Lung Cancer Patient-derived Xenograft Research Articles

Establishment of lung cancer patient-derived xenograft models and primary cell lines for lung cancer study

BackgroundThe overall 5-year survival rate of lung cancer is about 15% even with therapeutic drugs like tyrosine kinase inhibitors. Ideal models are urgently needed for exploring mechanisms and finding new drugs. Patient-derived xenografts (PDX) models and primary cells are both used to screen therapeutic regimens for cancer. However, PDX models and primary cells from the same patient are difficult to establish. Their consistency to the original tumor tissue is not well studied.Methods31 lung cancer patient tissues were procured to establish the lung cancer PDX models and primary cell lines. Tumor growth measurements, histological and immunohistochemistry analysis, Western blotting, EGFR and K-RAS mutation detection and gefitinib sensitive assay were performed to evaluate the characteristic of established PDX models. Immunofluorescence analysis, anchorage-independent cell growth, Western blotting and gefitinib sensitive assay were performed to assay the characteristic of established primary cell lines. The whole-exome sequencing was used to compare the characteristic of the patient’s tumor tissue, established PDX and primary cell line.ResultsTwenty-one lung cancer PDX models (67.74%, 21/31) and ten primary cell lines (32.25%, 10/31) were established from patients’ tumor tissues. The histology and pathological immunohistochemistry of PDX xenografts are consistent with the patients’ tumor samples. Various signal pathways were activated in different PDX models (n = 5) and primary cell lines (n = 2). EGFR mutation PDX model and primary cell line (LG1) were sensitive to gefitinib treatment. The expression of CK8/18, TTF1 and NapsinA in LG1 and LG50 primary cells were also positive. And the activated signal pathways were activated in LG1 and LG50 primary cell lines. Furthermore, the gene mutation in PDX tumor tissues and primary cell line (LG50) was consistent with the mutation in LG50 patient’s tumor tissues.ConclusionThese data suggested that established lung cancer PDX models and primary cell lines reserved mostly molecular characteristics of primary lung cancer and could provide a new tool to further understand the mechanisms and explore new therapeutic strategies.

Genomic and Functional Fidelity of Small Cell Lung Cancer Patient-Derived Xenografts.

Small cell lung cancer (SCLC) patient-derived xenografts (PDX) can be generated from biopsies or circulating tumor cells (CTC), though scarcity of tissue and low efficiency of tumor growth have previously limited these approaches. Applying an established clinical-translational pipeline for tissue collection and an automated microfluidic platform for CTC enrichment, we generated 17 biopsy-derived PDXs and 17 CTC-derived PDXs in a 2-year timeframe, at 89% and 38% efficiency, respectively. Whole-exome sequencing showed that somatic alterations are stably maintained between patient tumors and PDXs. Early-passage PDXs maintain the genomic and transcriptional profiles of the founder PDX. In vivo treatment with etoposide and platinum (EP) in 30 PDX models demonstrated greater sensitivity in PDXs from EP-naïve patients, and resistance to EP corresponded to increased expression of a MYC gene signature. Finally, serial CTC-derived PDXs generated from an individual patient at multiple time points accurately recapitulated the evolving drug sensitivities of that patient's disease. Collectively, this work highlights the translational potential of this strategy.Significance: Effective translational research utilizing SCLC PDX models requires both efficient generation of models from patients and fidelity of those models in representing patient tumor characteristics. We present approaches for efficient generation of PDXs from both biopsies and CTCs, and demonstrate that these models capture the mutational landscape and functional features of the donor tumors. Cancer Discov; 8(5); 600-15. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

Patient derived xenografts (PDX) predict an effective heparanase-based therapy for lung cancer.

Heparanase, the sole heparan sulfate (HS) degrading endoglycosidase, regulates multiple biological activities that enhance tumor growth, metastasis, angiogenesis, and inflammation. Heparanase accomplishes this by degrading HS and thereby facilitating cell invasion and regulating the bioavailability of heparin-binding proteins. HS mimicking compounds that inhibit heparanase enzymatic activity were examined in numerous preclinical cancer models. While these studies utilized established tumor cell lines, the current study utilized, for the first time, patient-derived xenografts (PDX) which better resemble the behavior and drug responsiveness of a given cancer patient. We have previously shown that heparanase levels are substantially elevated in lung cancer, correlating with reduced patients survival. Applying patient-derived lung cancer xenografts and a potent inhibitor of heparanase enzymatic activity (PG545) we investigated the significance of heparanase in the pathogenesis of lung cancer. PG545 was highly effective in lung cancer PDX, inhibiting tumor growth in >85% of the cases. Importantly, we show that PG545 was highly effective in PDX that did not respond to conventional chemotherapy (cisplatin) and vice versa. Moreover, we show that spontaneous metastasis to lymph nodes is markedly inhibited by PG545 but not by cisplatin. These results reflect the variability among patients and strongly imply that PG545 can be applied for lung cancer therapy in a personalized manner where conventional chemotherapy fails, thus highlighting the potential benefits of developing anti-heparanase treatment modalities for oncology.

Expression of GOLPH3 in patients with non-small cell lung cancer and xenografts models.

Increased expression of Golgi phosphoprotein 3 (GOLPH3) has been reported to be associated with several types of human cancer. Patient-derived cancer xenograft models have demonstrated great potential in preclinical studies. In the present study, the link between GOLPH3 expression and survival was examined in patients with non-small cell lung cancer (NSCLC). Patient-derived lung cancer xenograft models were established with two different methods. Lastly, the association between GOLPH3 expression and establishment of the xenograft models was explored. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry analysis were used to examine GOLPH3 expression in 60 NSCLC tissues and matched adjacent non-cancerous tissues (ANT). In addition, tumor pieces from the 60 NSCLC tissues were implanted in the subcutaneous layer and in the subrenal kidney capsule of nude mice. RT-qPCR, histopathology and immunohistochemistry were used to confirm the human origin of the xenograft tumors. RT-qPCR was also used to research the mutation status of GOLPH3 in the xenograft tumors. The results demonstrated that NSCLC tissues had higher expression of GOLPH3, at the mRNA and protein level, compared with ANT. High expression of GOLPH3 correlated with poor survival in patients with NSCLC. Successful engraftment was established for 27 tissues in the subrenal kidney capsule and for 16 in the subcutaneous layer of nude mice. The subrenal kidney capsule group demonstrated significantly higher engraftment rates than the subcutaneous layer group. In addition, higher GOLPH3 expression in the tumor tissues was significantly correlated with higher engraftment rates in mice. In both groups, few xenografts lost the GOLPH3 mutation. In summary, GOLPH3 may be an important diagnosis and prognosis indicator in patients with NSCLC. The genotype and phenotype of the xenograft tumors derived from patient lung cancer tissues exhibited significant similarities to the originating primary tumors. High GOLPH3 expression may promote the successful establishment of xenograft models for NSCLC.

Antitumor activity of kinetochore-associated protein 2 siRNA against lung cancer patient-derived tumor xenografts.

It has been widely reported that patient-derived tumor xenografts (PDXs) are more similar to tumor tissues than conventional cancer cell lines. Kinetochore-associated protein 2 (KNTC2) is known to be upregulated specifically in tumor tissues of cancer patients and is recognized as a potential target for cancer therapy. Previously, in vivo antitumor activities of KNTC2 short interfering RNA encapsulated into a lipid nanoparticle (KNTC2-LNP) were reported in orthotopic hepatocellular carcinoma mouse models. However, it remains unclear whether KNTC2-LNP exhibits antitumor activities against lung cancer PDXs. In the present study, the antitumor activities of KNTC2-LNP were clarified in a three-dimensional culture system and a subcutaneous tumor model of lung cancer PDX, LC-60, which was resistant to erlotinib. Growth inhibitory activities of KNTC2-LNP were associated with knockdown activities. Furthermore, KNTC2-LNP also exhibited in vivo antitumor activity against another lung cancer PDX, LC-45, which was sensitive to erlotinib. These results suggest that KNTC2 is a promising target for patients with lung cancer.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract A123: Delivery of a potent, selective, and efficacious RET inhibitor for the treatment of RET-driven lung adenocarcinoma

Abstract Background: Constitutive activation of RET kinase activity following mutation or rearrangement can lead to the development of cancers such as medullary thyroid carcinoma and lung adenocarcinoma. The currently approved therapeutics for these diseases are significantly compromised due to dose-limiting toxicities associated with off-target activity vs KDR (VEGFR2) and lack of potency vs anticipated secondary resistance (e.g., gatekeeper) mutations. Consequently there is considerable interest in the development of highly selective inhibitors targeting diverse RET alterations including the putative resistance mutation, V804M. Methods: We have established a robust screening cascade complemented by structure-enabled drug design and effective medicinal chemistry. Biochemical activity vs RET, KDR, and RETV804M protein was assessed using a HTRF assay. Cellular activity was quantified in BaF3 cells dependent on activity of RET, KDR, or RETV804M for proliferation. Tumor growth inhibition and supporting PK/PD studies were carried out in a number of disease-relevant models including a KIF5B-RET lung cancer patient-derived xenograft (PDX) model, a medullary thyroid carcinoma (MZ-CRC-1) xenograft model, and a lung cancer control (Calu-6) xenograft model. Results: Using this optimized, robust platform, we have identified a number of selective compounds offering a range of interesting biochemical and cellular profiles, targeting either, or both, RET and the gatekeeper mutant, RETV804M. We believe certain examples of these compounds offer the first cell-active RETV804M-selective derivatives. More importantly perhaps, we have also delivered a highly selective preclinical candidate compound demonstrating potency vs both RET fusion and RETV804M. This compound is well tolerated in vivo after oral dosing at up to 80mg/kg bid and, in a KIF5B-RET lung cancer PDX model, demonstrates efficacy at much lower doses: 50% tumor regression at 20mg/kg bid and 92% tumor growth inhibition at 10mg/kg bid. Importantly, this agent shows no efficacy in the (non-RET driven) Calu-6 xenograft model, demonstrating selective inhibition of the RET kinase domain. Conclusions: We believe that the identification of well-tolerated, selective RET inhibitors with potent activity against diverse RET alterations (including the anticipated resistance mutation, V804M) offers a clear therapeutic advantage over the present clinically approved compounds. Our most advanced compound fulfills all of these challenging criteria and has now entered preclinical development. Citation Format: Mandy Watson, Rebecca Newton, Ben Acton, Helen Small, Habiba Begum, Samantha Hitchin, Paul Kelly, Donald Ogilvie, Ian Waddell, Allan Jordan. Delivery of a potent, selective, and efficacious RET inhibitor for the treatment of RET-driven lung adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A123.

MET-GRB2 Signaling-Associated Complexes Correlate with Oncogenic MET Signaling and Sensitivity to MET Kinase Inhibitors.

Purpose: Targeting MET in cancer is hampered by lack of diagnostics that accurately reflect high MET signaling and dependence. We hypothesized that assays reflecting MET signaling associated protein complexes could redefine tumors dependent on MET and could add additional precision beyond genomic assessments.Experimental Design: We used biochemical approaches, cellular viability studies, and proximity ligation assays to assess MET dependence. We examined MET signaling complexes in lung cancer patient specimens (N = 406) and patient-derived xenograft (PDX) models of solid tumors (N = 308). We evaluated response to crizotinib in a MET-amplified cohort of PDX models of lung cancer (N = 6) and provide a case report of a lung cancer patient harboring a Δexon14 MET splice variant.Results: We found the interaction of MET with the adaptor protein GRB2 is necessary for oncogenic survival signaling by MET. MET-GRB2 complexes were identified only within MET-amplified PDX models and patient specimens but exhibit substantial variability. Lack of MET-GRB2 complexes was associated with lack of response to MET TKI in cell lines and PDX models. Presence of MET-GRB2 complexes can further subtype tumors with Δexon14 MET splice variants. Presence of these complexes correlated with response to crizotinib in one patient with Δexon14 MET lacking MET gene amplification.Conclusions: Proximity assays measuring MET-GRB2 signaling complexes provide novel insights into MET-mediated signaling and could complement current clinical genomics-based assay platforms. Clin Cancer Res; 23(22); 7084-96. ©2017 AACR.

RANK rewires energy homeostasis in lung cancer cells and drives primary lung cancer.

Lung cancer is the leading cause of cancer deaths. Besides smoking, epidemiological studies have linked female sex hormones to lung cancer in women; however, the underlying mechanisms remain unclear. Here we report that the receptor activator of nuclear factor-kB (RANK), the key regulator of osteoclastogenesis, is frequently expressed in primary lung tumors, an active RANK pathway correlates with decreased survival, and pharmacologic RANK inhibition reduces tumor growth in patient-derived lung cancer xenografts. Clonal genetic inactivation of KRasG12D in mouse lung epithelial cells markedly impairs the progression of KRasG12D -driven lung cancer, resulting in a significant survival advantage. Mechanistically, RANK rewires energy homeostasis in human and murine lung cancer cells and promotes expansion of lung cancer stem-like cells, which is blocked by inhibiting mitochondrial respiration. Our data also indicate survival differences in KRasG12D -driven lung cancer between male and female mice, and we show that female sex hormones can promote lung cancer progression via the RANK pathway. These data uncover a direct role for RANK in lung cancer and may explain why female sex hormones accelerate lung cancer development. Inhibition of RANK using the approved drug denosumab may be a therapeutic drug candidate for primary lung cancer.

Abstract 2092: A potent and selective RET inhibitor with efficacy in RET-driven mouse models of medullary thyroid carcinoma and lung adenocarcinoma

Abstract Background: The aim of this CRUK-MI Drug Discovery project is to deliver a RET-selective inhibitor for the treatment of cancers with RET activating mutations, which include 1-2% of lung adenocarcinomas and medullary thyroid cancers (MTC). Recent data supports the hypothesis that the efficacy of vandetanib and cabozantinib, clinically approved multi-kinase inhibitors, is limited by toxicities associated with potent activity against KDR. Therefore, a RET-selective inhibitor would represent a best-in-class agent for the treatment of these cancers. Methods: We have established a robust screening cascade to develop a potent, selective RET inhibitor and developed several in vivo models to evaluate compound PKPD and antitumor efficacy. Tumor growth inhibition and PKPD studies were carried out in BaF3 mouse allograft models overexpressing KIF5B-RET or RETV804M and other disease relevant models, including an MTC xenograft (MZ-CRC-1), a KIF5B-RET lung cancer patient derived xenograft (PDX) model (CTG-0838, Champions Oncology) and a lung cancer control xenograft (Calu-6). Results: Two orally bioavailable compounds displaying nanomolar RET potency and >10 fold selectivity over KDR in cellular assays were selected from the lead series and further evaluated in our in vivo PD and efficacy models. Both compounds demonstrated efficacy in the BaF3 KIF5B-RET driven model (71% and 103% tumor growth inhibition (TGI), respectively), accompanied by reduced levels of pRET in the tumor tissue. Following further lead optimisation; a compound displaying an improved DMPK profile and additional nanomolar potency versus the gatekeeper mutation (RETV804M) was identified and accelerated through our DMPK/in vivo cascade. We consider this additional activity versus RETV804M beneficial since mutations at the gatekeeper residue in other tyrosine kinases (e.g. EGFR) have been shown to mediate acquired drug resistance in the clinic. This compound demonstrated significant TGI of 58% and 82% respectively in the BaF3 KIF5B-RET and BaF3 RETV804M allograft models. Moreover, tumor growth in the lung cancer PDX model was strongly inhibited (95% TGI) and tumor regression induced in the MTC xenograft model (109% TGI). As expected, this potent and selective RET inhibitor was not active in the Calu-6 model, which is sensitive to KDR inhibition, whereas vandetanib, a potent KDR inhibitor, significantly inhibited tumor growth (84% TGI). Additional in vitro and in vivo DMPK analyses further support the nomination of this compound as a preclinical candidate. Conclusions: The identification of selective RET inhibitors with significant in vivo activity and minimal toxicity may overcome the limitations of the currently available clinical compounds. We have made considerable progress towards this goal and show here the compelling data supporting our nomination of a preclinical development compound. Citation Format: Mandy Watson, Helen Small, Ben Acton, Habiba Begum, Samantha Hitchin, Allan Jordan, Paul Kelly, Rebecca Newton, Ian Waddell, Gina Paris, Donald Ogilvie. A potent and selective RET inhibitor with efficacy in RET-driven mouse models of medullary thyroid carcinoma and lung adenocarcinoma [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 2092. doi:10.1158/1538-7445.AM2017-2092

Abstract 2333: Modulation of Bax and mTOR for cancer therapeutics

Abstract Pharmacologic manipulation of the serine (S)184 phosphorylation site of Bax protein to functionally regulate its proapoptotic activity is an attractive anticancer strategy. We recently identified three small molecule Bax agonists. SMBA1 was selected as the lead compound based on its chemical structure and its drug-like properties, from which a more effective analog, CYD-2-11, was generated. CYD-2-11 targets the structural pocket around S184 in the C-terminal tail of Bax, directly activating its proapoptotic activity via 6A7 conformational change and formation of Bax homo-oligomers in mitochondrial membranes. CYD-2-11 suppresses tumor growth in SCLC, NSCLC and patient-derived lung cancer xenografts as well as the genetically engineered mutant KRAS-driven lung cancer model with no significant normal tissue toxicity. Inhibition of mTOR by RAD001 enhances S184 Bax phosphorylation in lung cancer cell lines and tumor tissues from lung cancer patients treated with RAD001, which inactivates the propaoptotic function of Bax, contributing to rapalog-resistance. Combined CYD-2-11 and RAD001 treatment not only displays strong synergistic activity against lung cancer but also overcomes rapalog-resistance in vitro and in vivo. Therefore, a mechanism-driven combination of Bax agonist and mTOR inhibitor represents a highly attractive therapeutic strategy to improve lung cancer patient outcomes. Citation Format: Rui Li, Chunyong Ding, Jun Zhang, Maohua Xie, Dongkyoo Park, Ye Ding, Guo Chen, Guojing Zhang, Melissa Gilbert-Ross, Wei Zhou, Adam Marcus, Shi-Yong Sun, Zhuo Chen, Gabriel Sica, Suresh Ramalingam, Andrew Magis, Haian Fu, Fadlo Khuri, Walter Curran, Taofeek Owonikoko, Dong Shin, Jia Zhou, Xingming Deng. Modulation of Bax and mTOR for cancer therapeutics [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 2333. doi:10.1158/1538-7445.AM2017-2333

Abstract 4801: Collection of lung cancer patient-derived xenograft (PDX) models to evaluate precision therapeutic strategies

Abstract Background: Translational cancer research needs appropriated preclinical models to discover predictive biomarkers and evaluate precision therapeutic strategies. Conventional available preclinical models have proven not been optimal to this end, since cell lines based xenografts do not reconstitute the complexity of human cancer and carry acquired mutations not found in the original tumor. For these reasons our aim was generate and completely characterize a collection of non-small cell lung cancer (NSCLC) PDX models useful for translational lung cancer research. Materials and methods: Resected primary tumors from NSCLC patients were subcutaneous xenografted in athymic nude mice and expanded in successive groups of mice to get a perpetual live bank of each tumor. Every tumor, which successfully grew in mice, was analyzed by histologic and molecular techniques (NGS gene panel, exome and RNA Seq). Furthermore, a bank of frozen pieces of tumor was expanded and stored in order to generate later cohorts of tumor-bearing mice suitable for preclinical drug evaluation. Results: Our collection consists on 42 PDX models (26 SCC, 12ADC, 3LCC). We have characterized all of them histologically and molecularly with a 22 genes NGS panel. Furthermore, we have analyzed the exome and transcriptome of 33 models (20 SCC, 10 ADC and 3 LCC). All PDX models mostly retain the principal histologic and molecular characteristics of their donors and recapitulate the heterogeneity of human lung tumors. In our PDX collection we have sufficiently represented all the NSCLC histology and the most relevant molecular alterations in lung cancer in order to perform precision medicine evaluation studies. Conclusions: We have generated a collection of 42 PDX models of NSCLC characterized at the histological, genomic and transcriptomic level, which represents the most frequent histological and molecular subtypes of this type of lung cancer. This collection will be really useful to integrate drug screening with biomarker discovery and to evaluate precision therapeutic strategies. Our future aim will be to use this collection for biomarkers identification and preclinical evaluation of new therapeutic strategies targeted to bad prognostic lung tumors with suboptimal therapeutic approaches. Citation Format: Sonia Molina-Pinelo, Ricardo Melendez, Rocío Suarez, Laura García, Laura Ojeda, Nicolas Moreno, María V Guijarro, Luis Paz-Ares, Irene Ferrer. Collection of lung cancer patient-derived xenograft (PDX) models to evaluate precision therapeutic strategies [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 4801. doi:10.1158/1538-7445.AM2017-4801

Abstract 5770: in vitro PDX models: 3D cultured patient-derived tumors for compound evaluation

Abstract Background. Patient-derived xenograft (PDX) models in immune-compromised mice allow propagation of and compound testing in human-derived tumors in vivo. To expand the potential of these human-relevant PDX models, we sought to develop 3D in vitro culture methods for PDX-derived tumor cells that show in vivo-like growth characteristics, invasion and responses to therapeutics. In combination with advanced 3D image analysis methods, we created a unique high throughput in vitro PDX screening platform that not only allows efficient identification of active and selective molecules but also enables selection of the optimal PDX tumor models for subsequent validation of candidates in vivo. Results. Each PDX model has its own unique growth characteristics. Hydrogel and growth media composition were optimized to support growth of tumor tissues in vitro from cells derived from bladder, stomach, breast, pancreas, colon and lung cancer PDX tumors. Tumor tissues were cultured in a 384-well format and used to test chemotherapeutics (e.g. 5-FU, doxorubicin, paclitaxel, cisplatin), small molecules (e.g. erlotinib, lapatinib, trametinib, everolimus), antibodies (e.g. cetuximab, trastuzumab) and antibody-drug-conjugate (ADC, T-DM1) dose ranges. Using OcellO’s 3D image analysis platform, Ominer, tumoroid growth, cell proliferation, apoptosis, invasion, cell polarity, differentiation and other aspects of cell and tissue architecture were analyzed and the effects of compound exposure on tumoroids was determined. By performing feature training based on reference compounds, we selected ±10 morphological features (out of more than 500) to generate a phenotypic signature that described the unique phenotypic change induced by each compound. Different compounds that target the same molecule were found to induce a similar morphological change whereas compounds with off-target effects could be discriminated. This approach enabled a high resolution evaluation and comparison of compound activity in an automated manner. Conclusions. We established several PDX model-derived 3D tumor cultures in which standard-of-care and novel therapeutic agents (small molecules, antibodies and ADCs) can efficiently be screened, based on therapeutically relevant parameters and their changing morphological profile. This method enables both the in vitro selection of promising compounds in a pre-clinically relevant setting and the selection of optimum PDX tumor models for follow-up in vivo studies. This highly translational in vitro-in vivo PDX pipeline is expected to reduce attrition and increase efficiency in early drug-discovery. Citation Format: Sander Basten, Bram Herpers, Julia Schueler, Torsten Giesemann, Leo S. Price. in vitro PDX models: 3D cultured patient-derived tumors for compound evaluation [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 5770. doi:10.1158/1538-7445.AM2017-5770

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

Abstract 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

Targeting KRAS-dependent tumors with AZD4785, a high-affinity therapeutic antisense oligonucleotide inhibitor of KRAS.

Activating mutations in KRAS underlie the pathogenesis of up to 20% of human tumors, and KRAS is one of the most frequently mutated genes in cancer. Developing therapeutics to block KRAS activity has proven difficult, and no direct inhibitor of KRAS function has entered clinical trials. We describe the preclinical evaluation of AZD4785, a high-affinity constrained ethyl-containing therapeutic antisense oligonucleotide (ASO) targeting KRAS mRNA. AZD4785 potently and selectively depleted cellular KRAS mRNA and protein, resulting in inhibition of downstream effector pathways and antiproliferative effects selectively in KRAS mutant cells. AZD4785-mediated depletion of KRAS was not associated with feedback activation of the mitogen-activated protein kinase (MAPK) pathway, which is seen with RAS-MAPK pathway inhibitors. Systemic delivery of AZD4785 to mice bearing KRAS mutant non-small cell lung cancer cell line xenografts or patient-derived xenografts resulted in inhibition of KRAS expression in tumors and antitumor activity. The safety of this approach was demonstrated in mice and monkeys with KRAS ASOs that produced robust target knockdown in a broad set of tissues without any adverse effects. Together, these data suggest that AZD4785 is an attractive therapeutic for the treatment of KRAS-driven human cancers and warrants further development.

MA17.06 Landscape of Somatic Mutations Involving Lung Cancer Associated Genes in Non-Small Cell Lung Cancer (NSCLC) Patient-Derived Xenografts

Patient-derived tumor xenografts (PDXs) have high fidelity to their histological origins, and maintain the molecular heterogeneity and genetic aberrations of the donor patient tumors more faithfully than established in non-small cell lung cancer (NSCLC) cell lines. This study evaluated whether our panel of PDX models recapitulate known cancer-related gene mutations. Whole-exome sequencing was completed on 103 NSCLC PDX models, 47 adenocarcinoma (AdC) and 56 squamous (SqCC), with a mean coverage of 84x. After filtering for contaminating mouse reads, the exome data were aligned using the Burrows-Wheeler Aligner, processed using the standard GATK pipeline, and mutations were identified using MuTect. Additional filtering using dbSNP, ExAC and ESP was performed for cases without corresponding normal adjacent lung exome data (n = 80). The identified mutations were compared to 1260 frequently mutated cancer-related genes, which were compiled from a panel of cancer-related mutated genes (555) and a panel of lung cancer-specific mutated genes (1082). High rates of somatic mutations were observed in both AdC (mean of 12.4 mutations/megabase) and SqCC (mean of 11.7 mutations/megabase) PDX models. Compared to the rates observed in primary lung cancers in The Cancer Genome Atlas studies (mean of 8.9 mutations/megabase in AdC; 8.1 mutations/megabase in SqCC), these values appear higher, but may be inflated due to the lack of data from corresponding normal tissues. AdC models had a total of 953 mutated genes (median: 57 genes/model; range: 5-307), while SqCC models were characterized by 1007 mutated genes (median: 55 genes/model; range: 21-354). Specific mutation frequencies were compared to those determined in a recent study involving genomic alterations in human primary lung AdC and SqCC (Nature Genetics 2016; 48; 607–616). This comparison, based on mutated genes common in both studies, demonstrated significant correlation of the frequencies in 791 genes in AdC (ρ=0.78; p<2.2×10-16), as well as in 799 genes in SqCC (ρ=0.73; p<2.2×10-16). Three genes that were reported as significantly mutated in both AdC and SqCC primaries, and had higher mutation frequencies in SqCC, were also observed to be higher in our SqCC PDX models (TP53: 48.9% in AdC vs. 55.4% in SqCC; CDKN2A: 4.3% vs. 7.1% and PIK3CA: 2.1% vs. 23.2%); however, the statistical significance of these differences needs to be tested. Mutation landscapes in cancer genes are recapitulated in AdC and SqCC PDX models. The fidelity of these landscapes in matched patient primary tumour samples is being investigated.

P2.03b-077 EGFR/ALK+ Patient-Derived Xenografts from Advanced NSCLC for TKI Drug Selection & Resistance Development: The REAL-PDX Study: Topic: Biomarkers

Lung cancer patient-derived xenografts (PDX) have shown to be representative models for individual patient tumors. Theoretically, such models could inform the choice of subsequent lines of therapy, since PDX development, TKI resistance induction, and subsequent drug-screening can be completed before TKI resistance develops in the patient. The goal of Resistance modeling in EGFR and ALK Lung cancer (REAL)-PDX is to develop PDX models for real-time treatment selection of subsequent lines of therapy in advanced-stage NSCLC patients. Since August 2015, Princess Margaret Cancer Centre patients with EGFR/ALK+, as well as lifetime never-smoking lung cancer patients with unknown mutation status, were consented to have additional tumor sampling for PDX development during routine- or trial-related biopsies. Tumor sufficiency was confirmed prior to implantation into non-obese severe combined immunodeficient (NOD-SCID) mice, with successful engraftment defined as propagation beyond first passage; unsuccessful implantations had no palpable tumor after 6 months. 72/82 (88%) approached patients consented; 49/72 (68%) had adequate tumor tissue for implantation (71% stage III/IV): 46 adenocarcinomas, 2 squamous cell carcinoma, 1 LCNEC. 36/49 (73%) were lifetime never smokers. Patients received adjuvant chemotherapy (3), TKI therapy (15), both (5), or no treatment (26) prior to sampling. Tumor samples were taken from surgically resected lung (18), metastatic adrenal (1) and brain (2), CT-guided lung biopsies (5), endoscopic ultrasound-guided (EBUS) biopsies (6), and thoracentesis pleural fluid (17) specimens. Twenty-eight implanted tumors were EGFR+ (12 exon19 deletions, 2 exon19 deletion/T790M, 1 exon19 del/exon18 mutation, 12 L858R, and 1 L858R/T790M); 7 had ALK-rearrangements, and 1 had ROS1-rearrangement. Engraftment rates of 31 assessable implanted tumors were as follows: lung resections 12/12 (100%), metastatic resections 2/3 (67%), CT- or EBUS-guided biopsies 1/5 (20%), and pleural fluid 2/11 (18%); Engraftment rate was associated with no prior treatment (14/17 no treatment vs 3/14 any treatment, p=0.001). Of 17 assessable tumors with EGFR activating mutations, 9 engrafted (53%). Of 3 assessable tumors with ALK-rearrangement, 1 was successful (33%). PDX development of EGFR/ALK+ models for testing with novel therapeutics from various tumor biopsy sites is feasible and will provide valuable real-time information for subsequent treatment decisions in advanced NSCLC patients. Updated engraftment and drug screening data will be presented.

PUB031 PRIMAL: A Phase 1b Study of PEGPH20 plus Docetaxel in Patients with Previously Treated Hyaluronan (HA)-High Advanced NSCLC

Hyaluronan (HA) is a megadalton polysaccharide found in the tumor microenvironment (TME). HA accumulation in the TME increases tumor interstitial pressure which promotes vascular collapse and limits access of chemotherapy and immune cells to tumor sites. In animal models, HA-High tumors exhibit increased growth and metastasis, treatment resistance, and reduced survival. PEGylated recombinant human hyaluronidase (PEGPH20) enzymatically degrades tumor HA. Preclinical studies in non-small cell lung cancer (NSCLC) patient-derived xenografts showed that PEGPH20 enhanced the effect of docetaxel (Doc) in HA-High tumors by increasing tumor growth inhibition and prolonging survival. This Phase 1b study will evaluate the safety and activity of PEGPH20 plus Doc in patients with NSCLC in dose escalation and cohort expansion periods. Eligible patients are ≥18 years, ECOG PS 0-1 with stage IIIB/IV NSCLC, and have failed 1 previous platinum-based chemotherapy regimen (plus EGFR/ALK inhibitor treatment where indicated). Patients with brain metastases are eligible if clinically stable and not receiving corticosteroids. During dose escalation, up to 30 patients (regardless of HA status) receive PEGPH20 (1.6, 2.2, 2.8 μg/kg IV over 10 minutes) on day 1, followed 24 hours later by Doc (75 mg/m2 IV over 60 minutes) on Day 2 of each 21-day cycle. In the cohort expansion period, PEGPH20 will be administered at the recommended Phase 2 dose (RP2D) in up to 50 patients with HA-High tumors determined using a companion diagnostic assay currently being developed in collaboration with Ventana Medical Systems. During both periods, dexamethasone (8 mg orally BID) will be given for possible musculoskeletal events. Patients with a history of deep vein thrombosis will receive enoxaparin. Primary endpoints include the RP2D and safety and tolerability of the study combination. Secondary endpoints are objective response rate, duration of response, disease control rate, progression-free survival, and pharmacokinetic profiles of the study drugs. Exploratory endpoints include change in plasma HA levels, potential prognostic/predictive markers, and correlation between plasma/tumor biomarkers and clinical outcomes. Clinical trial identifier: NCT02346370. Section not applicable Section not applicable

395 - Assessment of EGFR-dependent signaling pathway targeting in EGFR-mutated and wild type non-small cell lung cancer patient-derived xenografts

395 - Assessment of EGFR-dependent signaling pathway targeting in EGFR-mutated and wild type non-small cell lung cancer patient-derived xenografts

396 - Assessment of Pi3K and/or MAPK signaling pathway targeting in KRAS- or PIK3CA-mutated and wild type non-small cell lung cancer patient-derived xenografts

396 - Assessment of Pi3K and/or MAPK signaling pathway targeting in KRAS- or PIK3CA-mutated and wild type non-small cell lung cancer patient-derived xenografts