Highlights d Levels of NAD+ and NAMPT are decreased in TILs compared to those of other T cells d Tubby is a transcriptional factor for NAMPT in T cells d NAMPT-mediated NAD+ production is essential for T cell activation by generating ATP d NAD+ supplementation enhances the tumor-killing function of T cells

Tyrosine phosphorylation, orchestrated by tyrosine kinases and phosphatases, modulates a multi-layered signaling network in a time- and space-dependent manner. Dysregulation of this post-translational modification is inevitably associated with pathological diseases. Our previous work has demonstrated that non-receptor tyrosine kinase FER is upregulated in ovarian cancer, knocking down which attenuates metastatic phenotypes. However, due to the limited number of known substrates in the ovarian cancer context, the molecular basis for its pro-proliferation activity remains enigmatic. Here, we employed mass spectrometry and biochemical approaches to identify insulin receptor substrate 4 (IRS4) as a novel substrate of FER. FER engaged its kinase domain to associate with the PH and PTB domains of IRS4. Using a proximity-based tagging system in ovarian carcinoma-derived OVCAR-5 cells, we determined that FER-mediated phosphorylation of Tyr779 enables IRS4 to recruit PIK3R2/p85β, the regulatory subunit of PI3K, and activate the PI3K-AKT pathway. Rescuing IRS4-null ovarian tumor cells with phosphorylation-defective mutant, but not WT IRS4 delayed ovarian tumor cell proliferation both in vitro and in vivo. Overall, we revealed a kinase-substrate mode between FER and IRS4, and the pharmacological inhibition of FER kinase may be beneficial for ovarian cancer patients with PI3K-AKT hyperactivation.

Tumor immunotherapies have provided clinical benefits, yet great potential remains for optimizing therapeutic effects. Here, we show that low NAD + levels restrict the function of 20 tumor infiltrating T lymphocytes (TILs). TILs harvested from human ovarian tumor tissues showed decreased NAD + levels compared with T cells from paired peripheral blood samples.The combination of whole-genome CRISPR and large-scale metabolic inhibitor screens implicated the NAD + biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT) is required for T cell activation. Further isotopic labeling and LC-MS studies confirmed that NAD + 25 depletion suppressed mitochondrial energy biosynthesis in T cells. Excitingly, NAD + supplementation significantly enhanced the tumor cell-killing efficacy of CAR-T cells ex vivo, and extended animal survive in both adoptive CAR-T model and immune checkpoint blockade treatment models in vivo. This study demonstrates an over-the-counter nutrient supplement NAD + could robustly boost the efficacy of T cell-based immunotherapy and provides insights 30 into the cellular basis of T cell metabolic reprogramming in treating cancers. One Sentence Summary:NAD + supplementation during cancer immunotherapies significantly enhances T cell activation and tumor killing capacity.Main Text: 35 Cancer immunotherapies including adoptive transfer of naturally-occurring tumor infiltrating lymphocytes (TIL) and genetically-engineered T cells, as well as the use of immune checkpoint inhibitors to boost the function of T cells have emerged as promising approaches to achieve durable clinical responses of otherwise treatment-refractory cancers 1-5 . Although cancer immunotherapies have been successfully utilized in the clinic for subsets of patients, there are 40 several limitations which prevent the broad use of these therapies for entire patient populations 6,7 . Given the function of T cells as key mediators for tumor destruction, their characteristics (e.g., durability, longevity, and killing efficiency, etc.) substantially determine the clinical outcomes of many immunotherapies [8][9][10][11] . Studies have established that successful clearance of tumors mediated by infiltrated T cells can be limited by physical barriers generated by stroma 45 3 cells 12 , immuno-suppressive networks 13 , and nutrient limitations within the microenvironment 14,15 . Thus, efforts to promote the stemness, proliferation and activation capacity of T cells should enable improvements to the efficacy of cancer immunotherapies.Recently, cellular metabolic processes have been reported to shape T cell differentiation and functional activity [16][17][18] . Modulation of metabolic process such as fatty acid catabolism can 50 improve T cell activation and therapeutic function [19][20][21][22][23][24] . It was also reported that there is a strong link between metabolic activity in tumor infiltrated T cells (TIL) and their effector function 25,26 , and stimulation mitochondrial biogenesis via enforced expression of PGC1α resulted in...

Telomeres protect chromosome ends from inappropriately activating the DNA damage and repair responses. Primary microcephaly is a key clinical feature of several human telomere disorder syndromes, but how microcephaly is linked to dysfunctional telomeres is not known. Here, we show that the microcephalin 1/BRCT-repeats inhibitor of hTERT (MCPH1/BRIT1) protein, mutated in primary microcephaly, specifically interacts with the TRFH domain of the telomere binding protein TRF2. The crystal structure of the MCPH1–TRF2 complex reveals that this interaction is mediated by the MCPH1 330YRLSP334 motif. TRF2-dependent recruitment of MCPH1 promotes localization of DNA damage factors and homology directed repair of dysfunctional telomeres lacking POT1-TPP1. Additionally, MCPH1 is involved in the replication stress response, promoting telomere replication fork progression and restart of stalled telomere replication forks. Our work uncovers a previously unrecognized role for MCPH1 in promoting telomere replication, providing evidence that telomere replication defects may contribute to the onset of microcephaly.

A critical aspect of understanding the regulation of signal transduction is not only to identify the protein-protein interactions that govern assembly of signaling pathways, but also to understand how those pathways are regulated in time and space. In this report, we have applied both gain-of-function and loss-of-function analyses to assess the role of the non-receptor protein tyrosine kinase FER in activation of the HGF Receptor protein tyrosine kinase MET. Overexpression of FER led to direct phosphorylation of several signaling sites in MET, including Tyr1349, but not the activation loop residues Tyr1234/5; in contrast, suppression of FER by RNAi revealed that phosphorylation of both a C-terminal signaling site (Tyr1349) and the activation loop (Tyr1234/5) were influenced by the function of this kinase. Adaptin β, a component of the adaptor protein complex 2 (AP-2) that links clathrin to receptors in coated vesicles, was recruited to MET following FER-mediated phosphorylation. Furthermore, we provide evidence to support a role of FER in maintaining plasma membrane distribution of MET and thereby delaying protein-tyrosine phosphatase PTP1B-mediated inactivation of the receptor. Simultaneous up-regulation of FER and down-regulation of PTP1B observed in ovarian carcinoma-derived cell lines would be expected to contribute to persistent activation of HGF-MET signaling, suggesting that targeting of both FER and MET may be an effective strategy for therapeutic intervention in ovarian cancer.

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Tyrosine phosphorylation, orchestrated by tyrosine kinases and phosphatases, modulates a multi-layered signaling network in a time and space dependent manner. Dysregulation of this post-translational modification is inevitably associated with pathological diseases. Our previous work has demonstrated that non-receptor tyrosine kinase FER is upregulated in ovarian cancer. Knockdown of the kinase attenuates metastatic phenotypes in tumor cells. Here we employed mass spectrometry and biochemical approaches to identify IRS4 as a novel substrate of FER. Using a proximity-based tagging system, we determined that FER-mediated phosphorylation of Tyr779 enables IRS4 to recruit PIK3R2/p85β, the regulatory subunit of PI-3K, and activate the PI3K-AKT pathway. Rescuing IRS4-null ovarian tumor cells with phosphorylation-defective mutant, but not WT IRS4, delayed tumor cell proliferation both in vitro and in vivo. Overall, we revealed a kinase-substrate regulatory mode between FER and IRS4, and the pharmacological inhibition of FER kinase may be beneficial for ovarian cancer patients with PI3K-AKT hyperactivation.