Howard M. Stern

Zebrafish as a cancer model system

The zebrafish, with its combination of forward genetics and vertebrate biology, has great potential as a cancer model system.

Cancer genetics and drug discovery in the zebrafish

Fish have a long history of use in cancer toxicology studies, because they develop neoplasms that are histologically similar to human cancers. Because of considerable progress in zebrafish genetics and genomics over the past few years, the zebrafish system has provided many useful tools for studying basic biological processes. These tools include forward genetic screens, transgenic models, specific gene disruptions and small-molecule screens. By combining carcinogenesis assays, genetic analyses and small-molecule screening techniques, the zebrafish is emerging as a powerful system for identifying novel cancer genes and for cancer drug discovery.

Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells

Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.

A Chemical Genetic Screen for Cell Cycle Inhibitors in Zebrafish Embryos

Chemical genetic screening is an effective strategy to identify compounds that alter a specific biological phenotype. As a complement to cell line screens, multicellular organism screens may reveal additional compounds. The zebrafish embryo is ideal for small molecule studies because of its small size and the ease of waterborne treatment. We first examined a broad range of known cell cycle compounds in embryos using the mitotic marker phospho‐histone H3. The majority of the known compounds exhibited the predicted cell cycle effect in embryos. To determine whether we could identify novel compounds, we screened a 16 320‐compound library for alterations of pH3. This screen revealed 14 compounds that had not been previously identified as having cell cycle activity despite numerous mitotic screens of the same library with mammalian cell lines. With six of the novel compounds, sensitivity was greater in embryos than cell lines, but activity was still detected in cell lines at higher doses. One compound had activity in zebrafish embryos and cell lines but not in mammalian cell lines. The remaining compounds exhibited activity only in embryos. These findings demonstrate that small molecule screens in zebrafish can identify compounds with novel activity and thus may be useful tools for chemical genetics and drug discovery.

DNA Methylation Profiling Defines Clinically Relevant Biological Subsets of Non–Small Cell Lung Cancer

Purpose: Non–small cell lung cancers (NSCLC) comprise multiple distinct biologic groups with different prognoses. For example, patients with epithelial-like tumors have a better prognosis and exhibit greater sensitivity to inhibitors of the epidermal growth factor receptor (EGFR) pathway than patients with mesenchymal-like tumors. Here, we test the hypothesis that epithelial-like NSCLCs can be distinguished from mesenchymal-like NSCLCs on the basis of global DNA methylation patterns. Experimental Design: To determine whether phenotypic subsets of NSCLCs can be defined on the basis of their DNA methylation patterns, we combined microfluidics-based gene expression analysis and genome-wide methylation profiling. We derived robust classifiers for both gene expression and methylation in cell lines and tested these classifiers in surgically resected NSCLC tumors. We validate our approach using quantitative reverse transcriptase PCR and methylation-specific PCR in formalin-fixed biopsies from patients with NSCLC who went on to fail front-line chemotherapy. Results: We show that patterns of methylation divide NSCLCs into epithelial-like and mesenchymal-like subsets as defined by gene expression and that these signatures are similarly correlated in NSCLC cell lines and tumors. We identify multiple differentially methylated regions, including one in ERBB2 and one in ZEB2, whose methylation status is strongly associated with an epithelial phenotype in NSCLC cell lines, surgically resected tumors, and formalin-fixed biopsies from patients with NSCLC who went on to fail front-line chemotherapy. Conclusions: Our data show that patterns of DNA methylation can divide NSCLCs into two phenotypically distinct subtypes of tumors and provide proof of principle that differences in DNA methylation can be used as a platform for predictive biomarker discovery and development. Clin Cancer Res; 18(8); 2360–73. ©2012 AACR.

The zon laboratory guide to positional cloning in zebrafish.

Zebrafish genome sequencing project has improved efficiency of positional cloning in zebrafish and traditional chromosome walking by isolating large insert genomic libraries has become a past. However, the genetic principles underlying the positional cloning still form the foundation for current chromosome walking using the genome sequence assemblies and related genomic sequence and clone information. This guide intends to summarize our accumulated experience in positional cloning using the current genomic resources and tools, and provide a practical guide to positional and/or candidate cloning of mutants of interest.

Disruption of PH–kinase domain interactions leads to oncogenic activation of AKT in human cancers

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain–kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH–KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH–KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH–KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH–KD interface.

Analysis of the Cell Cycle in Zebrafish Embryos

Publisher Summary This chapter discusses the analysis of the cell cycle in Zebrafish Embroys. The advantages of the zebrafish system that make it a powerful organism to study vertebrate development include external fertilization of oocytes, transparent embryos, and rapid embryonic development. These features also provide the opportunity to study early cell divisions and tissue-specific cellular proliferation. This chapter provides protocols for assays that characterize various phases of cell division in zebrafish embryos. It discusses the design of screens for mutations aVecting embryonic cell proliferation and a method to detect the novel compounds affecting the zebrafish cell cycle. Assays discussed in this chapter include: DNA content analysis by flow cytometry, whole-mount embryonic antibody staining, mitotic spindle analysis, 5-bromo-2-deoxyuridine (BrdU) incorporation, cell death analysis, and in situ hybridization with cell cycle regulatory genes. Finally, this chapter concludes with the genetic tractability of the zebrafish system that makes it an excellent organism, in which to pursue forward genetic screens for mutations or chemical screens for novel compounds that alter cell division using one or more of these cell cycle assays.

Construction and application of a zebrafish array comparative genomic hybridization platform

The zebrafish is emerging as a prominent model system for studying the genetics of human development and disease. Genetic alterations that underlie each mutant model can exist in the form of single base changes, balanced chromosomal rearrangements, or genetic imbalances. To detect genetic imbalances in an unbiased genome‐wide fashion, array comparative genomic hybridization (CGH) can be used. We have developed a 5‐Mb resolution array CGH platform specifically for the zebrafish. This platform contains 286 bacterial artificial chromosome (BAC) clones, enriched for orthologous sequences of human oncogenes and tumor suppressor genes. Each BAC clone has been end‐sequenced and cytogenetically assigned to a specific location within the zebrafish genome, allowing for ease of integration of array CGH data with the current version of the genome assembly. This platform has been applied to three zebrafish cancer models. Significant genomic imbalances were detected in each model, identifying different regions that may potentially play a role in tumorigenesis. Hence, this platform should be a useful resource for genetic dissection of additional zebrafish developmental and disease models as well as a benchmark for future array CGH platform development.

HSP90 Inhibition Enhances Antimitotic Drug-Induced Mitotic Arrest and Cell Death in Preclinical Models of Non-Small Cell Lung Cancer

HSP90 inhibitors are currently undergoing clinical evaluation in combination with antimitotic drugs in non-small cell lung cancer (NSCLC), but little is known about the cellular effects of this novel drug combination. Therefore, we investigated the molecular mechanism of action of IPI-504 (retaspimycin HCl), a potent and selective inhibitor of HSP90, in combination with the microtubule targeting agent (MTA) docetaxel, in preclinical models of NSCLC. We identified a subset of NSCLC cell lines in which these drugs act in synergy to enhance cell death. Xenograft models of NSCLC demonstrated tumor growth inhibition, and in some cases, regression in response to combination treatment. Treatment with IPI-504 enhanced the antimitotic effects of docetaxel leading to the hypothesis that the mitotic checkpoint is required for the response to drug combination. Supporting this hypothesis, overriding the checkpoint with an Aurora kinase inhibitor diminished the cell death synergy of IPI-504 and docetaxel. To investigate the molecular basis of synergy, an unbiased stable isotope labeling by amino acids in cell culture (SILAC) proteomic approach was employed. Several mitotic regulators, including components of the ubiquitin ligase, anaphase promoting complex (APC/C), were specifically down-regulated in response to combination treatment. Loss of APC/C by RNAi sensitized cells to docetaxel and enhanced its antimitotic effects. Treatment with a PLK1 inhibitor (BI2536) also sensitized cells to IPI-504, indicating that combination effects may be broadly applicable to other classes of mitotic inhibitors. Our data provide a preclinical rationale for testing the combination of IPI-504 and docetaxel in NSCLC.