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Mutations and amplification of the fms-like receptor tyrosine kinase FLT3 gene, such as those found in MV-4-11 cells, are common in acute myeloid leukemia (AML). Such mutations result in aberrant activation of FLT3, increased growth and survival of tumor cells, and the development of refractory forms of AML with poor patient outcomes. In addition, AXL receptor tyrosine kinase imparts chemotherapeutic resistance in AML. MV-4-11 is a human biphenotypic B myelomonocytic leukemia with t(4;11)(q21;q23) chromosomal translocation commonly found in AML patients; it possesses a homozygous in-frame tandem repeat insertion in the FLT3 allele resulting in constitutively phosphorylated FLT3. Commonly employed preclinical orthotopic models of AML are primarily evaluated based on morbidity and mortality, which provide limited real time information on tumor burden and dissemination. The purpose of this study is to develop a MV-4-11 mouse model that allows the quantification and anatomical localization of the AML cells by monitoring bioluminescent signal in live animals and to utilize this model to investigate the efficacy of a FLT3/AXL inhibitor, gilteritinib. This poster describes a) the production of luciferase-expressing human MV-4-11 (MV-4-11-luc) cells via lentiviral transduction, b) the characterization of MV-4-11-luc progression in two models of immunocompromised mice, c) the efficacy of the FLT3/AXL kinase inhibitor gilteritinib against systemic MV-4-11-luc compared with two standard AML therapies, cytarabine and cyclophosphamide and d) the development of a humanized MV-4-11 tumor bearing model in NCG mice. Bioluminescence imaging revealed dose dependent tumor growth localized to the hind limbs and peritoneal cavity, which disseminated and intensified over time. Characteristic moribundity associated with disease progression was observed in this model. By comparison, robust growth of both parental and luc-transduced MV-4-11 was also observed in the highly immunodeficient NCG mice with or without prior myeloablation. Efficacy studies in NCG mice bearing parental or luciferase-tagged MV-4-11 cells demonstrated potent anti-tumor activity using the FLT3/AXL inhibitor. Bioluminescent imaging of orthotopic MV-4-11-luc xenografts in immunocompromised mice is a quantitative and sensitive method that provides anatomical context for assessing the efficacy of AML therapeutics. Ongoing and future work is focused on a) genetic profiling of MV-4-11 tumors after treatment with gilteritinib, b) further characterization of a humanized MV-4-11 model to evaluate immune-modulating therapeutics, and c) utilizing the model to evaluate activity of cell therapies that target AML. Citation Format: Na Li, Vivek Mahajan, Lindsay Butler, Elizabeth Rainbolt, Shasta Kidder, Kay Meshaw, Diana Gietl, Susan Yeyeodu, Anya Avrutskaya, Jing Wan, Thi Bui, Chassidy Hall, David P. Harris. Inhibition of FLT3 and AXL by gilteritinib controls systemic tumor growth in luciferase-transduced MV-4-11 acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2933.

Greater than 50% of all cancer patients receive radiation therapy at some point during their cancer treatment to control solid tumor growth (Harrington 2011). In addition to the direct action of radiation on tumor cell proliferation, radiation also has the potential to stimulate an anti-tumor immune response (Weichselbaum 2017). The success of immune checkpoint therapies such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 inhibitory antibodies has driven large investment into the discovery of novel immune-oncology therapies. While the clinical response to immune checkpoint inhibitors can be profound for those patients that respond, there still exists a large percentage of patients that do not see benefit. In order to expand the number of patients that may respond to immune checkpoint inhibition, combination with radiation therapy needs to be tested and in fact a large number of preclinical and clinical studies are being conducted (for review see Asna 2019). Preclinical syngeneic tumor models are a critical means to assess new immunotherapies and drive preclinical programs. E0771 is a medullary breast adenocarcinoma cell line originally isolated from a spontaneous tumor and can be implanted subcutaneously in C57BL/6 mice (Ewens 2005). In order to characterize the E0771 syngeneic breast tumor model for future studies with radiation and immunotherapy, we assessed the efficacy changes in response to various schedules of radiation doses with and without either anti-PD-1 or anti-CTLA-4 antibody therapies. In addition, we analyzed the tumor infiltrating immune cell populations following radiation therapy. E0771 is sensitive to radiation therapy with a clear dose response. Radiation administered either as a single dose, hypofractionated, or hyperfractionated gave similar tumor responses. Flow cytometry analysis shows a higher infiltration of T regulatory cells compared with CD8+ T cells at baseline, which is exacerbated by irradiation. The CD8/Treg ratio is reversed with the addition of anti-CTLA-4 treatment to radiation and resulted in curative responses for 9 of 10 mice in the combination group. Citation Format: Patrick Fadden, Thi Bui, Kelli Davis, David Hurtado, Emily Sugg, Abigail Svancarek, Ian Belle, Chassidy Hall. Radiation therapy and immune checkpoint inhibitor combinations in the E0771 medullary breast adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1558.

Targeting CTLA-4 has shown remarkable long-term benefits and thus remains a valuable approach for combating cancers of many types. A number of preclinical models have been developed over the years to evaluate the efficacy of immune checkpoint blockade in promoting antitumor immunity. In particular, knock-in (KI) humanized mouse models offer the possibility to study clinical grade immune checkpoint inhibitors (ICI) in the context of a fully functional immune system. Here we show the response to ipilimumab in a newly developed hCTLA-4 KI humanized mouse model. Our results demonstrate significant tumor growth inhibition as well as complete tumor regressions in the MC38 colorectal cancer model following treatment with ipilimumab. We have extended these studies by re-challenging the tumor-free surviving animals with tumors cells implanted opposite to the original tumor site. We established that all re-challenged hCTLA-4 KI mice remained tumor free suggesting potent T cell memory was maintained. To identify the immune cells and cytokines that could be responsible for the durable responses, we used multiparameter flow cytometry to characterize and compare blood, spleen and tumors tissues from the control, ipilimumab and mCTLA-4 treated mice. Comprehensive multichromatic phenotyping and functional intracellular cytokine staining (ICS) using validated 18-color flow cytometry panels showed a significant increase in CD8+ T cell frequencies when mice were treated with anti-hCTLA-4 but not with the mouse counterpart or the isotype control. Importantly, hCTLA-4 blockade reduced the regulatory T cell (FoxP3+ Treg) frequency and increased leukocyte infiltration into the tumor in the hCTLA-4 treated mice but not in the other two treated groups. Although equivalent numbers of CD4+ and CD8+ total T lymphocytes where found in the blood of all groups after each treatment, the quality of the T cell responses was found to be significantly different. CD45+ lymphocytes from the blood analyzed from the hCTLA-4 treated mice showed significant increases in both central memory (CM) CD8+ and CD4+ T cells consistent with increased anti-tumor efficacy seen in these mice. Furthermore, treatment of MC38 tumor-bearing mice with ipilimumab enhanced significantly the secretion of IFNγ from CD8+ and CD4+ T cells and the proliferation of both T cell populations as shown by the upregulation of Ki67 staining. These results indicate that immune cells were actively proliferating and had the appropriate effector functions to impact tumor growth. Altogether, the data presented here demonstrates that hCTLA-4 KI humanized mice are a robust model for evaluating the immune-modulatory effects and the activity of clinical grade ICI against tumors. Citation Format: Elizabeth Reap, Fabiane SÔNEGO, Emily O'Koren, Martin Gaëlle, Anya Avrutskaya, Jacob Hauser, Robin Ball, Thi Bui, Ian Belle, Chassidy Hall, Paula L. Miliani De Marval, Kader Thiam. Enhanced anti-tumor lymphocyte function and frequencies measured by multichromatic flow cytometry in human CTLA-4 knock-in mice in a colorectal carcinoma model after treatment with ipilimumab [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5635.

Background: Data about the risk factors and cancer types in developing countries remains limited. We investigate for the first time the role of a number of risk factors (family cancer history, smoking, alcohol consumption, diabetes, hypertension) associated with pancreatic cancer among Vietnamese patients. Methods: Patients hospitalized at 4 hospitals (K, Bach Mai, Viet Duc, Thai Nguyen) and diagnosed with pancreatic cancer (confirmed by histopathology and/or MRI/CT scan) in the period from January 2017 to May 2019 were included in the study. Some risk factors of the patients were collected and compared with a healthy age- and gender-matched (1:1) control group. Results: We have identified 196 patients with diagnosis of pancreatic cancer of which 114 males and 82 females. The average age of the patient at the time of diagnosis was 58.28 years (standard deviation of 12.94, ranging from 25 to 87). A positive family history, smoking and hypertension disease of pancreatic cancer significantly reduced the risk (OR and 95% CI were 0.06 [0.02 - 0.18], 0.53 [0.27 - 1.00], 0.1 [0.04 - 0.25], respectively). A past or present history of diabetes significantly increased the cancer risk (OR = 2.76 [1.34 - 6.05]), especially in female (OR = 5.68 [1.49 - 28.69]). Current alcohol drinkers showed a significantly increased risk of pancreatic cancer (OR = 2.23 [1.20 - 4.25]). Conclusions: Our finding showed that diabetes significantly increased the risk of pancreatic cancer in Vietnam, especially in women. An alcohol intake habit also increased the risk of pancreatic cancer. Further studies are required for fully understood the other risk factors related to this type of cancer. Citation Format: Tu V. Dao, Thuan V. Tran, Dung K. Nguyen, Oanh T. Bui, Quang T. Nguyen, Huong T. Tran. Risk factors of pancreatic cancer in Vietnam: A matched case-control hospital-based study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2368.

Over the past decade there has been an increasing demand in the use of syngeneic models for evaluating the efficacy of checkpoint inhibition-based cancer immunotherapies. During tumor development, immune cells can became unresponsive to the presence of tumor cells due to chronic activation and expression of the programmed cell death protein-1 (PD-1) or the T-lymphocyte-associated antigen 4 (CTLA4) in T-cells or the presence of FoxP3+ Treg cells resulting in tumor immune-tolerance. Our previous studies have demonstrated that murine anti-PD-1 and CTLA-4 therapy can effectively re-activate the antitumor response against multiple syngeneic tumor models. While these models proved instrumental for evaluating murine immune-checkpoint inhibitors (ICI), there is a clear need for additional mouse models to evaluate the efficacy of ICI specific for human targets. To address this need, we describe the development of a humanized PD-1 knock-in (KI) mouse model. This mouse model has the advantage of expressing the human PD-1 protein in the context of a fully functional immune system. We also show the response to clinically relevant immune checkpoint inhibitors in two preclinical tumor models. We evaluated the anti-tumor activity of pembrolizumab in the MC38 colorectal carcinoma and the GL261 glioblastoma models. We observed significant tumor growth inhibition and growth delay in the MC38 tumor model when treated with pembrolizumab monotherapy, but not when treated with the murine counterpart (anti-PD-1 clone RPM1-14). To extend our validation studies to other tumor models, we implanted GL261 glioblastoma orthotopically in the brain of PD-1 KI mice and achieved a significant increased life span in the group treated with pembrolizumab compared to both the control group and the group treated with murine anti-PD-1 antibody. The immune profile from control and treated these animals were also characterized in these studies by flow cytometry. In summary, the results shown here underscore the value of the humanized PD-1 knock-in (KI) mouse model as a tool to evaluate human specific immune-checkpoint based therapeutics alone and in combination with other agents. Citation Format: Anya Avrutskaya, Fabiane Sonego, Jacob Hauser, Emily O’Koren, Gaëlle Martin, Julie Chaix, Robin Ball, Thi Bui, Ian Belle, Chassidy Hall, Kader Thiam, Paula L. Miliani De Marval. Validation of humanized PD-1 knock-in mice as an emerging model to evaluate human specific PD-1 therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-063.

To prevent uncontrolled immune cell activation and limit the potential for auto reactivity, T cell responses are subject to multiple levels of regulatory control. In peripheral tissues, tolerance is maintained by regulatory networks and immunosuppressive mechanisms which restrict T cell activity. These same mechanisms can however contribute to failure to mount productive and sustained anti-tumor responses. Therapeutic intervention with immune checkpoint blockade therapy (ICT) restrains negative regulation, re-engages the immune system and promotes enhanced effector responses. Following the success of immunotherapy in certain solid tumors, Nivolumab was the first checkpoint inhibitor approved for treatment of lymphoma, and this approach is now being evaluated in other types of hematological malignancies. Therefore, we developed and characterized an immunocompetent disseminated mouse model for AML using CD11blo CD49b+ PD-L1+, luciferase-transduced C1498 tumor cells. Tumor growth, evidenced by luciferase expression, was progressive and widespread with metastatic development in peripheral organs and tissues and led to increased myeloid (gMDSC, mMDSC, Macrophage, DC) populations in the tumor bearing tissues, characteristic of myeloid leukemia. An efficacious response to therapy was observed, with anti-PD-L1 showing greater overall efficacy than anti-PD-1 and enhancement with combination anti-CTLA-4 therapy. To gain a mechanistic understanding of efficacy, we performed an extensive spatial (blood, spleen, bone marrow, and ovaries) and temporal (after 1st, 3rd and 5th dose of ICT) immune cell profile analysis using flow cytometry, immuno-histochemistry and microarray gene expression in response to mono and combination ICT. In blood, the response to combination immunotherapy was characterized by an initial increase in CD4 T cells (conventional and Tregs), consistent with early priming events after 1st dose of ICT. As the response developed, T cells in blood and spleen expressed elevated Ki67, Granzyme B and CTLA-4 after 3rd and 5th dose of ICT, consistent with development of an established activated effector response. Correspondingly, the NK cell population decreased in spleen and increased in the blood correlating with mobilization of effector response. Similarly, we observed a decrease in CD4 and CD8 cells in spleen, suggesting egress from blood into the sites of neoplasm e.g. ovaries and liver. Besides the lymphoid effector response, we also observed an increase in inhibitory feedback in response to ICT in both blood and spleen. This reflected in increased mMDSC and macrophages population in the blood and an increased PD1+ and CTLA4+ CD4 and CD8 cells in spleen. Citation Format: Harris David, Lindsay Usher, Thi Bui, Ian Belle, Elizabeth Rainbolt, Shasta Kidder, Kay Meshaw, Chassidy Hall, Vivek Mahajan. Spatiotemporal changes in effector immunity in response to checkpoint blockade immune therapy in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-191.

Immune focused interventions remain a high priority for many drug discovery efforts, spurred on by the success of immune checkpoint therapies such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 inhibitory antibodies. Preclinical efficacy assessments of novel immune-oncology therapies requires a functional immune system which limits the usefulness of traditional xenograft models and drive the use of humanized (human immune cell engrafted animals) and syngeneic model systems. Syngeneic mouse tumors are popular model systems in which to evaluate these novel therapies due the presence of fully intact mouse immune system and ease of accessibility. The MC38 mouse colon cancer model is popular for efficacy assessment studies due to its responsiveness to typical immune checkpoint inhibitors. We endeavored to characterize the immune response to checkpoint inhibitors in MC38 tumors using flow cytometry, gene expression analysis, and immunohistochemistry (IHC). Gene expression analysis provided a signature of gene changes that correlate to immune driven changes in tumor growth and may be used in preclinical pharmacodynamics studies as evidence of mode of action for other novel immune-oncology therapies. In addition, gene overrepresentation analysis highlighted the presence and involvement of B cell populations in the MC38 tumor environment which was supported by IHC data that show distinctive B cell staining in what resemble tumor associated tertiary lymph structures. These finding suggests that MC38 may provide a good model system for evaluating therapies that can modulate or impact tumor infiltrating B cell populations. Citation Format: Patrick Fadden, Thi Bui, David Hurtado, Kelli Davis, Ian Belle, Kathrine Krontz, Jacob Hauser, Na Li, Kyle Takayama, Cristina Sokolowski, Edgar Wood. Characterization of the immune checkpoint inhibitor response in the MC38 murine colon cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-060.

The majority of the leukemia cases are diagnosed in adults, of which, acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) are the most common subtypes. Treatment options include chemotherapy, stem cell transplantation and more recently the use of checkpoint inhibitors such as anti-PD-1 and anti-CTLA-4 to enhance antitumor responses. In this study we developed a disseminated AML disease model using PD-L1+, C1498 tumor cells to evaluate the therapeutic response to immune checkpoint inhibitors, PD-1, PD-L1 and CTLA-4 and to examine the effect of treatment on infiltration of immune cell populations in selected tissues. C1498 cells were transduced with a lentivirus vector construct containing luciferase and GFP reporter genes. Upon establishment of a clonal population with stable reporter expression, C1498-GFP-Luc expressing cells were implanted intravenously into syngeneic albino C57BL/6 mice and growth of the leukemic tumor cells was monitored via in vivo bioluminescence imaging (BLI) using IVIS Spectrum CT®. In vehicle-treated animals tumor growth was progressive and most animals reached endpoint within 30 days. Luciferase expression was widespread with metastatic development in peripheral organs and tissues. An efficacious response to therapy directed against the PD-1/PD-L1 axis was observed, with anti-PD-L1 showing greater overall efficacy than anti-PD-1. Combination of either PD-1/PD-L1 with anti-CTLA-4 further improved the therapeutic efficacy as measured by prolonged survival and reduced tumor growth. To gain mechanistic insights into how these immunotherapies function in suppressing tumor growth we performed additional studies utilizing flow cytometry and immunohistochemistry to assess the composition and phenotype of immune cell infiltrates in hematological and non-hematological tissues. In summary, comprehensive immunological characterization of the C1498 model revealed the importance of PD1 / PD-L1 pathway in immune evasion and provides a rationale for therapeutic intervention with checkpoint blockade. Citation Format: Vivek Mahajan, Thi Bui, Ian Belle, Edgar Wood, Lindsay Usher, Elizabeth Rainbolt, Shasta Kidder, Aidan Synnott, David Harris. Therapeutic response to PD1/PDL1 axis in a disseminated C1498 syngeneic tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1152.

The success of immune checkpoint therapy with anti-PD-1 and anti-CTLA-4 antibodies has generated broad interest in immuno-based interventions for oncology. Preclinical efficacy assessment of these novel immune-oncology therapies requires a functional immune system and therefore is frequently performed in murine syngeneic tumor models. In addition to tumor growth inhibition, syngeneic tumor models provide a platform to investigate the mechanism of action for a given novel therapeutic target. Characterization and mechanism of action for immune-based cancer therapies in murine syngeneic tumor models is often assessed by flow cytometry; measuring changes in tumor infiltrating leukocyte (TIL) populations following treatment induction. We assessed whether transcriptomic gene expression analysis could identify additional relevant biomarkers for immunotherapy response. Mice bearing subcutaneous syngeneic colon cancer tumors were dosed intraperitoneally with either vehicle alone, anti-CTLA-4, anti-PD-1, or a combination of the two immune checkpoint inhibitors on days 1, 4, and 8. Tumors were harvested on day 9 and half from each group were assessed for TIL population composition by flow cytometry and half by Clariom D mouse transcriptome array analysis. We identified a number of immune cell-related genes with differential expression that correlated to the therapeutic response and were consistent with changes identified in TIL populations by flow cytometry. These results provide an additional metric for assessing immunotherapy response in vivo. In addition, these data suggest that a smaller, defined panel of gene targets may be developed as biomarkers for specific immune system responses to immune checkpoint therapies in murine syngeneic models. Citation Format: Patrick Fadden, Douglas Weitzel, Kelli Davis, Thi Bui, David Hurtado, Hannah Haines, Rhiannon Roark, Ashleigh Derrick, Beverly Godfrey, Ian Belle, Katherine Krontz, Cristina Sokolowski, Aidan Synnott, Edgar Wood. Immune checkpoint inhibitor induced tumor gene expression changes in murine syngeneic colon cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 712.