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IMPORTANCE Individuals genetically predisposed to pancreatic cancer may benefit from early detection. Genes that predispose to pancreatic cancer and the risks of pancreatic cancer associated with mutations in these genes are not well defined. OBJECTIVE To determine whether inherited germline mutations in cancer predisposition genes are associated with increased risks of pancreatic cancer. DESIGN, SETTING, AND PARTICIPANTS Case-control analysis to identify pancreatic cancer predisposition genes; longitudinal analysis of patients with pancreatic cancer for prognosis. The study included 3030 adults diagnosed as having pancreatic cancer and enrolled in a Mayo Clinic registry between October 12, 2000, and March 31, 2016, with last follow-up on June 22, 2017. Reference controls were 123 136 individuals with exome sequence data in the public Genome Aggregation Database and 53 105 in the Exome Aggregation Consortium database. EXPOSURES Individuals were classified based on carrying a deleterious mutation in cancer predisposition genes and having a personal or family history of cancer. MAIN OUTCOMES AND MEASURES Germline mutations in coding regions of 21 cancer predisposition genes were identified by sequencing of products from a custom multiplex polymerase chain reaction–based panel; associations of genes with pancreatic cancer were assessed by comparing frequency of mutations in genes of pancreatic cancer patients with those of reference controls. RESULTS Comparing 3030 case patients with pancreatic cancer (43.2% female; 95.6% non-Hispanic white; mean age at diagnosis, 65.3 [SD, 10.7] years) with reference controls, significant associations were observed between pancreatic cancer and mutations in CDKN2A (0.3% of cases and 0.02% of controls; odds ratio [OR], 12.33; 95% CI, 5.43–25.61); TP53 (0.2% of cases and 0.02% of controls; OR, 6.70; 95% CI, 2.52–14.95); MLH1 (0.13% of cases and 0.02% of controls; OR, 6.66; 95% CI, 1.94–17.53); BRCA2 (1.9% of cases and 0.3% of controls; OR, 6.20; 95% CI, 4.62–8.17); ATM (2.3% of cases and 0.37% of controls; OR, 5.71; 95% CI, 4.38–7.33); and BRCA1 (0.6% of cases and 0.2% of controls; OR, 2.58; 95% CI, 1.54–4.05). CONCLUSIONS AND RELEVANCE In this case-control study, mutations in 6 genes associated with pancreatic cancer were found in 5.5% of all0 pancreatic cancer patients, including 7.9% of patients with a family history of pancreatic cancer and 5.2% of patients without a family history of pancreatic cancer. Further research is needed for replication in other populations.

Despite the strides that immunotherapy has made in mediating tumor regression, the clinical effects are often transient, and therefore more durable responses still are needed. The temporary nature of the therapy-induced immune response can be attributed to tumor immune evasion mechanisms, mainly the effect of suppressive immune cells and, in particular, T regulatory cells (Treg). Although the depletion of Treg has been shown to be effective in enhancing immune responses, selective depletion of these suppressive cells without affecting other immune cells has not been very successful, and new agents are sought. We found that PI3K-Akt pathway inhibitors selectively inhibit Treg with minimal effect on conventional T cells (Tconv). Our results clearly show selective in vitro inhibition of activation (as represented by a decrease in downstream signaling) and proliferation of Treg in comparison to Tconv when treated with different Akt and PI3K inhibitors. This effect has been observed in both human and murine CD4 T cells. In vivo treatment with these inhibitors resulted in a significant and selective reduction in Treg both in naïve and tumor-bearing mice. Furthermore, these PI3K-Akt inhibitors led to a significant therapeutic antitumor effect, which was shown to be Treg-dependent. Here, we report the use of PI3K-Akt pathway inhibitors as potent agents for the selective depletion of suppressive Treg. We show that these inhibitors are able to enhance the antitumor immune response and are therefore promising clinical reagents for Treg-depletion.

Programmed death-1 receptor (PD-1) is expressed on T cells following TCR activation. Binding of this receptor to its cognate ligands, programmed death ligand (PDL)-1 and PDL-2, down-regulates signals by the TCR, promoting T-cell anergy and apoptosis, thus leading to immune suppression. Here, we find that using an anti-PD-1 antibody (CT-011) with Treg-cell depletion by low-dose cyclophosphamide (CPM), combined with a tumor vaccine, induces synergistic antigen-specific immune responses and reveals novel activities of each agent in this combination. This strategy led to complete regression of established tumors in a significant percentage of treated animals, with survival prolongation. We show for the first time that combining CT-011 and CPM significantly increases the number of vaccine-induced tumor-infiltrating CD8 1 T cells, with simultaneous decrease in infiltrating Treg cells. Interestingly, we find that CT-011 prolongs Treg-cell inhibition induced by CPM, leading to a sustainable significant synergistic decrease of splenic and tumor-infiltrated Treg cells. Surprisingly, we find that the anti-tumor effect elicited by the combination of CT-011 and CPM is dependent on both CD8 1 and CD4 1 T-cell responses, although the antigen we used is a class I MHCrestricted peptide. Thus, we describe a novel and effective therapeutic approach by combining multiple strategies to target several tumor-mediated immune inhibitory mechanisms.Key words: Cancer immunotherapy . Programmed death-1 receptor . Vaccine IntroductionImmune suppression/evasion is one of the major impediments to the development of effective immune therapy for cancer. Programmed death-1 receptor (PD-1) is a member of the B7 family that is expressed on activated T cells and is found to play an important role in immune evasion. On binding its cognate ligands programmed death ligand (PDL)-1 or PDL-2, PD-1 downregulates signaling by the T-cell receptor (TCR), inducing T-cell anergy and apoptosis and thus leading to immune suppression [1][2][3][4][5][6]. Many human malignancies up-regulate PDL-1, and this upregulation has been directly correlated with immune suppression and poor prognosis in several types of cancer [4,[7][8][9][10][11]. The PD-1/PDL-1 interaction leads to suppression and apoptosis of [27,[29][30][31][32][33].Here, we hypothesize that combining inhibition of Treg cells with strategies that block the PD-1/PDL-1 interaction and vaccine would result in a potent anti-tumor immunotherapeutic strategy. CT-011 is a novel humanized IgG1 k recombinant monoclonal anti-PD-1 antibody that has been shown to promote anti-tumor immunity in animal models in a Phase I clinical trial in hematological malignancies [34].We found that PD-1 blockade with low-dose CPM, given in combination with vaccine, synergistically induces strong antigenspecific immune responses and increases CD8 1 and CD4 1 Foxp3À T-cell infiltration into the tumor, leading to a potent antitumor effect. Interestingly, we demonstrated that the efficacy of the combination relies not only on CD8 1 but...

The transcription factor E2F-1 is implicated in the activation of S-phase genes as well as induction of apoptosis, and is regulated by interactions with Rb and by cell cycle-dependent alterations in E2F-1 abundance. We earlier demonstrated a pivotal role for poly(ADPribose) polymerase-1 (PARP-1) in the regulation of E2F-1 expression and promoter activity during S-phase re-entry when quiescent cells re-enter the cell cycle. We now investigate the putative mechanism(s) by which PARP-1 may upregulate E2F-1 promoter activity during S-phase re-entry. DNase-1 footprint assays with purified PARP-1 showed that PARP-1 did not directly bind the E2F-1 promoter in a sequence-specific manner. In contrast to p53, a positive acceptor in poly(ADP-ribosyl)ation reactions, E2F-1 was not poly(ADP-ribosyl)ated by wildtype PARP-1 in vitro, indicating that PARP-1 does not exert a dual effect on E2F-1 transcriptional activation. Protein-binding reactions and coimmunoprecipitation experiments with purified PARP-1 and E2F-1, however, revealed that PARP-1 binds to E2F-1 in vitro. More significantly, physical association of PARP-1 and E2F-1 in vivo also occurred in wild-type fibroblasts 5 h after re-entry into S phase, coincident with the increase in E2F-1 promoter activity and expression of E2F-1-responsive Sphase genes cyclin A and c-Myc. Mapping of the interaction domains revealed that full-length PARP-1 as well as PARP-1 mutants lacking either the catalytic active site or the DNA-binding domain equally bind E2F-1, whereas a PARP-1 mutant lacking the automodification domain does not, suggesting that the protein interaction site is located in this central domain. Finally, gel shift analysis with end-blocked E2F-1 promoter sequence probes verified that the binding of PARP-1 to E2F-1 enhances binding to the E2F-1 promoter, indicating that PARP-1 acts as a positive cofactor of E2F-1-mediated transcription.

To modulate T-cell function for cancer therapy, one challenge is to selectively attenuate regulatory but not conventional CD4 T-cell subsets [regulatory T cell (Treg) and conventional T cell (Tconv)]. In this study, we show how a functional dichotomy in Class IA PI3K isoforms in these two subsets of CD4 T cells can be exploited to target Treg while leaving Tconv intact. Studies employing isoform-specific PI3K inhibitors and a PI3Kδ-deficient mouse strain revealed that PI3Kα and PI3Kβ were functionally redundant with PI3Kδ in Tconv. Conversely, PI3Kδ was functionally critical in Treg, acting there to control T-cell receptor signaling, cell proliferation, and survival. Notably, in a murine model of lung cancer, coadministration of a PI3Kδ-specific inhibitor with a tumor-specific vaccine decreased numbers of suppressive Treg and increased numbers of vaccine-induced CD8 T cells within the tumor microenvironment, eliciting potent antitumor efficacy. Overall, our results offer a mechanistic rationale to employ PI3Kδ inhibitors to selectively target Treg and improve cancer immunotherapy. .

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Carcinoembryonic antigen (CEA) is a tumor marker that is associated with metastasis, poor response to chemotherapy of colorectal cancer (CRC), and anoikis, a form of apoptosis caused by cell detachment from matrix that is dependent on TRAIL-R2 (DR5) and caspase-8 activation in CRC. Although CEA is a homophilic binding protein that may provide survival signals through homotypical cell aggregation, we now report that CEA binds TRAIL-R2 (DR5) directly in twohybrid assays to decrease anoikis through the extrinsic pathway. Deletion of the PELPK sequence (delPELPK) of CEA (delPELPK CEA) restores sensitivity to anoikis while it maintains its cell aggregation function. Wild-type (WT) CEA also increases experimental hepatic metastasis, whereas the delPELPK CEA does not. Thus, membrane CEA interacts with DR5 to inhibit anoikis and increase metastatic potential in CRC. [Cancer Res 2007;67(10):4774-82]