Citations

Abstract The role of the transcription factor GLI1 in pancreatic tumor initiation promoting the progression of preneoplastic lesions into tumors is well established. However, its function at later stages of pancreatic carcinogenesis remains poorly understood. To address this issue, we crossed the GLI1 knockout animal (GKO) with cre-dependent pancreatic activation of oncogenic KRAS concomitant with loss of the tumor suppressor TP53 (KPC). Interestingly, in this model, GLI1 played a tumor protective function, where survival of GKO/KPC mice was reduced compared to KPC littermates. Both cohorts developed pancreatic cancer without significant histopathological differences in survival studies. However, analysis of mice using ultrasound-based imaging at earlier time-points showed increased tumor burden in GKO/KPC mice. These animals have larger tumors, decreased body weight, increased lactate dehydrogenase production, and leukopenia. Expression studies identified FAS and FAS ligand as potential mediators of this phenomenon. The FAS /FASL axis, an apoptotic inducer, plays a role in the progression of pancreatic cancer, where its expression is usually lost or significantly reduced in advanced stages of the disease. Chromatin immunoprecipitation assays identified FAS and FASL as direct targets of GLI1 where GKO/KPC mice showed lower levels of this ligand compared to KPC animals. Furthermore, GLI1 overexpression and knockdown studies demonstrate a key regulatory role for GLI1 in both FAS and FASL expression. Finally, decreased levels of apoptosis were detected in the tumor tissue in the absence of GLI1 by TUNEL staining. Together, these findings define a novel pathway regulated by GLI1 controlling pancreatic tumor progression and provide a new theoretical framework to help the design and analysis of trials targeting GLI1-related pathways. Citation Format: Lisa D. Mills, Lizhi Zhang, Ronald J. Marler, Phyllis A. Svingen, Maite Fernandez-Barena, Maneesh Dave, William Bamlet, Robert McWilliams, William A. Faubion, Martin E. Fernandez-Zapico. Loss of GLI1 accelerates pancreatic cancer progression in the KPC mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A76.

KLF10 has recently elicited significant attention as a transcriptional regulator of transforming growth factor-β1 (TGF-β1) signaling in CD4+ T cells. In the current study, we demonstrate a novel role for KLF10 in the regulation of TGF-β receptor II (TGF-βRII) expression with functional relevance in antiviral immune response. Specifically, we show that KLF10-deficient mice have an increased number of effector/memory CD8+ T cells, display higher levels of the T helper type 1 cell-associated transcription factor T-bet, and produce more IFN-γ following in vitro stimulation. In addition, KLF10−/− CD8+ T cells show enhanced proliferation in vitro and homeostatic proliferation in vivo. Freshly isolated CD8+ T cells from the spleen of adult mice express lower levels of surface TGF-βRII (TβRII). Congruently, in vitro activation of KLF10-deficient CD8+ T cells upregulate TGF-βRII to a lesser extent compared with wild-type (WT) CD8+ T cells, which results in attenuated Smad2 phosphorylation following TGF-β1 stimulation compared with WT CD8+ T cells. Moreover, we demonstrate that KLF10 directly binds to the TGF-βRII promoter in T cells, leading to enhanced gene expression. In vivo viral infection with Daniel's strain Theiler's murine encephalomyelitis virus (TMEV) also led to lower expression of TGF-βRII among viral-specific KLF10−/− CD8+ T cells and a higher percentage of IFN-γ-producing CD8+ T cells in the spleen. Collectively, our data reveal a critical role for KLF10 in the transcriptional activation of TGF-βRII in CD8+ T cells. Thus, KLF10 regulation of TGF-βRII in this cell subset may likely play a critical role in viral and tumor immune responses for which the integrity of the TGF-β1/TGF-βRII signaling pathway is crucial.

Inducible gene expression, which requires chromatin remodeling on gene promoters, underlies the epigenetically inherited differentiation program of most immune cells. However, chromatin-mediated mechanisms that underlie these events in T regulatory cells remain to be fully characterized. Here, we report that inducibility of FOXP3, a key transcription factor for the development of T regulatory cells, depends upon Kruppel-like factor 10 (KLF10) interacting with two antagonistic histone-modifying systems. We utilized chromatin immunoprecipitation, genome-integrated reporter assays, and functional domain KLF10 mutant proteins, to characterize reciprocal interactions between this transcription factor and either the Sin3-histone deacetylase complex or the histone acetyltransferase, p300/CBP-associated factor (PCAF). We characterize a Sin3-interacting repressor domain on the NH2 terminus of KLF10, which works to limit the activating function of this transcription factor. Indeed, inactivation of this Sin3-interacting domain renders KLF10 able to physically associate with PCAF as to induce FOXP3 gene transcription. We show that this biochemical data derived from studying our genome-integrated reporter cell system are recapitulated in primary murine lymphocytes. Collectively, these results advance our understanding of how a single transcription factor, namely KLF10, functions as a toggle to integrate antagonistic signals regulating FOXP3 and, thus, immune activation.

Abstract Although functionally relevant TLRs can be expressed on human T regulatory (Treg) cells, little is known about the transcriptional control of their expression. We hypothesized that the transcription factor forkhead box P3 (FOXP3) regulates the expression of TLR family members in human Treg cells. Using primary human T cells and a reporter assay in Jurkat T cell lines, we dissected the regulation of TLR10, a TLR highly expressed in human Treg cells. We determined that TLR10 was expressed in human Treg cells through quantitative PCR, Western blotting, and flow cytometry. DNA binding of FOXP3 to a suspected cis-regulatory region in proximity to the transcription start site of TLR10 was established through EMSA and chromatin immunoprecipitation. Transcriptional control of TLR10 by FOXP3 was determined through luciferase reporter assays in Jurkat T cell lines. Relevance of FOXP3 to TLR10 gene transcription in primary T cells was established through the transfection of primary CD4+CD25−FOXP3− T cells with a FOXP3 expression vector, which resulted in prompt production of TLR10 mRNA. Enhanced expression of TLR10 protein in primary Treg cells was induced in a calcium-dependent fashion through TCR activation. The suspected promotional cooperation between FOXP3 and NF-AT was established in the abolition of the luciferase signal upon transfection of a mutant FOXP3 devoid of NF-AT-binding activity. These results suggest that human Treg cells express TLR10, and this expression is regulated through a cooperative complex of FOXP3 and NF-AT.

ABSTRACTObjectives:Inflammatory bowel disease (IBD) is heritable, but a total of 163 variants commonly implicated in IBD pathogenesis account for only 25% of the heritability. Rare, highly penetrant genetic variants may also explain mendelian forms of IBD and some of the missing heritability. To test the hypothesis that rare loss‐of‐function mutations can be causative, we performed whole exome sequencing (WES) on 5 members of a 2‐generation family of European ancestry presenting with an early‐onset and atypical form of IBD.Methods:WES was performed for all of the 5 family members; the mother and 3 male offspring were affected, whereas the father was unaffected. Mapping, annotation, and filtering criteria were used to reduce candidate variants. For functional testing we performed forkhead box P3 (FOXP3) staining and a T‐cell suppression assay.Results:We identified a novel missense variant in exon 6 of the X‐linked FOXP3 gene. The c.694A>C substitution in FOXP3 results in a cysteine‐to‐glycine change at the protein position 232 that is completely conserved among all vertebrates. This variant (heterozygous in the mother and hemizygous in all 3 affected sons) did not impair FOXP3 protein expression, but significantly reduced the ability of the host's T regulatory cells to suppress an inappropriate autoimmune response. The variant results in a milder immune dysregulation, polyendocrinopathy, enteropathy, and X‐linked phenotype with early‐onset IBD.Conclusions:Our study illustrates the successful application of WES for making a definitive molecular diagnosis in a case of multiply affected families, with atypical IBD‐like phenotype. Our results also have important implications for disease biology and disease‐directed therapeutic development.

Abstract The expression of pathogen recognition receptors in human FOXP3+ T regulatory cells is established, yet the function of these receptors is currently obscure. In the process of studying the function of both peripheral and lamina propria FOXP3+ lymphocytes in patients with the human inflammatory bowel disease Crohn’s disease, we observed a clear deficiency in the quantity of FOXP3+ lymphocytes in patients with disease-associated polymorphisms in the pathogen recognition receptor gene NOD2. Subsequently, we determined that the NOD2 ligand, muramyl dipeptide (MDP), activates NF-κB in primary human FOXP3+ T cells. This activation is functionally relevant, as MDP-stimulated human FOXP3+ T cells are protected from death receptor Fas-mediated apoptosis. Importantly, apoptosis protection was not evident in MDP-stimulated FOXP3+ T cells isolated from a patient with the disease-associated polymorphism. Thus, we propose that one function of pathogen recognition receptors in human T regulatory cells is the protection against death receptor-mediated apoptosis in a Fas ligand-rich environment, such as that of the inflamed intestinal subepithelial space.

Abstract Purpose: According to some studies, susceptibility of cells to anticancer drug-induced apoptosis is markedly inhibited by targeted deletion of genes encoding apoptotic protease activating factor 1 (Apaf-1) or certain caspases. Information about levels of these polypeptides in common cancer cell types and any possible correlation with drug sensitivity in the absence of gene deletion is currently fragmentary. Experimental Design: Immunoblotting was used to estimate levels of Apaf-1 as well as procaspase-2, -3, -6, -7, -8, and -9 in the 60-cell-line panel used for drug screening by the National Cancer Institute. Sensitivity of the same lines to >80,000 compounds was determined with 48-hour sulforhodamine B binding assays. Additional 6-day assays were performed for selected agents. Results: Levels of Apaf-1 and procaspases varied widely. Apaf-1 and procaspase-9, which are implicated in caspase activation after treatment of cells with various anticancer drugs, were detectable in all of the cell lines, with levels of Apaf-1 ranging from ∼1 × 105 to 2 × 106 molecules per cell and procaspase-9 from ∼5 × 103 to ∼1.6 × 105 molecules per cell. Procaspase-8 levels ranged from 1.7 × 105 to 8 × 106 molecules per cell. Procaspase-3, a major effector caspase, varied from undetectable to ∼1.6 × 106 molecules per cell. Correlations between levels of these polypeptides and sensitivity to any of a variety of experimental or conventional antineoplastic agents in either 2-day or 6-day cytotoxicity assays were weak at best. Conclusions: With the exception of caspase-3, all of the components of the core cell-death machinery are expressed in all of the cell lines examined. Despite variations in expression, levels of any one component are not a major determinant of drug sensitivity in these cells in vitro.

Abstract Purpose: To assess the maximum tolerated dose, toxicities, pharmacokinetics, and antileukemic activity of topotecan and carboplatin in adults with recurrent or refractory acute leukemias. Experimental Design: Patients received topotecan and carboplatin by 5-day continuous infusion at nine dose levels. Patients achieving a complete remission received up to two additional courses for consolidation. Plasma topotecan and ultrafilterable platinum were assayed on days 1 to 5. In addition, pretreatment levels of various polypeptides in leukemic cells were examined by immunoblotting to assess possible correlations with response. Results: Fifty-one patients received a total of 69 courses of therapy. Dose-limiting toxicity consisted of grade 4/5 typhlitis and grade 3/4 mucositis after one course of therapy or grade 4 neutropenia and thrombocytopenia lasting >50 days when a second course was administered on day 21. Among 45 evaluable patients, there were 7 complete remissions, 2 partial remissions, 1 incomplete complete remission, and 1 reversion to chronic-phase chronic myelogenous leukemia. Topotecan steady-state plasma concentrations increased with dose. No accumulation of topotecan or ultrafilterable platinum occurred between days 1 and 5 of therapy. Leukemic cell levels of topoisomerase I, checkpoint kinase 1, checkpoint kinase 2, and Mcl-1 correlated with proliferating cell nuclear antigen but not with response. In contrast, low Bcl-2 expression correlated with response (P = 0.014, Mann-Whitney U test). Conclusions: The maximum tolerated dose was 1.6 mg/m2/d topotecan plus 150 mg/m2/d carboplatin. The complete remission rate in a heavily pretreated population was 16% (33% at the highest three dose levels). Responses seem to correlate with low pretreatment blast cell Bcl-2 expression.

Abstract The adenosine triphosphate binding-site–directed agent STI571 and the tyrphostin adaphostin are undergoing evaluation as bcr/abl kinase inhibitors. The current study compared the effects of these agents on the survival of K562 cells, bcr/abl-transduced FDC-P1 cells, and myeloid progenitors from patients with chronic myelogenous leukemia (CML) compared with healthy donors. Treatment of K562 cells with 10 μM adaphostin resulted in decreased p210bcr/ablpolypeptide levels in the first 6 hours, followed by caspase activation and accumulation of apoptotic cells in less than 12 hours. By 24 hours, 90% of the cells were apoptotic and unable to form colonies. In contrast, 20 μM STI571 caused rapid inhibition of bcr/abl autophosphorylation without p210bcr/abl degradation. Although this was followed by the inhibition of Stat5 phosphorylation and the down-regulation of Bcl-xL and Mcl-1, only 7% ± 3% and 25% ± 9% of cells were apoptotic at 16 and 24 hours, respectively. Instead, the cytotoxic effects of STI571 became more pronounced with prolonged exposure, with IC90values greater than 20 μM and 1.0 ± 0.6 μM after 24 and 48 hours, respectively. Consistent with these results, 24-hour adaphostin exposure inhibited CML granulocyte colony-forming units (CFU-G) (median IC50, 12 μM) but not normal CFU-G (median IC50, greater than 20 μM), whereas 24-hour STI571 treatment had no effect on CML or normal CFU-G. Additional experiments revealed that STI571-resistant K562 cells remained sensitive to adaphostin. Moreover, the combination of STI571 + adaphostin induced more cytotoxicity in K562 cells and in CML CFU-G than either agent alone did. Collectively, these results identify adaphostin as a mechanistically distinct CML-selective agent that retains activity in STI571-resistant cell lines.