Aidan Synnott

Abstract 3362: Systematic evaluation of immune checkpoint inhibitors

With the increasing success and subsequent interest in tumor immunology, we recognized a growing need for well-characterized preclinical models. While the literature contains many different experimental models, the available data includes a variety of reagents and the side by side comparative evaluation of therapeutics in multiple models is relatively rare. Lechner et al., (J. Immunother. 36:477-489, 2013) addressed this issue and reported on a comparative analysis of a set of six syngeneic models. Our effort looked to expand this paradigm towards a comparative evaluation of the responsiveness of a collection of syngeneic models to antibody based checkpoint inhibitor therapeutics. Specifically, we examined the response of the Colon26, MC38, B16F10, Lewis Lung, Madison109, EMT-6, and 4T1 models to anti-CTLA-4, anti-PD-1 and anti-PDL-1 monotherapies as well as capturing the response to combination therapies of anti-CTLA-4 with anti-PD-1 or anti-PDL-1.. Our results clearly show a differential response across this set of models both in regard to CTLA-4 targeted therapy as well as PD-1/PDL-1 therapy. In an attempt to expand the number of checkpoint inhibitor responsive models, we are performing a second phase of work evaluating the impact of immuno-modulatory radiation therapy upon these baseline responses. In initiating these studies and evaluating the literature, it became clear that there were multiple choices in regard to anti-CTLA-4 preclinical reagents. We have also performed a series of studies evaluating the efficacy of multiple anti-CTLA-4 clones as an extension of previously published work which described the variable ability of anti-CTLA-4 clones to deplete tumor Treg populations (Simpson et al. J. Exp. Med. 210(9) 1695-1710, 2013). Our anti-tumor data correlates well with these cell based observations. This differential allows one to match efficacy with model and expands the reagents available for evaluating combination therapies. Citation Format: Sheri Barnes, Paula Miliani de Marval, Jacob Hauser, Tracy Brainard, Daniel Small, Aidan J. Synnott, Robert J. Mullin. Systematic evaluation of immune checkpoint inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3362. doi:10.1158/1538-7445.AM2015-3362

Targeting the cyclin dependent kinase and retinoblastoma axis overcomes standard of care resistance in BRAF V600E -mutant melanoma

Patient-derived tumor xenograft (PDTX) mouse models were used to discover new therapies for naïve and drug resistant BRAFV600E-mutant melanoma. Tumor histology, oncogenic protein expression, and antitumor activity were comparable between patient and PDTX-matched models thereby validating PDTXs as predictive preclinical models of therapeutic response in patients. PDTX models responsive and non-responsive to BRAF/MEK standard of care (SOC) therapy were used to identify efficacious combination therapies. One such combination includes a CDK4/6 inhibitor that blocks cell cycle progression. The rationale for this is that the retinoblastoma protein (pRb) is 95% wildtype in BRAF mutant melanoma. We discovered that 77/77 stage IV metastatic melanoma tissues were positive for inactive phosphorylated pRb (pRb-Ser780). Rb is hyperphosphorylated and inactivated by CDK4/6:cyclin D1 and when restored to its hypophosphorylated active form blocks cell cycle progression. The addition of a CDK4/6 inhibitor to SOC therapy was superior to SOC. Importantly, triple therapy in an upfront treatment and salvage therapy setting provided sustained durable response. We also showed that CDK4/6 blockade resensitized drug resistant melanoma to SOC therapy. Durable response was associated with sustained suppression of pRb-Ser780. Thus, reactivation of pRb may prove to be a clinical biomarker of response and the mechanism responsible for durable response. In light of recent clinical trial data using this triple therapy against BRAFV600E-mutant melanoma, our findings demonstrating superior and prolonged durable response in PDTX models portend use of this therapeutic strategy against naïve and SOC resistant BRAF V600E-mutant metastatic melanoma coupled with pRB-Ser780 as a biomarker of response.

Combination immune checkpoint inhibitors for the treatment of human colon carcinoma in NSG mice engrafted with human PBMC

PK and Efficacy studies: Female NSGTM mice (Jackson laboratory), were 8-10 weeks were engrafted with human peripheral blood mononuclear cells (PBMC) at 3x107 cell/animal (Hemacare; PBMC-HLA-A*01/01, PBMC-HLA-A*01/02; PBMCHLAA*03/24). Seven days post engraftment, animals were implanted with 5x106 RKO tumor cells in 50% Matrigel, subcutaneously in the right flank. Dosing began on Day 1 in mice with established RKO tumors (group mean 70 mm3). The study endpoint was a tumor volume of 1500 mm3 in the control group for the RKO I study and Day 18 in the RKO II study in which samples were collected for flow cytometry analysis. Treatment outcome was based on percent tumor growth inhibition (%TGI), defined as the percent difference between the median tumor volumes (MTVs) of treated and control mice. Percent TGI was calculated using the following formula: %TGI = [1-(MTVdrug treated/MTVcontrol)] x 100. The results of two independent studies are shown here. Statistical Significance was determined using the MannWhitney U test. Flow Cytometry Analysis Peripheral blood was collected in tubes containing K2EDTA and immune cell populations were analyzed by flow cytometry. All data were collected on a FACSCanto II (BD) and analyzed with FlowJo software (Tree Star, Inc.). Initial sequential gating on singlets (FSC-H vs. FSCA), leukocytes (SSC-A vs. FSC-A) and live cells was performed followed by identification of immune cells populations based upon the following phenotype markers: hCD45+; CD4 (hCD45+CD3+CD4+), CD8 (hCD45+CD3+CD8+), T cell populations were further defined as effector (EFF) and effector memory (EM) based upon CCR7 and CD45RO expression. The cell lines A375, A2058, SK-MEL-24, SK-MEL5, HT-1080, Colo205, RKO, HCT116, MDA-MB-231, Calu6, HCC827, SKOV3, were analyzed by flow cytometry for the expression of PD-L1 and PDL-2. 1 ABSTRACT

Abstract 2215: Pembrolizumab and ipilimumab reduce human RKO colon carcinoma tumor growth in HLA matched CD34-NSG humanized mice

Over the past decade there has been an increasing demand for preclinical models useful for evaluating the efficacy of checkpoint inhibition-based cancer immunotherapies. The recently developed humanized mouse models composed of various types of human immune cells has the potential to recapitulate the human anti-tumor response modulated by checkpoint inhibitors. Tumor cells may develop multiple mechanisms to evade immune surveillance, including PD-L1 upregulation which suppresses T-cell effector responses towards the tumor. In the present study, we evaluated the efficacy of the checkpoint inhibitors pembrolizumab (anti-PD-1) and ipilimumab (anti-CTLA-4) in the human RKO colon carcinoma xenograft model in CD34-NSG humanized mice. These mice were engrafted with human hematopoietic stem cells (HSC) that matched the HLA haplotype of the RKO cells (HLA-A*01). Flow cytometry analysis confirmed high level of expression of PD-L1 in RKO colon carcinoma cells. The results from this study revealed that pembrolizumab and ipilimumab monotherapies significantly inhibited tumor growth. Surprisingly, combination therapy did not provide additive or synergistic effects and resulted in the same level of efficacy as the individual regimens. In order to understand the impact of these therapies on distinct immune cell populations, we analyzed the distribution of CD4 and CD8 T lymphocytes, Treg cells, NK cells and B cells in peripheral blood, spleen and tumor samples from tumor bearing humanized mice, via flow cytometry. We found that these checkpoint inhibitors can enhance effector functions of CD4+ and CD8+ T cells associated with increased expression of IFN-Γ. A parallel study with these checkpoint inhibitors carried out with humanized mice engrafted with HSC of the same haplotype as the RKO cells but from a different donor resulted in identical anti-tumor efficacy. In summary, these studies validate the use of humanized mouse models to test the tumor response to immune-checkpoint based therapies as it shows significant anti-tumor growth as a result of pembrolizumab and ipilimumab monotherapies associated with activation of T lymphocytes. Citation Format: Marcio Lasaro, Jason M. Davis, Mark Ellison, Martin R. Graf, Vivek Mahajan, Aidan Synnott, Robert J. Mullin, Paula L. Miliani De Marval. Pembrolizumab and ipilimumab reduce human RKO colon carcinoma tumor growth in HLA matched CD34-NSG humanized mice. [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 2215.

Abstract C110: Evaluation of immune cell populations in checkpoint inhibitor responsive syngeneic models

Given the increasing success of cancer immunotherapies, we recognized a growing need for well-characterized preclinical models to enable drug discovery efforts. Syngeneic mouse tumor models offer advantages over conventional xenograft models for immunotherapy studies due to the presence of a fully functional immune system. We recently completed a comparative evaluation of the responsiveness of a collection of syngeneic models to two antibody based immunocheckpoint inhibitor therapeutics (anti-CTLA4, anti-PD1). This body of work resulted in the identification of several models responsive to checkpoint inhibition; CT26, Colon26, MC38, EMT-6, and A20. Our results clearly show differential tumor growth responses across this set of models following treatment with CTLA-4 and PD-1 targeted therapy. To further characterize these responses, we have performed a series of studies to identify and track immune cell populations by flow cytometry in blood, spleen, and tumor samples from mice harboring each responsive model. In these studies, we have investigated whether the modulation of CD4+, CD8+, regulatory CD4+ T cells (Treg), and myeloid derived suppressor cells (MDSC) populations correlates with the impact of immunocheckpoint inhibitors on tumor growth over time in untreated and challenged animals. These correlations of flow analyses with our antitumor data can point to the utility of cell-based endpoints as additional tools for evaluating combination outcomes. Citation Format: Sheri Barnes, Marcio Lasaro, Jacob Hauser, Robert J. Mullin, Aidan Synnott. Evaluation of immune cell populations in checkpoint inhibitor responsive syngeneic models. [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 C110.

Abstract 2417: Characterization of novel patient derived melanoma xenografts and cell lines in response to targeted therapies

The success of clinical trials for patients with malignant melanoma (MM) designed from the “promising” results using pre-clinical models is minimal. With its increase in incidence, there is a dire need to develop both pre-clinical models comparable to clinical practice that have the capability to translate from bench-to-bedside and novel therapeutic agents for this life-threatening form of skin cancer. To address this concern, we have developed a panel of pre-clinical MM models including patient matched cell lines, patient cell-line derived xenograft models and patient tumor tissue-derived xenograft models. Patient derived xenograft (PDX) models are purported to most closely replicate a patient9s response to therapy. Since the development of patient-derived cell lines and tumors grown in mice were more efficacious, their genetic, phenotypic, and therapeutic responses were compared to those of their corresponding patient-matched PDX model. Each model was molecularly characterized to confirm a match to that of the originating patient tumor tissue via protein immunohistochemistry (IHC), mRNA quantitative polymerase chain reaction (qPCR), DNA short tandem repeat (STR) analysis, and Sanger sequencing. Each pre-clinical model provided a genetic match to their respective patient tumor sample. All pre-clinical models were positive for the melanoma-specific markers (S-100 beta, HMB-45, and Melan-A) confirming a melanoma phenotype. Soft agar studies and tumor growth of our PDX and patient cell-line derived xenograft models confirmed tumorigenicity within our models providing the foundation to perform therapeutic studies. Mutation status data (i.e. BRAF, RAS), patient therapeutic clinical history and oncogene overexpression analysis (pERK, pAKT, PI3K, pmTOR, etc.) provide the platform and rationale behind the use of specific therapeutic agents per model. BRAF, NRAS and KRAS mutations were identified along with elevated pERK, pAKT and pmTOR in the models. Targeted therapies (trametinib/MEK inhibitor; vemurafenib/BRAF inhibitor; MK-2206/pAKT inhibitor; and everolimus/mTOR inhibitor) demonstrated dose dependent inhibition of cell proliferation in each of the models based upon oncogenic expression or mutations. In vivo experiments are in progress, which will allow comparison of the three models. The next steps include combination therapy to identify antitumor synergy targeting oncogenic signaling pathways. Citation Format: Antoneicka L. Harris, Laura Marlow, Adam Mathias, Louis Dawson, William Durham, Kenneth Meshaw, Robert Mullin, Daniel Small, Aidan Synnott, Kevin Wu, Dragana Milosevic, Brian Netzel, Stefan Grebe, Svetomir Markovic, John Copland. Characterization of novel patient derived melanoma xenografts and cell lines in response to targeted therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2417. doi:10.1158/1538-7445.AM2015-2417

Abstract 1475: Therapeutic responses in a novel patient-derived xenograft mouse model for rare acinar cell pancreatic carcinoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Acinar cell carcinoma of the pancreas (ACC) is an uncommon malignancy, accounting for less than 1% of all pancreatic neoplasms. Because of its rarity, only a few retrospective studies are available to help guide management. We previously reported the case of a patient with metastatic ACC who achieved prolonged survival with doxorubicin as a result of personalized treatment designed in part on the basis of molecular and in-vitro data collected on analysis of the tumor and primary cells in culture developed from the liver metastasis. We now report the characterization of a patient derived xenograft (PDX) mouse model originating from this patients ACC liver metastasis tissue. Antitumor activity of multiple drugs (5-FU, irinotecan, oxaliplatin, gemcitabine, bevacizumab, erlotinib, doxorubicin and imatinib) used as single agent therapy is demonstrated. Of the targeted and cytotoxic therapies used, oxaliplatin produced a dramatic and prolonged response to therapy even after withdrawal of treatment. Bevacizumab produced a significant response, as well. Serum lipase and tissue amylase levels correlated with the antitumor response to therapy. Thus, we have developed and characterized an ACC PDX model that may be used in drug discovery for the treatment of this rare cancer for which no standard of care exists. Note: This abstract was not presented at the meeting. Citation Format: Laura A. Marlow, Adam C. Mathias, Louis K. Dawson, William F. Durham, Kenneth A. Meshaw, Robert J. Mullin, Aidan J. Synnott, Ricardo Paz-Fumagalli, Murli Krishna, Daniel Von Hoff, Daniel L. Small, Gerardo Colon-Otero, John A. Copland. Therapeutic responses in a novel patient-derived xenograft mouse model for rare acinar cell pancreatic carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1475. doi:10.1158/1538-7445.AM2015-1475

Abstract 1458: Characterization of novel thyroid PDX models and their response to combination therapies

Patient derived xenografts (PDX) mouse models for many types of cancer have demonstrated therapeutic responses similar to those seen in patients. Thus, resected patient tumor tissue directly implanted into immune compromised mice followed by therapy and tumor growth analysis is thought to be the closest preclinical model to predict patient therapeutic responses. To date, few models are available for the different histotypes of thyroid cancer derived from follicular thyrocytes; these include papillary, follicular, Hurthle cell, squamous and anaplastic thyroid carcinoma. We have developed eight PDX models in athymic nude mice representing all of these subtypes. The models have been extensively characterized for mutational status (i.e. BRAF, telomerase, RAS, PTEN, TP53, RB, etc) as well as validation by short tandem repeat (STR) analysis to match that of the originating patient tumor tissue. Squamous and anaplastic thyroid cancers are rare tumor types with no FDA approved therapies. Each model demonstrated their own unique responses to radiation, cytotoxic or molecular targeted therapies such as doxorubicin, sorafenib (tyrosine kinase inhibitor), cisplatin, paclitaxel, sunitinib (tyrosine kinase inhibitor), erlotinib (EGFR inhibitor), trametinib (MEK inhibitor) and vemurafenib (BRAF inhibitor). We expect that these models may provide useful in vivo models for thyroid cancer research as well as models for therapeutic guidance based upon histotype, mutational status and response to therapies. Citation Format: Laura A. Marlow, Adam C. Mathias, Louis K. Dawson, William F. Durham, Kenneth A. Meshaw, Robert J. Mullin, Daniel L. Small, Aidan J. Synnott, Dragana Milosevic, Brian C. Netzel, Stefan K. Grebe, Kevin Wu, Robert C. Smallridge, John A. Copland. Characterization of novel thyroid PDX models and their response to combination therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1458. doi:10.1158/1538-7445.AM2015-1458