Characterization of an Mtbp Hypomorphic Allele in a Diethylnitrosamine-Induced Liver Carcinogenesis Model.

MTBP is implicated in cell cycle progression, DNA replication, and cancer metastasis. However, the function of MTBP remains enigmatic and is dependent on cellular contexts and its cellular localization. To understand the in vivo physiological role of MTBP, it is important to generate Mtbp knockout mice. However, complete deletion of the Mtbp gene in mice results in early embryonic lethality, while its heterozygous deletion shows modest biological phenotypes, including enhanced cancer metastasis. To overcome this and better characterize the in vivo physiological function of MTBP, we, for the first time, generated mice that carry an Mtbp hypomorphic allele (MtbpH) in which Mtbp protein is expressed at approximately 30% of that in the wild-type allele. We treated wild-type, Mtbp+/-, and MtbpH/- mice with a liver carcinogen, diethylnitrosamine (DEN), and found that the MtbpH/- mice showed worse overall survival when compared to the wild-type mice. Consistent with previous reports using human liver cancer cells, mouse embryonic fibroblasts (MEFs) from the MtbpH/- mice showed an increase in the nuclear localization of p-Erk1/2 and migratory potential. Thus, MtbpH/- mice and cells from MtbpH/- mice are valuable to understand the in vivo physiological role of Mtbp and validate the diverse functions of MTBP that have been observed in human cells.

Abrogation of Rb Tumor Suppression Initiates GBM in Differentiated Astrocytes by Driving a Progenitor Cell Program.

Glioblastoma (GBM) remains lethal with no effective treatments. Despite the comprehensive identification of commonly perturbed molecular pathways, little is known about the disease's etiology, particularly in early stages. Several studies indicate that GBM is initiated in neural progenitor and/or stem cells. Here, we report that differentiated astrocytes are susceptible to GBM development when initiated by perturbation of the RB pathway, which induces a progenitor phenotype. In vitro and in vivo inactivation of Rb tumor suppression (TS) induces cortical astrocytes to proliferate rapidly, express progenitor markers, repress differentiation markers, and form self-renewing neurospheres that are susceptible to multi-lineage differentiation. This phenotype is sufficient to cause grade II astrocytomas which stochastically progress to GBM. Together with previous findings, these results demonstrate that cell susceptibility to GBM depends on the initiating driver.

Distinct Biomarker Profiles and TCR Sequence Diversity Characterize the Response to PD-L1 Blockade in a Mouse Melanoma Model.

Only a subset of patients responds to immune checkpoint blockade (ICB) in melanoma. A preclinical model recapitulating the clinical activity of ICB would provide a valuable platform for mechanistic studies. We used melanoma tumors arising from an Hgftg;Cdk4R24C/R24C genetically engineered mouse (GEM) model to evaluate the efficacy of an anti-mouse PD-L1 antibody similar to the anti-human PD-L1 antibodies durvalumab and atezolizumab. Consistent with clinical observations for ICB in melanoma, anti-PD-L1 treatment elicited complete and durable response in a subset of melanoma-bearing mice. We also observed tumor growth delay or regression followed by recurrence. For early treatment assessment, we analyzed gene expression profiles, T-cell infiltration, and T-cell receptor (TCR) signatures in regressing tumors compared with tumors exhibiting no response to anti-PD-L1 treatment. We found that CD8+ T-cell tumor infiltration corresponded to response to treatment, and that anti-PD-L1 gene signature response indicated an increase in antigen processing and presentation, cytokine-cytokine receptor interaction, and natural killer cell-mediated cytotoxicity. TCR sequence data suggest that an anti-PD-L1-mediated melanoma regression response requires not only an expansion of the TCR repertoire that is unique to individual mice, but also tumor access to the appropriate TCRs. Thus, this melanoma model recapitulated the variable response to ICB observed in patients and exhibited biomarkers that differentiate between early response and resistance to treatment, providing a valuable platform for prediction of successful immunotherapy. IMPLICATIONS: Our melanoma model recapitulates the variable response to anti-PD-L1 observed in patients and exhibits biomarkers that characterize early antibody response, including expansion of the TCR repertoire.

Multimodel preclinical platform predicts clinical response of melanoma to immunotherapy.

Although immunotherapy has revolutionized cancer treatment, only a subset of patients demonstrate durable clinical benefit. Definitive predictive biomarkers and targets to overcome resistance remain unidentified, underscoring the urgency to develop reliable immunocompetent models for mechanistic assessment. Here we characterize a panel of syngeneic mouse models, representing a variety of molecular and phenotypic subtypes of human melanomas and exhibiting their diverse range of responses to immune checkpoint blockade (ICB). Comparative analysis of genomic, transcriptomic and tumor-infiltrating immune cell profiles demonstrated alignment with clinical observations and validated the correlation of T cell dysfunction and exclusion programs with resistance. Notably, genome-wide expression analysis uncovered a melanocytic plasticity signature predictive of patient outcome in response to ICB, suggesting that the multipotency and differentiation status of melanoma can determine ICB benefit. Our comparative preclinical platform recapitulates melanoma clinical behavior and can be employed to identify mechanisms and treatment strategies to improve patient care.

Inactivation of TRP53, PTEN, RB1, and/or CDH1 in the ovarian surface epithelium induces ovarian cancer transformation and metastasis.

Ovarian cancer (OvCa) remains the most common cause of death from gynecological malignancies. Genetically engineered mouse models have been used to study initiation, origin, progression, and/or mechanisms of OvCa. Based on the clinical features of OvCa, we examined a quadruple combination of pathway perturbations including PTEN, TRP53, RB1, and/or CDH1. To characterize the cancer-promoting events in the ovarian surface epithelium (OSE), Amhr2cre/+ mice were used to ablate floxed alleles of Pten, Trp53, and Cdh1, which were crossed with TgK19GT121 mice to inactivate RB1 in KRT19-expressing cells. Inactivation of PTEN, TRP53, and RB1 with or without CDH1 led to the development of type I low-grade OvCa with enlarged serous papillary carcinomas and some high-grade serous carcinomas (HGSCs) in older mice. Initiation of epithelial hyperplasia and micropapillary carcinoma started earlier at 1 month in the triple mutations of Trp53, Pten, and Rb1 mice as compared to 2 months in quadruple mutations of Trp53, Pten, Rb1, and Cdh1 mice, whereas both genotypes eventually developed enlarged proliferating tumors that invaded into the ovary at 3-4 months. Mice with triple and quadruple mutations developed HGSC and/or metastatic tumors, which disseminated into the peritoneal cavity at 4-6 months. In summary, inactivation of PTEN, TRP53, and RB1 initiates OvCa from the OSE. Additional ablation of CDH1 further increased persistence of tumor dissemination and ascites fluid accumulation enhancing peritoneal metastasis.

RB inactivation in keratin 18 positive thymic epithelial cells promotes non-cell autonomous T cell hyperproliferation in genetically engineered mice.

Thymic epithelial cells (TEC), as part of thymic stroma, provide essential growth factors/cytokines and self-antigens to support T cell development and selection. Deletion of Rb family proteins in adult thymic stroma leads to T cell hyperplasia in vivo. To determine whether deletion of Rb specifically in keratin (K) 18 positive TEC was sufficient for thymocyte hyperplasia, we conditionally inactivated Rb and its family members p107 and p130 in K18+ TEC in genetically engineered mice (TgK18GT121; K18 mice). We found that thymocyte hyperproliferation was induced in mice with Rb inactivation in K18+ TEC, while normal T cell development was maintained; suggesting that inactivation of Rb specifically in K18+ TEC was sufficient and responsible for the phenotype. Transplantation of wild type bone marrow cells into mice with Rb inactivation in K18+ TEC resulted in donor T lymphocyte hyperplasia confirming the non-cell autonomous requirement for Rb proteins in K18+ TEC in regulating T cell proliferation. Our data suggests that thymic epithelial cells play an important role in regulating lymphoid proliferation and thymus size.

Aurora-A promotes the establishment of spindle assembly checkpoint by priming the Haspin-Aurora-B feedback loop in late G2 phase.

Aurora-A kinase functions mainly in centrosome maturation, separation and spindle formation. It has also been found to be amplified or overexpressed in a range of solid tumors, which is linked with tumor progression and poor prognosis. Importantly, Aurora-A inhibitors are being studied in a number of ongoing clinical trials. However, whether and how Aurora-A has a role in the regulation of the mitotic checkpoint is controversial. Additionally, the function of nuclear-accumulated Aurora-A in late G2 phase is not clear. Here we show that knockout, inhibition or blockade of the nuclear entry of Aurora-A severely decreased the centromere localization of Aurora-B and the phosphorylation of histone H3 threonine 3 (H3T3-ph) mediated by the kinase Haspin in late G2 phase. We further reveal that nuclear-accumulated Aurora-A phosphorylates Haspin at multiple sites at its N-terminus and that this promotes H3T3-ph and the rapid recruitment to the centromere of the chromosomal passenger complex. In addition, Aurora-A facilitates the association of Aurora-B with their common substrates: Haspin and Plk1. Notably, these functions of Aurora-A are mostly independent of Plk1. Thus we demonstrate that, in late G2 and prophase, Aurora-A phosphorylates Haspin to trigger the Haspin-H3T3-ph-Aurora-B positive feedback loop that supports the timely establishment of the chromosomal passenger complex and the mitotic checkpoint before spindle assembly.

Concepts in Cancer Modeling: A Brief History.

Modeling, an experimental approach to investigate complex biological systems, has significantly contributed to our understanding of cancer. Although extensive cancer research has been conducted utilizing animal models for elucidating mechanisms and developing therapeutics, the concepts in a good model design and its application have not been well elaborated. In this review, we discuss the theory underlying biological modeling and the process of producing a valuable and relevant animal model. Several renowned examples in the history of cancer research will be used to illustrate how modeling can be translatable to clinical applications. Finally, we will also discuss how the advances in cancer genomics and cancer modeling will influence each other going forward. Cancer Res; 76(20); 5921-5. ©2016 AACR.

Preclinical mouse cancer models: a maze of opportunities and challenges.

Significant advances have been made in developing novel therapeutics for cancer treatment, and targeted therapies have revolutionized the treatment of some cancers. Despite the promise, only about five percent of new cancer drugs are approved, and most fail due to lack of efficacy. The indication is that current preclinical methods are limited in predicting successful outcomes. Such failure exacts enormous cost, both financial and in the quality of human life. This Primer explores the current status, promise, and challenges of preclinical evaluation in advanced mouse cancer models and briefly addresses emerging models for early-stage preclinical development.

The genetics of splicing in neuroblastoma.

UNLABELLED: Regulation of mRNA splicing, a critical and tightly regulated cellular function, underlies the majority of proteomic diversity and is frequently disrupted in disease. Using an integrative genomics approach, we combined both genomic data and exon-level transcriptome data in two somatic tissues (cerebella and peripheral ganglia) from a transgenic mouse model of neuroblastoma, a tumor that arises from the peripheral neural crest. Here, we describe splicing quantitative trait loci associated with differential splicing across the genome that we use to identify genes with previously unknown functions within the splicing pathway and to define de novo intronic splicing motifs that influence splicing from hundreds of bases away. Our results show that these splicing motifs represent sites for functional recurrent mutations and highlight novel candidate genes in human cancers, including childhood neuroblastoma. SIGNIFICANCE: Somatic mutations with predictable downstream effects are largely relegated to coding regions, which comprise less than 2% of the human genome. Using an unbiased in vivo analysis of a mouse model of neuroblastoma, we have identified intronic splicing motifs that translate into sites for recurrent somatic mutations in human cancers.

A preclinical orthotopic model for glioblastoma recapitulates key features of human tumors and demonstrates sensitivity to a combination of MEK and PI3K pathway inhibitors.

Current therapies for glioblastoma multiforme (GBM), the highest grade malignant brain tumor, are mostly ineffective, and better preclinical model systems are needed to increase the successful translation of drug discovery efforts into the clinic. Previous work describes a genetically engineered mouse (GEM) model that contains perturbations in the most frequently dysregulated networks in GBM (driven by RB, KRAS and/or PI3K signaling and PTEN) that induce development of Grade IV astrocytoma with properties of the human disease. Here, we developed and characterized an orthotopic mouse model derived from the GEM that retains the features of the GEM model in an immunocompetent background; however, this model is also tractable and efficient for preclinical evaluation of candidate therapeutic regimens. Orthotopic brain tumors are highly proliferative, invasive and vascular, and express histology markers characteristic of human GBM. Primary tumor cells were examined for sensitivity to chemotherapeutics and targeted drugs. PI3K and MAPK pathway inhibitors, when used as single agents, inhibited cell proliferation but did not result in significant apoptosis. However, in combination, these inhibitors resulted in a substantial increase in cell death. Moreover, these findings translated into the in vivo orthotopic model: PI3K or MAPK inhibitor treatment regimens resulted in incomplete pathway suppression and feedback loops, whereas dual treatment delayed tumor growth through increased apoptosis and decreased tumor cell proliferation. Analysis of downstream pathway components revealed a cooperative effect on target downregulation. These concordant results, together with the morphologic similarities to the human GBM disease characteristics of the model, validate it as a new platform for the evaluation of GBM treatment.

Expression quantitative trait loci and receptor pharmacology implicate Arg1 and the GABA-A receptor as therapeutic targets in neuroblastoma.

The development of targeted therapeutics for neuroblastoma, the third most common tumor in children, has been limited by a poor understanding of growth signaling mechanisms unique to the peripheral nerve precursors from which tumors arise. In this study, we combined genetics with gene-expression analysis in the peripheral sympathetic nervous system to implicate arginase 1 and GABA signaling in tumor formation in vivo. In human neuroblastoma cells, either blockade of ARG1 or benzodiazepine-mediated activation of GABA-A receptors induced apoptosis and inhibited mitogenic signaling through AKT and MAPK. These results suggest that ARG1 and GABA influence both neural development and neuroblastoma and that benzodiazepines in clinical use may have potential applications for neuroblastoma therapy.

"Glowing head" mice: a genetic tool enabling reliable preclinical image-based evaluation of cancers in immunocompetent allografts.

Preclinical therapeutic assessment currently relies on the growth response of established human cell lines xenografted into immunocompromised mice, a strategy that is generally not predictive of clinical outcomes. Immunocompetent genetically engineered mouse (GEM)-derived tumor allograft models offer highly tractable preclinical alternatives and facilitate analysis of clinically promising immunomodulatory agents. Imageable reporters are essential for accurately tracking tumor growth and response, particularly for metastases. Unfortunately, reporters such as luciferase and GFP are foreign antigens in immunocompetent mice, potentially hindering tumor growth and confounding therapeutic responses. Here we assessed the value of reporter-tolerized GEMs as allograft recipients by targeting minimal expression of a luciferase-GFP fusion reporter to the anterior pituitary gland (dubbed the "Glowing Head" or GH mouse). The luciferase-GFP reporter expressed in tumor cells induced adverse immune responses in wildtype mouse, but not in GH mouse, as transplantation hosts. The antigenicity of optical reporters resulted in a decrease in both the growth and metastatic potential of the labeled tumor in wildtype mice as compared to the GH mice. Moreover, reporter expression can also alter the tumor response to chemotherapy or targeted therapy in a context-dependent manner. Thus the GH mice and experimental approaches vetted herein provide concept validation and a strategy for effective, reproducible preclinical evaluation of growth and response kinetics for traceable tumors.

Vitamin D receptor-mediated stromal reprogramming suppresses pancreatitis and enhances pancreatic cancer therapy.

The poor clinical outcome in pancreatic ductal adenocarcinoma (PDA) is attributed to intrinsic chemoresistance and a growth-permissive tumor microenvironment. Conversion of quiescent to activated pancreatic stellate cells (PSCs) drives the severe stromal reaction that characterizes PDA. Here, we reveal that the vitamin D receptor (VDR) is expressed in stroma from human pancreatic tumors and that treatment with the VDR ligand calcipotriol markedly reduced markers of inflammation and fibrosis in pancreatitis and human tumor stroma. We show that VDR acts as a master transcriptional regulator of PSCs to reprise the quiescent state, resulting in induced stromal remodeling, increased intratumoral gemcitabine, reduced tumor volume, and a 57% increase in survival compared to chemotherapy alone. This work describes a molecular strategy through which transcriptional reprogramming of tumor stroma enables chemotherapeutic response and suggests vitamin D priming as an adjunct in PDA therapy. PAPERFLICK:

Akt-dependent activation of mTORC1 complex involves phosphorylation of mTOR (mammalian target of rapamycin) by IκB kinase α (IKKα).

The serine/threonine protein kinase Akt promotes cell survival, growth, and proliferation through phosphorylation of different downstream substrates. A key effector of Akt is the mammalian target of rapamycin (mTOR). Akt is known to stimulate mTORC1 activity through phosphorylation of tuberous sclerosis complex 2 (TSC2) and PRAS40, both negative regulators of mTOR activity. We previously reported that IκB kinase α (IKKα), a component of the kinase complex that leads to NF-κB activation, plays an important role in promoting mTORC1 activity downstream of activated Akt. Here, we demonstrate IKKα-dependent regulation of mTORC1 using multiple PTEN null cancer cell lines and an animal model with deletion of IKKα. Importantly, IKKα is shown to phosphorylate mTOR at serine 1415 in a manner dependent on Akt to promote mTORC1 activity. These results demonstrate that IKKα is an effector of Akt in promoting mTORC1 activity.

Pathway-specific engineered mouse allograft models functionally recapitulate human serous epithelial ovarian cancer.

The high mortality rate from ovarian cancers can be attributed to late-stage diagnosis and lack of effective treatment. Despite enormous effort to develop better targeted therapies, platinum-based chemotherapy still remains the standard of care for ovarian cancer patients, and resistance occurs at a high rate. One of the rate limiting factors for translation of new drug discoveries into clinical treatments has been the lack of suitable preclinical cancer models with high predictive value. We previously generated genetically engineered mouse (GEM) models based on perturbation of Tp53 and Rb with or without Brca1 or Brca2 that develop serous epithelial ovarian cancer (SEOC) closely resembling the human disease on histologic and molecular levels. Here, we describe an adaptation of these GEM models to orthotopic allografts that uniformly develop tumors with short latency and are ideally suited for routine preclinical studies. Ovarian tumors deficient in Brca1 respond to treatment with cisplatin and olaparib, a PARP inhibitor, whereas Brca1-wild type tumors are non-responsive to treatment, recapitulating the relative sensitivities observed in patients. These mouse models provide the opportunity for evaluation of effective therapeutics, including prediction of differential responses in Brca1-wild type and Brca1-deficient tumors and development of relevant biomarkers.

Carcinoma initiation via RB tumor suppressor inactivation: a versatile approach to epithelial subtype-dependent cancer initiation in diverse tissues.

Carcinomas arise in a complex microenvironment consisting of multiple distinct epithelial lineages surrounded by a variety of stromal cell types. Understanding cancer etiologies requires evaluating the relationship among cell types during disease initiation and through progression. Genetically engineered mouse (GEM) models facilitate the prospective examination of early oncogenic events, which is not possible in humans. Since most solid tumors harbor aberrations in the RB network, we developed an inducible GEM approach for the establishment and assessment of carcinoma initiation in a diverse range of epithelial tissues and subtypes upon inactivation of RB-mediated tumor suppression (RB-TS). The system allows independent assessment of epithelial subtypes that express either cytokeratins (K) 18 or 19. By Cre-dependent expression of a protein that dominantly inactivates RB and functionally redundant proteins p107 and p130, neoplasia could be initiated in either K18 or K19 expressing cells of numerous tissues. By design, because only a single pathway aberration was engineered, carcinomas developed stochastically only after long latency. Hence, this system, which allows for directed cell type-specific carcinoma initiation, facilitates further definition of events that can progress neoplasms to aggressive cancers via engineered, carcinogen-induced and/or spontaneous evolution.

Requirements for Aurora-A in tissue regeneration and tumor development in adult mammals.

Aurora-A is a kinase involved in the formation and maturation of the mitotic spindle and chromosome segregation. This kinase is frequently overexpressed in human cancer, and its activity may confer resistance to antitumoral drugs such as Taxol. Inhibition of Aurora-A results in mitotic defects, and this kinase is considered as an attractive therapeutic target for cancer. Nevertheless, the specific requirements for this kinase in adult mammalian tissues remain unclear. Conditional genetic ablation of Aurora-A in adult tissues results in polyploid cells that display a DNA-damage-like response characterized by the upregulation of p53 and the cell-cycle inhibitor p21(Cip1). This is accompanied by apoptotic, differentiation, or senescence markers in a tissue-specific manner. Therapeutic elimination of Aurora-A prevents the progression of skin and mammary gland tumors. However, this is not due to significant levels of apoptosis or senescence, but because Aurora-A-deficient tumors accumulate polyploid cells with limited proliferative potential. Thus, Aurora-A is required for tumor formation in vivo, and the differential response observed in various tissues might have relevant implications in current therapeutic strategies aimed at inhibiting this kinase in the treatment of human cancer.

Evolutionary etiology of high-grade astrocytomas.

Glioblastoma (GBM), the most common brain malignancy, remains fatal with no effective treatment. Analyses of common aberrations in GBM suggest major regulatory pathways associated with disease etiology. However, 90% of GBMs are diagnosed at an advanced stage (primary GBMs), providing no access to early disease stages for assessing disease progression events. As such, both understanding of disease mechanisms and the development of biomarkers and therapeutics for effective disease management are limited. Here, we describe an adult-inducible astrocyte-specific system in genetically engineered mice that queries causation in disease evolution of regulatory networks perturbed in human GBM. Events yielding disease, both engineered and spontaneous, indicate ordered grade-specific perturbations that yield high-grade astrocytomas (anaplastic astrocytomas and GBMs). Impaired retinoblastoma protein RB tumor suppression yields grade II histopathology. Additional activation of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) network drives progression to grade III disease, and further inactivation of phosphatase and tensin homolog (PTEN) yields GBM. Spontaneous missense mutation of tumor suppressor Trp53 arises subsequent to KRAS activation, but before grade III progression. The stochastic appearance of mutations identical to those observed in humans, particularly the same spectrum of p53 amino acid changes, supports the validity of engineered lesions and the ensuing interpretations of etiology. Absence of isocitrate dehydrogenase 1 (IDH1) mutation, asymptomatic low grade disease, and rapid emergence of GBM combined with a mesenchymal transcriptome signature reflect characteristics of primary GBM and provide insight into causal relationships.