Daniel Ciznadija

TRIM3, a tumor suppressor linked to regulation of p21(Waf1/Cip1.).

The TRIM family of genes is largely studied because of their roles in development, differentiation and host cell antiviral defenses; however, roles in cancer biology are emerging. Loss of heterozygosity of the TRIM3 locus in ∼20% of human glioblastomas raised the possibility that this NHL-domain containing member of the TRIM gene family might be a mammalian tumor suppressor. Consistent with this, reducing TRIM3 expression increased the incidence of and accelerated the development of platelet-derived growth factor -induced glioma in mice. Furthermore, TRIM3 can bind to the cdk inhibitor p21WAF1/CIP1. Thus, we conclude that TRIM3 is a tumor suppressor mapping to chromosome 11p15.5 and that it might block tumor growth by sequestering p21 and preventing it from facilitating the accumulation of cyclin D1–cdk4.

Cyclin D1 and cdk4 mediate development of neurologically destructive oligodendroglioma

Although the molecular changes that characterize gliomas have been studied, the pathogenesis of tumor development remains unclear. p21 contributes to gliomagenesis by stabilizing cyclin D1-cdk4 kinase complexes, suggesting that cyclin D1 and cdk4 may also be required for glial tumor development. In this study, we used a mouse model to attempt to confirm this hypothesis, finding that cyclin D1 and cdk4 played active roles in not only the tumor but also the tumor microenvironment. Loss of cdk4 blocked tumor development, but loss of cyclin D1 did not prevent gliomas from developing. Instead, loss of cyclin D1 impeded progression to higher stages of malignancy. Enforcing expression of cyclin D1 was insufficient to correct the progression defect observed in cyclin D1-deficient animals. In contrast, restoration of cdk4 in the cdk4-deficient animals restored cell proliferation and tumor formation, although at lower tumor grades. Notably, the failure of tumors in the cyclin D1- and cdk4-deficient animals to progress to higher grades was correlated with a failure to fully activate microglia in the tumor microenvironment. Moreover, when platelet-derived growth factor-transformed glial cells were engrafted orthotopically into the mice, the tumors that formed progressed to high grades in wild-type mice but not cyclin D1-deficient animals. Together, our findings establish that the cyclin D1-cdk4 axis is not only critical in glial tumor cells but also in stromal-derived cells in the surrounding tumor microenvironment that are vital to sustain tumor outgrowth.

Tyrosine Phosphorylation of the p21 Cyclin-dependent Kinase Inhibitor Facilitates the Development of Proneural Glioma

Background: Phosphorylation of Tyr in the 310 helix of p27 reduces its inhibitory activity on cyclin-CDK complexes. Results: Mutation of this site to Phe reduces the tumor-promoting activity of p21 in the RCAS-PDGF-HA/nestin-TvA mouse model of proneural gliomagenesis. Conclusion: Tyr phosphorylation of p21 contributes to its oncogenic role. Significance: This mouse model establishes the significance of this modification for the nuclear accumulation of cyclin D1-CDK4. Phosphorylation of Tyr-88/Tyr-89 in the 310 helix of p27 reduces its cyclin-dependent kinase (CDK) inhibitory activity. This modification does not affect the interaction of p27 with cyclin-CDK complexes but does interfere with van der Waals and hydrogen bond contacts between p27 and amino acids in the catalytic cleft of the CDK. Thus, it had been suggested that phosphorylation of this site could switch the tumor-suppressive CDK inhibitory activity to an oncogenic activity. Here, we examined this hypothesis in the RCAS-PDGF-HA/nestin-TvA proneural glioma mouse model, in which p21 facilitates accumulation of nuclear cyclin D1-CDK4 and promotes tumor development. In these tumor cells, approximately one-third of the p21 is phosphorylated at Tyr-76 in the 310 helix. Mutation of this residue to glutamate reduced inhibitory activity in vitro. Mutation of this residue to phenylalanine reduced the tumor-promoting activity of p21 in the animal model, whereas glutamate or alanine substitution allowed tumor formation. Consequently, we conclude that tyrosine phosphorylation contributes to the conversion of CDK inhibitors from tumor-suppressive roles to oncogenic roles.

Hdm2- and proteasome-dependent turnover limits p21 accumulation during S phase

Double-strand DNA breaks detected in different phases of the cell cycle induce molecularly distinct checkpoints downstream of the ATM kinase. p53 is known to induce arrest of cells in G1 and occasionally G2 phase but not S phase following ionizing radiation, a time at which the MRN complex and cdc25-dependent mechanisms induce arrest. Our understanding of how cell cycle phase modulates pathway choice and the reasons certain pathways might be favored at different times is limited. In this report, we examined how cell cycle phase affects the activation of the p53 checkpoint and its ability to induce accumulation of the cdk2 inhibitor p21. Using flow cytometric tools and centrifugal elutriation, we found that the p53 response to ionizing radiation is largely intact in all phases of the cell cycle; however, the accumulation of p21 protein is limited to the G1 and G2 phase of the cell cycle because of the activity of a proteasome-dependent p21 turnover pathway in S-phase cells. We found that the turnover of p21 was independent of the SCFskp2 E3 ligase but could be inhibited, at least in part, by reducing hdm2, although this depended on the cell type studied. Our results suggest that there are several redundant pathways active in S-phase cells that can prevent the accumulation of p21.

Immunohistochemical Assessment of Signal Transduction and Cell-Cycle Networks in Neural Tumors

The ability to detect transient changes in molecular networks lies at the heart of cancer biology research. This is especially apparent during tumorigenesis, where initiating mutations typically affect mitogens and cell-cycle molecules such as PDGF or retinoblastoma protein (Rb). One of the primary consequences of such processes is the inappropriate stimulation of downstream targets, normally through posttranslational modification. Immunohistochemistry (IHC) provides an important tool for assessing such changes in situ, permitting different aspects of tumor biology to be examined as a tissue undergoes transformation. Nevertheless, this can be difficult to achieve, particularly in complex environments like the brain. Here, we provide the automated methodology we have employed for the successful detection of phosphorylation of S6 ribosomal protein (S6-RP) and the retinoblastoma protein (Rb) in response to PDGF stimulation in a mouse model of glial brain tumor development.

Abstract 1648: ImmunoGraft®platform for the evaluation of Immuno-Oncology agents in PDX tumors models

Recent breakthroughs in Immunotherapy have given new hope to treating previously untreatable tumor types and provide a better tolerated alternative to standard agents. There is an unmet need for a pre-clinical platform to test potential immune-oncology therapeutics that would also provide a tool to examine the mechanisms of response to better predict clinical outcomes. We have previously presented the Champions ImmunoGraft®, an innovative pre-clinical model enabling immunotherapeutic agents to be evaluated for efficacy in solid tumors. This platform is more reflective of the human tumor microenvironment (both immune and non-immune cell-based) and may be one of the most translationally-relevant models to date for screening therapies targeting the immune system. However, optimization of humanization and PDX implantation protocols that allow a broader reconstitution of cell linage and higher engraftment rate are necessary to further improve the pre-clinical evaluation of immune-oncology therapeutics and enhance the value of this modality for patient’s benefit. To this end, immune-compromised NOG (PrkdcscidIl2rgtm1Sug) mice were reconstituted (humanized) with human CD34+ cells using optimized procedure and blood was collected at different time points post engraftment to check for the major leukocyte linages. At ten weeks after humanization, mature human CD45+ cells comprised close to 50% of the leukocytes detected in the circulation and secondary lymphoid tissues of the humanized animals. As a result of improved methodology of the reconstitution protocol we have achieved an 85% success rate of humanization with a shortened engraftment period utilizing fewer CD34+ cells that maintain humanization reconfirmed at 22 weeks post reconstitution. Champions TumorGraft® database contains more than one-thousand clinically relevant well-annotated PDX models. We have complied data from extensive genetic and protein expression analyses to design applicable ImmunoGraft® studies for testing of Immuno-Oncology agents. We have identified tumors expressing key markers that may predict response to immunotherapy agents including PDL-1, CD40, IDO-1 and IDO-2 as well as tumors that harbor a high mutational load. Here we present the efficacy and clinically relevant endpoints of two well-studied checkpoint inhibitors Ipilimumab and Pembrolizumab in this highly translational PDX platform. Citation Format: David Cerna, Daniel Ciznadija, Bhavna Verma, David Sidransky, Evgeny Izumchenko, Angela Davies, Maria Mancini. ImmunoGraft® platform for the evaluation of Immuno-Oncology agents in PDX tumors models [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 1648. doi:10.1158/1538-7445.AM2017-1648

Patient-derived xenografts effectively capture patient clinical responses to oncology therapy

1. A cohort of 90 PDX models reflecting clinical trial populations The high proportion of experimental oncology agents failing to demonstrate sufficient clinical activity represents a continuing challenge and results in part from a failure to appropriately identify responsive patient cohorts. Technologies that reflect the intrinsic heterogeneity characterizing human cancers and which also enable multiple experimental regimens to be evaluated simultaneously to identify those most likely to be clinically beneficial in specific patient populations are needed. Drug sensitivity screening in patient-derived xenografts (PDXs) is a viable solution, but requires such models to accurately reflect patient clinical outcomes. In this study we examined the capacity of PDXs to replicate 126 patient responses across a heterogeneous population of 90 solid tumors and treatments and report performance metrics highlighting the clinical utility of this tool. Patient-derived xenografts effectively capture patient clinical responses to oncology therapy

Abstract B40: Mouse clinical trials: integrating PDX models of sarcoma subtypes with genomics to replicate patient responses to cancer therapeutics

Objective: Sarcomas are clinically and genetically heterogeneous tumors that are often difficult to treat. Patient-derived xenograft (PDX or TumorGraft) models have been shown to accurately reflect the characteristics of patient tumors and may be useful tools for developing personalized treatment strategies and deployment in mouse clinical trials assessing novel therapies. We evaluated the accuracy of PDX models in reproducing clinical responses to standard and experimental drugs used for sarcoma treatment. Methods: Fresh tumor tissue (comprising 172 distinct explants) was collected by surgery or biopsy from 150 patients with sarcoma and implanted into immunodeficient mice. Tumors successfully engrafting were screened using next-generation sequencing technology to identify key genomic alterations with therapeutic implications. PDX sensitivity to standard of care and experimental agents was evaluated and tumor growth inhibition/regression values and clinical RECIST outcomes determined. Drug screening results were correlated with individual patient outcomes. Results: Of the 172 implanted tumors, 145 have completed the implantation process, with 86 (59%) successfully establishing a PDX model. Engraftment rate depended on sarcoma subtype and specimen origin (surgical explant versus biopsy). Next generation sequencing of models from major sarcoma subtypes (Ewing sarcoma, leiomyosarcoma, liposarcoma, osteosarcoma, and rhabdomyosarcoma) highlighted alterations in 454 genes, including those informing treatment selection such as PIK3CA, MET, and CDK4. A total of 26 PDX models from 25 patients across the major sarcoma subtypes were screened in 148 drug tests employing 64 FDA-approved drugs/combinations such as ifosfamide, and gemcitabine/docetaxel, and 26 experimental therapies in clinical trial. In 13/13 (100%) cases with available data, a significant correlation between patient clinical response and PDX model outcome was noted (p=0.0004; Fisher9s exact test). Conclusions: Given the close match between patient clinical responses and PDX model outcomes, these results validate the concept of mouse clinical trials for determining the efficacy of novel therapies in sarcoma prior to broad application in expensive human trials. Moreover, the retention of alterations in key genes influencing therapeutic decision-making suggests a use for PDX models in functionally validating genomic hypotheses in a pre-clinical setting. Citation Format: Amanda Katz, Raphael E. Pollock, Leonard H. Wexler, Carlos Rodriguez-Galindo, Jonathan C. Trent, Robert Maki, Jennifer Jaskowiak, Lindsay Ryland, Daniel Ciznadija, Angela Davies, Keren Paz. Mouse clinical trials: integrating PDX models of sarcoma subtypes with genomics to replicate patient responses to cancer therapeutics. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B40.

Abstract A8: The ImmunoGraft: A humanized mouse model for translational assessment of immunotherapy in solid tumors

Background: Therapeutics reactivating the immune system have demonstrated promise, with durable objective responses in patients with a variety of solid tumors. Despite these successes, current animal models do not reliably identify immunotherapeutic targets with the greatest clinical potential, due in part to differences between human and murine immune systems. Hence, development of robust preclinical tools to test such drugs against human tumors in the context of an allogeneic immune system remains an imperative. We have previously demonstrated the generation of its ImmunoGraftTM platform, whereby two technologies, the patient-derived xenograft (PDX) and humanized mice (immunodeficient mice reconstituted with a human immune system), are combined in a single platform. We now report on the utility of the ImmunoGraftTM for assessing the effect of immune-modulating agents in solid tumors. Materials and Methods: Immune-compromised NOG (PrkdcscidIl2rgtm1Sug) mice were reconstituted with human CD34+ cells and monitored for the expansion of human immune cells (humanized). Humanized mice were engrafted with solid tumors that had been subjected to histocompatibility typing and characterized for a number of molecular markers, including PD-L1 expression. Tumor growth in the ImmunoGraftsTM was compared against non-humanized counterparts, as well as the level of immune reconstitution. Finally, ImmunoGraftsTM were treated with drugs blocking the immune checkpoints CTLA4 and PD1 and human immune activation and tumor growth inhibition evaluated. Results: Mature human CD45+ cells comprised close to 50% of the leukocytes detected in the circulation and lymphoid organs of humanized mice. Solid tumors, including NSCLC, melanoma, and head and neck cancer, were successfully engrafted in the humanized mice. Moderate to high expression of PD-L1 was found in approximately 80% of these tumors. ImmunoGraftsTM treated with anti-CTLA4 or anti-PD1 antibodies exhibited systemic immune responses characterized by robust proliferation of splenic and circulating huCD3+ T cells, as well as activated huCD4+ Th1 cells. There was also an increase in tumor-infiltrating huCD8+ cytotoxic T lymphocytes and huCD68+ macrophages, along with elevated secretion of human-specific cytokines. Tumor growth inhibition, and in some instances tumor regression, was demonstrated in treated ImmunoGraftsTM. The magnitude of growth inhibition correlated with the level of immune activation. Conclusion: The ImmunoGraftTM is an innovative pre-clinical model enabling immunotherapeutic agents to be evaluated for efficacy in solid tumors. This platform is more reflective of the human tumor microenvironment (both immune and non-immune cell-based) and may be one of the most translationally-relevant models to date for screening therapies targeting the immune system. To gauge the clinical potential of the ImmunoGraftTM, a retrospective analysis is currently ongoing using PDX models developed from patients treated with immuno-oncology drugs. The ImmunoGraftTMhas the potential to revolutionize translational drug discovery and development for immunotherapeutic agents in oncology. Citation Format: Gilson Baia, David Vasquez, David Cerna, Daniel Ciznadija, David Sidransky, Amanda Katz, Keren Paz. The ImmunoGraft: A humanized mouse model for translational assessment of immunotherapy in solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A8.

Abstract A14: Molecular fidelity of patient derived xenograft (PDX) models to original human tumor and to the cancer genome atlas (TCGA)

Background: Patient-derived xenograft (PDX) models, also known as Champions TumorGraft® models, maintain the complex intra-tumoral biology of the primary tumor. Over 250 of the Champions models, ranging over a wide variety of solid tumors and passaging generations, have been analyzed using whole exome sequencing (WES) and RNA sequencing (RNAseq). SNPs, InDels and copy number alterations (CNAs) data have been generated for each model, following the Genome Analysis Toolkit (GATK). While several publications compare small numbers of PDX models and human tumors on the molecular level, this is the first known comprehensive analysis whereby the molecular fidelity of the PDX platform is corroborated across several cancer types and throughout different mouse generations. Method and Results: First, we compared PDXs to their human original counterparts using a preliminary group of four PDX models with available matching human patient WES data. Patient tumor source included dedifferentiated liposarcoma, synovial sarcoma, renal cell carcinoma and squamous cell carcinoma of the lung. PDX passages ranged from 2 to 4. We compared called mutations and a high percentage of identified human tumor mutations were present in the PDX models (42-82%), with the lowest scoring model also showing signs of normal contamination in the human tumor sample. For CNAs in oncogenic sites, we saw an average of 65% of human tumor alterations recurring in the PDX models. This was observed, despite inherent difficulties due to exome- based CNA analysis methods. Encouraged by the individual patient results, we subjected our largest (per cancer type) PDX cohorts to a molecular comparison with the equivalent TCGA cohorts. More than 200 of the sequenced models, grouped into colorectal adenocarcinoma (COADREAD), lung adenocarcinoma (LUAD), breast carcinoma (BRCA), head and neck squamous cell carcinoma (HNSC) and ovarian serous carcinoma (OV) cohorts were compared. We applied mutation category (MC) and significantly mutated genes (SMG) analysis, as well as comparison of mutation population frequencies for TCGA SMG. Results showed high correlation between the TCGA and the Champions PDX cohorts, although the level of matching varied between cancer types. For instance, COADREAD was highly correlative, while other cancer types, such as BRCA, showed bias toward CpG site mutations. In SMG analysis and population frequency analysis, major SMGs recur across the cohorts, while, as expected, weaker signals from the TCGA were often missed in the smaller cohorts. Conclusions: Detailed comparison of several PDX models to the human tumor counterpart demonstrated high fidelity, not only at the gene level but also the mutation and CNA level. Cohort comparisons were correlative as well, but a certain bias was discerned in both MC and SMG analyses. There could be several causes for this, including statistical artifacts due to small cohort sizes, clinical and demographic differences between the Champions and TCGA patient profiles, or biological factors such as clonal selection and engraftment pressure. Further analysis is ongoing to better understand the model at a molecular level and maximize its utility as a robust translational research tool. Citation Format: Ido Sloma, Ido Ben-zvi, Tin Khor, Daniel Ciznadija, Amanda Katz, David Vasquez, Jennifer Jaskowiak, Lindsay Ryland, Angela Davies, David Sidransky, Keren Paz. Accurate molecular fidelity of patient-derived xenograft (PDX) models to original human tumors and to The Cancer Genome Atlas (TCGA). [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A21.