Search

The cell viability assay with different order of drug treatment.

Supplementary Figure Legends

Supplementary Figure Legends

Nuclear p53 plays a critical role in synergistic cytotoxic effect in RKO cells.

Carfilzomib and KPT330 exhibited synergistic cytotoxicity in HCT116 cells.

Bortezomib induced nuclear export of p53 in SW480 and SW620 cells.

Bortezomib and KPT330 co-treatment inhibit patients-derived xenografts in nude mice.

Limited T-cell persistence restrains chimeric antigen receptor (CAR)-T-cell therapy in solid tumors. To improve persistence, T cells have been engineered to secrete proinflammatory cytokines, but other possible methods have been understudied. Runx3 has been considered a master regulator of T-cell development, cytotoxic T-lymphocyte (CTL) differentiation and tissue-resident memory T (Trm)-cell formation. A study using a transgenic mouse model revealed that overexpression of Runx3 promoted T-cell persistence in solid tumors. Here, we generated CAR-T cells overexpressing Runx3 (Run-CAR-T cells) and found that Run-CAR-T cells had long-lasting antitumor activities and achieved better tumor control than conventional CAR (Con-CAR)-T cells. We observed that more Run-CAR-T cells circulated in the peripheral blood and accumulated in tumor tissue, indicating that Runx3 coexpression improved CAR-T-cell persistence in vivo. Tumor-infiltrating Run-CAR-T cells showed less cell death with enhanced proliferative and effector activities. Consistently, in vitro studies indicated that AICD was also decreased in Run-CAR-T cells via downregulation of tumor necrosis factor (TNF) secretion. Further studies revealed that Runx3 could bind to the TNF promoter and suppress its gene transcription after T-cell activation. In conclusion, Runx3-armored CAR-T cells showed increased antitumor activities and could be a new modality for the treatment of solid tumors.

BACKGROUND: Syngeneic mouse tumor models are widely used as preclinical models for immuno-oncology (I/O) research. However, their response to immune checkpoint inhibitors (ICIs) in vivo is limited, despite the increase in tumor infiltrating lymphocytes (TILs), which is potentially due to low intrinsic immunogenicity. Expression of chicken ovalbumin (OVA) engineered into syngeneic tumors has previously been shown to render the cells more immunogenic and increases the response to ICIs such as anti-PD-1. In this study we investigated the potential to increase the response to ICIs through OVA expression and characterized the model for TILs and T cell-mediated immunity compared with parent cell line. METHODS: A series of murine cells were transduced by lentiviral vector (pLVX-EF1a_IRES-PURO) with a chicken OVA coding cDNA. OVA expression was confirmed by western blot following single clone selection under puromycin. MC38-OVA, B16-F10-OVA, CT26.WT-OVA and EMT6-OVA cells were subcutaneously inoculated into immunocompetent C57BL/6 or BALB/c mice to evaluate the tumorigenicity and their in vivo response to anti-PD-1(10mg/kg, IP) treatment. In the study, blood, spleen and tumors were collected at termination for FACS analysis of the immune cell populations and the OVA-specific T cells. The effects of CD4+ and CD8+ T lymphocyte depletion was investigated using anti-CD4 and anti-CD8 antibodies in the PD-1 cured EMT6-OVA mice. Adoptive cell transfer (ACT) was evaluated by re-challenge studies in both OVA expressing and WT tumor-bearing mice with isolated T cells derived from OVA untreated mice, anti-PD-1 cured mice or OT-I mice. RESULTS: Anti-PD-1 treatment resulted in complete regression in most mice bearing CT26.WT-OVA, MC38-OVA and EMT6-OVA, whereas an improvement in the response was shown in B16-F10-OVA in vivo. T cell and NK cells expansion was observed after anti-PD-1 treatment in peripheral blood with increased T cells, Treg cells and NK cells in both tumor-draining lymph nodes and spleen. In the re-challenge study anti-PD-1 cured mice generated robust tumor specific memory T cell, which successfully inhibited OVA expressing tumor growth following ACT. CD3+ T cells from EMT6-OVA-bearing mice showed anti-tumor immunity in vivo. CD4 and CD8 depletion studies provided further insight into the T cell role in PD-1 treatment. CONCLUSIONS: The panel of OVA expressing murine cell lines displayed increased T cell-mediated immunity and enhanced anti-PD-1 responses compared to WT models for different cancer indications in vivo, providing novel immunogenic syngeneic models for preclinical immunotherapy evaluation. Citation Format: Chenpan Nie, Yi Zhang, Jia Zheng, Annie X. An, Jun Zhou, Henry Q.X. Li, Jingjing Wang. Characterization of OVA-expressing immunogenic syngeneic tumor models for immune checkpoint inhibitor evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4221.

BACKGROUND: Despite the progress and development of targeted agents and Immunotherapeutics, breast cancer is still the leading cause of death among women worldwide. At present, targeted therapy for breast cancers include endocrine blockage therapy for estrogen receptor positive (ER+) tumors and trastuzumab or lapatinib for HER2 positive (HER2+) tumors and CDK inhibitors for HER negative (HER-) tumors as well as PARP inhibitors for BRCA1/2 mutations. There is still a high unmet need for triple negative breast cancer (TNBC) and relapsed breast cancers with specific mutations. Breast cancer is a highly heterogeneous disease and reliable breast cancer models are needed to develop new clinical therapies and better reflect tumor biology. Patient-derived xenograft (PDX) models have been widely used for understanding tumor characteristics and represent human heterogeneity more faithfully and thus are suitable for mouse clinical studies for predicting drug efficacy in the clinic. Here we report the establishment and characterization of a panel of breast cancer (BC) PDX for preclinical research. METHODS: Tumor samples from patients’ breast tumor or metastastic lesion obtained via surgery, biopsy, or pleural effusions/ascites were engrafted subcutaneously in immunodeficient mice to establish PDX models. The models were classified and characterized by histopathology, immunohistochemistry (IHC), and breast cancer specific biomarkers such as estrogen receptor (ER), progesterone receptor (PR) and HER2. Genomic analysis of these models was performed by next generation sequencing (NGS). In these breast cancer models, the effects of estrogen on tumor growth and targeted endocrine therapy such as Fulvestrant were evaluated. The change of tumor volume over time was used to determine the growth rate and response to treatment in mice. RESULTS: A series of BC PDX models derived from primary patient breast tumor or metastatic lesions were successfully established and characterized. Key characteristics of parental tumors, such as histopathology, clinical markers, gene expression and copy number, as well as estrogen dependence was retained. The IHC data indicated various expression levels of ER, PR and HER2 across the panel, with TNBC being the dominant subtype. ESR1 mutation, which may play an important role in BC progression and endocrine therapy resistance, was also identified in an estrogen-independent model. In vivo, these models showed distinct tumor growth rates and different pharmacodynamic responses to standard of care agents. For example, ESR1 mutant model was responsive to Fulvestrant. CONCLUSIONS: The established BC PDX panel represent a range or ER/PR/HER2 expression status from primary and advanced cancer providing a series of preclinical models for breast cancer research and potential for mouse clinical trial enrollment. Citation Format: Leilei Chen, Xueying Yang, Likun Zhang, Xiaobo Chen, Henry Q.X. Li, Jingjing Wang. Establishment and characterization of patient-derived breast cancer models for cancer studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3115.

Although proteasome inhibitors such as bortezomib had significant therapeutic effects in multiple myeloma and mantel cell lymphoma, they exhibited minimal clinical activity as a monotherapy for solid tumors, including colorectal cancer. We found in this study that proteasome inhibition induced a remarkable nuclear exportation of ubiquitinated proteins. Inhibition of CRM1, the nuclear export carrier protein, hampered protein export and synergistically enhanced the cytotoxic action of bortezomib on colon cancer cells containing wild-type p53, which underwent G2–M cell-cycle block and apoptosis. Further analysis indicated that tumor suppressor p53 was one of the proteins exported from nuclei upon proteasome inhibition, and in the presence of CRM1 inhibitor KPT330, nuclear p53, and expression of its target genes were increased markedly. Moreover, knockdown of p53 significantly reduced the synergistic cytotoxic action of bortezomib and KPT330 on p53+/+ HCT116 cells. In mice, KPT330 markedly augmented the antitumor action of bortezomib against HCT116 xenografts as well as patient-derived xenografts that harbored functional p53. These results indicate that nuclear p53 is a major mediator in the synergistic antitumor effect of bortezomib and KPT330, and provides a rationale for the use of proteasome inhibitor together with nuclear export blocker in the treatment of colorectal cancer. It is conceivable that targeting nuclear exportation may serve as a novel strategy to overcome resistance and raise chemotherapeutic efficacy, especially for the drugs that activate the p53 system. Mol Cancer Ther; 16(4); 717–28. ©2016 AACR.

Patient derived xenograft (PDX) is widely used in preclinical cancer research for drug efficacy evaluation, biomarker discovery and validation, drug mechanism and resistant mechanism study, tumor biology studies and so on. Many PDX libraries are now available to researchers1-4. A good library is composed of PDXs with high-quality characterization for genomics, pharmacology, histology, and clinical information associated with the originating patient tumors. Various technologies are used for genomic profiling of PDX models. For example, RNAseq is used for transcriptomic profiling to obtain data on gene expression, RNA-level mutations, gene fusion; whole exome sequencing (WES) is used to infer DNA-level mutations and gene copy numbers. In this study, we compared mutations inferred by RNAseq and WES for 600 PDX models with both datasets. We found that about 50% of mutations detected by WES were uncovered by RNAseq. The other half are missing because these mutation alleles were covered by insufficient reads ( Overall, our study shows that RNAseq data is reliable to infer mutations in highly expressed genes and can be readily used for such when WES data is not available. References 1. Budinská E, Popovici V. The EurOPDX consortium: Sharing patient tumor-derived xenografts for collaborative multicentric preclinical trials. Molecular cancer therapeutics 2014;12:A8-A 2. Gao H, Korn JM, Ferretti S, Monahan JE, Wang Y, Singh M, et al. High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response. Nature medicine 2015;21:1318-25 3. Ledford, H. (2016). Us cancer institute to overhaul tumour cell lines. Nature, 530. 4. Guo, S., Qian, W., Cai, J., Zhang, L., Wery, J. P., & Li, Q. (2016). Molecular pathology of patient tumors, patient derived xenografts and cancer cell lines. Cancer Research, 76(16), 4619. Citation Format: Wubin Qian, Henry Q.X. Li, Sheng Guo. Mutation detection in patient-derived xenografts by WES and RNAseq [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 1051.

NSCLC is a common cancer with the highest mortality and few effective treatment options, except for the patients with EGFR (Erlotinib, Iressa) and ALK (Crizotinib) activating mutations. Recent studies indicated that some patients with the activating EGFR mutations but without pre-exposure to EGFR tyrosine kinase inhibitors (TKIs) are resistant to TKIs. The proposed mechanisms (1) include: T790M gatekeeper mutation preventing accessing the kinase activation site, Kras mutation, pTEN loss (2), or c-met amplification (3) that activates ERBB3 signaling (4). Patient Derived Xenograft (PDX) models have been suggested to more closely represent patient tumors (5, 6). We established several PDXs, including HuPrime® LU858, LU859, LU357, LU387, LU1901 and LU2503, using tumor tissues from individual NSCLC patients. LU858/859 contain the “classic” activating EGFR mutation, L858R; LU1868 contain L858R/T790M double mutations; LU357/LUF387 contain the common exon 20 insertion (2319-2320 AACCCCCAC); and LU1901/2503 have wild type EGFR. To our surprise, the two models with single L858R mutation, just like the model with double mutations, respond to the erlotinib poorly, as seen in some of clinic cases (1). Since they do not have T790M and Kras mutation, we examined them for c-met status, finding that their c-met gene are significantly amplified with also increased c-met/HGF expression as measured by at mRNA and protein levels (IHC), consistent with those seen in clinic (3). Furthermore, LU357 is sensitive to erlotinib while LU387 is not, corresponding to no c-met amplification in LU357 but significant amplification in LU387. All these suggest that the observed erlotinib resistance in these models could potentially be attributed to c-met signaling activation via c-met/HGF autocrine. To this end, we treated LU858, LU859 and LU2503 (also with c-met amplification) with c-met inhibitor, Crizotinib. Indeed, Crizotinib caused significant tumor response as expected in all these models. In particular, the combination treatment of LU858 and LU859 with erlotinib caused complete tumor regression by overcoming the resistance, suggesting a new potential effective treatment option. The results using PDX models can potentially help to design a prospective human study to demonstrate improved efficacy by the combination treatment via patient stratification to include the naïve NSCLC patient subjects with c-met amplification/EGFR mutation. 1. A. F. Gazdar, Oncogene 28 Suppl 1, S24, 2009 2. M. L. Sos et al., Cancer Res 69, 3256, 2009 3. T. Kubo et al., Int J Cancer 124, 1778, 2009 4. A. B. Turke et al., Cancer Cell 17, 77, 2010. 5. L. Ding et al., Nature 464, 999, 2010 6. E. Marangoni et al., Clin Cancer Res 13, 3989, 2007 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2344. doi:1538-7445.AM2012-2344

c-Met is a receptor tyrosine kinase that interacts with its ligand, HGF, secreted from surrounding stroma (paracrine signaling). This interaction is involved in cell growth, invasion, metastases and angiogenesis of a number of solid tumors. c-met gene amplification and increased gene expression have been frequently associated with these tumors. c-met is thus considered as an important cancer target for therapy of the cancers that their growth depends on activated c-met/HGF signaling1, including many HCC. As for any investigational c-met inhibitors, a highly relevant c-met/HGF experimental cancer model is critical to evaluate its efficacy and associated changes in biomarkers for predicting the likely responders. While patient derived xenograft (PDX) models, without any in vitro manipulation, mirror patients’ histopathological and genetic profiles2,3, the successfully engrafted PDX models usually lost its human stroma in mice and thus its c-met dependency. This is due to the lack of paracrine c-met/HGF mechanism in mice (no cross reactivity between mouse and human), and the tumors with such c-met/HGF paracrine dependency are selected against. The engrafted models might have changed its properties from the original human tumors by relying on other signaling pathways for survival, and thus not suitable for evaluating c-met inhibitors. Apparently, neither of these scenarios can be adequately used to evaluate c-met inhibitors. One alternative to this is engraftment of primary human tumor into HGF-transgenic immunocompromised mice, which would support the growth of c-met dependent PDX via paracrine mechanism. We recently screened a number of PDX HCC models using a c-met inhibitor and identified two responders, indicating their reliance on c-met/HGF signaling for growth, among many non-responders. The non-responders likely grow by relying on signaling pathway other than c-met/HGF. The two responders, on the other hand, likely grow through c-met/HGF autocrine mechanism. To further investigate this, we performed genomic profiling and IHC analysis of c-met and HGF on these two responders and other non-responders. Our results demonstrated that these two responders have amplification of HGF gene and express high levels of both c-met and HGF in both mRNA and protein (IHC). This is in contrast to no gene amplification and low level expression of both genes for non-responders. All these are consistent with that these two responders grow in mice via c-met/HGF autocrine mechanism. In conclusion, these two HCC models could be very useful models for evaluating c-met inhibitors. 1. Nat Rev Drug Discov 7, 504-516 (2008). 2. Nature 464, 999-1005 (2010). 3. Clin Cancer Res 13, 3989-3998 (2007). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1322. doi:1538-7445.AM2012-1322