Recruitment of effector T cells to inflamed peripheral tissues is regulated by chemokines and their receptors, but the factors regulating recruitment to tumors remain largely undefined. Ionizing radiation (IR) therapy is a common treatment modality for breast and other cancers. Used as a cytocidal agent for proliferating cancer cells, IR in combination with immunotherapy has been shown to promote immune-mediated tumor destruction in preclinical studies. In this study we demonstrate that IR markedly enhanced the secretion by mouse and human breast cancer cells of CXCL16, a chemokine that binds to CXCR6 on Th1 and activated CD8 effector T cells, and plays an important role in their recruitment to sites of inflammation. Using a poorly immunogenic mouse model of breast cancer, we found that irradiation increased the migration of CD8+CXCR6+ activated T cells to tumors in vitro and in vivo. CXCR6-deficient mice showed reduced infiltration of tumors by activated CD8 T cells and impaired tumor regression following treatment with local IR to the tumor and Abs blocking the negative regulator of T cell activation, CTLA-4. These results provide the first evidence that IR can induce the secretion by cancer cells of proinflammatory chemotactic factors that recruit antitumor effector T cells. The ability of IR to convert tumors into “inflamed” peripheral tissues could be exploited to overcome obstacles at the effector phase of the antitumor immune response and improve the therapeutic efficacy of immunotherapy.

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The mTOR signaling complex integrates signals from growth factors and nutrient availability to control cell growth and proliferation, in part through effects on the protein-synthetic machinery. Protein synthesis rates fluctuate throughout the cell cycle but diminish significantly during the G 2 /M transition. The fate of the mTOR complex and its role in coordinating cell growth and proliferation signals with protein synthesis during mitosis remain unknown. Here we demonstrate that the mTOR complex 1 (mTORC1) pathway, which stimulates protein synthesis, is actually hyperactive during mitosis despite decreased protein synthesis and reduced activity of mTORC1 upstream activators. We describe previously unknown G 2 /M-specific phosphorylation of a component of mTORC1, the protein raptor, and demonstrate that mitotic raptor phosphorylation alters mTORC1 function during mitosis. Phosphopeptide mapping and mutational analysis demonstrate that mitotic phosphorylation of raptor facilitates cell cycle transit through G 2 /M. Phosphorylation-deficient mutants of raptor cause cells to delay in G 2 /M, whereas depletion of raptor causes cells to accumulate in G 1 . We identify cyclindependent kinase 1 (cdk1 [cdc2]) and glycogen synthase kinase 3 (GSK3) pathways as two probable mitosisregulated protein kinase pathways involved in mitosis-specific raptor phosphorylation and altered mTORC1 activity. In addition, mitotic raptor promotes translation by internal ribosome entry sites (IRES) on mRNA during mitosis and is demonstrated to be associated with rapamycin resistance. These data suggest that this pathway may play a role in increased IRES-dependent mRNA translation during mitosis and in rapamycin insensitivity.

The cellular response to DNA damage is mediated through multiple pathways that regulate and coordinate DNA repair, cell cycle arrest, and cell death. We show that the DNA damage response (DDR) induced by ionizing radiation (IR) is coordinated in breast cancer cells by selective mRNA translation mediated by high levels of translation initiation factor eIF4G1 (eukaryotic initiation factor 4γ1). Increased expression of eIF4G1, common in breast cancers, was found to selectively increase translation of mRNAs involved in cell survival and the DDR, preventing autophagy and apoptosis [ Survivin , hypoxia inducible factor 1α ( HIF1 α ), X-linked inhibitor of apoptosis ( XIAP )], promoting cell cycle arrest [growth arrest and DNA damage protein 45a ( GADD45a ), protein 53 ( p53 ), ATR-interacting protein ( ATRIP ), Check point kinase 1 ( Chk1 )] and DNA repair [p53 binding protein 1 ( 53BP1 ), breast cancer associated proteins 1, 2 ( BRCA1/2 ), Poly-ADP ribose polymerase ( PARP ), replication factor c2–5 ( Rfc2-5 ), ataxia telangiectasia mutated gene 1 ( ATM ), meiotic recombination protein 11 ( MRE-11 ), and others]. Reduced expression of eIF4G1, but not its homolog eIF4G2, greatly sensitizes cells to DNA damage by IR, induces cell death by both apoptosis and autophagy, and significantly delays resolution of DNA damage foci with little reduction of overall protein synthesis. Although some mRNAs selectively translated by higher levels of eIF4G1 were found to use internal ribosome entry site (IRES)-mediated alternate translation, most do not. The latter group shows significantly reduced dependence on eIF4E for translation, facilitated by an enhanced requirement for eIF4G1. Increased expression of eIF4G1 therefore promotes specialized translation of survival, growth arrest, and DDR mRNAs that are important in cell survival and DNA repair following genotoxic DNA damage.

BackgroundThe adenosine/uridine-rich element (ARE)-binding protein AUF1 functions to regulate the inflammatory response through the targeted degradation of cytokine and other mRNAs that contain specific AREs in their 3' noncoding region (3' NCR). To investigate the role of AUF1 in the immune system, we characterized the lymphoid compartments of AUF1-deficient mice.ResultsMice lacking AUF1 exhibit an altered proportion and size of splenic B cell subsets. We show prominent apoptosis in splenic B cell follicles and reduced expression of Bcl-2, A1, and Bcl-XL correlate with increased turnover and significant reduction in the number and proportion of splenic FO B cells in AUF1-deficient mice. In addition, AUF1-deficient mice exhibit a sharp decrease in splenic size and lymphocyte cellularity. Bone marrow transfer studies demonstrate that AUF1 deficiency induces cell-autonomous defects in mature B cell subsets but not in the overall number of splenocytes. Reconstitution of irradiated adult AUF1-deficient mice with wild-type bone marrow restores the proportion of FO and marginal zone (MZ) B cells, but does not rescue the decrease in the number of splenocytes. Functionally, AUF1-deficient mice mount an attenuated response to T cell-independent (TI) antigen, which correlates with impaired MZ B cell function.ConclusionThese data indicate that AUF1 is important in the maintenance of splenic FO B cells and adequate humoral immune responses.

Purpose: Enfortumab vedotin (EV) is an antibody–drug conjugate (ADC) targeting NECTIN4 (encoded by the PVRL4/NECTIN4 gene) approved for treatment-refractory metastatic urothelial cancer. Factors that mediate sensitivity or resistance to EV are unknown. In this study, we sought to (i) examine heterogeneity of NECTIN4 gene expression across molecular subtypes of bladder cancer and (ii) determine whether NECTIN4 expression mediates EV sensitivity or resistance. Experimental Design: Molecular subtyping and NECTIN4 expression data from seven muscle-invasive bladder cancer clinical cohorts (n = 1,915 total specimens) were used to assess NECTIN4 expression across molecular subtypes. The outcome of the transcriptomic analysis was relative NECTIN4 expression in the consensus molecular subtypes of bladder cancer. Expression of NECTIN4 was validated in bladder cancer cell lines. NECTIN4 was stably overexpressed or knocked down in basal and luminal bladder cancer cell lines and EV drug sensitivity assays were performed, as measured by cell proliferation and clonogenic assays. Results: NECTIN4 expression is heterogenous across molecular subtypes of bladder cancer and significantly enriched in luminal subtypes. NECTIN4 expression is positively correlated with luminal markers GATA3, FOXA1, and PPARG across all cohorts. NECTIN4 expression is both necessary and sufficient for EV sensitivity in luminal and basal subtypes of urothelial bladder cancer cells. Downregulation of NECTIN4 leads to EV resistance. Conclusions: Sensitivity to EV is mediated by expression of NECTIN4, which is enriched in luminal subtypes of bladder cancer. These findings may have implications for biomarker development, patient selection, and the inclusion of molecular subtyping in ongoing and future EV clinical trials. See related commentary by Teo and Rosenberg, p. 4950

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Many studies have positioned Notch signaling at various critical junctions during T-cell development. There is, however, debate regarding the role of Notch in the CD4 versus CD8 lineage commitment. Because there are 4 Notch receptors and RBP-J-independent Notch signaling has been reported, we decided to eliminate ␥-secretase activity once its activity is required for all forms of Notch signaling. T-cell-specific elimination of ␥-secretase was carried out by crossing presenilin-1 (PS1) floxed mice with CD4-Cre mice and PS2 KO mice, generating PS KO mice. Thymic CD4 ؉ CD8 ؉ double-positive (DP) cells from these mice were strikingly resistant to apoptosis by anti-CD3 treatment in vivo and expressed more Bcl-X L than control thymocytes, and deletion of only one allele of Bcl-X L gene restored wildtype levels of sensitivity to apoptosis. In addition, these PS KO animals displayed a significant decrease in the number of CD8 ؉ T cells in the periphery, and these IntroductionNotch signaling was first identified in strains of Drosophila flies that exhibit Notching at their wing edges, and subsequent analyses showed that Notch signals regulate cell fate, cell numbers via effects on proliferation and survival, and cell position, all of them dependent of dose, timing, and context of the Notch signal. 1 Nearly all aspects of invertebrate Notch signaling are recapitulated in mammals in which this pathway plays multiple roles in normal development and disease. 1,2 Engagement of Notch receptor by its ligands results in a series of proteolytic cleavage events that cause the release of the Notch intracellular domain (NIC). The final processing step of Notch is achieved by a ␥-secretase complex where presenilins (PSs) play a major role. 3 Indeed, the absence of PS function through genetic ablation or using ␥-secretase inhibitors induces a phenocopy of Notch loss of function. [4][5][6][7][8][9] Once NIC is in the nucleus, it interacts with RBP-J resulting in the transcription of several Notch targets such as hes, hrt, and deltex family members, or pre-T␣, to name a few. 10,11 It is important to note that Notch activities independent of RBP-J but linked to other proteins such as deltex have been described. 2,[12][13][14][15] Despite the crucial role of presenilins in the Notch pathway, PSs could also be directly involved in MAPK activation/ suppression, so it is also possible that any PS effect on the differentiation, activation, cell growth, and death of T cells could be independent of Notch signaling.Many studies have positioned Notch receptor-ligand interactions at various critical junctions in T-cell development, most notably in the T versus B lineage choice, a Notch role for which there is complete agreement. 16,17 In other set of studies, Notch1 signals have also been shown to promote the ␣␤ over the ␥␦ T lineage. 18 However, the effect of the Notch pathway in CD4 versus CD8 T-cell lineage commitment is controversial because overexpression of NIC1 or NIC2, but not NIC3, decreases the CD4/CD8 ratio in the thymus, 19-23 althoug...