Patient-derived AML mouse models with FLT3-ITD and IDH1 mutations

Poster Session – Animal Models, Thursday 1 December 2016 328 Poster (Board P007) Autophagy dependence and effect of autophagy inhibition on chemosensitivity in canine osteosarcoma cell lines C. Carnicello1 , K. Collins1 , D. Duval1 , D. Gustafson1 . 1 Colorado State University, Flint Animal Cancer Center, Fort Collins, USA Background: The process of autophagy is being actively studied to determine its role in tumor cell death in response to anticancer drug therapy. A number of studies have shown that autophagic processes can play a role in the sensitivity of human osteosarcoma (hOSA) cells to chemotherapy. Naturally occurring osteosarcoma in dogs is 30−50 times more prevalent than that seen in human adolescents and the diseases are strikingly similar in terms of etiology, pathology and molecular signatures. Thus canine osteosarcoma (cOSA) is generally regarded as an excellent translational model for testing therapeutic modalities for potential use in hOSA. For that purpose, these studies determined the autophagy dependence of 6 cOSA cell lines as well as whether autophagy inhibition can be used in combination with cytotoxic chemotherapy as a strategy in treating cOSA. Materials and Methods: Six cOSA cell lines that stably express nuclear RFP (Abrams, D17, Gracie, McKinley, Moresco and OSA8) were screened for autophagy dependence by measuring sensitivity to hydroxychloroquine (HCQ) by Incucyte® ZOOM live cell imaging. Sensitivity to doxorubicin (DOX) and combined HCQ and DOX were also measured. Basal autophagic flux was measured using CYTO-ID® Autophagy detection kit and accumulation of LC3II following nutritional stress and autophagy inhibition by HCQ. Results: Basal autophagic flux was detected in all cOSA cell lines. Sensitivity to HCQ, DOX and the combination is shown in the table below as measured by Dm and combination index (CI) values. The data show that cOSA cells are relatively sensitive to HCQ with Dm values ranging from 8.3 to 28.4 mM. CI values show that HCQ and DOX combined effects on cOSA cells are additive.

Abrams D17 Gracie McKinley Moresco OSA8

HCQ Dm (mM)

DOX Dm (ng/ml)

CI value

8.3 19.6 9.0 9.1 28.4 10.6

27.1 3.9 6.4 15.5 17.2 9.6

0.84–1.56 0.88–1.65 0.93–1.26 0.75–1.13 0.70–1.38 1.10–1.89

Conclusions: cOSA cell lines are sensitive to HCQ and DOX at concentrations that are achievable in dogs based on comparing measured tumor tissue trough levels of HCQ in dogs (Autophagy 10:1415, 2014) to measured Dm values and to DOX AUC in dogs treated at 30 mg/m2 (J Vet Intern Med 24:579, 2010). This suggests that autophagy inhibition by HCQ may be a viable strategy for enhancing chemotherapy response to DOX in cOSA and merits further study. No conflict of interest. 329 Poster (Board P008) Establishment and characterization of pairs of patient-derived cells and xenograft models of gastrointestinal stromal tumors resistant to standard tyrosine kinase inhibitors Y.S. Na1 , M.H. Ryu2 , S.R. Park2 , J.K. Lee1 , C.W. Lee1 , J.M. Park1 , Y.K. Shin3 , J.L. Ku3 , S.Y. Lee1 , S.M. Ahn4 , Y.K. Kang2 . 1 ASAN Institute for Life Sciences, ASAN Medical Center, Department of Oncology, Seoul, South Korea; 2 Asan Medical Center, University of Ulsan College of Medicine, Department of Oncology, Seoul, South Korea; 3 Korean Cell Line Bank, Cancer Research Institute, Seoul National University College of Medicine, Laboratory of Cell Biology, Seoul, South Korea; 4 Gachon University, Department of BioNano Technology, Incheon, South Korea Background: Standard of therapy for patients with gastrointestinal stromal tumor (GIST) includes imatinib, sunitinib and regorafenib. However, most patients eventually develop resistance to these tyrosine kinase inhibitors (TKIs), and novel agents are needed for these patients. We established pairs of patient-derived cells (PDC) and xenograft (PDX) models of TKIresistant GISTs for the effective drug treatment. Methods: PDXs have been established in NOD-SCID mice by implanting metastatic and/or unresectable GIST fragments which were failed to treat with at least imatinib and/or sunitinib. PDCs have also been established from the same patient’s tumor as mentioned above or the tumor of PDX. Mutation was detected by whole exome sequencing or Comprehensive cancer panel (CCP) of patients’ tumors and validated by Sanger sequencing of tumors of PDCs and PDXs. The drug sensitivity assay (imatininb, sunitinib, and regorafenib) was conducted in established PDC and PDX models after sequential passaging to BALB/c nude mouse. Results: The genomic similarities between PDC, PDX, and primary patients’ tumors have been confirmed using short tandem repeat

Poster abstracts S109 (STR) analysis. Three pairs of GIST PDC and PDX models were established and confirmed the following mutations; an imatinib-resistant GIST harboring KIT exon 11 (p.W557_K558del) mutations (AMC-G1 and -GX8), an imatinib/sunitinib-resistant GIST harboring KIT exon 11 (p.565– 577GNNYVVIDPTQLP>Q) and 17 (p.D820Y) mutations (AMC-G2m and -GX3), and an imatinib/sunitinib-resistant GIST harboring KIT exon 11 (p.558–560KVV>I) and 17 (p.Y823D) mutations (AMC-G3 and -GX10). The mutation status of GIST PDCs and PDXs was consistent with primary tumors. Additional somatic mutations include FGFR3 (p.G65R), NOTCH1 (p.V2024I), SDHA (p.G184R), and TP53 (p.Y107*) in AMC-G1 and -GX8, TP53 (p.P34fs) in AMC-G2m and -GX3, and NOTCH4 (p.16insL) and SDHA (p.G184R) in AMC-G3 and -GX10. The GIST PDCs and PDXs showed TKI sensitivity profiles comparable to clinical responses in patients except the resistance to sunitinib in AMC-G1 and -GX8. Conclusion: We have established 3 pairs of TKI-resistant GIST PDC and PDX models harboring a variety of KIT and additional mutations. The established pairs of GIST PDC and PDX models would play a role for further studies on mechanisms of resistance to TKI and evaluation of novel targeted therapies in GIST. No conflict of interest. 331 Poster (Board P010) Characterisation of the tumour-infiltrating leucocyte population in staged syngeneic models S. Alzabin1 . 1 Epistem Ltd, Contract Research Services, Manchester, United Kingdom Background: Recent advances in cancer therapy focus on harnessing the body’s immune system to destroy cancer cells by targeting mechanisms of tumour evasion. Immune checkpoints define a class of inhibitory receptors and/or respective ligands that are expressed either by the tumour cell or by leucocytes to attenuate the ability of effector immune cells to attack tumour cells. In order to assess the efficacy of new immune-checkpoint targeted therapies against cancer, a number of immune-intact murine tumour models have been developed and employed for pre-clinical testing. A combination of in-life tumour volumetric readouts and ex vivo analyses of cell types with associated protein readouts define drug efficacy against cancer and associated anti-cancer immune mechanisms. Our efforts have focused on utilising our understanding of the complexities of the immune system to refine existing syngeneic oncology models in order to provide a template for more effective preclinical testing. The tumour infiltrating leucocyte (TIL) population, peripheral blood cells (PBCs) and splenocytes from three common syngeneic models, B16, CT26, and 4T1, have been characterised for the presence of immune mediators in a time-dependent manner. Materials and Methods: Female Balb/C or C57BL/6 mice aged were inoculated with B16, CT26 or 4T1 cells at 0.5×106 to 5×106 cells per mouse. Animals were euthanised at various time points post tumour cell implantation. Blood was collected, the spleen and tumour were resected. Single cell suspensions were prepared from tumour, PBCs and spleen. Cells were labelled for analysis of leucocyte populations using a combination of antibodies to evaluate the frequency of cells at various disease states. Results and Conclusion: Implantation of 0.5×106 cells resulted in a more chronic immune response, whilst the injection of 2−5×106 cells resulted in an acute profile. This was significantly reflected in the types of immune mediators within the tumour and in the periphery, where for example, effector CD4+ T cells were enriched when the tumours were taken at a later stage in all 3 models examined, with the most enrichment in the B16 model. The data indicate that the TIL profile was not only dependent on the tumour cell type but also on the tumour stage/growth kinetics. Our approach and characterisation highlight the heterogeneity of the immune response in cancer, and provides a clear rationale for the re-assessment of animal models for immuno-oncology drug development. This data is crucial to allow an informed decision on the most appropriate model to evaluate the efficacy of therapy against a specific target. Further, immune targeting therapies are likely to be deployed in combination with other agents where determination of the effect of these adjunct therapies on the TIL population will be crucial for appropriate timing of immune intervention. No conflict of interest. 332 Poster (Board P011) Patient-derived AML mouse models with FLT3-ITD and IDH1 mutations P. Kaur1 , M. Brehm2 , D. Greiner2 , L. Shultz3 , M. Cheng1 , J. Keck1 , D. Cai1 . 1 The Jackson Laboratory, In Vivo Services, Sacramento, USA; 2 University of Massachusetts Medical School, Program in Molecular Medicine, Worcester, USA; 3 The Jackson Laboratory, Cancer Center, Bar Harbor, USA Acute myeloid leukemia (AML) is characterized by an uncontrolled proliferation of functionally immature hematopoietic cells with an increasing incidence in adults 65 years and older. AML is genetically diverse and presents

S110 Poster abstracts a challenge to develop targeted therapies. Here we describe two genetically defined AML mouse models that are derived from patient leukemia cells isolated from leukapheresis samples. J000096994 is FLT3-ITD positive and J000096854 is FLT3-ITD positive and IDH1 positive. Both AML lines efficiently engrafted in the triple transgenic NSG™-SGM3 mice that express human IL-3, GM-CSF and SCF to support the stable engraftment of myeloid lineages. However, under the same condition J000096994 did not engraft in NSG™ mice without expression of the cytokines and J000096854 engrafted in NSG™ at a lower level than in NSG™-SGM3 mice. In the characterization of both AML lines, J000096854 exhibited faster engraftment kinetics than that of J000096994. Over 1% the cells in the peripheral blood of NSG™-SGM3 mice were human CD33+ at 6 and 10 weeks post engraftment of the J000096854 and J000096994 AML cells, respectively. The level of human CD33+ cells in peripheral blood reached 20% for J000096854 at 10 weeks post engraftment while it reached over 10% for J000096994 at 16 weeks. While there was a difference in the engraftment kinetics in peripheral blood between the two models, the engraftment levels of human CD33+ cells in spleen and bone marrow were comparable at endpoint. Moreover, AML cells were able to be serially transferred to recipient NSG™-SGM3 mice. Both models displayed positive response to cytarabine (ara-C) treatment with levels of human CD33+ cells reduced to the nadir two weeks after the 5-day treatment cycle was initiated. This presentation will demonstrate that two patient-derived AML models have been established in NSG™-SGM3 mice and that they have been characterized to show distinct engraftment kinetics and positive response to an AML standard of care drug ara-C. These AML models may be useful tools for testing experimental therapeutics that target FLT3 and IDH1 for treating AML. No conflict of interest. 333 Poster (Board P012) The Em-TCL-1 mouse model of chronic lymphocytic leukemia H.S. Chin1,2 , A. Egle3 , S.L. Khaw1,2,4 , M.F. Van Delft1,2 , D.C. Huang1,2 . 1 Walter and Eliza Hall Institute, Cancer & Heamatology, Melbourne, Victoria, Australia; 2 University of Melbourne, Medical Biology, Melbourne, Australia; 3 Salzburg Cancer Research Institute, Department of Internal Medicine III, Salzburg, Austria; 4 Royal Children’s Hospital, Royal Children’s Hospital, Parkville, Australia Background: Chronic lymphocytic leukemia (CLL) is a heterogenous disease characterized by the accumulation of CD5+ /CD19+ B cells in all lymphoid compartments. Apart from hematopoietic stem cell transplantation, current treatment options are not curative, and in many patients, the disease eventually relapses or becomes resistant to standard therapies. Preclinical evaluation of novel therapeutic approaches for CLL has been limited by the our inability to propagate patient samples in in vitro culture for prolonged periods and the lack of representative cell lines or mouse models. Croce et al. developed and characterised the Em-TCL-1 mouse model, where the oncogene TCL-1 is overexpressed in B cells, as a putative CLL model. The aim of our studies is to further characterise the mouse model using next-gen sequencing approaches. Materials and Methods: Next-gen sequencing was performed using RNA isolated from CD5+/CD19+ cells from the spleens of transgenic mice. We compared the transcriptome profiles of murine leukemic cells with normal B cell subsets and a cohort of human CLL patients (Ferreira et al., 2014). Results: We show that the molecular signatures derived from CD5+ /CD19+ leukemic cells from Em-TCL-1 mice are distinct from other normal B cell subsets. Strikingly, molecular signatures of these leukemias demonstrate minimal overlap with those of the those of most human population (Ferreira et al., 2014). Nonetheless, more stringent analysis revealed that the mouse model does bear a close resemblance to a subgroup of human CLL characterized by high TCL1 expression. TCL1 overexpression has been associated with adverse outcome in CLL patients. Interestingly, unlike prototypical CLL disease, dysregulation of BCL-2 family protein expression was not detected in leukemic cells from Em-TCL-1 transgenic mice, which appear refractory to the inhibition of BCL2, at least in vitro. Moreover, the leukemias isolated from sick mice showed a surprisingly wide range of sensitivity to standard-of-care agents such as fludarabine. Conclusions: Taken together, our studies suggest that the leukemias which develop in Em-TCL-1 transgenic mice represent a highly aggressive, chemorefractory subtype of CLL. The goals of our ongoing studies are to further characterize this model of CLL and utilize it to test novel therapeutic approaches for treating CLL, particularly chemorefractory disease. References Ferreira, P.G., Jares, P., Rico, D., Gomez-Lopez, G., Martinez-Trillos, A., Villamor, N., Ecker, S., Gonzalez-Perez, A., Knowles, D.G., Monlong, J., et al. (2014). Transcriptome characterization by RNA sequencing identifies a major molecular and clinical subdivision in chronic lymphocytic leukemia. Genome research 24, 212–226. No conflict of interest.

Poster Session – Animal Models, Thursday 1 December 2016 334 Poster (Board P013) Genomic characterization of immune targets in patient-derived xenograft models for translational assessment of immunotherapy D. Cerna1 , D. Ciznadija1 , I. Ben zvi2 , I. Sloma2 , D. Sidransky3 , A. Davies4 , N. Goodwin1 . 1 Champions Oncology, Translational Oncology, Baltimore, MD, USA; 2 Champions Oncology, Bioinformatics, Baltimore, MD, USA; 3 Johns Hopkins University School of Medicine, Medicine, Baltimore, USA; 4 Champions Oncology, Medical Affairs, Baltimore, MD, USA Background: Although oncology therapies reactivating the immune response by targeting checkpoints such as PD1 and PD-L1 are now FDA approved, development of therapeutics regulating other immune targets remains an area of active research. Differences between human and murine immune systems have generally limited the use of animal models in this setting. Whilst patient-derived xenograft (PDX) models are robust pre-clinical tools, whether they reflect human immune marker expression patterns remains unclear. Here we describe the genomic characterization of immunotherapy targets across a large panel of PDX models. This dataset is essential for planning downstream translational studies with PDX models in humanized mouse systems for evaluating immuno-oncology (IO) agents. Materials and Methods: RNA sequencing was used on PDX models to determine expression of genes considered targets for IO agents, including PD-L1, LAG3, IDO1 and 2, MUC1, and MUC4. Given the reported correlation between mutation load and response to IO agents, the total number of germline and somatic mutations (single nucleotide variations/small indels) in each PDX model was calculated from whole exome sequencing data. HLA locus analysis by sequence-based typing (SBT) was performed for graft-host alignment. Results: A total of 327 PDX models across 8 tumor types (melanoma, lung (NSCLC/SCLC), ovarian, breast, pancreatic, head and neck, sarcoma, and colorectal) were sequenced. Overall, MUC4 was the most prevalently expressed target, showing >2-fold expression (relative to all PDX models) in 18% of PDX models, followed by PD-L1 (15%) and IDO1 (13%). Across specific tumor types, PD-L1 was most prominently expressed in lung (41% of models), IDO1 and LAG3 in ovarian (33% of models for both genes), and MUC4 in lung and pancreatic (~33% of models for both genes). Melanoma and lung PDX cohorts had the highest percentage of models showing >2-fold expression of at least one IO target (78% and 67% respectively). Melanoma, gastric, and colorectal PDX demonstrated the greatest confluence of mutations, averaging >800 mutations/model. In contrast, sarcoma had fewer, averaging <400 mutations/model. In keeping with population data, HLA-A2 (45%) was the most commonly identified HLA antigen, followed by HLA-A1 (19%) and HLA-A3 (15%). Conclusion: PDX models are robust translational models; however, evidence of their utility for evaluating IO agents has been limited. We have now described the differential expression of various new and wellcharacterized immune targets in a large number of solid tumor PDX models. Such models may be reasonable surrogates for screening novel immunotherapies in mice the context of a human immune system. Conflict of interest: Ownership: David Sidransky, David Cerna, Angela Davies, Daniel Ciznadija, Neal Goodwin, Ido Sloma, Ido Ben Zvi. Board of Directors: David Sidransky. 335 Poster (Board P014) Patient-derived xenograft (PDX) models of BRCA-deficient and BRCA-like ovarian tumors reflect clinical responses to PARP inhibition A. Davies1 , J. Hou2 , L.M. Shih3 , J.D. Wright2 , D. Ciznadija4 , A. Katz4 , D. Sidransky3 . 1 Champions Oncology, Medical Affairs, Baltimore, USA; 2 Columbia University Medical Center, Obstetrics and Gynecology, New York, USA; 3 Johns Hopkins University School of Medicine, Medicine, Baltimore, USA; 4 Champions Oncology, Translational Oncology, Baltimore, USA Background: PARP inhibition in BRCA-deficient/BRCA-like ovarian tumors can lead to synthetic lethality. The FDA has approved a PARP inhibitor to treat BRCA-deficient ovarian carcinomas and other drugs targeting PARPs (PARPi) are in development, generating further interest for this target. PDX models could enable evaluation of PARPi resistance/response mechanisms, improving patient stratification protocols and optimizing therapeutic strategies. In this pilot study, we evaluated responses to olaparib and carboplatin/paclitaxel in BRCA-deficient/BRCA-like ovarian PDX models and correlated responses to clinical trial outcomes. Materials and Methods: PDX models developed from ovarian patient tumors were subjected to next-generation sequencing to identify genomic alterations in BRCA 1/2 and regulators of alternative DNA repair processes that contribute to a BRCA-like phenotype (e.g. ATM, RAD51, FANCA, and FANCD2). Models were screened against the FDA-approved PARPi,