Benedikt Bosbach

Somatic cells initiate primordial follicle activation and govern the development of dormant oocytes in mice.

BACKGROUND The majority of oocytes in the mammalian ovary are dormant oocytes that are enclosed in primordial follicles by several somatic cells, which we refer to as primordial follicle granulosa cells (pfGCs). Very little is known, however, about how the pfGCs control the activation of primordial follicles and the developmental fates of dormant oocytes. RESULTS By targeting molecules in pfGCs with several mutant mouse models, we demonstrate that the somatic pfGCs initiate the activation of primordial follicles and govern the quiescence or awakening of dormant oocytes. Inhibition of mTORC1 signaling in pfGCs prevents the differentiation of pfGCs into granulosa cells, and this arrests the dormant oocytes in their quiescent states, leading to oocyte death. Overactivation of mTORC1 signaling in pfGCs accelerates the differentiation of pfGCs into granulosa cells and causes premature activation of all dormant oocytes and primordial follicles. We further show that pfGCs trigger the awakening of dormant oocytes through KIT ligand (KITL), and we present an essential communication network between the somatic cells and germ cells that is based on signaling between the mTORC1-KITL cascade in pfGCs and KIT-PI3K signaling in oocytes. CONCLUSIONS Our findings provide a relatively complete picture of how mammalian primordial follicles are activated. The microenvironment surrounding primordial follicles can activate mTORC1-KITL signaling in pfGCs, and these cells trigger the awakening of dormant oocytes and complete the process of follicular activation. Such communication between the microenvironment, somatic cells, and germ cells is essential to maintaining the proper reproductive lifespan in mammals.

Deletions linked to TP53 loss drive cancer through p53-independent mechanisms

Mutations disabling the TP53 tumour suppressor gene represent the most frequent events in human cancer and typically occur through a two-hit mechanism involving a missense mutation in one allele and a ‘loss of heterozygosity’ deletion encompassing the other. While TP53 missense mutations can also contribute gain-of-function activities that impact tumour progression, it remains unclear whether the deletion event, which frequently includes many genes, impacts tumorigenesis beyond TP53 loss alone. Here we show that somatic heterozygous deletion of mouse chromosome 11B3, a 4-megabase region syntenic to human 17p13.1, produces a greater effect on lymphoma and leukaemia development than Trp53 deletion. Mechanistically, the effect of 11B3 loss on tumorigenesis involves co-deleted genes such as Eif5a and Alox15b (also known as Alox8), the suppression of which cooperates with Trp53 loss to produce more aggressive disease. Our results imply that the selective advantage produced by human chromosome 17p deletion reflects the combined impact of TP53 loss and the reduced dosage of linked tumour suppressor genes.

PTEN action in leukaemia dictated by the tissue microenvironment

PTEN encodes a lipid phosphatase that is underexpressed in many cancers owing to deletions, mutations or gene silencing. PTEN dephosphorylates phosphatidylinositol (3,4,5)-triphosphate, thereby opposing the activity of class I phosphatidylinositol 3-kinases that mediate growth- and survival-factor signalling through phosphatidylinositol 3-kinase effectors such as AKT and mTOR. To determine whether continued PTEN inactivation is required to maintain malignancy, here we generate an RNA interference-based transgenic mouse model that allows tetracycline-dependent regulation of PTEN in a time- and tissue-specific manner. Postnatal Pten knockdown in the haematopoietic compartment produced highly disseminated T-cell acute lymphoblastic leukaemia. Notably, reactivation of PTEN mainly reduced T-cell leukaemia dissemination but had little effect on tumour load in haematopoietic organs. Leukaemia infiltration into the intestine was dependent on CCR9 G-protein-coupled receptor signalling, which was amplified by PTEN loss. Our results suggest that in the absence of PTEN, G-protein-coupled receptors may have an unanticipated role in driving tumour growth and invasion in an unsupportive environment. They further reveal that the role of PTEN loss in tumour maintenance is not invariant and can be influenced by the tissue microenvironment, thereby producing a form of intratumoral heterogeneity that is independent of cancer genotype.

A modular and flexible ESC-based mouse model of pancreatic cancer

Genetically engineered mouse models (GEMMs) have greatly expanded our knowledge of pancreatic ductal adenocarcinoma (PDAC) and serve as a critical tool to identify and evaluate new treatment strategies. However, the cost and time required to generate conventional pancreatic cancer GEMMs limits their use for investigating novel genetic interactions in tumor development and maintenance. To address this problem, we developed flexible embryonic stem cell (ESC)-based GEMMs that facilitate the rapid generation of genetically defined multiallelic chimeric mice without further strain intercrossing. The ESCs harbor a latent Kras mutant (a nearly ubiquitous feature of pancreatic cancer), a homing cassette, and other genetic elements needed for rapid insertion and conditional expression of tetracycline-controlled transgenes, including fluorescence-coupled shRNAs capable of efficiently silencing gene function by RNAi. This system produces a disease that recapitulates the progression of pancreatic cancer in human patients and enables the study and visualization of the impact of gene perturbation at any stage of pancreas cancer progression. We describe the use of this approach to dissect temporal roles for the tumor suppressor Pten and the oncogene c-Myc in pancreatic cancer development and maintenance.

Imatinib resistance and microcytic erythrocytosis in a KitV558Δ;T669I/+ gatekeeper-mutant mouse model of gastrointestinal stromal tumor

Most gastrointestinal stromal tumors (GISTs) harbor a gain-of-function mutation in the Kit receptor. GIST patients treated with the tyrosine kinase inhibitor imatinib frequently develop imatinib resistance as a result of second-site Kit mutations. To investigate the consequences of second-site Kit mutations on GIST development and imatinib sensitivity, we engineered a mouse model carrying in the endogenous Kit locus both the KitV558Δ mutation found in a familial case of GIST and the KitT669I (human KITT670I) “gatekeeper” mutation found in imatinib-resistant GIST patients. Similar to KitV558∆/+ mice, KitV558∆;T669I/+ mice developed gastric and colonic interstitial cell of Cajal hyperplasia as well as cecal GIST. In contrast to the single-mutant KitV558∆/+ control mice, treatment of the KitV558∆;T669I/+ mice with either imatinib or dasatinib failed to inhibit oncogenic Kit signaling and GIST growth. However, this resistance could be overcome by treatment of KitV558∆;T669I/+ mice with sunitinib or sorafenib. Although tumor lesions were smaller in KitV558∆;T669I/+ mice than in single-mutant mice, both interstitial cell of Cajal hyperplasia and mast cell hyperplasia were exacerbated in KitV558∆;T669I/+ mice. Strikingly, the KitV558∆;T669I/+ mice developed a pronounced polycythemia vera-like erythrocytosis in conjunction with microcytosis. This mouse model should be useful for preclinical studies of drug candidates designed to overcome imatinib resistance in GIST and to investigate the consequences of oncogenic KIT signaling in hematopoietic as well as other cell lineages.

DNAJB1–PRKACA fusion kinase interacts with β-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma

Significance Efforts to understand and treat fibrolamellar hepatocellular carcinoma (FL-HCC) have been confounded by a lack of models that accurately reflect the genetics and biology of the disease. Here we demonstrate that the Dnajb1–Prkaca gene fusion drives tumorigenesis in mice, and that fusion to DNAJB1 drives FL-HCC initiation more effectively than wild-type PRKACA overexpression. The requirement of the PRKACA kinase domain in tumor initiation establishes the potential utility of kinase inhibitors targeting the fusion. By identifying genetic and environmental factors that can enhance the consistency and aggressiveness of disease progression, we reveal biological characteristics of the disease and advance a robust platform for future preclinical studies. A segmental deletion resulting in DNAJB1–PRKACA gene fusion is now recognized as the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. Here we implement CRISPR-Cas9 genome editing and transposon-mediated somatic gene transfer to demonstrate that expression of either the endogenous fusion protein or a chimeric cDNA leads to the formation of indolent liver tumors in mice that closely resemble human FL-HCC. Notably, overexpression of the wild-type PRKACA was unable to fully recapitulate the oncogenic activity of DNAJB1–PRKACA, implying that FL-HCC does not simply result from enhanced PRKACA expression. Tumorigenesis was significantly enhanced by genetic activation of β-catenin, an observation supported by evidence of recurrent Wnt pathway mutations in human FL-HCC, as well as treatment with the hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which causes tissue injury, inflammation, and fibrosis. Our study validates the DNAJB1–PRKACA fusion kinase as an oncogenic driver and candidate drug target for FL-HCC, and establishes a practical model for preclinical studies to identify strategies to treat this disease.

KIT Receptor Gain‐of‐Function in Hematopoiesis Enhances Stem Cell Self‐Renewal and Promotes Progenitor Cell Expansion

The KIT receptor tyrosine kinase has important roles in hematopoiesis. We have recently produced a mouse model for imatinib resistant gastrointestinal stromal tumor (GIST) carrying the KitV558Δ and KitT669I (human KITT670I) mutations found in imatinib‐resistant GIST. The KitV558Δ;T669I/+ mice developed microcytic erythrocytosis with an increase in erythroid progenitor numbers, a phenotype previously seen only in mouse models of polycythemia vera with alterations in Epo or Jak2. Significantly, the increased hematocrit observed in KitV558Δ;T669I/+ mice normalized upon splenectomy. In accordance with increased erythroid progenitors, myeloerythroid progenitor numbers were also elevated in the KitV558Δ;T669I/+ mice. Hematopoietic stem cell (HSC) numbers in the bone marrow (BM) of KitV558Δ;T669I/+ mice were unchanged in comparison to wild‐type mice. However, increased HSC numbers were observed in fetal livers and the spleen and peripheral blood of adult KitV558Δ;T669I/+ mice. Importantly, HSC from KitV558Δ;T669I/+ BM had a competitive advantage over wild‐type HSC. In response to 5‐fluorouracil treatment, elevated numbers of dividing Lin−Sca+ cells were found in the KitV558Δ;T669I/+ BM compared to wild type. Our study demonstrates that signaling from the KitV558Δ;T669I/+ receptor has important consequences in hematopoiesis enhancing HSC self‐renewal and resulting in increased erythropoiesis. STEM Cells 2013;31:1683‐ ‐1695

DNAJB1-PRKACA fusion kinase drives tumorigenesis and interacts with β-catenin and the liver regenerative response.

A segmental deletion resulting in DNAJB1-PRKACA gene fusion is now recognized as the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. Here, we implement CRISPR/Cas9 genome editing and transposon-mediated somatic gene transfer to demonstrate that expression of both the endogenous fusion protein or a chimeric cDNA leads to the formation of indolent liver tumors in mice that closely resemble human FL-HCC. Notably, overexpression of the wild type PRKACA was unable to fully recapitulate the oncogenic activity of DNAJB1-PRKACA, implying that FL-HCC does not simply result from enhanced PRKACA expression. Tumorigenesis was significantly enhanced by genetic activation of β-catenin, an observation supported by evidence of recurrent Wnt pathway mutations in human FL-HCC, as well as treatment with hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), which causes tissue injury, inflammation and fibrosis. Our study validates the DNAJB1-PRKACA fusion kinase as an oncogenic driver and candidate drug target for FL-HCC. Practical and scalable mouse models of this disease serve as a resource to further explore tumor development and treatment. Significance Limited resources are available for the treatment of FL-HCC and a lack of research tools have led to an absence of experimental characterization of the disease. Here, we describe a rapidly scalable method to generate synchronous cohorts of mice bearing FL-HCC tumors. We demonstrate that the Dnajb1-Prkaca gene fusion drives tumorigenesis in mice, and that fusion to DNAJB1 drives FL-HCC initiation more strongly than wild type PRKACA overexpression. The requirement of the PRKACA kinase domain reinforces the potential utility of kinase inhibitors targeting the fusion. By identifying genetic and environmental factors that can enhance the consistency and aggressiveness of disease progression, we reveal biological characteristics of the disease and advance a platform for future pre-clinical studies.A segmental deletion resulting in DNAJB1-PRKACA gene fusion is now recognized as the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. Here, we implement CRISPR/Cas9 genome editing and transposon-mediated somatic gene transfer to demonstrate that expression of both the endogenous fusion protein or a chimeric cDNA leads to the formation of indolent liver tumors in mice that closely resemble human FL-HCC. Notably, overexpression of the wild type PRKACA was unable to fully recapitulate the oncogenic activity of DNAJB1-PRKACA, implying that FL-HCC does not simply result from enhanced PRKACA expression. Tumorigenesis was significantly enhanced by genetic activation of β-catenin, an observation supported by evidence of recurrent Wnt pathway mutations in human FL-HCC, as well as treatment with hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), which causes tissue injury, inflammation and fibrosis. Our study validates the DNAJB1-PRKACA fusion kinase as an oncogenic driver and candidate drug target for FL-HCC and establishes a practical model for preclinical studies to identify strategies to treat this disease.

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor

Significance Oncogenic receptor tyrosine kinases (RTKs) are important drug targets in the clinical setting. While RTK inhibitors have become important tools in the clinic, as has been demonstrated with chronic myelogenous leukemia and gastrointestinal stromal tumors (GIST), drug resistance invariably develops. The tools and rationale for the treatment of RTK drug resistance are limited, and success is of short duration. The identification of secondary intracellular drug targets is thus of critical importance. By using new Kit-GIST mouse models in which specific Kit signaling cascades are inhibited, we show the importance of PI3 kinase signaling in tumor development, as well as the utility of PI3 kinase inhibition in the treatment of primary and imatinib-resistant GIST. These studies provide a rationale for targeting dominant molecular pathways in tumors driven by oncogenic kinases. Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic KitV558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant KitV558Δ/+ mice, double-mutant KitV558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in KitV558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing KitV558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha–restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant KitV558Δ;T669I/+ tumors.

Abstract 1038: The KIT-V654A second site mutation confers perinatal lethality and increased oncogenesis in a mouse model of GIST

Tyrosine kinase inhibitors have revolutionized the treatment of gastrointestinal stromal tumor (GIST), which are mostly driven by mutations in the receptor tyrosine kinase KIT. However, resistance commonly develops, which is associated with second site mutations in KIT. Here, we created the first genetically engineered mouse model of the most common second site mutation, KIT-V654A (mouse KIT-V653A), to study in vivo its oncogenic properties and mechanisms of resistance in order to develop next-generation GIST therapies. The knock-in strategy consisted of inserting into the endogenous murine Kit locus a targeting vector containing both the KitV558Δ exon 11 mutation and the KitV653A exon 13 mutation under the control of a floxed neomycin-resistance gene-expression cassette which acts similar to a loxP-STOP-loxP cassette. Treatments included imatinib 45 mg/kg i.p., sunitinib 40 mg/kg p.o., and cabozantinib 60 mg/kg p.o. Tumors were assessed by histology, immunohistochemistry, and western blot. In contrast to our previously published GIST model with a single mutant KitV558Δ allele, the double knock-in KitV558Δ; V653A-neo allele caused perinatal lethality when activated in pre-implantation embryos with a germline EIIa-Cre approach. We therefore sought to restrict the induction of the KitV558Δ;V653A double mutation to the interstitial cells of Cajal (ICC), the cell of origin of GIST, via the essential ICC lineage survival factor ETV1. The KitV558Δ; V653A-neo/+ mouse was crossed with the Etv1Cre-ERT2/+ mouse expressing Cre recombinase under the Etv1-specific promoter. Systemic administration of tamoxifen at birth in KitV558Δ; V653A-neo/+; Etv1Cre-ERT2/+ mice resulted in cecal GIST development with full penetrance. The tumors were histologically similar to human GIST, stained positive for KIT, and displayed active p-KIT signaling. As expected, high-dose imatinib treatment did not inhibit p-KIT Y719 in these GISTs and did not induce a histologic response. Sunitinib and cabozantinib each had significant anti-tumor effects, as assessed by decreased Ki67 index and increased histologic response. Compared to single mutant KitV558Δ-neo/+; Etv1Cre-ERT2/+ mice, double mutant KitV558Δ; V653A-neo/+; Etv1Cre-ERT2/+ mice developed larger cecal GISTs and had decreased survival. The KitV558Δ; V653A-neo/+; Etv1Cre-ERT2/+ mouse is the first in vivo model of the most common secondary mutation in GIST and the first in vivo demonstration that cell-autonomous expression of mutant KIT in the ICC lineage gives rise to GIST. We have found cabozantinib, an FDA-approved drug, to be an effective therapy in GISTs harboring the V653A mutation. Furthermore, our results suggest the addition of the KIT-V653A second site mutation leads to increased oncogenesis. Citation Format: Jennifer Q. Zhang, Benedikt Bosbach, Cristina Antonescu, Peter Besmer, Ronald DeMatteo. The KIT-V654A second site mutation confers perinatal lethality and increased oncogenesis in a mouse model of GIST [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1038. doi:10.1158/1538-7445.AM2017-1038