Rene Opavsky

Immature truncated O-glycophenotype of cancer directly induces oncogenic features

Significance Cancer cells characteristically express proteins with immature O-glycosylation, but how and why cancer cells express immature O-glycans has remained poorly understood. Here, we report that one prevalent mechanism in pancreatic cancer is epigenetic silencing, rather than somatic mutations in a key chaperone, core 1 β3-Gal-T-specific molecular chaperone (COSMC), required for mature elongated O-glycosylation. We also demonstrate, with the use of well-defined cell systems generated by precise gene editing, that the aberrant O-glycophenotype by itself induces oncogenic features with enhanced growth and invasion. Our study suggests that the characteristic aberrant O-glycophenotype is critical for the development and behavior of cancer and further provides support for immunotherapeutic strategies that target aberrant O-glycans. Aberrant expression of immature truncated O-glycans is a characteristic feature observed on virtually all epithelial cancer cells, and a very high frequency is observed in early epithelial premalignant lesions that precede the development of adenocarcinomas. Expression of the truncated O-glycan structures Tn and sialyl-Tn is strongly associated with poor prognosis and overall low survival. The genetic and biosynthetic mechanisms leading to accumulation of truncated O-glycans are not fully understood and include mutation or dysregulation of glycosyltransferases involved in elongation of O-glycans, as well as relocation of glycosyltransferases controlling initiation of O-glycosylation from Golgi to endoplasmic reticulum. Truncated O-glycans have been proposed to play functional roles for cancer-cell invasiveness, but our understanding of the biological functions of aberrant glycosylation in cancer is still highly limited. Here, we used exome sequencing of most glycosyltransferases in a large series of primary and metastatic pancreatic cancers to rule out somatic mutations as a cause of expression of truncated O-glycans. Instead, we found hypermethylation of core 1 β3-Gal-T-specific molecular chaperone, a key chaperone for O-glycan elongation, as the most prevalent cause. We next used gene editing to produce isogenic cell systems with and without homogenous truncated O-glycans that enabled, to our knowledge, the first polyomic and side-by-side evaluation of the cancer O-glycophenotype in an organotypic tissue model and in xenografts. The results strongly suggest that truncation of O-glycans directly induces oncogenic features of cell growth and invasion. The study provides support for targeting cancer-specific truncated O-glycans with immunotherapeutic measures.

E2f1-3 Control E2F-target Expression and Cellular Proliferation via a p53-dependent Negative Feedback Loop

ABSTRACT E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1, E2f2, and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21CIP1. Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1-, E2f2-, and E2f3-deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21CIP1 and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.

Loss of Dnmt3b function upregulates the tumor modifier Ment and accelerates mouse lymphomagenesis

DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b-/- lymphomas, but not in Dnmt3b-/- pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b-/- lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b+/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies.

Chronic lymphocytic leukemia cells in a lymph node microenvironment depict molecular signature associated with an aggressive disease.

Chronic lymphocytic leukemia (CLL) cells survive longer in vivo than in vitro, suggesting that the tissue microenvironment provides prosurvival signals to tumor cells. Primary and secondary lymphoid tissues are involved in the pathogenesis of CLL, and the role of these tissue microenvironments has not been explored completely. To elucidate host-tumor interactions, we performed gene expression profiling (GEP) of purified CLL cells from peripheral blood (PB; n = 20), bone marrow (BM; n = 18), and lymph node (LN; n = 15) and validated key pathway genes by real-time polymerase chain reaction, immunohistochemistry and/or TCL1 trans-genic mice. Gene signatures representing several pathways critical for survival and activation of B cells were altered in CLL cells from different tissue compartments. Molecules associated with the B-cell receptor (BCR), B cell-activating factor/a proliferation-inducing ligand (BAFF/APRIL), nuclear factor (NF)-κB pathway and immune suppression signature were enriched in LN-CLL, suggesting LNs as the primary site for tumor growth. Immune suppression genes may help LN-CLL cells to modulate antigen-presenting and T-cell behavior to suppress antitumor activity. PB CLL cells overexpressed chemokine receptors, and their cognate ligands were enriched in LN and BM, suggesting that a chemokine gradient instructs B cells to migrate toward LN or BM. Of several chemokine ligands, the expression of CCL3 was associated with poor prognostic factors. The BM gene signature was enriched with antiapoptotic, cytoskeleton and adhesion molecules. Interestingly, PB cells from lymphadenopathy patients shared GEP with LN cells. In Eμ-TCL1 transgenic mice (the mouse model of the disease), a high percentage of leukemic cells from the lymphoid compartment express key BCR and NF-κB molecules. Together, our findings demonstrate that the lymphoid microenvironment promotes survival, proliferation and progression of CLL cells via chronic activation of BCR, BAFF/APRIL and NF-κB activation while suppressing the immune response.

Dormant Cancer Cells Contribute to Residual Disease in a Model of Reversible Pancreatic Cancer

The initiation and progression of pancreatic ductal adenocarcinoma (PDAC) is governed by a series of genetic and epigenetic changes, but it is still unknown whether these alterations are required for the maintenance of primary and metastatic PDAC. We show here that the c-Myc oncogene is upregulated throughout the entire process of neoplastic progression in human PDAC and in genetically engineered mice that express mutant Kras. To experimentally address whether c-Myc is essential for the growth and survival of cancer cells, we developed a novel mouse model that allows a temporally and spatially controlled expression of this oncogene in pancreatic progenitors and derived lineages of the exocrine pancreas. Unlike previous reports, upregulation of c-Myc was sufficient to induce the formation of adenocarcinomas after a short latency without additional genetic manipulation of cell survival pathways. Deficiency in Cdkn2a increased the rate of metastasis but had no effect on tumor latency or c-Myc-mediated cancer maintenance. Despite a macroscopically complete regression of primary, metastatic, and transplantable tumors following the ablation of c-Myc, some cancer cells remained dormant. A significant number of these residual neoplastic cells expressed cancer stem cell markers, and re-expression of exogenous c-Myc in these cells led to rapid cancer recurrence. Collectively, the results of this study suggest that c-Myc plays a significant role in the progression and maintenance of PDAC, but besides targeting this oncogene or its downstream effectors, additional therapeutic strategies are necessary to eradicate residual cancer cells to prevent disease recurrence.

Tumor suppressor functions of Dnmt3a and Dnmt3b in the prevention of malignant mouse lymphopoiesis

Tumor suppressor functions of Dnmt3a and Dnmt3b in the prevention of malignant mouse lymphopoiesis

Essential Role for Dnmt1 in the Prevention and Maintenance of MYC-Induced T-Cell Lymphomas

ABSTRACT DNA cytosine methylation is an epigenetic modification involved in the transcriptional repression of genes controlling a variety of physiological processes, including hematopoiesis. DNA methyltransferase 1 (Dnmt1) is a key enzyme involved in the somatic inheritance of DNA methylation and thus plays a critical role in epigenomic stability. Aberrant methylation contributes to the pathogenesis of human cancer and of hematologic malignancies in particular. To gain deeper insight into the function of Dnmt1 in lymphoid malignancies, we genetically inactivated Dnmt1 in a mouse model of MYC-induced T-cell lymphomagenesis. We show that loss of Dnmt1 delays lymphomagenesis by suppressing normal hematopoiesis and impairing tumor cell proliferation. Acute inactivation of Dnmt1 in primary lymphoma cells rapidly induced apoptosis, indicating that Dnmt1 is required to sustain T-cell lymphomas. Using high-resolution genome-wide profiling, we identified differentially methylated regions between control and Dnmt1-deficient lymphomas, demonstrating a locus-specific function for Dnmt1 in both maintenance and de novo promoter methylation. Dnmt1 activity is independent of the presence of Dnmt3a or Dnmt3b in de novo promoter methylation of the H2-Ab1 gene. Collectively, these data show for the first time that Dnmt1 is critical for the prevention and maintenance of T-cell lymphomas and contributes to aberrant methylation by both de novo and maintenance methylation.

Methylation-independent repression of Dnmt3b contributes to oncogenic activity of Dnmt3a in mouse MYC-induced T-cell lymphomagenesis

DNA methyltransferase 3A (DNMT3A) catalyzes cytosine methylation of mammalian genomic DNA. In addition to myeloid malignancies, mutations in DNMT3A have been recently reported in T-cell lymphoma and leukemia, implying a possible involvement in the pathogenesis of human diseases. However, the role of Dnmt3a in T-cell transformation in vivo is poorly understood. Here we analyzed the functional consequences of Dnmt3a inactivation in a mouse model of MYC-induced T-cell lymphomagenesis (MTCL). Loss of Dnmt3a delayed tumorigenesis by suppressing cellular proliferation during disease progression. Gene expression profiling and pathway analysis identified upregulation of 17 putative tumor suppressor genes, including DNA methyltransferase Dnmt3b, in Dnmt3a-deficient lymphomas as molecular events potentially responsible for the delayed lymphomagenesis in Dnmt3aΔ/Δ mice. Interestingly, promoter and gene body methylation of these genes was not substantially changed between control and Dnmt3a-deficient lymphomas, suggesting that Dnmt3a may inhibit their expression in a methylation-independent manner. Re-expression of both wild type and catalytically inactive Dnmt3a in Dnmt3aΔ/Δ lymphoma cells in vitro inhibited Dnmt3b expression, indicating that Dnmt3b upregulation may be directly repressed by Dnmt3a. Importantly, genetic inactivation of Dnmt3b accelerated lymphomagenesis in Dnmt3aΔ/Δ mice, demonstrating that upregulation of Dnmt3b is a relevant molecular change in Dnmt3a-deficient lymphomas that inhibits disease progression. Collectively, our data demonstrate an unexpected oncogenic role for Dnmt3a in MTCL through methylation-independent repression of Dnmt3b and possibly other tumor suppressor genes.

Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma

DNA methyltransferase 3A (DNMT3A) is an enzyme involved in DNA methylation that is frequently mutated in human hematologic malignancies. We have previously shown that inactivation of Dnmt3a in hematopoietic cells results in chronic lymphocytic leukemia in mice. Here we show that 12% of Dnmt3a-deficient mice develop CD8+ mature peripheral T cell lymphomas (PTCL) and 29% of mice are affected by both diseases. 10% of Dnmt3a+/- mice develop lymphomas, suggesting that Dnmt3a is a haploinsufficient tumor suppressor in PTCL. DNA methylation was deregulated genome-wide with 10-fold more hypo- than hypermethylated promoters and enhancers, demonstrating that hypomethylation is a major event in the development of PTCL. Hypomethylated promoters were enriched for binding sites of transcription factors AML1, NF-κB and OCT1, implying the transcription factors potential involvement in Dnmt3a-associated methylation. Whereas 71 hypomethylated genes showed an increased expression in PTCL, only 3 hypermethylated genes were silenced, suggesting that cancer-specific hypomethylation has broader effects on the transcriptome of cancer cells than hypermethylation. Interestingly, transcriptomes of Dnmt3a+/- and Dnmt3aΔ/Δ lymphomas were largely conserved and significantly overlapped with those of human tumors. Importantly, we observed downregulation of tumor suppressor p53 in Dnmt3a+/- and Dnmt3aΔ/Δ lymphomas as well as in pre-tumor thymocytes from 9 months old but not 6 weeks old Dnmt3a+/- tumor-free mice, suggesting that p53 downregulation is chronologically an intermediate event in tumorigenesis. Decrease in p53 is likely an important event in tumorigenesis because its overexpression inhibited proliferation in mouse PTCL cell lines, suggesting that low levels of p53 are important for tumor maintenance. Altogether, our data link the haploinsufficient tumor suppressor function of Dnmt3a in the prevention of mouse mature CD8+ PTCL indirectly to a bona fide tumor suppressor of T cell malignancies p53.

Inactivation of Rb and E2f8 synergizes to trigger stressed DNA replication during erythroid terminal differentiation

ABSTRACT Rb is critical for promoting cell cycle exit in cells undergoing terminal differentiation. Here we show that during erythroid terminal differentiation, Rb plays a previously unappreciated and unorthodox role in promoting DNA replication and cell cycle progression. Specifically, inactivation of Rb in erythroid cells led to stressed DNA replication, increased DNA damage, and impaired cell cycle progression, culminating in defective terminal differentiation and anemia. Importantly, all of these defects associated with Rb loss were exacerbated by the concomitant inactivation of E2f8. Gene expression profiling and chromatin immunoprecipitation (ChIP) revealed that Rb and E2F8 cosuppressed a large array of E2F target genes that are critical for DNA replication and cell cycle progression. Remarkably, inactivation of E2f2 rescued the erythropoietic defects resulting from Rb and E2f8 deficiencies. Interestingly, real-time quantitative PCR (qPCR) on E2F2 ChIPs indicated that inactivation of Rb and E2f8 synergizes to increase E2F2 binding to its target gene promoters. Taken together, we propose that Rb and E2F8 collaborate to promote DNA replication and erythroid terminal differentiation by preventing E2F2-mediated aberrant transcriptional activation through the ability of Rb to bind and sequester E2F2 and the ability of E2F8 to compete with E2F2 for E2f-binding sites on target gene promoters.