS, depleted cellular ATP, and altered cellular metabolism pathways that compromise their survival and function. Furthermore, we demonstrate that the function of LKB1 in Tregs is largely independent of the AMP-activated protein kinase, but is mediated by the MAP/microtubule affinity-regulating kinases and salt-inducible kinases. Our results define a metabolic checkpoint in Tregs that couples metabolic regulation to immune homeostasis and tolerance.

Assessing Potential Genotoxicity of Sleeping Beauty Transposition Events in T-Cell Immunotherapy by Supercomputer-Based High Throughput Profiling,

Abstract 4174 The first-in-human clinical trial using Sleeping Beauty (SB) transposition for T-cell immunotherapy is under way at the University of Texas MD Anderson Cancer Center. To understand potential genotoxic risks involved in our non-viral SB transposition-based immunotherapy, we employed high throughput sequencing (Illumina) and profiling (BlueBioU supercomputer) of the SB insertion sites in T cells. Our SB clinical vector inserted 99.999% of the time at expected TA dinucleotide sites, with 44% of the insertions localizing to intragenic loci and the remainder 56% intergenic. The vast majority (96.5%) of intragenic insertions are intronic while the majority (>60%) of intergenic transpositions fall within non-coding repeat regions. By linking microarray gene expression profile to the vector insertion profile, we find that the number of transcriptional start sites (TSS) hit is proportional to the expected ‘open' conformational loci for the starting population of quiescent T cells. Compared to immunotherapy infusing T cells genetically modified with retrovirus, the integration profiles for SB-modified T cells are favored as: 1) there are more insertions within intergenic regions (56% SB vs. 44% in retroviral), 2) insertions concentrated at TA sites which have a different genomic distribution profile than retroviral insertion sites, 3) TSS associated with quiescent T cells were favored reflecting the electro-transfer of SB DNA plasmids into non-proliferating T cells vs. retroviral protocols that require transduction of activated T cells. Scanning for potential danger loci (e.g. oncogene, tumor suppressor, miRNA etc.), SB integration profiles compare favorably with retroviral insertions. In addition, we identified potential “safe harbor” genetic loci for future targeted modifications. Opportunities in immunotherapy and other personalized medicine modalities will be fully realized when the actual and perceived risks are understood and perhaps mitigated. In the absence of targeted insertions by clinical vectors, the next best option is random integration with an evenly distributed insertion profile across the whole genome to minimize hot spot and clonal expansion of therapeutic T cells. We now seek to proactively profile the T-cell products prior to infusion and follow up sampling to help clinicians in data-driven decision making in a time-sensitive manner. Our methods and findings are consistent with the human application of SB system and the harnessing high throughput supercomputing resources to help clinicians mitigate potential risks. Disclosures: No relevant conflicts of interest to declare.

Stable transduction of quiescent T cells without induction of cycle progression by a novel lentiviral vector pseudotyped with measles virus glycoproteins

A major limitation of current lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent cells such as primary T cells, which hampers their application for gene therapy. Here we generated high-titer LVs incorporating Edmonston measles virus (MV) glycoproteins H and F on their surface. They allowed efficient transduction through the MV receptors, SLAM and CD46, both present on blood T cells. Indeed, these H/F-displaying vectors outperformed by far VSV-G-LVs for the transduction of IL-7–prestimulated T cells. More importantly, a single exposure to these H/F-LVs allowed efficient gene transfer in quiescent T cells, which are not permissive for VSV-G-LVs that need cell-cycle entry into the G1b phase for efficient transduction. High-level transduction of resting memory (50%) and naive (11%) T cells with H/F-LVs, which seemed to occur mainly through SLAM, was not at cost of cell-cycle entry or of target T-cell activation. Finally, the naive or memory phenotypes of transduced resting T cells were maintained and no changes in cytokine profiles were detected, suggesting that T-cell populations were not skewed. Thus, H/F-LV transduction of resting T cells overcomes the limitation of current lentiviral vectors and may improve the efficacy of T cell–based gene therapy.

T Cells Demonstrate Enhanced Specificity for CD19+ Malignancies When Stimulated with IL-21.

T cells genetically modified to express CD19-specific chimeric antigen receptors (CARs) are being evaluated in early-phase clinical trials in patients with B-lineage malignancies. Therapeutic success is predicted by ability of infused CAR+ T cells to both persist and kill in an antigen-dependent fashion. The first of these dual goals can be achieved by altering the CAR molecule to provide T-cell survival signals through a chimeric CD28 endodomain (designated CD19RCD28). We now report that altering the culturing microenvironment with IL-21 improves antigen-dependent cytolysis of T cells when propagated on CD19+ artificial antigen presenting cells (aAPC) derived from K562. To test whether IL-21 acts in conjunction with CD28 signaling to support acquisition of redirected effector functions we electro-transferred quiescent T cells from peripheral blood with Sleeping Beauty system DNA plasmids to introduce CD19RCD28 CAR transposon. Selective outgrowth of CAR+ T cells was achieved on CD19+ aAPC that provide co-stimulation with the addition of exogenous IL-2 and/or IL-21. When IL-21 was present there was preferential numeric expansion of CD19-specific CD8+ T cells which lysed and produced IFN-g in response to CD19 (Figure). Furthermore, the CD8+CAR+ T cells displayed a central memory (CM) cell surface phenotype characterized as CD62L+ and CD28+. In contrast, genetically modified T cells propagated with exogenous IL-2 resulted in predominately CD19-specific CM CD4+ T cells. Thus, cytokines can be used to tailor the CD8/CD4 ratio of CAR+ T cells derived from peripheral blood. These data demonstrate that the dual goals of persistence and lysis can be achieved by altering CAR and the cytokine milieu and have implications for infusing CAR+ T cells in next-generation immunotherapy trials. Figure Figure

Twisted gastrulation (Tsg) is regulated by Tob and enhances TGF-β signaling in activated T lymphocytes

Quiescent T cells express Tob, an APRO gene family member, which functions as a transcriptional regulator. Subtractive hybridization identified Twisted gastrulation (Tsg) as one of the genes suppressed by Tob. Tsg is a secreted protein that interacts with Drosophila decapentaplegic (Dpp) and its vertebrate orthologs BMP2/4 and regulates morphogenetic effects in embryos. Here, we report the expression and function of Tsg in human T cells. Tsg mRNA was almost undetectable in unstimulated T cells and was up-regulated after activation by TCR/CD3 and either CD28, IL-2, or PMA. Tsg protein had no effect on responses of primary T cells to TCR/CD3 stimulation but had a potent inhibitory effect on proliferation and cytokine production of primed alloreactive CD4+ cells. Surprisingly, Tsg did not affect phosphorylation of the BMP-specific Smad1 but induced phosphorylation of the TGF-β–specific Smad2 and mediated DNA binding on Smad3/4 consensus-binding sites, suggesting that it acted downstream of TGF-β. In vitro association assays revealed a direct interaction of Tsg and TGF-β proteins. Thus, Tsg functions as an agonist synergizing with TGF-β to inhibit T-cell activation. Modulation of Tsg signaling may represent a novel target for molecular intervention toward control of aberrant T-cell responses during ongoing graft-versus-host disease (GVHD) and autoimmune diseases.