Qingyang Gu

HIP1–ALK, A Novel ALK Fusion Variant that Responds to Crizotinib

Introduction: The aim of this study was to identify anaplastic lymphoma kinase (ALK) rearrangements in lung cancer patient-derived xenograft (PDX) models and to explore their responses to crizotinib. Methods: Screening of 99 lung cancer PDX models by the NanoString ALK fusion assay identified two ALK-rearranged non–small-cell lung cancer (NSCLC) tumors, including one harboring a previously known echinoderm microtubule-associated protein-like 4 (EML4)–ALK fusion and another containing an unknown ALK fusion variant. Expression array, RNA-Seq, reverse transcription polymerase chain reaction, and direct sequencing were then conducted to confirm the rearrangements and to identify the novel fusion partner in the xenograft and/or the primary patient tumor. Finally, pharmacological studies were performed in PDX models to evaluate their responses to ALK inhibitor crizotinib. Results: Two ALK-rearranged NSCLC PDX models were identified: one carried a well-known EML4–ALK variant 3a/b and the other harbored a novel huntingtin interacting protein 1 (HIP1)–ALK fusion gene. Exon 28 of the HIP1 gene located on chromosome 7 was fused to exon 20 of the ALK gene located on chromosome 2. Both cases were clinically diagnosed as squamous cell carcinoma. Compared with the other lung cancer PDX models, both ALK-rearranged models displayed elevated ALK mRNA expression. Furthermore, in vivo efficacy studies demonstrated that, similar to the EML4–ALK-positive model, the HIP1–ALK-containing PDX model was sensitive to treatment with crizotinib. Conclusions: Discovery of HIP1 as a fusion partner of ALK in NSCLC is a novel finding. In addition, the HIP1–ALK-rearranged tumor is sensitive to treatment with crizotinib in vivo, implicating HIP1–ALKas an oncogenic driver of lung tumorigenesis. Collectively, our results indicate that HIP1–ALK-positive NSCLC may benefit from clinical applications of crizotinib.

Aldehyde dehydrogenase‐2 (ALDH2) opposes hepatocellular carcinoma progression by regulating AMP‐activated protein kinase signaling in mice

Potential biomarkers that can be used to determine prognosis and perform targeted therapies are urgently needed to treat patients with hepatocellular carcinoma (HCC). To meet this need, we performed a screen to identify functional genes associated with hepatocellular carcinogenesis and its progression at the transcriptome and proteome levels. We identified aldehyde dedydrogenase‐2 (ALDH2) as a gene of interest for further study. ALDH2 levels were significantly lower at the mRNA and protein level in tumor tissues than in normal tissues, and they were even lower in tissues that exhibited increased migratory capacity. A study of clinical associations showed that ALDH2 is correlated with survival and multiple migration‐associated clinicopathological traits, including the presence of metastasis and portal vein tumor thrombus. The result of overexpressing or knocking down ALDH2 showed that this gene inhibited migration and invasion both in vivo and in vitro. We also found that ALDH2 altered the redox status of cells by regulating acetaldehyde levels and that it further activated the AMP‐activated protein kinase (AMPK) signaling pathway. Conclusion: Decreased levels of ALDH2 may indicate a poor prognosis in HCC patients, while forcing the expression of ALDH2 in HCC cells inhibited their aggressive behavior in vitro and in mice largely by modulating the activity of the ALDH2‐acetaldehyde‐redox‐AMPK axis. Therefore, identifying ALDH2 expression levels in HCC might be a useful strategy for classifying HCC patients and for developing potential therapeutic strategies that specifically target metastatic HCC. (Hepatology 2017;65:1628‐1644).

The Hippo pathway effector TAZ induces TEAD-dependent liver inflammation and tumors

Transcriptional cofactors YAP and TAZ have distinct roles in promoting proinflammatory cytokine production and tumorigenesis. TAZ drives inflammation Key effectors of the Hippo pathway, YAP and TAZ, are overexpressed in various cancers. Loss of upstream kinases that inhibit the activity of these transcriptional coactivators promotes inflammation. In patient-derived xenografts and TCGA data sets, Hagenbeek et al. found that only TAZ expression correlated strongly with inflammatory cytokine transcript abundance. Expression of hyperactivated TAZ, but not YAP, in the livers of mice augmented transcription factor TEAD-mediated systemic inflammation and tissue infiltration by myeloid cells. RNA-seq analysis identified distinct gene signatures in tumor cells driven by activated YAP or TAZ, suggesting that these Hippo pathway effectors have nonredundant functions. The Hippo signaling pathway regulates organ size and plays critical roles in maintaining tissue growth, homeostasis, and regeneration. Dysregulated in a wide spectrum of cancers, in mammals, this pathway is regulated by two key effectors, YAP and TAZ, that may functionally overlap. We found that TAZ promoted liver inflammation and tumor development. The expression of TAZ, but not YAP, in human liver tumors positively correlated with the expression of proinflammatory cytokines. Hyperactivated TAZ induced substantial myeloid cell infiltration into the liver and the secretion of proinflammatory cytokines through a TEAD-dependent mechanism. Furthermore, tumors with hyperactivated YAP and TAZ had distinct transcriptional signatures, which included the increased expression of inflammatory cytokines in TAZ-driven tumors. Our study elucidated a previously uncharacterized link between TAZ activity and inflammatory responses that influence tumor development in the liver.

Establishing a patient-derived xenograft model of human myxoid and round-cell liposarcoma

Myxoid and round cell liposarcoma (MRCL) is a common type of soft tissue sarcoma. The lack of patient-derived tumor xenograft models that are highly consistent with human tumors has limited the drug experiments for this disease. Hence, we aimed to develop and validate a patient-derived tumor xenograft model of MRCL. A tumor sample from a patient with MRCL was implanted subcutaneously in an immunodeficient mouse shortly after resection to establish a patient-derived tumor xenograft model. After the tumor grew, it was resected and divided into several pieces for re-implantation and tumor passage. After passage 1, 3, and 5 (i.e. P1, P3, and P5, respectively), tumor morphology and the presence of the FUS-DDIT3 gene fusion were consistent with those of the original patient tumor. Short tandem repeat analysis demonstrated consistency from P1 to P5. Whole exome sequencing also showed that P5 tumors harbored many of the same gene mutations present in the original patient tumor, one of which was a PIK3CA mutation. PF-04691502 significantly inhibited tumor growth in P5 models (tumor volumes of 492.62 ± 652.80 vs 3303.81 ± 1480.79 mm3, P < 0.001, in treated vs control tumors, respectively) after 29 days of treatment. In conclusion, we have successfully established the first patient-derived xenograft model of MRCL. In addition to surgery, PI3K/mTOR inhibitors could potentially be used for the treatment of PIK3CA-positive MRCLs.Myxoid and round cell liposarcoma (MRCL) is a common type of soft tissue sarcoma. The lack of patient-derived tumor xenograft models that are highly consistent with human tumors has limited the drug experiments for this disease. Hence, we aimed to develop and validate a patient-derived tumor xenograft model of MRCL. A tumor sample from a patient with MRCL was implanted subcutaneously in an immunodeficient mouse shortly after resection to establish a patient-derived tumor xenograft model. After the tumor grew, it was resected and divided into several pieces for re-implantation and tumor passage. After passage 1, 3, and 5 (i.e. P1, P3, and P5, respectively), tumor morphology and the presence of the FUS-DDIT3 gene fusion were consistent with those of the original patient tumor. Short tandem repeat analysis demonstrated consistency from P1 to P5. Whole exome sequencing also showed that P5 tumors harbored many of the same gene mutations present in the original patient tumor, one of which was a PIK3CA mutation. PF-04691502 significantly inhibited tumor growth in P5 models (tumor volumes of 492.62 ± 652.80 vs 3303.81 ± 1480.79 mm3, P < 0.001, in treated vs control tumors, respectively) after 29 days of treatment. In conclusion, we have successfully established the first patient-derived xenograft model of MRCL. In addition to surgery, PI3K/mTOR inhibitors could potentially be used for the treatment of PIK3CA-positive MRCLs.

Abstract 3852: Validation of synthetic lethality of PARP and NAMPT dual inhibition in a small cell lung cancer PDX model

Small cell lung cancer (SCLC) is a highly malignant cancer type with a 5-year survival rate of less than 10%. Different from non-small cell lung cancer (NSCLC), no effective target therapies have been approved to treat SCLC. Poly (ADP) ribose polymerase (PARP) overexpression in SCLC has prompted great efforts to evaluate the role of PARP inhibitors in clinic. To maximize their therapeutic value it is urgent in need to explore the best combination strategies between PARP inhibitors and other pathway modulators. In this study we identified a SCLC patient-derived xenograft (PDX) model with high PARP level and low nicotinamide phosphoribosyltransferase (NAMPT) level. Furthermore, synergistic effect of PARP and NAMPT dual inhibition was demonstrated, supporting translational research of PARP inhibitors and NAMPT inhibitors in this model. The mRNA expression of PARP as well as its 30 most-studied synthetic lethality genes was compared between SCLC and NSCLC in both PDXs and cancer cell lines. We found that NAMPT levels decreased most significantly in SCLC compared with NSCLC across the 30 synthetic lethality genes of PARP. As NAMPT is the rate-limiting enzyme for the synthesis of the PARP substrate β-NAD+, we hypothesized that low β-NAD+ level due to low NAMPT level might render SCLC sensitive to PARP inhibition. LU-01-0547, a SCLC PDX model with high PARP and low NAMPT expression, was identified here. To test our hypothesis, we investigated the preclinical efficacy of ABT888, a PARP inhibitor, with or without FK866, a NAMPT inhibitor, in this subcutaneous xenograft model. The single-agent efficacy of ABT888 was demonstrated (TGI=96%@100 mg/kg, BID). At a sub-optimal dose (50 mg/kg, BID), treatment of ABT888 alone produced little activity in the same model, while its combination with a NAMPT inhibitor, FK866, significantly boosted the antitumor response indicating the synergistic effect (TGI=117%, ORR=6/6, CR=4/6). When nicotinic acid (NA) was administered to promote β-NAD+ biosynthesis via the de novo pathway other than the NAMPT-mediated transformation, the PARP inhibitor’s effectiveness was greatly antagonized (TGI=70% vs. 13%), suggesting β-NAD+ level might correlate with PARP inhibitor sensitivity. In conclusion, this study identified a SCLC PDX model with high PARP and low NAMPT expression. Synergistic inhibition of PARP and NAMPT in this model was demonstrated superior to either treatment alone in efficacy, which warranted its future application in drug discovery. To our knowledge, this was the first in vivo evidence of synthetic lethality in SCLC PDX, supporting future clinical test of combination of PARP and NAMPT inhibitors in SCLC patients. Citation Format: Zhixiang Zhang, Dongfang Li, Chen Chen, Bo Zhang, Xuzhen Tang, Hao Ye, Qingyang Gu, Qunsheng Ji. Validation of synthetic lethality of PARP and NAMPT dual inhibition in a small cell lung cancer PDX model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3852. doi:10.1158/1538-7445.AM2017-3852

Abstract 3849: HuPBL humanized models as a tool to evaluate the efficacy and immune regulation of immune checkpoint inhibitors

After the success of PD-1, PD-L1 and CTLA-4 antibodies, a number of immune-checkpoint inhibitors are already in advanced stages. Meanwhile, tumor infiltrating lymphocytes (TIL) are well recognized as features of tumor progression and keys to effective cancer immunotherapy. Therefore, there is an urgent need to establish an effective pre-clinical platform for the evaluation of therapeutical outcomes in the human immune-system and the understanding of TILs. Either traditional syngeneic models or human target knockin models have their drawbacks: syngeneic models disable the development of antibodies that do not recognize mouse receptors; neither of them can achieve a good clinical prediction due to their inherent property as a murine system. To get a reconstitution of the human immune system in murine models, we have established several customized Human peripheral blood lymphocytes (hu-PBL) humanized models in NOG (NOD/Shi-scid/IL-2Rγnull) mice either systemically or focalized in tumors. Those models were validated by immune-checkpoint inhibitors for efficacy and by conducting TIL analysis with the support of sophisticated Flow Cytometry-Based Approach. Here we report that the engrafted human cells can expand and infiltrate in tumors in response to xenogenic stimulation with functional Th and Tc detected. The cell lineages are mainly CD3+ T lymphocytes (90%) and to lesser extent NK/NKT cells; the redistribution in tumors is unique not only compared to splenic and peripheral blood T cells, but also varies among different cancer cell lines when the same donor was applied. In addition, cell surface expression of immune checkpoint targets showed unique redistributions. We further investigated the correlation between the efficacy and the immune-regulation in these models: Models with more dense infiltration at the early phase usually grew more slowly than its NON-PBMC control, which also are prone to be better responders to immunotherapy due to the higher Effector cell: Target cell ratio (E:T ratio). When compared the efficacy of 2 different hPD-1 test antibodies in A375 systemic humanized model, the one with better efficacy also achieved stronger inhibition on PD-1 expression in TILs. Treatment with the clinically used Opdivo showed not only different responses in HCC827, A375 and A431 xenograft models but also unique profiles in TILs subsets and related immune-checkpoint expression. These models can be used for the purpose of pre-clinical immune-checkpoint inhibitor and bi-specific antibody screening under human background, as well as for further investigations regarding distinct functions of TILs subsets and target expression. Citation Format: Xuzhen Tang, Xianzhi Zhai, Hui Qi, Qin Hong, Qiyao Zhang, Lei Jin, Shaoyu Yan, Zhuozhi Wang, Yong Zheng, Qingyang Gu, Norman Zhang, Jing Li, Qunsheng Ji. HuPBL humanized models as a tool to evaluate the efficacy and immune regulation of immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3849. doi:10.1158/1538-7445.AM2017-3849

Abstract 82: Genomic profiling of PDX models across 14 tumor types reveals novel targeted therapeutic opportunities

Introduction: Large efforts have been made to detect somatic alterations and corresponding transcriptome changes in cancer patients, which are expected to bring about significant improvements in precision cancer medicine. Here we report a comprehensive list of driver genes and new targets identified in a large pan-cancer cohort of 550 Patient Derived Xenograft (PDX) models across 14 tumor types. Material and methods: For each PDX model with passages 2-4, we performed comprehensive genomic analyses, including whole exome sequencing (WES), SNP6.0 array, and RNASeq (or microarray). The genomic data were used for extrapolating genomic SNV, CNV, mRNA expression levels, and for identifying gene fusions. Genomic profiling data were generated in internal and analyzed by in-house validated pipelines. Results: 220 PDXs were identified to have actionable driver gene mutations /and/or amplifications among 550 models. We identified novel genomic alterations in a number of the PDX models. Significant portion of the models were validated with targeted therapeutic agents (TKI, Abs etc.) and certain cytotoxic agents. All processed data could be accessed via OncoWuXi database: http://onco.wuxiapptec.com and corresponding OncoWuXi APP (App store or onco.wuxiapptec.com/oncowuxi.apk Conclusions: We have established a large panel of PDX models with comprehensive genomic annotation and identified novel targets, with open access database and App. These models could serve as a platform for Mouse Clinical Trial and for pre-clinical development of next generation targeted therapeutic drugs. Citation Format: Hua Dong, Zhixiang Zhang, Baoyuan Zhang, Xuzhen Tang, Xiangnan Qiang, Yuxin Qin, Shuqun Yang, Yunbiao Yan, Norman Zhang, Qingyang Gu, Qunsheng Ji. Genomic profiling of PDX models across 14 tumor types reveals novel targeted therapeutic opportunities. [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 82.

Abstract 4759: Identification of ALK, ROS1, FGFR2 and NRG1 fusions and validation with targeted inhibitors in lung and ovarian PDX models

Chromosomal rearrangement mediated gene fusions and activation of oncogenic signaling pathways have become druggable targets for therapeutic intervention, with successful development of Imatinib as an inhibitor of Bcr-Abl in treatment of Philadelphia chromosome positive chronic myelogenous leukemia. A recent study by Stransky et. al. reported systemic analysis of the transcriptome of nearly 7000 human tumor samples from the Cancer Genome Atlas and revealed the landscape of near twenty kinase fusions across twenty solid tumor types. Identification of corresponding PDX models that carry given kinase fusions would be of paramount interest for preclinical validation of candidate inhibitors. We have established about 1000 PDX models across 20 tumor types. Here we report the identification of ALK, ROS1, FGFR2 fusions in lung cancer PDX models and a NRG1 fusion in the ovarian cancer models by RNA using Sequencing data combined with the Chimerascan and Star-Fusion software for fusion calling.. The gene fusions, as validated by PCR and Sanger Sequencing, led to over-expression of the affected kinases, supporting the notion that kinase activation may be the oncogenic driver in the corresponding tumors. We further validated that ALK inhibitors Crizotinib, AP26113 and LDK378 exhibited robust antitumor activities in the ALK fusion model. Current works on validation of ROS1, FGFR2 and NRG1 inhibitors in the pertinent fusion models are under progression. These models should provide means for pre-clinical validation of novel kinase inhibitors and for experimenting novel combinatorial therapeutic strategies. Citation Format: Xuzhen Tang, Hua Dong, Yibo Gao, Zhixiang Zhang, Xiangnan Qiang, Baoyuan Zhang, Yuxin Qin, Shuqun Yang, Yunbiao Yan, Qingyang Gu, Norman Zhang, Jie He, Qunsheng Ji. Identification of ALK, ROS1, FGFR2 and NRG1 fusions and validation with targeted inhibitors in lung and ovarian PDX models. [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 4759.

Abstract 688: Activating JAK1-S703I mutation may predict the sensitivity of JAK-STAT inhibition in hepatocellular carcinoma patient-derived xenograft tumor models

Hepatocellular carcinoma (HCC) is the fifth most common type of cancer worldwide. However, current therapeutic approaches for this epidemic disease are limited and its 5-year survival rate has not been improved in the past decades. Patient-derived xenograft (PDX) tumor models have become an excellent in vivo system for understanding of disease biology and drug discovery. In order to identify new therapeutic targets for HCC, whole-exome sequencing (WES) was performed on more than 60 HCC PDX models. Among them, four models exhibited point mutations in JAK1 (Janus Kinase) gene, which were confirmed by Sanger sequencing. To explore the transforming capability, these mutations were then introduced into 293T and Ba/F3 cells by transient transfection and retroviral infection, respectively. The results demonstrated that JAK1S703I mutation was able to activate JAK-STAT (Signal Transducer and Activator of Transcription) signaling pathway and drive cell proliferation in the absence of cytokine stimulation in vitro. Furthermore, a modest sensitivity to the treatment of a JAK1/2 inhibitor, Ruxolitinib, was observed in JAK1S703I mutant PDX model, but not in other mutant or wild type models. Pharmacodynamic analysis showed that phosphorylation of STAT3 in the Ruxolitinib-treated tumor tissues was significantly inhibited. Collectively, our results suggested that JAK1S703I is an activating mutation for JAK-STAT signaling pathway in vitro and in vivo. JAK-STAT pathway might represent a new therapeutic opportunity for HCC. Monotherapy using a more potent inhibitor and combinatory therapy may be further explored in JAK1S703I mutant PDX model. Citation Format: Shuqun Yang, Chonglin Luo, Qingyang Gu, Qiang Xu, Hongye Sun, Ziliang Qian, Yexiong Tan, Hao Wu, Yuxin Qin, Yuhong Shen, Xiaowei Xu, Shu-Hui Chen, Chi-Chung Chan, Hongyang Wang, Mao Mao, Douglas D. Fang. Activating JAK1-S703I mutation may predict the sensitivity of JAK-STAT inhibition in hepatocellular carcinoma patient-derived xenograft tumor models. [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 688. doi:10.1158/1538-7445.AM2015-688

Abstract 2839: Antitumor activities of FGFR inhibitors in FGFR1-overexpressing hepatocellular carcinoma patient-derived xenograft tumor models

Hepatocellular carcinoma (HCC) is intrinsically resistant to conventional chemotherapies. Developing effective therapeutics becomes critical to tackle the unmet medical need. The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling axis plays an important role in liver development and homeostasis in adults. Deregulation of the FGF/FGFR signaling pathway through genetic modification or overexpression of the receptors (or their ligands) has been increasingly recognized in HCC. Accordingly, therapeutic intervention of the pathway has been extensively explored. In this study, analysis of a panel of patient-derived xenograft (PDX) HCC models by Affymetrix human gene expression arrays revealed overexpression of FGFR1 in 13 out of 43 (30%) models. Antitumor activities of FGFR inhibitors, including lenvatinib, dovitinib, ENMD-2076, and LY2874455, were then tested in five models. Lenvatinib exhibited the most potent activity in all models, whereas LY2874455 failed to show the effect in one of models. IC50s for FGFR1 inhibition of ENMD-2076, lenvatinib, dovitinib and LY2874455 were 145 nM, 134.5 nM, 53.5 nM and 0.6 nM, respectively. In SNU-16 and NCI-H520 cell lines, IC50s of dovitinib, lenvatinib, ENMD-2076, and LY2874455 were 76/1355 nM, 94/4770 nM, 306/2280 nM, and 0.3/664 nM, respectively. Furthermore, analyses of PDX tissues by Affymetrix SNP Array 6.0 arrays revealed no amplification of FGFR1/2 genes in four models except for amplifications of FGFR3 gene in two models. Whole exome sequencing did not detect nonsynonymous genetic alterations, including SNPs and indels, in FGFR1/2 genes in these xenografts except for a few genetic variations in FGFR3/4 genes. Overall, due to the multi-targeting mechanisms of these inhibitors, in vitro inhibitory effects of the compounds on FGFR1 enzyme and cell proliferation may not correlate with the in vivo anti-tumor activities. Overexpression of FGFR1 gene may serve as a predictive biomarker for therapeutic intervention of the FGF/FGFR pathway in HCC by using lenvatinib, dovitinib and ENMD-2076. Further studies are required to define additional biomarkers for LY2874455. Thus, our findings provide the rationale for the clinical development of FGFR inhibitors in a subset of HCC patients expressing a high level of FGFR1 gene. Citation Format: Douglas D. Fang, Bin Zhang, Weiguo Xu, Kang Yan, Qingyang Gu, Qiang Xu, Yexiong Tan, Hongye Sun, Qin Luo, Weifeng Mao, Chiho Li, Jiangpeng Liao, Guibai Liang, Shu-Hui Chen, Chi-Chung Chan. Antitumor activities of FGFR inhibitors in FGFR1-overexpressing hepatocellular carcinoma patient-derived xenograft tumor models. [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 2839. doi:10.1158/1538-7445.AM2014-2839