Markus Reschke

The BRAF Pseudogene Functions as a Competitive Endogenous RNA and Induces Lymphoma In Vivo

Research over the past decade has suggested important roles for pseudogenes in physiology and disease. In vitro experiments demonstrated that pseudogenes contribute to cell transformation through several mechanisms. However, in vivo evidence for a causal role of pseudogenes in cancer development is lacking. Here, we report that mice engineered to overexpress either the full-length murine B-Raf pseudogene Braf-rs1 or its pseudo CDS or 3 UTR develop an aggressive malignancy resembling human diffuse large B cell lymphoma. We show that Braf-rs1 and its human ortholog, BRAFP1, elicit their oncogenic activity, at least in part, as competitive endogenous RNAs (ceRNAs) that elevate BRAF expression and MAPK activation in vitro and in vivo. Notably, we find that transcriptional or genomic aberrations of BRAFP1 occur frequently in multiple human cancers, including B cell lymphomas. Our engineered mouse models demonstrate the oncogenic potential of pseudogenes and indicate that ceRNA-mediated microRNA sequestration may contribute to the development of cancer.

Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance.

Mutations in the metabolic enzymes isocitrate dehydrogenase-1 (IDH1) and IDH2 that produce the oncometabolite D-2-hydroxyglutarate (2-HG) occur frequently in human acute myeloid leukemia (AML). 2-HG modulates numerous biological pathways implicated in malignant transformation, but the contribution of mutant IDH proteins to maintenance and progression of AML inxa0vivo is currently unknown. To answer this crucial question we have generated transgenic mice that express IDH2(R140Q) in an on/off- and tissue-specific manner using a tetracycline-inducible system. We found that IDH2(R140Q) can cooperate with overexpression of HoxA9 and Meis1a and with mutations in FMS-like tyrosine kinase 3 (FLT3) to drive acute leukemia inxa0vivo. Critically, we show that genetic deinduction of mutant IDH2 in leukemic cells inxa0vivo has profound effects on their growth and/or maintenance. Our data demonstrate the proto-oncogenic role of mutant IDH2 and support its relevance as a therapeutic target for the treatment of human AML.

Active Pin1 is a key target of all- trans retinoic acid in acute promyelocytic leukemia and breast cancer

A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency for inhibiting Pin1 function in vivo. By using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)—a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but whose drug target remains elusive—inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the protein encoded by the fusion oncogene PML–RARA and treats APL in APL cell and animal models as well as in human patients. ATRA-induced Pin1 ablation also potently inhibits triple-negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors.

Characterization and analysis of the composition and dynamics of the mammalian riboproteome

Increasing evidence points to an important role for the ribosome in the regulation of biological processes and as a target for deregulation in disease. Here, we describe a SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry approach to probing mammalian riboproteomes. Using a panel of cell lines, as well as genetic and pharmacological perturbations, we obtained a comparative characterization of the cellular riboproteome. This analysis identified a set of riboproteome components, consisting of a diverse array of proteins with a strong enrichment for RNA-binding proteins. Importantly, this global analysis uncovers a high incidence of genetic alterations to riboproteome components in cancer, with a distinct bias toward genetic amplification. We further validated association with polyribosomes for several riboproteome components and demonstrate that enrichment at the riboproteome can depend on cell type, genetics, or cellular stimulus. Our results have important implications for the understanding of how ribosomes function and provide a platform for uncovering regulators of translation.

The RNA binding protein ESRP1 fine-tunes the expression of pluripotency-related factors in mouse embryonic stem cells

In pluripotent stem cells, there is increasing evidence for crosstalk between post-transcriptional and transcriptional networks, offering multifold steps at which pluripotency can be controlled. In addition to well-studied transcription factors, chromatin modifiers and miRNAs, RNA-binding proteins are emerging as fundamental players in pluripotency regulation. Here, we report a new role for the RNA-binding protein ESRP1 in the control of pluripotency. Knockdown of Esrp1 in mouse embryonic stem cells induces, other than the well-documented epithelial to mesenchymal-like state, also an increase in expression of the core transcription factors Oct4, Nanog and Sox2, thereby enhancing self-renewal of these cells. Esrp1-depleted embryonic stem cells displayed impaired early differentiation in vitro and formed larger teratomas in vivo when compared to control embryonic stem cells. We also show that ESRP1 binds to Oct4 and Sox2 mRNAs and decreases their polysomal loading. ESRP1 thus acts as a physiological regulator of the finely-tuned balance between self-renewal and commitment to a restricted developmental fate. Importantly, both mouse and human epithelial stem cells highly express ESRP1, pinpointing the importance of this RNA-binding protein in stem cell biology.

NPMc+ cooperates with Flt3/ITD mutations to cause acute leukemia recapitulating human disease

Cytoplasmic nucleophosmin (NPMc(+)) mutations and FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations are two of the most common known molecular alterations in acute myeloid leukemia (AML); they frequently occur together, suggesting cooperative leukemogenesis. To explore the specific relationship between NPMc+ and FLT3/ITD in vivo, we crossed Flt3/ITD knock-in mice with transgenic NPMc+ mice. Mice with both mutations develop a transplantable leukemia of either myeloid or lymphoid lineage, definitively demonstrating cooperation between Flt3/ITD and NPMc+. In mice with myeloid leukemia, functionally significant loss of heterozygosity of the wild-type Flt3 allele is common, similar to what is observed in human FLT3/ITD+ AML, providing further in vivo evidence of the importance of loss of wild-type FLT3 in leukemic initiation and progression. Additionally, in vitro clonogenic assays reveal that the combination of Flt3/ITD and NPMc+ mutations causes a profound monocytic expansion, in excess of that seen with either mutation alone consistent with the predominance of myelomonocytic phenotype in human FLT3/ITD+/NPMc+ AML. This in vivo model of Flt3/ITD+/NPMc+ leukemia closely recapitulates human disease and will therefore serve as a tool for the investigation of the biology of this common disease entity.

Diverse genetic-driven immune landscapes dictate tumor progression through distinct mechanisms

Multiple immune-cell types can infiltrate tumors and promote progression and metastasis through different mechanisms, including immunosuppression. How distinct genetic alterations in tumors affect the composition of the immune landscape is currently unclear. Here, we characterized the immune-cell composition of prostate cancers driven by the loss of the critical tumor suppressor gene Pten, either alone or in combination with the loss of Trp53, Zbtb7a or Pml. We observed a striking quantitative and qualitative heterogeneity that was directly dependent on the specific genetic events in the tumor and ranged from cold, noninflamed tumors to massively infiltrated landscapes—results with important therapeutic implications. Further, we showed these qualitative differences in transcriptomic analysis of human prostate cancer samples. These data suggest that patient stratification on the basis of integrated genotypic–immunophenotypic analyses may be necessary for successful clinical trials and tailored precision immunological therapies.

Found in translation of mTOR signaling.

The mammalian target of rapamycin (mTOR) protein kinase regulates a wide variety of cellular processes, including protein synthesis, yet the downstream translational program under the control of mTOR is poorly understood. Two recent studies by Hsieh et al. and Thoreen et al. now start to address this issue, and uncover a subset of genes translationally regulated by oncogenic mTOR signaling that may contribute to tumorigenesis.

Loss of LDAH associated with prostate cancer and hearing loss

&NA; Great strides in gene discovery have been made using a multitude of methods to associate phenotypes with genetic variants, but there still remains a substantial gap between observed symptoms and identified genetic defects. Herein, we use the convergence of various genetic and genomic techniques to investigate the underpinnings of a constellation of phenotypes that include prostate cancer (PCa) and sensorineural hearing loss (SNHL) in a human subject. Through interrogation of the subjects de novo, germline, balanced chromosomal translocation, we first identify a correlation between his disorders and a poorly annotated gene known as lipid droplet associated hydrolase (LDAH). Using data repositories of both germline and somatic variants, we identify convergent genomic evidence that substantiates a correlation between loss of LDAH and PCa. This correlation is validated through both in vitro and in vivo models that show loss of LDAH results in increased risk of PCa and, to a lesser extent, SNHL. By leveraging convergent evidence in emerging genomic data, we hypothesize that loss of LDAH is involved in PCa and other phenotypes observed in support of a genotype‐phenotype association in an n‐of‐one human subject.

Abstract A37: The BRAF pseudogene is a proto-oncogenic competitive endogenous RNA.

Noncoding RNAs have long been viewed as non-functional genomic relicts of evolution, but recetn findings have implicated their importance in physiology and disease. Recently, in vitro experiments demonstrated that the pseudogenes of PTEN and KRAS operate as natural miRNA decoys (competitive endogenous RNAs or ceRNAs) that regulate the expression of their parental genes. However, in vivo evidence for a causal role of pseudogenes in cancer development is lacking. To investigate whether the BRAF pseudogene (BRAFps) possesses oncogenic properties we generated transgenic mice carrying a Tet-inducible BRAF pseudogene allele. Global BRAFps overexpression resulted in the development of aggressive B-cell lymphoma after 6-12 months. These tumors were characterized by a profound expansion of B-lymphocytes in the spleen, as well as splenomegaly, lymphadenopathy and infiltration of the kidneys, lungs, and liver by neoplastic cells. The BRAFps-induced lymphoma was polyclonal, transplantable, dependent on continuous BRAFps expression, and cooperated with heterozygous loss of PTEN to accelerate disease onset. Mechanistically, we propose that BRAFps functions as a ceRNA that sequesters miRNAs from BRAF and possibly other targets. Indeed, overexpression of BRAFps results in elevated levels of BRAF in a Dicer-dependent manner. This, in turn, increased BRAF-dependent MAPK signaling and proliferation. To further validate the ceRNA activity of BRAFps, we engineered mice to express only the 39UTR or CDS of BRAFps as each portion of the pseudogene may individually engage in miRNA-mediated crosstalk with BRAF. Notably, both BRAFps-CDS and BRAFps-39UTR increased spleen and lymph node weights 6 months after induction. Interestingly, BRAFps-39UTR elicited a lymphoma phenotype similar to full length BRAFps, while mice expressing BRAFps-CDS developed a more indolent form of this phenotype, suggesting that lymphomagenesis is primarily mediated by the BRAFps 39UTR. BRAFps transcript was undetectable in primary human B-cells, but was aberrantly expressed in primary human DLBCL and human DLBCL cell lines. Expression of BRAF and BRAFps was positively correlated in human primary DLBCL and human DLBCL cell lines. In addition, gains or amplifications of the genomic locus containing BRAFps were found in various human cancer types. Overexpression of BRAFps in human lymphoma cells elevated BRAF levels, MAPK activation, proliferation and growth in xenografts. Our results demonstrate for the first time the oncogenic potential of a pseudogene in an engineered mouse model and indicate that ceRNA- mediated regulation is an important regulatory mechanism of gene expression in vivo. Citation Format: Florian A. Karreth, Markus Reschke, Chapuy Bjoern, Margaret Shipp, Roberto Chiarle, Pier Paolo Pandolfi. The BRAF pseudogene is a proto-oncogenic competitive endogenous RNA. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A37.