Chansey J. Veinotte

Translational Activation of HIF1α by YB-1 Promotes Sarcoma Metastasis

Metastatic dissemination is the leading cause of death in cancer patients, which is particularly evident for high-risk sarcomas such as Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma. Previous research identified a crucial role for YB-1 in the epithelial-to-mesenchymal transition (EMT) and metastasis of epithelial malignancies. Based on clinical data and two distinct animal models, we now report that YB-1 is also a major metastatic driver in high-risk sarcomas. Our data establish YB-1 as a critical regulator of hypoxia-inducible factor 1α (HIF1α) expression in sarcoma cells. YB-1 enhances HIF1α protein expression by directly binding to and activating translation of HIF1A messages. This leads to HIF1α-mediated sarcoma cell invasion and enhanced metastatic capacity in vivo, highlighting a translationally regulated YB-1-HIF1α axis in sarcoma metastasis.

Hooking the big one: the potential of zebrafish xenotransplantation to reform cancer drug screening in the genomic era

The current preclinical pipeline for drug discovery can be cumbersome and costly, which limits the number of compounds that can effectively be transitioned to use as therapies. Chemical screens in zebrafish have uncovered new uses for existing drugs and identified promising new compounds from large libraries. Xenotransplantation of human cancer cells into zebrafish embryos builds on this work and enables direct evaluation of patient-derived tumor specimens in vivo in a rapid and cost-effective manner. The short time frame needed for xenotransplantation studies means that the zebrafish can serve as an early preclinical drug screening tool and can also help personalize cancer therapy by providing real-time data on the response of the human cells to treatment. In this Review, we summarize the use of zebrafish embryos in drug screening and highlight the potential for xenotransplantation approaches to be adopted as a preclinical tool to identify and prioritize therapies for further clinical evaluation. We also discuss some of the limitations of using zebrafish xenografts and the benefits of using them in concert with murine xenografts in drug optimization.

Focused chemical genomics using zebrafish xenotransplantation as a pre-clinical therapeutic platform for T-cell acute lymphoblastic leukemia

Cancer therapeutics is evolving to precision medicine, with the goal of matching targeted compounds with molecular aberrations underlying a patient’s cancer. While murine models offer a pre-clinical tool, associated costs and time are not compatible with actionable patient-directed interventions. Using the paradigm of T-cell acute lymphoblastic leukemia, a high-risk disease with defined molecular underpinnings, we developed a zebrafish human cancer xenotransplantation model to inform therapeutic decisions. Using a focused chemical genomic approach, we demonstrate that xenografted cell lines harboring mutations in the NOTCH1 and PI3K/AKT pathways respond concordantly to their targeted therapies, patient-derived T-cell acute lymphoblastic leukemia can be successfully engrafted in zebrafish and specific drug responses can be quantitatively determined. Using this approach, we identified a mutation sensitive to γ-secretase inhibition in a xenograft from a child with T-cell acute lymphoblastic leukemia, confirmed by Sanger sequencing and validated as a gain-of-function NOTCH1 mutation. The zebrafish xenotransplantation platform provides a novel cost-effective means of tailoring leukemia therapy in real time.

The Zebrafish Xenograft Platform: Evolution of a Novel Cancer Model and Preclinical Screening Tool

Animal xenografts of human cancers represent a key preclinical tool in the field of cancer research. While mouse xenografts have long been the gold standard, investigators have begun to use zebrafish (Danio rerio) xenotransplantation as a relatively rapid, robust and cost-effective in vivo model of human cancers. There are several important methodological considerations in the design of an informative and efficient zebrafish xenotransplantation experiment. Various transgenic fish strains have been created that facilitate microscopic observation, ranging from the completely transparent casper fish to the Tg(fli1:eGFP) fish that expresses fluorescent GFP protein in its vascular tissue. While human cancer cell lines have been used extensively in zebrafish xenotransplantation studies, several reports have also used primary patient samples as the donor material. The zebrafish is ideally suited for transplanting primary patient material by virtue of the relatively low number of cells required for each embryo (between 50 and 300 cells), the absence of an adaptive immune system in the early zebrafish embryo, and the short experimental timeframe (5-7 days). Following xenotransplantation into the fish, cells can be tracked using in vivo or ex vivo measures of cell proliferation and migration, facilitated by fluorescence or human-specific protein expression. Importantly, assays have been developed that allow for the reliable detection of in vivo human cancer cell growth or inhibition following administration of drugs of interest. The zebrafish xenotransplantation model is a unique and effective tool for the study of cancer cell biology.

In Vivo Validation of PAPSS1 (3′-phosphoadenosine 5′-phosphosulfate synthase 1) as a Cisplatin-sensitizing Therapeutic Target

Purpose: Our previous screening efforts found that inhibition of PAPSS1 increases the potency of DNA-damaging agents in non–small cell lung cancer (NSCLC) cell lines. Here, we explored the clinical relevance of PAPSS1 and further investigated it as a therapeutic target in preclinical model systems. Experimental Design: PAPSS1 expression and cisplatin IC50 values were assessed in 52 lung adenocarcinoma cell lines. Effects of PAPSS1 inhibition on A549 cisplatin sensitivity under hypoxic and starvation conditions, in 3D spheroids, as well as in zebrafish and mouse xenografts, were evaluated. Finally, the association between PAPSS1 expression levels and survival in patients treated with standard chemotherapy was assessed. Results: Our results show a positive correlation between low PAPSS1 expression and increased cisplatin sensitivity in lung adenocarcinoma. In vitro, the potentiation effect was greatest when A549 cells were serum-starved under hypoxic conditions. When treated with low-dose cisplatin, PAPSS1-deficient A549 spheroids showed a 58% reduction in size compared with control cells. In vivo, PAPSS1 suppression and low-dose cisplatin treatment inhibited proliferation of lung tumor cells in zebrafish xenografts and significantly delayed development of subcutaneous tumors in mice. Clinical data suggest that NSCLC and ovarian cancer patients with low PAPSS1 expression survive longer following platinum-based chemotherapy. Conclusions: These results suggest that PAPSS1 inhibition enhances cisplatin activity in multiple preclinical model systems and that low PAPSS1 expression may serve as a biomarker for platin sensitivity in cancer patients. Developing strategies to target PAPSS1 activity in conjunction with platinum-based chemotherapy may offer an approach to improving treatment outcomes. Clin Cancer Res; 23(21); 6555–66. ©2017 AACR.

Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9

We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.

Successful optimization of CRISPR/Cas9-mediated defined point mutation knock-in using allele-specific PCR assays in zebrafish

Single-stranded oligodeoxynucleotides (ssODN) are donor templates for homology-directed repair-based knock-in of point mutations using CRISPR/Cas9. To optimize the efficiency of ssODN-based knock-ins in zebrafish, we developed allele-specific PCR (AS-PCR) assays for introducing point mutations in tp53, cdh5 and lmna as case studies. In these point mutation strategies we introduced the codon mutations, sgRNA site mutations and restriction sites which can be detected by AS-PCR with the primers matching their respective alleles in combination with a common primer. We employed the anti-sense asymmetric oligo design as the main optimization as well as phosphorothioate oligo modification and also observed that proximity of the mutation site to the Cas9 cut site improves the efficiency when knock-ins into different genes were compared. We improved the efficiencies of two tp53 knock-ins using anti-sense asymmetric ultramer oligos (126-nt in length with homology arms of 36 and 90 nucleotides, anti-sense to the sgRNA) by 3-10 fold, the optimizations which resulted in successful founders for both tp53 knock-ins with transmission rates of 20-40 %. The initially low knock-in efficiency for tp53 mutants was likely due to the distance between the Cas9 cut site and mutations since cdh5 G767S knock-in located at the cut site had much higher founder identification and germline transmission rates. The phosphorothioate oligo modifications was used for a lamin A/C (lmna) knock-in strategy and it resulted in 40 % overall improvement in knock-in efficiency and greater knock-in consistency. We also determined that AS-PCR detected false-positive knock-ins which constituted 25-80 % of total in different strategies and developed a workflow to screen out the founders and F1 zebrafish carrying these undesirable modifications. In summary, we provide a complementary set of optimizations for CRISPR/Cas9-based ssODN knock-ins in zebrafish using a novel combination of methods.

Abstract 3792: PAPSS1 (3’-phosphoadenosine 5’-phosphosulfate synthase 1) inhibition sensitizes non-small cell lung cancer to cisplatin treatment in vivo

We previously reported that 3’-phosphoadenosine-5’-phosphosulfate (PAPS) synthase 1 (PAPSS1), an enzyme that synthesizes the biologically active form of sulfate (PAPS) for all sulfation reactions, is a novel therapeutic target that when suppressed enhances the activity of multiple DNA damaging agents in NSCLC cells. PAPSS1 was the lead hit in a synthetic lethal screen completed using chemotherapy-naive NSCLC cells exposed to the IC10 of cisplatin (CDDP). PAPSS1 silencing was more effective in potentiating CDDP activity than our positive control (BRACA2). Here, we evaluated PAPSS1 as a CDDP-sensitizing target in three different model systems: 3D spheroids, zebrafish xenografts, and a mouse xenograft model. siRNA-transfected A549 cells were seeded in round bottom ultra-low attachment plates for spheroid formation. Spheroids were formed over a period of three days and then treated with CDDP. The spheroids were imaged using the IncuCyte ZOOM® Live Cell Imaging system every 3 hours for 8 days to monitor changes in spheroid size. To evaluate PAPSS1 in zebrafish, transfected A549 cells were microinjected into the yolk sack of zebrafish embryos and then maintained in CDDP-containing media for 48 hours. The human cells were harvested from 20 fish per treatment group and counted to determine the change in cell number as a measure of tumor growth in vivo. For mouse studies, RAG2M mice were inoculated subcutaneously with 5×106 parental, non-targeting shRNA, or shPAPSS1-expressing A549 cells. The mice were treated 7 days later with 3 mg/kg CDDP (IV, Q4Dx3). Tumor size was measured using an electronic caliper and tumor volumes were calculated using the equation (lxw2)/2. PAPSS1-silenced cells formed spheroids of comparable size as the scramble control. CDDP (12.5μM) was effective against both control and PAPSS1-silenced spheroids with a reduction of 31% and 46% in spheroid size, respectively. PAPSS1-knockdown spheroids were significantly more sensitive to CDDP even when added at an 8-fold lower dose (1.56 μM). At this concentration, the control spheroids grew about 37% in size while the size of the PAPSS1-silenced spheroids was reduced by 21% (p Citation Format: Ada W.Y. Leung, Chansey J. Veinotte, Nicole Melong, Ian Backstrom, Corinna Warburton, Edie Dullaghan, Jason N. Berman, Marcel B. Bally. PAPSS1 (3’-phosphoadenosine 5’-phosphosulfate synthase 1) inhibition sensitizes non-small cell lung cancer to cisplatin treatment in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3792.

Abstract 1398: Using zebrafish xenotransplantation to study the role of Y-Box binding protein (YB-1) in the metastasis of Ewing family tumors

Sarcomas are malignant cancers of soft tissue or bone predominantly affecting children and adolescents. The most common subtypes are osteosarcoma and Ewing family tumours (EFTs). The most unfavorable prognostic factor is the presence of metastases, which accounts for 9 out of 10 sarcoma cancer deaths. Identifying factors and/or drugs that have an impact on metastatic spread have tremendous potential to affect outcome by reducing disease burden to the primary site, which can be more effectively treated by surgery and radiation. Pertinent animal models are critical for translating in vitro findings to clinical trials. Xenotransplantation of human cancer cells into transparent zebrafish embryos provides a novel in vivo platform for visualizing tumor micro-environment interactions contributing to sarcoma proliferation and spread in real time, which is not easily provided by other animal models. We recently demonstrated the effectiveness of the zebrafish xenotransplantation model for the study of specific drug-tumor interactions for both chronic myelogenous leukemia and acute promyelocytic leukemia and used a rapid and novel ex-vivo proliferation assay to quantify therapeutic responses (Corkery et al, BJH 2011). We have now applied this technology to EFTs. Human EFT TC-32 cells were fluorescently labeled with CmDiI, and microinjected into the yolk sac of two day old casper embryos, a double pigment mutant that prevents any auto-fluorescence that might interfere with image quality. EFT cells successfully engrafted, survived and proliferated over 96 hours post-injection (hpi). Migration of cells from the yolk sac to the tail occurred between 48 and 144 hpi with evidence of vascular extravasation and tissue infiltration. Y-box binding protein 1 (YB-1) is implicated in the metastatic spread of epithelial cancers due to its key role in promoting an epithelial-to-mesenchymal transition (EMT). In contrast to parental TC-32 cells, xenografted YB-1 knockdown (KD) TC-32 cells showed absent or significantly delayed migration, suggesting that YB-1 also regulates this process in zebrafish xenografts. Moreover, using transgenic fli-EGFP casper embryos that display fluorescent vasculature, we saw evidence of vascular recruitment into the tumor mass in WT TC-32 cells but not in YB-1 KD, potentially implicating angiogenesis as a mechanism that contributes to tumor spread in YB-1 expressing sarcomas. Exposure of TC-32 xenografted embryos to 5-40 Gy of ionizing radiation effectively reduced cell proliferation in a dose-dependent manner. These studies highlight the utility of the zebrafish xenograft model to elucidate the mechanisms underlying the metastatic behavior of EFTs and position this system as an in vivo tool for drug discovery to identify novel anti-proliferative and anti-metastatic agents to improve outcome in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1398. doi:1538-7445.AM2012-1398