Non-small Cell Lung Cancer Xenograft Models Research Articles

TdIF1: a putative oncogene in NSCLC tumor progression

TdT-interacting factor 1 (TdIF1) is a ubiquitously expressed DNA- and protein-binding protein that directly binds to terminal deoxynucleotidyl transferase (TdT) polymerase. Little is known about the functional role of TdIF1 in cancer cellular signaling, nor has it previously been identified as aberrant in any type of cancer. We report here for the first time that TdIF1 is abundantly expressed in clinical lung cancer patients and that high expression of TdIF1 is associated with poor patient prognosis. We further established that TdIF1 is highly expressed in human non-small cell lung cancer (NSCLC) cell lines compared to a normal lung cell line. shRNA-mediated gene silencing of TdIF1 resulted in the suppression of proliferation and anchorage-independent colony formation of the A549 adenocarcinoma cell line. Moreover, when these TdIF1-silenced cells were used to establish a mouse xenograft model of human NSCLC, tumor size was greatly reduced. These data suggest that TdIF1 is a potent regulator of lung tumor development. Several cell cycle-related and tumor growth signaling pathways, including the p53 and HDAC1/2 pathways, were identified as participating in the TdIF1 signaling network by in silico analysis. Microarray, transcriptome and protein-level analyses validated p53 and HDAC1/2 modulation upon TdIF1 downregulation in an NSCLC cellular model. Moreover, several other cell cycle regulators were affected at the transcript level by TdIF1 silencing, including an increase in CDKN1A/p21 transcripts. Taken together, these results indicate that TdIF1 is a bona fide tumor-promoting factor in NSCLC and a potential target for therapy.

PARP inhibitors synergize with gemcitabine by potentiating DNA damage in non-small-cell lung cancer.

Poly (ADP-ribose) polymerase (PARP) inhibitors have demonstrated great promise in the treatment of patients with deficiencies in homologous recombination (HR) DNA repair, such as those with loss of BRCA1 or BRCA2 function. However, emerging studies suggest that PARP inhibition can also target HR-competent cancers, such as non-small-cell lung cancer (NSCLC), and that the therapeutic effect of PARP inhibition may be improved by combination with chemotherapy agents. In our study, it was found that PARP inhibitors talazoparib (BMN-673) and olaparib (AZD-2281) both had synergistic activity with the common first-line chemotherapeutic gemcitabine in a panel of lung cancer cell lines. Furthermore, the combination demonstrated significant in vivo antitumor activity in an H23 xenograft model of NSCLC compared to either agent as monotherapy. This synergism occurred without loss of HR repair efficiency. Instead, the combination induced synergistic single-strand DNA breaks, leading to accumulation of toxic double-strand DNA lesions in vitro and in vivo. Our study elucidates the underlying mechanisms of synergistic activity of PARP inhibitors and gemcitabine, providing a strong motivation to pursue this combination as an improved therapeutic regimen.

Abstract B23: Novel epidermal growth factor receptor inhibitors cross the blood-brain barrier and inhibit the growth of metastatic non-small cell lung cancer

Abstract Deaths from solid tumors are often not due to the primary lesion but to metastatic disease at distal sites such as the lung, liver, and brain. Patients with non-small cell lung cancer (NSCLC) experience brain metastases, a poor prognostic marker, at an incidence rate of 30-50%. A significant proportion of the metastatic tumors express activating mutations of the EGFR, including exon 19 deletions as well as point mutations within the enzyme active site (L858R), which confer increased sensitivity to EGFR inhibitors. However, current small-molecule therapeutics poorly inhibit the growth of NSCLC brain metastases due to difficulty crossing the blood-brain barrier and harmful off-target effects. Thus, NSCLC patients with brain metastases have access to very few emerging treatment options. We have synthesized a novel class of compounds that irreversibly inhibit the epidermal growth factor receptor (EGFR) in the nanomolar range in vitro, and demonstrate a high degree of selectivity for EGFR mutants found frequently in NSCLC, in particular exon 19 deletions and L858R mutations, while sparing wild-type (WT) EGFR. Two compounds, LL-001 and LL-019, from Capella Therapeutics, Inc., inhibited L858R EGFR-mediated autophosphorylation and phosphorylation of downstream targets Akt and ERK MAP kinase at a concentration of approximately 100nM and blocked the kinase activity of an EGFR mutant, T790M, which confers resistance to first-generation EGFR inhibitors. In contrast, these compounds only weakly inhibited WT EGFR and failed to inhibit either the insulin receptor or insulin-like receptor-I, suggesting that these inhibitors would produce minimal off-target side effects in treated patients. Both compounds induced full remission of subcutaneous tumors with the human NSCLC cell line H1975 (T790M/L858R+). Most notably, pharmacokinetic studies showed that these compounds, when administered at a concentration of 150mg/kg in mice, showed no signs of toxicity and were found in the brain at concentrations ranging from 12-171 times higher than the GI50 for inhibition of EGFR+ NSCLC cell line proliferation in vitro. To determine whether these compounds could treat NSCLC brain metastases, we developed intracranial and intracardiac injection orthotopic xenograft models of brain metastatic NSCLC. LL-001 and LL-019 could induce remission of brain tumors in mice injected intracranially with the human NSCLC cell line HCC827-luciferase, which expresses an exon 19 deletion mutant of EGFR. Likewise, both compounds inhibited the growth of the human NSCLC cell line PC9M in an intracardiac model of brain metastatic NSCLC, and greatly extended the survival of mice compared to those receiving vehicle alone. Our results indicate that LL-001 and LL-019 can cross the blood-brain barrier at levels sufficient to inhibit the growth of brain metastatic lung cancer, with minimal deleterious off-target effects. These compounds show promise for the treatment of critically underserved NSCLC patients with brain metastases. Citation Format: Michelle Muldong, Christina Jamieson, Yun Oliver Long, Ida Deichaite, David W. Anderson, Alan Lewis, Nicholas A. Cacalano. Novel epidermal growth factor receptor inhibitors cross the blood-brain barrier and inhibit the growth of metastatic non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B23.

Polyphyllin I modulates MALAT1/STAT3 signaling to induce apoptosis in gefitinib-resistant non-small cell lung cancer

Non-small cell lung cancer (NSCLC) patients harboring EGFR mutation who initially respond to EGFR-TKI will gradually develop acquired resistance. There is still a challenge to treat EGFR-TKI resistant NSCLC patients. Polyphyllin I (PP I), a steroidal saponin isolated from Paris polyphylla., has been exhibited antitumor activities against various carcinomas. However, its mechanism in treating EGFR-TKI resistant NSCLC has not been well elucidated. In this study, we found that PP I suppressed the cell viability and induced apoptosis of gefitinib-resistant NSCLC cells and xenograft models. These therapeutic efficacies were associated with down-regulated level of MALAT1, leading to inactivation of STAT3 signaling pathway. The cell viability inhibition and apoptosis inducing in gefitinib-resistant NSCLC triggered by PP I were abolished by MALAT1 overexpression, while the cell viability inhibition and apoptosis inducing triggered by PP I were potentiated by MALAT1 knockdown. These findings suggest that, in vitro and in vivo, PP I inhibits the viability and induces apoptosis of gefitinib-resistant NSCLC by down-regulating MALAT1 and inactivating STAT3 signaling pathway. Thus, PPI could serve a promising therapeutic agent for the treatment of gefitinib-resistant NSCLC.

Abstract 5467: Efficacy of the ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 in patient-derived RAS-mutant NSCLC models

Abstract Introduction: RAS mutations are present in 30% of non-small cell lung cancer (NSCLC) and, despite activated MAPK signaling, MEK inhibitors have disappointed in clinical trials. MAPK pathway reactivation and activation of anti-apoptotic signaling via the PI3K/mTOR axis are major resistance mechanisms, among others. ERK inhibitors are believed to overcome some of the restrictions of MEK inhibitors, and combinations with PI3K/mTOR inhibitors could potentially increase therapeutic efficacy. Past trials of MEK and PI3K inhibitor combinations, however, showed that toxicities and side effects are likely to be expected also for the ERK-PI3K/mTOR inhibitor combination. Therefore, more specific and potent drugs as well as alternating dosing schedules may overcome toxicities and improve treatment outcome. Methods: We so far established five patient-derived RAS-mutant NSCLC cell lines (1/5 NRASQ61K, 1/5 KRASQ61K, 2/5 KRASG12C, 1/5 KRASG12D) and xenograft models (PDX), characterized them by next-generation sequencing (DFCI Oncopanel) and tested the antiproliferative (CellTiter-Glo® assay) and apoptosis-inducing (Caspase3/7 activity, IncuCyte® technology) efficacy of LY3214996 (ERK inhibitor) and LY3023414 (PI3K/mTOR inhibitor) in vitro. Effects on signal transduction were investigated by Western blot analysis. Results: The ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 exhibit single-agent antiproliferative and apoptosis-inducing activity in patient-derived RAS-mutant NSCLC cell lines (for LY3214996: 1/5 sensitive (IC50<1μM), 2/5 intermediate sensitive (IC50<5μM), 2/5 resistant (IC50>10μM); for LY3023414: all IC50 <3μM, 3/5 <1μM). Despite reactivation of MAPK signaling (pMEK) upstream of ERK during LY3214996 treatment, ERK downstream targets (cMyc, DUSP4, SPRY2) were strongly suppressed. In sensitive cell lines, LY3214996 induced PARP cleavage and Bim accumulation in a dose- and time-dependent manner. Sensitivities to ERK and PI3K/mTOR inhibition varied between cell lines, indicating different pathway dependencies for proliferation and cancer cell survival. Conclusions and Outlook: Both LY3214996 and LY3023414 have single-agent in vitro efficacy in patient-derived NSCLC models with different RAS mutations and co-mutational landscapes. Single-agent in vivo studies and tolerability studies of drug combinations are ongoing, and treatment predictors and intrinsic/adaptive resistance mechanisms will be investigated via RNAseq/GSEA and phospho-receptor tyrosine kinase arrays. Citation Format: Jens Köhler, Prafulla C. Gokhale, Jiaqi Li, Margaret K. Wilkens, Hong Tiv, Shripad V. Bhagwat, Greg Donoho, Patrick Brueck, Ramon V. Tiu, Amanda J. Redig, Emily S. Chambers, Atsuko Ogino, Jihyun Choi, Man Xu, Paul Kirschmeier, Pasi A. Jänne. Efficacy of the ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 in patient-derived RAS-mutant NSCLC models [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 5467.

Abstract 1522: The CDK4/6 inhibitor G1T38 enhances response to targeted therapies in preclinical models of non-small cell lung cancer

Abstract The recent FDA approvals of palbociclib, ribociclib, and abemaciclib in breast cancer validate cyclin-dependent kinases 4 and 6 (CDK4/6) as key therapeutic targets, and warrant investigations in other tumor types with frequently observed alterations in the CDK4/6 pathway. Towards this end, we assessed the preclinical antitumor efficacy of G1T38, an oral, potent, and selective small-molecule CDK4/6 inhibitor in clinical development, in models of non-small cell lung cancer (NSCLC). Importantly, preclinical studies have shown that G1T38 has a differentiated and potential best-in-class profile since it can be dosed continuously without causing severe neutropenia, which could result in better tumor efficacy. Initial efficacy screening of G1T38 in 60 patient-derived NSCLC xenografts revealed significant tumor growth inhibition (TGI), primarily in lung adenocarcinomas harboring frequently observed oncogenic alterations in KRAS, EGFR, BRAF, and ALK. In vitro analyses show that CDK4/6 inhibition enhances the antiproliferative effect of inhibitors targeting these oncogenes (or their signaling pathways), suggesting a possible role for G1T38 in augmenting response and/or delaying acquired resistance to agents currently utilized in the clinic. These results were validated in vivo in mouse NSCLC xenograft models with defined oncogenic alterations, including EGFR mutations. For example, in H1975 xenografts harboring EGFR L858R/T790M (the T790M mutation is responsible for over 50% of cases of acquired resistance to first-generation EGFR inhibitors), combination of G1T38 with erlotinib resulted in 77% TGI after 18 days–a significant improvement over either therapy alone. Moreover, in H1975 xenografts, G1T38 delayed resistance to the second-generation EGFR inhibitor afatinib, and upon development of resistance to afatinib monotherapy, addition of G1T38 to afatanib treatment resulted in stabilization of tumor growth. G1T38 also augmented the response of EGFR-mutant NSCLC xenografts to the third-generation EGFR inhibitor osimertinib, significantly enhancing TGI compared to either therapy alone. As a result, a safety, pharmacokinetics, and efficacy study of G1T38 + osimertinib (G1T38-03) is scheduled for initiation in 1Q18 in patients with EGFR-mutant, T790M-positive NSCLC. Additional efforts are ongoing to investigate the combination of G1T38 with targeted therapies in NSCLC murine models harboring alterations in KRAS, ALK, and BRAF. Together, our results suggest a compelling rationale for utilizing G1T38 as a backbone for multiple targeted therapy combination regimens in NSCLC. Citation Format: Jessica A. Sorrentino, Daniel M. Freed, John E. Bisi, Jay C. Strum, Patrick J. Roberts. The CDK4/6 inhibitor G1T38 enhances response to targeted therapies in preclinical models of non-small cell lung cancer [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 1522.

Using Radiolabeled 3'-Deoxy-3'-18F-Fluorothymidine with PET to Monitor the Effect of Dexamethasone on Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality in the United States, and pemetrexed-based therapies are regularly used to treat nonsquamous NSCLC. Despite widespread use, pemetrexed has a modest effect on progression-free survival, with varying efficacy between individuals. Recent work has indicated that dexamethasone, given to prevent pemetrexed toxicity, is able to protect a subset of NSCLC cells from pemetrexed cytotoxicity by temporarily suppressing the S phase of the cell cycle. Therefore, dexamethasone might block treatment efficacy in a subpopulation of patients and might be contributing to the variable response to pemetrexed. Methods: Differences in retention of the experimental PET tracer 3'-deoxy-3'-fluorothymidine (FLT) were used to monitor S-phase suppression by dexamethasone in NSCLC cell models, animals with tumor xenografts, and patients with advanced cancer. Results: Significant reductions in tracer uptake were observed after 24 h of dexamethasone treatment in NSCLC cell lines and xenograft models expressing high levels of glucocorticoid receptor α, coincident with pemetrexed resistance visualized by attenuation of the flare effect associated with pemetrexed activity. Two of 4 patients imaged in a pilot study with 18F-FLT PET after dexamethasone treatment demonstrated reductions in tracer uptake from baseline, with a variable response between individual tumor lesions. Conclusion:18F-FLT PET represents a useful method for the noninvasive monitoring of dexamethasone-mediated S-phase suppression in NSCLC and might provide a way to individualize chemotherapy in patients receiving pemetrexed-based regimens.

Suppressing autophagy enhances disulfiram/copper-induced apoptosis in non-small cell lung cancer

Autophagy, a cellular survival mechanism, is thought to allow the recycling of cellular breakdown products when cancer cells are subjected to chemotherapy, thus decreasing drug-induced apoptosis. Disulfiram (DSF), a drug widely used to control alcoholism, possesses anticancer activity by inducing apoptosis in vitro and in vivo in a copper (Cu)-dependent manner. Our previous studies proved that DSF/Cu exerts increased anti-tumor effects on non-small cell lung cancer (NSCLC) xenograft models, and inhibits NSCLC recurrence driven by ALDH-positive cancer stem cells. The present study is designed to investigate whether DSF/Cu can induce autophagy in NSCLC cells and to determine the relationship between autophagy and apoptosis. First, we observed that DSF/Cu induced significant cytotoxicity and caspase-dependent apoptosis in NSCLC cells, accompanied by the formation of vacuoles in the cytoplasm. Next, we investigated the levels of autophagic markers, including LC3II (microtubule-associated protein 1 light chain 3), ATG5 and p62 with or without chloroquine by Western blot. In addition, we observed co-localization of LC3 and the lysosomal protein LAMP2 (lysosome-associated membrane protein 2) after treatment with DSF/Cu. The results showed that DSF/Cu induced autophagy. Finally, we demonstrated that DSF/Cu-induced apoptosis was greatly enhanced when autophagy was suppressed with Atg5 siRNA or 3-MA in NSCLC cells. This synergistic effect of DSF/Cu and 3-MA was further confirmed in the NSCLC xenograft model. Taken together, our results show that DSF/Cu stimulates autophagy in NSCLC cells, which is an impediment to DSF/Cu-induced apoptosis.

Abstract A018: Patient-derived xenograft (PDX) models of NSCLC reflect clinical drug responses and predict effective treatments for patients

Abstract Background: Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related mortality and prognosis remains poor despite the availability of numerous therapies. Integration of drug screening and sequencing in PDX models may allow for improved understanding of mechanisms of resistance (de novo and acquired), identification of biomarkers, and optimization of therapeutic strategies for NSCLC patients. In this study, we evaluated the response of NSCLC PDX models to multiple therapies and correlated responses to known clinical outcomes and molecular characteristics. Methods: PDX models were developed from 86 patients with NSCLC and evaluated by next-generation sequencing for genomic alterations (mutations, amplifications/deletions, fusions, and gene expression changes). Models were screened against different therapies including first-line platinum and nonplatinum doublets and triplets, second-line single agent docetaxel and pemetrexed (second-line therapies), and EGFR-targeted inhibitors. Tumor regression (TR) values and RECIST criteria were determined and correlated with known literature-based response rates (RR) as well as individual patient outcomes. Results: Eighty-eight PDX models from 86 patients were interrogated. To date, 63 (72%) models have been sequenced and 39 screened against standard-of-care therapies. There was robust concordance in mutational and allelic frequency profiles between patient tumors and corresponding PDX models. Based on PDX tumor growth, regression (CR/PR RECIST) was observed for at least one regimen in 50% (12/24) of models screened against first-line therapies and 25% (2/8) screened against second-line docetaxel or pemetrexed. No tumor regression was observed in any NSCLC model treated with the EGFR inhibitor, erlotinib (0%, 0/15), despite EGFR amplification and the absence of KRAS mutations. Nine PDX responses could be correlated to clinical outcomes, with 89% (8/9) accurately reflecting patient responses to the same treatment. Conclusion: Our study demonstrated the strong alignment between PDX model response to standard-of-care therapies and patient clinical outcomes, which highlights the potential application of PDX models for translational modeling and utilizing cohorts of PDX models for clinical trial simulation. The responses of these models to different lines of therapy reflected corresponding patient outcomes both at an individual and population level. Comprehensive sequencing (WES and RNA) and standard-of-care drug testing of these PDX models is planned and could allow a deeper understanding of such mechanisms. In this context, application of PDX models to drug development and stratification of clinical trial patients for treatment will continue to evolve. Citation Format: Daniel Ciznadija, Igor Astaturov, Haiying Cheng, Nir Peled, Jennifer Jaskowiak, Angela Davies, David Sidransky. Patient-derived xenograft (PDX) models of NSCLC reflect clinical drug responses and predict effective treatments for patients [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 A018.

Biochanin A regulates the growth and migration of NSCLC through suppressing the VEGF/VEGFR2 signaling pathway.

Non-small cell lung cancer (NSCLC) is the most common lung cancer with 15.9% of predicted 5-year survival rate which represents extremely poor prognosis. In this study, we aimed to investigate the effects of Biochanin A (BIO-A) on NSCLC both in vitro and in vivo and to explore its underlying mechanism. We found that BIO-A could effectively inhibit the proliferation of NSCLC cell line A549 in a dose-dependent manner through down-regulating Ki-67 and VEGF, induce apoptosis by activation of cleaved-Caspase-3 and cleaved-Caspase-9, and suppress cell migration by downregulating of MMP-2 and VEGF. Mechanistically, the results of western blot indicated that BIO-A exerted its anti-growth ability through blocking VEGF/VEGFR2 signaling pathway. Moreover, BIOA significantly inhibited the growth of tumor in NSCLC xenograft model. Taken together, our investigation has suggested that BIO-A exhibits potent antitumor activities against NSCLC both in vitro and in vivo and provided a molecular basis for BIO-A potential applications in the treatment of NSCLC and other VEGF-induced diseases.

Upregulation of IL-11, an IL-6 Family Cytokine, Promotes Tumor Progression and Correlates with Poor Prognosis in Non-Small Cell Lung Cancer

Background/Aims: Cytokines are key players in tumorigenesis and are potential targets in cancer treatment. Although IL-6 has attracted considerable attention, interleukin 11 (IL-11), another member of the IL-6 family, has long been overlooked, and little is known regarding its specific function in non-small cell lung cancer (NSCLC). In this study, we explored IL-11’s role in NSCLC and the detailed mechanism behind it. Methods: Cell proliferation in response to IL-11 was determined by colony formation, BrdU incorporation and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Cell motility was measured by Transwell and wound healing assays. NSCLC xenograft models were used to confirm oncogenic function of IL-11 in vivo. Immunohistochemical staining and western blot assay were performed to detect epithelial–mesenchymal transition (EMT) markers and cell signaling pathway alterations. Eighteen NSCLC patients and 5 normal lung samples were collected together with data from an online database to determine the link between IL-11 expression and malignant progression. Results: We observed that IL-11 was upregulated in NSCLC samples compared with normal tissue samples and correlated with poor prognosis. Data from in vitro and in vivo models indicated that IL-11 promotes cell proliferation and tumorigenesis. Cell migration and invasion were also enhanced by IL-11. Epithelial–mesenchymal transition (EMT) was also observed after IL-11 incubation. Furthermore, IL-11 activated AKT and STAT3 in our experimental models. In addition, we observed that hypoxia induced IL-11 expression in NSCLC cells. Deferoxamine (DFX) or dimethyloxalylglycine (DMOG) induced hypoxia-inducible factor 1-alpha (HIF1α) upregulation, which enhanced IL-11 expression in NSCLC cells. Conclusions: Taken together, our results indicate that IL-11 is an oncogene in NSCLC, and elucidating the mechanism behind it may provide insights for NSCLC treatment.

Upregulation of P21-Activated Kinase 1 (PAK1)/CREB Axis in Squamous Non-Small Cell Lung Carcinoma

Background/Aims: p21-activated Ser/Thr kinase 1 (PAK1) is essential for the genesis and development of many cancers. The purpose of this study was to investigate the role of the PAK1–cyclic AMP response element-binding (CREB) axis in non-small cell lung cancer (NSCLC) tumorigenesis and its related mechanisms. Methods: Western blot assay and immunohistochemical staining were employed to investigate the PAK1 and CREB expression in the tissue microarray of human squamous NSCLC. Co-immunoprecipitation and immunofluorescence confocal assays were performed to determine the link between PAK1 and CREB. NSCLC xenograft models were used to study oncogenic function of PAK1 in vivo. Results: We observed that PAK1 and CREB expression levels were significantly elevated in human squamous NSCLC-tissue specimens, compared with those in adjacent normal bronchial or bronchiolar epithelial-tissue specimens, as well as their phosphorylated forms, based on western blotting. We showed in vitro that PAK1 knockdown by small-interfering RNA (siRNA) blocked CREB phosphorylation, whereas plasmid-based PAK1 overexpression resulted in CREB phosphorylation at Ser133, based on western blotting. In addition, PAK1 interacted with CREB in co-immunoprecipitation assays. Additionally, our in vitro findings detected by flow cytometry revealed that PAK1 silencing attenuated cell cycle progression, inducing apoptosis. Inhibition of PAK1 expression reduced tumor sizes and masses by modulating CREB expression and activation in xenograft models. Conclusion: These results suggest a novel mechanism whereby the PAK1–CREB axis drives carcinogenesis of squamous-cell carcinomas, and have important implications in the development of targeted therapeutics for squamous-cell lung cancer.

Abstract B173: Preclinical characterization and antitumor efficacy of DS-5010, a highly potent and selective RET inhibitor

Abstract Background: RET gene rearrangement has been detected in several cancers including 1-2% and 50% of non-small cell lung cancer (NSCLC) and papillary thyroid carcinoma, respectively, and it is known as a driver mutation. Several US Food and Drug Administration (FDA)-approved multi-tyrosine kinase inhibitors (MTKIs) have shown inhibitory effects on RET kinase activity and have been tested in several clinical studies. However, MTKIs do not appear potent enough to show clinical benefits. The FDA-approved MTKIs have been reported to exhibit dose-limiting toxicity (DLT) at doses below those suppressing RET kinase activity. Kinase insert domain receptor (KDR) inhibition especially leads to anti-angiogenesis-related DLT such as hypertension. Thus, the development of highly potent and selective second-generation RET inhibitors is desired. DS-5010 is an orally available small-molecule RET inhibitor that shows a specific and highly potent activity against RET and gatekeeper-mutated RET (RET-GKm) and slight KDR activity. In this study, we characterized the in vitro and in vivo activities of DS-5010. Results: In biochemical assays of 106 kinases, RET and platelet-derived growth factor receptor (PDGFR) alpha/beta were inhibited more than 80% by 193 nM DS-5010. The half-maximal inhibitory concentration (IC50) values of DS-5010 against RET, RET-GKm (V804L) were single digit nano-molar even under a condition of high concentration of ATP; besides it against KDR was more than 1000 nM. In a Ba/F3-RET subcutaneous tumor model, DS-5010 dosing at 10 mg/kg twice daily (bid) induced tumor regression. Moreover, DS-5010 exhibited a similar antitumor effect in a Ba/F3-RET-GKm (V804L) subcutaneous tumor model. In contrast, the FDA-approved MTKIs (cabozantinib, vandetanib, and alectinib) showed no significant antitumor effect on a Ba/F3-RET-GKm (V804L) subcutaneous tumor model. In an LC2/ad NSCLC xenograft model, which has the RET-CCDC6 fusion gene, DS-5010 dosing at 1 mg/kg thrice daily (tid) induced tumor regression. To predict acquired mutations against FDA-approved MTKIs, resistant clones were established by prolonged incubation of Ba/F3-RET cells with cabozantinib. A sequence analysis revealed that all the resistant clones possessed V804E mutation in the RET kinase domain and DS-5010 inhibited cell proliferation of Ba/F3-RET (V804E) mutation in the low nano-molar range. However, the FDA-approved MTKIs failed to show strong growth inhibitory effects (half-maximal growth inhibition [GI50]: 1584-5381 nM). Conclusion: These results indicate that DS-5010 has potent in vitro and in vivo activities against RET and RET-GKm, suggesting the potential usefulness of the compound for targeted therapy of cancers with RET gene rearrangements and mutations. Moreover, its potential effectiveness against acquired MTKI-resistant cells was also demonstrated. We are currently performing investigational new drug-enabling studies of DS-5010. Citation Format: Yasuyuki Kaneta, Takahiro Komatsu, Masashi Miyamoto, Megumi Goto, Hidenori Namiki, Yoshihiro Shibata, Hideaki Kageji, Hiroaki Inagaki, Kiyoshi Nakayama, Yuichi Tominaga, Takeshi Isoyama. Preclinical characterization and antitumor efficacy of DS-5010, a highly potent and selective RET inhibitor [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 B173.

Structure-based design and synthesis of 2,4-diaminopyrimidines as EGFR L858R/T790M selective inhibitors for NSCLC

Mutated epidermal growth factor receptor (EGFR) is a major driver of non-small cell lung cancer (NSCLC). The EGFRT790M secondary mutation has become a leading cause of clinically-acquired resistance to gefitinib and erlotinib. Herein, we present a structure-based design approach to increase the potency and selectivity of the previously reported reversible EGFR inhibitor 7, at the kinase and cellular levels. Three-step structure-activity relationship exploration led to promising compounds 19e and 19h with unique chemical structure and binding mode from the other third-generation tyrosine kinase inhibitors. In a human NSCLC xenograft model, 19e and 19h exhibited dose-dependent tumor growth suppression without toxicity. These selective inhibitors are promising drug candidates for EGFRT790M-driven NSCLC.

Apatinib enhances antitumour activity of EGFR-TKIs in non-small cell lung cancer with EGFR-TKI resistance

BackgroundEpidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs)-rechallenged therapy for EGFR-mutant non-small cell lung cancer (NSCLC) patients who acquired resistance showed moderate efficacy. Considering the high interrelation between EGFR and vascular endothelial growth factor/vascular endothelial growth factor receptor (VEGF/VEGFR) pathways, we firstly evaluated EGFR-TKI combined with apatinib (a highly selective VEGFR2 inhibitor) in EGFR-TKI-resistant model and patients. MethodsEffects of apatinib, gefitinib and gefitinib plus apatinib were assessed on four NSCLC cell lines (A549 with wild-type EGFR, H1975 harbouring L858R and T790M, H1650 and HCC827 harbouring E746_A750 deletion) and xenograft model of acquired resistance that was established by injecting H1975 cells. Furthermore, we retrospectively evaluated EGFR-TKI rechallenge with apatinib in 16 patients. ResultsGefitinib plus apatinib strengthened the effect of gefitinib and apatinib alone on the four NSCLC cell lines, and H1975 was the most susceptible one. Co-administration delayed the tumour growth than mono-therapy in the xenograft model and had better effect on inhibiting the activation of EGFR and VEGFR2 and expression of CD31 (an angiogenesis marker) and vascular endothelial growth factor A (an important pro-angiogenesis factor in the tumour microenvironment). Changes in protein expression of protein kinase B/mammalian target of rapamycin and extracellular signal-regulated kinase pathways demonstrated the potent inhibitory effect on the pro-survival signalling pathways by combined therapy. EGFR-TKI rechallenge with apatinib achieved a median progression-free survival of 4.60 months (95% confidence interval, 2.23–12.52 months) in the patients. ConclusionsApatinib significantly potentiated the antitumour effect of gefitinib in NSCLC with T790M-related EGFR-TKI resistance both in vivo and vitro. EGFR-TKI rechallenge with apatinib might represent a new option for NSCLC with T790M or unknown resistance mechanism.

Abstract 2089: Evaluation of fruquintinib, a potent and selective oral VEGFR inhibitor, in combination with targeted therapies or immune checkpoint inhibitors in preclinical tumor models

Abstract The development of therapies targeting tumor angiogenesis, tumor driver gene alterations and tumor immune evasion has made tremendous advancement in improving overall survival. However, efficacy may be limited and resistance often develops rapidly when targeting a single axis of tumorigenesis. Therefore, it is worthwhile to explore rational combination of therapies based on tumor-specific features. Fruquintinib is a potent and selective oral VEGFR inhibitor currently in Phase III clinical trials for non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC). We report here the evaluation of anti-tumor effect of fruquintinib in preclinical animal tumor models in combination with therapies targeting tumor driver gene alterations such as EGFR and c-MET or with immune checkpoints. In NSCLC xenograft models with EGFR activation such as activating mutations, gene amplification or protein overexpression, fruquintinib plus an EGFR tyrosine kinase inhibitor such as gefitinib or theliatinib (HMPL-309) was found to be more efficacious than either monotherapy. For instance, in PC-9 subcutaneous tumor model carrying EGFR exon 19 deletion, single agent treatment with fruquintinib at 2 mg/kg and gefitinib at 5 mg/kg produced the tumor growth inhibition (TGI) of 58% and 63%, respectively, while the combination treatment resulted in a TGI of 100% and tumor regression was observed in 11 of 16 mice treated with combinational therapy. In multiple xenograft models derived from lung cancer or renal cell cancer with c-MET activation (amplification or over-expression), addition of fruquintinib to a c-MET inhibitor savolitinib (AZD6094, HMPL-504) also improved the tumor growth inhibition substantially. At the end of the efficacy studies, CD31 and phosphorylation of EGFR, c-MET, AKT and ERK were analyzed with immunohistochemistry and western blotting method in tumor tissues. The results suggested that the enhanced anti-tumor effect in combination therapy could be attributed to the simultaneous blockade of cell signaling in tumor cells (EGFR or c-MET) and VEGFR suppression in the tumor microenvironment. Up-regulation of the immune inhibitory checkpoints induced by VEGF is one of the important mechanisms for tumor cells to escape immune surveillance. In a syngeneic murine tumor model, co-administration of fruquintinib and anti-PD-L1 antibody was found to provide improved anti-tumor effect compared to fruquintinib or anti-PD-L1 single agent alone. Studies to understand the mechanism responsible for the combination effect are under way. All combinations with fruquintinib described above were well tolerated. The efficacy observed in these models suggested that simultaneous blockade of tumor angiogenesis and tumor cell signaling or immune evasion may be a promising approach in improving treatment outcomes. Citation Format: Yongxin Ren, Qiaoling Sun, Jingwen Long, Shiming Fan, Renxiang Tang, Wei Zhang, Xuelei Ge, Jianxing Tang, Linfang Wang, Dongxia Shi, Hongbo Chen, Min Cheng, Weiguo Qing, Weiguo Su. Evaluation of fruquintinib, a potent and selective oral VEGFR inhibitor, in combination with targeted therapies or immune checkpoint inhibitors in preclinical tumor models [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 2089. doi:10.1158/1538-7445.AM2017-2089

Abstract 4615: Development of an antibody-drug conjugate with broad anticancer activity

Abstract Treatment for patients with advanced solid tumors that include triple negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), and castrate resistant prostate cancer (CRPC), as well as Mantle Cell Lymphoma (MCL), while increasing survival is not curative. We have identified a membrane bound protease, “activated” matriptase, as an attractive target antigen for highly selective antibody delivery of cytotoxins as activated matriptase expression is restricted to epithelial tumors and some B-cell lymphomas. We generated a novel ADC by linking M69, a mouse antibody specific to activated matriptase, to monomethyl auristatin E (MMAE) via a PEGylated, releasable di-peptide linker as a proof-of-principle prototype. Both in cell lines and in human xenograft models of TNBC, NSCLC, CRPC and MCL, the conjugate was found to exhibit potent anticancer activity against all of these tumor types without toxicity. Encouraged by these results, we are also exploring the use of this ADC against gastric and pancreatic cancer, and in combination with chemotherapy and immunotherapy. As this is a mouse antibody, we are also generating a chimeric antibody for toxicity studies in primates. Citation Format: Siang-Yo Lin, Zoltan Szekely, Chen-Yong Lin, Joseph R. Bertino, Gulam Mohmad Rather. Development of an antibody-drug conjugate with broad anticancer activity [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 4615. doi:10.1158/1538-7445.AM2017-4615

Abstract 4046: Targeting jak/stat adaptive mechanism with jak1 inhibitor azd4205 reduces residual disease and prolongs benefit of osimertinib

Abstract EGFR kinase inhibitors have provided tremendous benefit for non-small cell lung cancer (NSCLC) patients with EGFR driver mutations. However, many patients fail to respond or have responses of limited duration. Accumulating evidence suggests that the JAK1/STAT3 axis plays a role in tumor escape, and that combined inhibition of EGFR and JAK1 may drive more complete and durable responses. Here we report AZD4205, a potent ATP-competitive inhibitor that inhibits JAK1 with a Ki of 2.8 nM and exhibits excellent selectivity vs. other JAK family kinases and across the kinome. Combination of AZD4205 (dosed at 12.5, 25 or 50 mg/kg BID, PO) with Osimertinib (doses ranging from 1 to 25 mg/kg QD, PO) led to enhanced in vivo anti-tumor activity in several NSCLC xenograft models driven by mutant EGFR: H1975 (L858R/T790M), PC-9 (del19), and HCC827 (del19). In the H1975 model, after 18 days of treatment beginning with 189mm3 established tumors, treatment with osimertinib at 2.5 mg/kg led to tumor growth inhibition of 92% relative to vehicle. Addition of AZD4205 at 12.5 and 50 mg/kg led to 30% and 69% tumor regression, respectively. AZD4205 dosed at 12.5 and 50 mg/kg BID resulted in pSTAT3 (Y705) knockdown of 72% and 91% at peak plasma concentration (2H) respectively, and 42% and 86% at 8H. In the PC-9 model, after 22 days of treatment beginning with 234mm3 established tumors, osimertinib at 1 mg/kg induced an average 41% regression, and increased to 74% with the addition of 50 mg/kg BID AZD4205. The volume of vehicle treated tumors increased by 3.7-fold over this treatment period. In the HCC827 model, beginning with 127mm3 established tumors, osimertinib at 25 mg/kg induced a complete response in 100% of mice after 11 days of treatment, but tumors regrew to the average starting volume ~24 days after stopping treatment. When 50 mg/kg of AZD4205 was added to the osimertinib treatment, regrowth was delayed by an additional ~7 days. Combination activity was also observed in the PDX (patient-derived xenograft) model LG1049 (del19 and T790M EGFR mutations). Beginning with 189mm3 established tumors, osimertinib at 25 mg/kg induced an average 39% regression after 28 days of treatment, which increased to 56% with the addition of 25 mg/kg AZD4205 on a 4 days on / 3 days off / week schedule. AZD4205 treatment led to 67% knockdown of pSTAT3 after 4H of treatment in this model. Our experiments establish AZD4205 as a potent and selective JAK1 inhibitor with desirable biophysical properties. While additional work is needed to build a patient enrichment strategy, the data generated supports targeting the adaptive JAK1/STAT3 axis with JAK1 inhibitor AZD4205 in clinic as a strategy to reduce residual disease and prolong the benefit of Osimertinib in lung cancer patients with EGFR activating mutations. Citation Format: Huawei (Ray) Chen, Geraldine Bebernitz, Kirsten Bell, Erica Anderson, Nanhua Deng, Jason Kettle, Paul Lyne, Richard Woessner. Targeting jak/stat adaptive mechanism with jak1 inhibitor azd4205 reduces residual disease and prolongs benefit of osimertinib [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 4046. doi:10.1158/1538-7445.AM2017-4046

Abstract 317: Combination of a novel ERK1/2 inhibitor (LY3214996) with CDK4 and CDK6 inhibitor (abemaciclib) enhances antitumor efficacy in KRAS mutant non-small cell lung cancer (NSCLC)

Abstract ERK1/2, a key downstream effector of RAS mutations, is involved in the signaling network which drives cell proliferation, survival, metastasis and cancer resistance to drug treatment (including MEK and BRAF inhibitors). Lung cancer is a leading cause of cancer death worldwide. KRAS mutation present in up to 30% of NSCLC patients is associated with a poor prognosis and represents an unmet medical need. In KRAS mutant NSCLC, enhanced ERK activation cooperates with dysregulation of the cell cycle checkpoint (e.g., cyclin D, CDK4 and CDK6 complex), and contributes to tumor progression; thus, the simultaneous inhibition of ERK and the CDK4/6 pathway is hypothesized to augment tumor growth inhibition. LY3214996, a novel and highly selective small molecule inhibitor of ERK1 and ERK2, is currently in phase I clinical trial and has been shown to inhibit cell proliferation in RAS or BRAF mutant tumor cells in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4 and CDK6-selective inhibitor is currently in phase III studies for ER positive breast cancer and KRAS mutant NSCLC. In this study we explore the potential efficacy of combined inhibition of ERK1/2 and CDK4 and CDK6 in KRAS mutant NSCLC. The combination of LY3214996 and abemaciclib synergistically inhibited cell proliferation in 85% of KRAS mutant cells in an unbiased NSCLC panel. Combination treatment with LY3214996 and abemaciclib significantly decreased levels of phospho- p90RSK, phospho-Rb, phospho-S6 and Ki67; and synergistically inhibited cell proliferation and survival in KRAS mutant NSCLC cell lines including NCI-H2122 (G-12C), A549 (G-12S) and NCI-H441 (G-12V). Subsequent in vivo studies showed that the combination treatment with LY3214996 and abemaciclib was well tolerated and led to more robust tumor growth inhibition or regression in all KRAS mutant NSCLC xenograft models (H2122, A549 and H441) compared with either single agent treatment (p≤0.002). Furthermore, in xenograft tumors the combination of LY3214996 and abemaciclib resulted in more significant reduction of phospho-p90RSK, phospho-Rb, phospho-S6 and Ki67 in H2122 tumors compared with either single agent. Overall, the combined inhibition of ERK1/2 and CDK4 and CDK6 was tolerated and enhanced antitumor efficacy in several KRAS mutant NSCLC preclinical models. These data support the feasibility of combining ERK inhibitor LY3214996 with CDK4 and CDK6 inhibitor abemaciclib as a promising strategy for the treatment of KRAS mutant NSCLC patients, and provides the rationale for the combination study in the on-going phase I LY3214996 clinic trial (NCT02857270). Citation Format: Wenjuan Wu, Shripad V. Bhagwat, Constance King, Susan Pratt, Xueqian Gong, Julie Stewart, Bonita Jones, Robert Flack, Richard Beckman, Beverly Falcon, Jason Manro, William T. McMillen, Ramon V. Tiu, Sheng-Bin Peng, Christoph Reinhard, Sajan Joseph, Sean Buchanan. Combination of a novel ERK1/2 inhibitor (LY3214996) with CDK4 and CDK6 inhibitor (abemaciclib) enhances antitumor efficacy in KRAS mutant non-small cell lung cancer (NSCLC) [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 317. doi:10.1158/1538-7445.AM2017-317

Bevacizumab counteracts VEGF-dependent resistance to erlotinib in an EGFR-mutated NSCLC xenograft model.

Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), shows superior efficacy in patients with non-small cell lung cancer (NSCLC) harboring activating EGFR mutations (EGFR Mut+). However, almost all tumors eventually develop resistance to erlotinib. Recently, the Phase II JO25567 study reported significant prolongation of progression-free survival (PFS) by erlotinib plus bevacizumab combination compared with erlotinib in EGFR Mut+ NSCLC. Herein, we established a preclinical model which became refractory to erlotinib after long-term administration and elucidated the mode of action of this combination. In this model, tumor regrowth occurred after remarkable shrinkage by erlotinib; regrowth was successfully inhibited by erlotinib plus bevacizumab. Tumor vascular endothelial growth factor (VEGF) was greatly reduced by erlotinib in the erlotinib-sensitive phase but significantly increased in the erlotinib-refractory phase despite continued treatment with erlotinib. Although EGFR phosphorylation remained suppressed in the erlotinib-refractory phase, phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT, and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) were markedly higher than in the erlotinib-sensitive phase; among these, pERK was suppressed by erlotinib plus bevacizumab. MVD was decreased significantly more with erlotinib plus bevacizumab than with each drug alone. In conclusion, the erlotinib plus bevacizumab combination demonstrated promising efficacy in the B901L xenograft model of EGFR Mut+ NSCLC. Re-induction of VEGF and subsequent direct or indirect VEGF-dependent tumor growth was suggested as a major mechanism of erlotinib resistance, and erlotinib plus bevacizumab achieved remarkably prolonged antitumor activity in this model.