Xiaoyu An

AC220 and AraC cause differential inhibitory dynamics in patient-derived M5-AML with FLT3-ITD and, thus, ultimately distinct therapeutic outcomes

Engrafting the bone marrow cells of a patient with M5 acute myeloid leukemia into immunocompromised mice (AM7577) resulted in serially transferrable stable AML and eventual mortality. The disease starts in the bone marrow and then expands to peripheral areas, which is typical of M5 leukemogenesis, where high leukemic burden in blood is coincident with symptoms/mortality. The leukemic cells in the mice had myeloid morphology, phenotypes, and genotypes (including the internal tandem duplication of FMS-like tyrosine kinase receptor 3 gene [FLT3-ITD]) similar to those of the original patient. Autocrine mechanisms of human granulocyte-macrophage colony-stimulating factor/interleukin-3 likely support AM7577 growth in mice. Treatment with FLT3 TKI (AC220) caused complete remission in peripheral blood, spleen, and bone, along with relief of symptoms and extended life, hinting that FLT3-ITD may be a key leukemogenic driver maintaining the disease. Interestingly, withdrawal of AC220 (high dose) did not result in relapse of disease, suggesting cure. These results, however, are in contrast to cytarabine (AraC) induction treatment: First, although AraC significantly suppresses the diseases in blood, and to a lesser degree in bone marrow and spleen, the suppression is temporary and does not prevent eventual onset of disease/death. Second, the withdrawal of AraC always resulted in rapid relapse in peripheral blood and eventually death. Our observation in this patient-derived model may provide useful information for clinical applications of the two drugs.

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of todays immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models. Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7-9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300-1,000 mm3 by 17 days. Only tumors of 400-600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Abstract 1016: Transcriptomic analysis of bulk tissues of large PDX collection as a novel platform discovering new TME target/drug

Cancers are collections of diverse diseases of genetic and immunological abnormalities. The heterogeneous tumor microenvironment (TME), including immune components, and their interactions with tumor cells play critical roles in tumor progression and response to pharmaceutics, particularly immuno-oncology (I/O) therapy. However, investigating TME-specific components is rather challenging for the difficulty to separate stroma from tumor cells, either physically via microdissection or in silico via bioinformatics. Patient derived xenograft (PDX) may be a new system to investigate TME 1 , where human and mouse content can readily be separated in silico 2 . We have transcriptome-sequenced ~1600 bulk tumor tissues from subcutaneous PDXs grown in athymic mice 3 . By aligning reads to human and mouse genomes, we found that the average mouse-to-human sequencing read ratio is around 11% (5~20%), consistent with the previous report 2 . After removal of the low-expressed and less-variable genes and by deconvolution analysis of gene expression data, we identified all types of TME components, including adaptive and innate immune cells. The corresponding fractions vary across cancer types and individual models. Co-regulation analysis identified a huge number of intra-species interactions and also, a smaller number of inter-species interactions that vary greatly among different cancer types. The cross-species interactions observed are likely implicated in the growth of these tumors, and their numbers may also reveal the degree of the dependence of tumor growth on TME, which should be reversely correlated to the transplantation take-rate of corresponding type of PDX. Indeed, we have demonstrated this reverse-correlations (# interactions: take-rate %) with statistical-significance (p-value = 0.034) across our PDX collections, including melanoma (406:27%), lung (146:50%), colorectal (CRC) (157:68%) and pancreatic cancer (32:80%). The cancer type with the highest take-rate and lowest # interactions is pancreatic cancer that also has highest KRAS mutation rate (>90%), hinting the role of KRAS mutation in tumor growth independency on TME. This is further confirmed in KRAS mutant CRC (1:96%) vs. wild-type (98:53%). Moreover, some putative cross-species co-regulations in specific cancers were also observed in human tumors (e.g. in TCGA dataset), indicating potential importance in TME-tumor interaction and tumor development in human. Further investigation of each of these interactions may reveal novel TME-related disease pathways and thus novel targeting strategy for cancer therapy. In conclusion, transcriptomic analysis of large number of bulk PDXs provides a novel and unique platform to study TME, likely to facilitate new discovery of disease pathways and strategy to treat cancers involving the TME mechanism, particularly I/O strategy. Citation Format: Jia Xue, Wubin Qian, Sheng Guo, Xiaoyu An, Xuesong Ouyang, Henry Q. Li. Transcriptomic analysis of bulk tissues of large PDX collection as a novel platform discovering new TME target/drug [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 1016.

Abstract A30: Modeling an immunotherapy of NK mechanism on a NSCLC patient derived xenograft

The recent clinical successes of immune checkpoint inhibitors have fueled the intense interest in novel immuno-oncology (I/O) therapeutics. The lack of relevant animal models remains a major hurdle in understanding the mechanism of action and evaluating the efficacy of such therapeutics. Patient derived xenograft (PDX), considered to most closely mimic patient tumors in both histo- and molecular pathology1,2, is however rarely used in I/O studies because it grow only in immune-compromised hosts. In reality, many PDXs grow well in nude mice where certain immune functions remain intact, excluding T-cells/T-cell functions. Therefore, PDX could still potentially be of practical use for studying T-cell independent I/O therapy. This study set out to evaluate a biologics for the treatment of a patient derived xenograft disease, by activating mouse natural killer (NK). NK and CD8 T cells are two major immune effector cell types that mediate cytotoxicity to tumor cells in vivo. One of the immunomodulatory agents, an anti-PD-L1 antibody-based IL-15 immunocytokine, was recently tested as a novel I/O treatment of cancer3. This molecule was engineered to be cross-species for both human and mouse PD-L1 and IL-15 that antagonize PD1/PD-L1 checkpoint-mediated immune suppression and also target the PD-L1-expressing tumor with IL-15 stimulated NK and CD8 T effector cells into local tumor sites, thus synergizing tumor-located anti-tumor immunity. In fact, our previous studies have demonstrated a greatly enhanced anti-tumor activity in the PDL-1-expressing syngeneic mouse tumor models via the mobilization of tumor infiltrating lymphocyte (TILs), over the PD-L1 antibody alone3. Since IL-15 also stimulates NKs in addition to T-cells, we reasoned that this bifunctional agent could also have potential activity against a PD-L1-expressing PDX in nude mice where NK remains functional. LU1901 is previously described NSCLC-PDX1, and was confirmed to express high level of PD-L1 by both RNAseq and flow analysis, after screening of a large panel of PDXs. We implanted LU1901 in Balb/c nude mice, and started to treat the mice by the bifunctional agent when the tumor reached to 150 mm3. Our result clearly demonstrated a significant inhibition of LU1901 growth by the bifunctional agent in nude mice, in the apparent absence of T-cells. When the treated tumors were examined at the termination, significantly infiltrate NK cells were found inside the treated tumors, as measured by both flow cytometry and immunohistochemistry (IHC). The number of infiltrating NK also statistically correlates to the amplitude of the tumor responses. Together, our data suggest that one of important mechanisms of action of this bifunctional agent relies on the tumor-targeting NK activation, and also that PDX could be a useful model suitable for in vivo efficacy analysis of T-cell independent immunotherapy. References 1. Yang, M., et al. Overcoming erlotinib resistance with tailored treatment regimen in patient-derived xenografts from naive Asian NSCLC patients. International journal of cancer. Journal international du cancer 132, E74-84 (2013). 2. Guo, S., Wubin Qian, Jie Cai, Likun Zhang, Jean-Pierre Wery, Qi-Xiang Li. Molecular pathology of patient tumors, patient derived xenografts and cancer cell lines EORTC-NCI-AACR. (2015). 3. Yan Wu, Zhaojing Zhong, Stella Martomo, Dan Lu, Zhanna Polonskaya, Xenia Luna, Haifan Zhang, Zhikai Zhang, Zhun Wang*, Leo Liu*, Jeegar Patel, James Tonra, Henry Li*, Larry Witte, Sam Waksal, Zhenping Zhu. Anti-PD-L1 antibody-based IL-15 immunocytokine has enhanced antitumor immunity. EORTC-NCI-AACR Abstract (2015). Citation Format: Henry Q. Li, Zhun Wang, Xiaoyu An, Jinping Liu, Likun Zhang, Jean-Pierre Wery, Yan Wu, James Tonra, Sam Waksal, Zhenping Zhu. Modeling an immunotherapy of NK mechanism on a NSCLC patient derived xenograft. [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 A30.

Abstract 3219: Prior vaccination is critical to PD1/PDL-1 treatment efficacy in a mouse breast cancer allograft

We previously generated the MuPrime® mBR6004 model, by engrafting the breast adenocarcinoma derived from MMTV-PyVT transgenic mice (GEMM)1 into the syngeneic FVB/N mice2,3. The allograft grows robustly, maintains the histopathology features of the original GEMM, and metastasizes to the lungs in all examined cases when implanted orthotopically2. Additionally, histopathology shows that the models is HER2++, but ER-/PR-. We profiled drug responses to standard of care (SoC)2 and checkpoint inhibitors3. Interestingly, among all checkpoints inhibitors tested, our model is insensitive to PD1/PDL-1 inhibitors, but responds to CTLA-4 inhibitors3. To further explore the underlying mechanism of response, we performed extensive tumor immuno-profiling for the presence of infiltrating immune cells, e.g. TIL, CTL, Treg, immune-suppressive macrophages, NK, etc3. We observed a good pharmacodynamic (PD) correlation between the presence of tumor infiltrating T-cells, particularly CD8+ TIL, and NK and a positive response to therapy, regardless the treatment type. Given this preliminary observation, we attempted animal vaccination with tumor lysates prior to tumor engraftment or treatments. Interestingly, while vaccination had minimal effects on the engraftment take rate (100% take for all animals) and on the growth kinetics of the engraftments, it had profound effects on the tumor response to anti-PD1/anti-PDL-1 antibody treatments, significantly enhancing tumor response to these agents in a model that was otherwise unresponsive. Our results suggest that the immunological status of the animal, at least with regard to the specific anti-tumor immunity, is critically important to determine the response to checkpoint inhibitors. Our observations suggest that vaccination is critical for the overall success of immunotherapy (I/O) treatments utilizing checkpoint inhibitors as well other treatment strategies. References 1. Guy, C.T., Cardiff, R.D. & Muller, W.J. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Molecular and cellular biology 12, 954-961 (1992). 2. Annie Xiaoyu An1, Jinping Liu1, Jie Cai1, Jean Pierre Wery1, and Henry Q.X. Li1,. Building mouse tumor derived allogragfts for immune-oncology research. (2015). 3. Zhun Wang1, A.X.A., 2*, Jinping Liu1, Gavin Jiagui Qu1, Likun Zhang, Jie Cai1, Bin Chen1, Davy Xuesong Ouyang1, Jean Pierre Wery1, and Henry Q.X. Li1,2. Response to checkpoint inhibition by GEMM breast cancer allograft EORTC-AACR-NCI Abstract (2015). Citation Format: Zhun Wang, Xiaoyu An, Jinping Liu, Xuesong Ouyang, Jiagui Qu, Likun Zhang, Jie Cai, Jean-Pierre Wery, Henry Li. Prior vaccination is critical to PD1/PDL-1 treatment efficacy in a mouse breast cancer allograft. [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 3219.

Abstract B97: Response to checkpoint inhibition by GEMM breast cancer allograft

Background. Cancer patient9s response to immunotherapy, e.g. checkpoint inhibitors, differ greatly, likely due to their genetic composition (e.g. neo-mutation) or immunological profile of tumors. Experimental models may facilitate the identification of responders or of the approach to enhance response. Syngeneic mouse tumor models have been widely used as experimental model for testing surrogate immunotherapy, but limited by the fact that only a few are available and respond to the current checkpoint inhibitors. Allografts of mouse spontaneous tumors (MuPrimeTM) may be used as new type of I/O model with advantages: 1) its primary natures with “stem cell diseases” and relevant tumor microenvironment as seen in PDX1-5; 2) diverse cancer types and deriving from wider range of available spontaneous mouse tumors. Method. We created a MuPrime, MBR6004, by allografting the breast adenocarcinoma derived from GEMM-MMTV-PyVT in FVB/N mice1. We then characterized the model by examine it subcutaneous and orthotopic growth, and its ability to metastasis. We also transcriptome-sequenced and immune-profiled the tumor, and assessed its response to different treatments, including I/O therapy. Results. MBR6004 was found to over-express HER2 but negative for ER and PR. It maintains original primary tumour histopathology, grows robustly, and is confirmed to express its original transgene. It is also resistant to docetaxel. The orthotopic implantation resulted in lung metastasis. We can detected and quantitatively confirmed tumor-infiltrating immune cells, e.g. TIL, CTL, Treg, immune-suppression macrophage, NK, etc. We confirmed that the tumor cell expressed relatively low PD-L1, but inducible, per flow analysis. Our preliminary data indicated that it partially respond to anti-mouse PD1 and anti-mouse CTLA-4 antibodies when mice are preconditioned. Currently, we are investigating whether the responses are associated with the reduced Treg and increased CD8+ TIL in tumors, and also whether the combination therapy can enhance its tumor response synergistically. Conclusion. Our data seem to suggest that MuPrime could be another type of I/O model alternative to commonly used syngeneic cell line derived models at present. References 1. Guy, C.T., Cardiff, R.D. & Muller, W.J. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Molecular and cellular biology 12, 954-961 (1992). 2. Guy, C.T., et al. Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc Natl Acad Sci U S A 89, 10578-10582 (1992). 3. Moser, A.R., Dove, W.F., Roth, K.A. & Gordon, J.I. The Min (multiple intestinal neoplasia) mutation: its effect on gut epithelial cell differentiation and interaction with a modifier system. The Journal of cell biology 116, 1517-1526 (1992). 4. Moser, A.R., et al. ApcMin, a mutation in the murine Apc gene, predisposes to mammary carcinomas and focal alveolar hyperplasias. Proc Natl Acad Sci U S A 90, 8977-8981 (1993). 5. Moser, A.R., Pitot, H.C. & Dove, W.F. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 247, 322-324 (1990). Citation Format: Zhun Wang, Xiaoyu An, Jinping Liu, Jie Cai, Likun Zhang, Jiagui Qu, Davy Ouyang, Bin Chen, Jean-Pierre Wery, Henry Li. Response to checkpoint inhibition by GEMM breast cancer allograft. [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 B97.

Abstract A11: HuGEMM-h/mPD1 mouse models for assessing anti-human PD1 therapeutics

Background. Blockage of immune checkpoints, e.g. by anti-PD1 antibody, becomes an important new cancer therapy1. Experimental models are important to evaluate new investigational therapy or new combination strategy. Syngeneic mouse tumor models have been widely utilized as an experimental model for testing surrogate immune-oncology (I/O) therapy by using its competent mouse immunity2, but cannot be used for testing human biologic therapeutics, due to the species specificity. The direct replacing mouse therapeutic target by human counterpart in mouse while maintaining the normal mouse immune-functions could be a potential practical preclinical approach to evaluate human biologic therapeutics in vivo. Methods. We have engineered a chimeric human/mouse PD1 gene (h/mPD1) composed of human exome 2&3 and mouse exome 1&4. We expressed the recombinant proteins and tested their bindings to their binding partner of PDL1 of both mouse and human origins, and also anti-human PD1 antibody. We knock-in the recombinant gene into C57Bl/6 mouse to create homozygous HuGEMM-h/mPD1 mouse, which is tested for growth of MC38 syngeneic mouse cell line tumor graft and for the growth inhibition by anti-human PDL1 antibody. Results. Our data demonstrated that chimeric protein h/mPD1 can interchangeably interact with mPDL1 or hPDL1 efficiently as efficiently as mouse PD1, and it also recognizes anti-human PD1 antibody as expected. The binding of anti-human PD1 antibody blocks its binding to mouse or human PDL1. The knock-in mice express the chimeric gene in the T-cells of the engineered mice both in vivo and ex vivo, however at significantly lower levels than mouse PD1 in the wild type C57BL/6 mice (1/10) under induction. When syngeneic MC38 cell line was subcutaneously engrafted in HuGEMM-h/mPD1 mice, the tumor were found to grow significantly slower with increased T-cell infiltration, as compared to those in the wild type mice. MC38 tumor did not respond to anti-human PD1 antibody well either. These observations can apparently be attributed to the low level PD1 and the associated high autoimmunity that also inhibits tumor growth. Interestingly, a specific condition can be artificially created to enhance MC38 tumor growth in the chimeric mice, likely contributed by the enhanced h/mPD1 expression. The enhanced tumor growth seems to also be suppressible, at least partially, by anti-human PD1 antibody as shown in our preliminary study. Currently, we are re-engineering our chimeric gene (version 2) in order to increase their expression to the wild type gene level, so to create a model for more optimal drug evaluation. In the meantime, we are also engineering HuGEMM-hu-CKPT (e.g. CTLA4 PDL-1, OX40, 4-1BB, etc.) for evaluating other checkpoint inhibitors. Conclusions. Our data suggests the conditioned version 1-HuGEMM-h/mPD1 mouse can be explored to evaluate anti-human antibody. References 1. Pardoll, D.M. The blockade of immune checkpoints in cancer immunotherapy. Nature reviews. Cancer 12, 252-264 (2012). 2. Allard, B., Pommey, S., Smyth, M.J. & Stagg, J. Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs. Clinical cancer research : an official journal of the American Association for Cancer Research 19, 5626-5635 (2013). Citation Format: Zhun Wang, Bin Cai, GANG Chen, Jinping Liu, Xiaoyu An, Zhengsheng Wang, Davy Ouyang, Jean-Pierre Wery, Jay Liu, Xin Dong, Henry Li. HuGEMM-h/mPD1 mouse models for assessing anti-human PD1 therapeutics. [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 A11.

Abstract 1472: Building comprehensive and fully annotated patient tumor derived xenogragft (PDX) library mirroring cancer patient population

Patient derived xenografts (PDXs) mirrors patients’ pathology and genetic profiles, thus valued as predictive experimental models for studying oncogenesis and personalized treatments. Cancer is not a single disease but diseases of complex genetic components and oncogenic processes. Limited number of PDX models with minimal genetic characterization is insufficient to meet current research needs. For this, we have built the largest and most comprehensive PDX library with full genetic profiles. By far, our PDX library contains over 1,100 models derived from patients of both Asian and Western origins, covering over 20 major cancer types, including large panels (over 100 models each) of NSLCL(1), CRC(2), gastric(3), HCC(4), and pancreatic, and smaller panels ( Our large library of different disease panels are particularly useful in conducting mouse clinical trial (MCT of HuTrial TM ) (2, 5, 12), which can be used to discover predictive biomarker (2, 5, 13) and guide clinical study design. Citation Format: Jie Cai, Dawei Chen, Rajendra Kumari, Sheng Guo, Jie Yang, Mengmeng Yang, Andrew McKenzie, Zhun Wang, Xuesong Huang, Xiaoyu An, Jinping Liu, Jean-Pierre Wery, Henry Li. Building comprehensive and fully annotated patient tumor derived xenogragft (PDX) library mirroring cancer patient population. [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 1472. doi:10.1158/1538-7445.AM2015-1472

Abstract 2281: Building mouse tumor derived allogragfts for immune-oncology research

Immuno-oncology is an area under intensive investigation, however, many questions remain to be answered due to lack of sufficient number of adequate experimental models: 1) why only subsets of patients respond? 2) who will respond (signature)? 3) what can we do to make more patients to respond? Patient derived xenograft (PDX or HuPrime®) mirrors patients’ histopathological/genetic profiles, considered more predictive than traditional human cell line derived xenograft (CDX) and also syngenic mouse cell derived models, due to the primary natures of PDX. On the other hand, both xenografts (PDX/CDX) grow in absence of immunity, thus unsuitable for immune-oncology research. Limited types/numbers of available syngenic cells also restrict its application. Genetically engineered mutant mouse cancer model (GEMM) are diverse (types/numbers)1-5, grow in immune-environment, and are primary tumors. However, GEMM has logistic limitation for pharmacology research for high cost and high variations in tumor development (spontaneous) in type/time. We set out to create a library of primary mouse tumor allografts (MuPrimeTM) as standard experimental models, aiming at overcoming these limitations. We established a number of such allografts and are profiling them for growth, histopathology, genomic (RNAseq), and standard of care (SOC). We are also charactering their immune-oncology properties: tumor-infiltrating immune cells, including different subtypes of T-cells (CTL, Treg), as well as their functional activation/suppression and tumor responses to immune-modulating agents. MBR6004 is an allograft derived from a breast adenocarcinoma of GEMM-MMTV-PyVT in FVB/N mice1, which expressed medium level Her2 but negative for ER and PR, and is resistant to Docetaxel(SOC). It expresses low-level PD-L1(CD274)/L2 and seems insensitive to PD-1 antibody treatment. MCR6013, an allograft of a colon adenocarcinoma derived from GEMM-C57BL/6J-ApcMin/JNju3-5, are currently being profiled. MPR6003, an allograft of transgenic adenocarcinoma mouse prostate (TRAMP) in C57BL/6 mice6-8, grow both orthotopically and subcutaneously, and are also being profiled. M6011, an allograft derived from a spontaneous tumor in Balb/c mouse with high Her2 expression, has apparent infiltration of T-cells, including Treg, but negative for PD-L1. We are currently characterizing its histopathology and other property. MLY1014, an allograft of a spontaneous lymphoma derived from Balb/c, is also being profiled. In general, these allografts keep the original primary tumor histopathology, but distinct from conventional syngenic cell line derived tumors, a contrast seen between PDX vs. CDX. The models grow robustly after engraftment, thus ideal for pharmacology evaluation. Our ongoing studies will provide a useful platform with normal immunity for drug evaluation, particularly for immune-oncology agents. Citation Format: Xiaoyu An, Jinping Liu, Jie Cai, Jean-Pierre Wery, Henry Qixiang Li. Building mouse tumor derived allogragfts for immune-oncology research. [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 2281. doi:10.1158/1538-7445.AM2015-2281

Abstract 3123: Modeling anti-leukemic therapy by patient derived AML xenografts with distinct phenotypes/geneotypes

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We have recently successfully engrafted leukemic cells from bone marrow of 3 AML patients, subtypes of M5 (AM7577), 2 (AM8096) and 6 (AM8070), into immunocompromised NOD/SCID mice. All three displayed aggressive AML diseases but with distinct characteristics. AM7577 displays typical M5 disease subtype, which starts at bone marrow and gradually expands to peripheral (spleen, lymph nodes and peripheral blood). AM7577 also harbors interesting genotype, including mutations of IDH2-R140Q, FLT3-ITD, DNMT3A R882H and NPM1. AM8096, derived from a recurrent AML-M2 patient sample, also displayed aggressive disease with 100% mortality, but many distinct pathological phenotypes: first, although with abundant leukemic cells in bone, the peripheral symptom is significant minor, characterized by lower leukemic counts in peripheral blood and only slightly enlarged spleen and smaller lymph nodes, as compared to AM7577; second, the leukemic cell morphology is also rather different where AM8096 demonstrates less differentiated phenotype; third, the genotype, in contrast to AM7577, is wild-types for all the above oncogenes. AM8070, on the extreme contrary, has severe bone marrow disease, but with few peripheral leukemia. While we have not identified the likely leukemogenesis driver gene for these models, we are currently performing RNAseq for all three models in order to explore the underlying molecular pathogenesis. In addition, we are investigating the model response to standard of care (SOC, AraC). We found that the treatment resulted in rapid control of the disease as shown by reduction (disappearance) of leukemic cells in peripheral blood, significantly relieved disease symptoms and extended life. However, when drug administration was stopped, the disease rapidly re-emerged. In order to investigate the underlying mechanism of the rapid disease relapse without obvious development of resistance, we examined the drug effect on leukemia at different relevant leukemic organs: bone, spleen, lymph nodes and blood. Surprisingly, we found that the drug has no effect on bone leukemic load, while completely suppressed leukemia in blood, with partial effect in spleen and lymph nodes. Our current working hypothesis is that the failure to suppress leukemia in bone, including LICs, are likely responsible for the observed rapid relapse of the disease immediately after drug withdrawal. In summary, the established PDX AML models could serve as useful experimental models to investigate the diverse leukemogenesis and to model anti-leukemia therapies. Citation Format: Jinping Liu, Xiaoyu An, Na Wang, Di Wang, Liang Huang, Ran Wu, Jie Cai, Jean-Pierre Wery, Henry Li. Modeling anti-leukemic therapy by patient derived AML xenografts with distinct phenotypes/geneotypes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3123. doi:10.1158/1538-7445.AM2014-3123