Lung Tumor Xenograft Model Research Articles

Abstract 513: Suppressing YBX1 with a small molecule inhibitor sensitizes lung cancer to chemotherapy

Abstract Y-box binding protein-1 (YBX1) is a multifunctional protein that regulates genes by binding to their DNA and/or RNA. YBX1 is known to contribute to tumorigenesis, disease progression and resistance by stabilizing the mRNAs of genes that are involved in cell growth and survival. High expression of YBX1 has been associated with poor clinical outcomes and decreased overall survival in over 20 cancer types including lung cancer. Given its critical roles in cancer progression and significant correlation with poor cancer prognosis, we hypothesized that inhibition of YBX1 may sensitize the cancer cells to chemotherapeutic agents. To profile the relationship between YBX1 and chemo-response, we aim to investigate the synergistic effect of YBX1 inhibition with standard of care chemotherapy drugs including cisplatin, in lung cancer using animal models and omics analysis. Our studies have found that SU056, a small molecule YBX1 inhibitor, effectively inhibits YBX1 expression in human lung adenocarcinoma cell lines and halts their cell proliferation and clonogenic potential, whereas overexpression of YBX1 promotes cell proliferation. SU056-mediated YBX1 inhibition also led to G2/M cell cycle arrest in these cells followed by apoptotic cell death. These results were further confirmed in A549 lung tumor xenograft model, where inhibitor treatment delayed tumor progression. We will present collective data from in vitro and in vivo studies with YBX1 inhibitor, combination studies with known chemotherapeutic drugs and omics analysis to suggest that targeting YBX1 has therapeutic potential to increase the efficacy of chemotherapeutic agents. Citation Format: Jee Min Lee, Sanjay Malhotra. Suppressing YBX1 with a small molecule inhibitor sensitizes lung cancer to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 513.

MiR-29a-3p inhibits the malignant characteristics of non-small cell lung cancer cells by reducing the activity of the Wnt/β-catenin signaling pathway.

MicroRNAs (miRNAs) can influence non-small cell lung cancer (NSCLC) in a tumor-suppressive and oncogenic manner. The present study aimed to investigate the effects and underlying mechanisms of miR-29a-3p in NSCLC. NSCLC cell lines (A549, H1299, and H460) and a normal lung epithelial cell line (BEAS-2B) were used. Additionally, a mouse lung tumor xenograft model was established using A549 cells and used to determine the effects of miR-29a-3p on NSCLC in vivo. Tumor volumes were measured every week. The expression of miR-29a-3p in cells and lung tissues were detected by RT-qPCR. Cell proliferation was detected using Cell Counting Kit-8 and EdU assays. Migration and invasion were assessed using wound healing and Transwell invasion assays, respectively. Ki-67 expression was detected using immunohistochemical staining. The expression levels of Wnt3a and β-catenin were determined using western blotting. miR-29a-3p expression was significantly downregulated in NSCLC cells and mice. In contrast to miR-29a-3p knockdown, miR-29a-3p overexpression decreased NSCLC cell proliferation, migration, and invasion as well as tumor growth in in the NSCLC mouse model. Moreover, miR-29a-3p overexpression decreased the protein expression levels of Wnt3a and β-catenin. The inhibitory effects of miR-29a-3p on NSCLC cells were reversed by LiCl (an activator of the Wnt signaling pathway). In conclusion, miR-29a-3p prevented NSCLC tumor growth and cell proliferation, migration, and invasion by inhibiting the Wnt/β-catenin signaling pathway. This finding offers novel insights into the prognosis and treatment of NSCLC.

Abstract P240: Benchmarking the novel dual-MEK inhibitor, IMM-1-104, head-to-head and in combination with sotorasib (AMG-510) in the MIA PaCa-2 (KRAS-G12C) pancreatic cancer xenograft model

Abstract Background: Approximately 9 out of 10 patients diagnosed with pancreatic cancer have tumors driven by a single activating mutation in KRAS. These tumors remain unaddressable by current therapies and collectively represent a high unmet clinical need. KRAS is the most commonly mutated form of RAS and represents approximately 85% of all RAS mutant human tumors with HRAS and NRAS accounting for the remaining 15%. Sotorasib (AMG-510), a covalent KRAS-G12C inhibitor, recently received accelerated regulatory approval for KRAS-G12C lung cancer in the United States. While the G12C mutation has been documented in up to 15% of all KRAS mutant tumors, it is rarely observed (<1%) in human pancreatic cancer, which could severely limit this type of new inhibitor in certain histologies. IMM-1-104 is a novel dual-MEK inhibitor that resists CRAF-bypass (goal: improve activity against RAS mutant tumors), and is designed to have a short plasma drug half-life, which leads to deep, cyclic inhibition of the MAPK pathway (goal: improved tolerability). Materials & Methods: The novel, orally bioavailable dual-MEK inhibitor, IMM-1-104, was evaluated for antitumor activity, as a single agent (50, 100, 150 mg/kg BID p.o.) in a head-to-head format against sotorasib (10, 30, 100 mg/kg QD p.o.) or in combination (50, 100, 150 mg/kg BID p.o. IMM-1-104 with 30 mg/kg QD p.o. sotorasib) in the MIA PaCa-2 KRAS-G12C mutant pancreatic tumor xenograft model. Tumor-bearing animals were treated for 21 days of oral dosing, before drug treatments were terminated and animals monitored for additional 3 weeks. Results: IMM-1-104 and sotorasib were well-tolerated as single agents and in combination with each other across all dose levels tested. Both drugs demonstrated dose-dependent antitumor activity with tumor regressions noted at higher dose levels. IMM-1-104 in combination with sotorasib produced deep regressions that were sustained longer than either drug alone. Conclusions: The dual-MEK mechanism and short half-life of IMM-1-104 has demonstrated broad activity with low toxicity across multiple RAS and RAF mutant preclinical tumor models. Tumor regressions were observed in NRAS mutant melanoma (SK-MEL-2) and KRAS mutant colorectal (Colon-26) rodent tumor models, and tumor stasis was observed in KRAS mutant lung (A549) and BRAF mutant melanoma (A375) tumor xenograft models. Here, we report that IMM-1-104 treatment resulted in tumor regressions similar to that observed for sotorasib in the recently benchmarked KRAS-G12C mutant pancreatic cancer xenograft model (MIA PaCa-2). IMM-1-104 in combination with sotorasib promoted deep, durable tumor regressions, when compared to either drug alone. Therefore, future drug-drug combinations with upstream inhibitors such as sotorasib may afford greater durability in combination for patients with KRAS-G12C and other select tumor types. In totality, these data suggest the potential for broad, single agent activity of IMM-1-104 in tumors with inappropriately elevated MAPK signaling, including a large percentage of KRAS mutant pancreatic cancer. Citation Format: Peter J. King, Amy E. Axel, Kevin D. Fowler, Sarah E. Kolitz, Scott Barrett, Benjamin J. Zeskind, Brett M. Hall. Benchmarking the novel dual-MEK inhibitor, IMM-1-104, head-to-head and in combination with sotorasib (AMG-510) in the MIA PaCa-2 (KRAS-G12C) pancreatic cancer xenograft model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P240.

The Pro-Survival Oct4/Stat1/Mcl-1 Axis Is Associated with Poor Prognosis in Lung Adenocarcinoma Patients.

The embryonic stem cell marker Oct4 is expressed in several human cancers and is positively correlated with a poor outcome in cancer patients. However, its physiological role in cancer progression remains poorly understood. Tumor cells block apoptosis to escape cell death so that they can proliferate indefinitely, leading to ineffective therapy for cancer patients. In this study, we investigated whether Oct4 regulates the apoptosis pathway and contributes to poor prognosis in patients with lung adenocarcinoma. Our results revealed that Oct4 expression is correlated with Stat1 expression in lung adenocarcinoma patients and Oct4 is directly bound to the Stat1 promoter to transactivate Stat1 in lung adenocarcinoma cells. Expression of the Stat1 downstream gene Mcl-1 increased in Oct4-overexpressing cancer cells, while Stat1 knockdown in Oct4-overexpressing cancer cells sensitized them to cisplatin-induced apoptosis. Furthermore, Oct4 promoted Stat1 expression and tumor growth, whereas silencing of Stat1 reduced Oct4-induced tumor growth in human lung tumor xenograft models. Taken together, we demonstrate that Oct4 is a pro-survival factor by inducing Stat1 expression and that the Oct4/Stat1/Mcl-1 axis may be a potential therapeutic target for lung adenocarcinoma.

Formylated honokiol analogs showed antitumor activity against lung carcinoma.

Honokiol, a biphenolic neolignan with inappreciable toxicity isolated from Magnolia officinalis, has been reported to have antiangiogenic and antitumor properties in several tumor cell lines and tumor xenograft models. In our previous study, structural modification by chemical synthesis has been carried out to develop novel honokiol derivatives to improve antitumor activity and clarify the structure-activity relationship. Honokiol analogs, especially 3,5'-diformylated honokiol HK-(CHO)2, have been found to moderately block the newly grown segmental vessels from the dorsal aorta in the transgenic zebrafish-based assay, show antiangiogenic property, and exert medium cytotoxicity against two lung cell lines (Lewis lung carcinoma LL/2 cells and human non-small-cell lung cancer A549 cells). However, the in-vitro and in-vivo antitumor effects of formylated honokiol derivatives against lung carcinoma remained poorly understood. In the study, two formylated honokiol derivatives also showed potent antitumor effects in the Lewis lung carcinoma cells, K-ras-dirived lung adenocarcinoma mice, and a mouse lung tumor xenograft model, with HK-(CHO)2 being most efficacious. The potential mechanism was inhibiting cell proliferation and inducing apoptosis in lung cancer by the regulation of vascular endothelial growth factor A expression. These results further suggested that HK-(CHO)2 might be potential candidates for the treatment of lung carcinoma.

Selective small-chemical inhibitors of protein arginine methyltransferase 5 with anti-lung cancer activity.

Protein arginine methyltransferase 5 (PRMT5) plays critical roles in a wide variety of biological processes, including tumorigenesis. By screening a library of small chemical compounds, we identified eight compounds that selectively inhibit the PRMT5 enzymatic activity, with IC50 values ranging from 0.1 to 6 μM. Molecular docking simulation and site-directed mutagenesis indicated that identified compounds target the substrate-binding site in PRMT5. Treatment of lung cancer cells with identified inhibitors led to inhibition of the symmetrical arginine methylation of SmD3 and histones and the cellular proliferation. Oral administration of the inhibitor demonstrated antitumor activity in a lung tumor xenograft model. Thus, identified PRMT5-specific small-molecule inhibitors would help elucidate the biological roles of PRMT5 and serve as lead compounds for future drug development.

Abstract 1167: Development of BT1718, a novel Bicycle Drug Conjugate for the treatment of lung cancer

Abstract BT1718 is a Bicycle Drug Conjugate (BDC®) comprising a constrained bicyclic peptide (Bicycle®) that binds with high affinity and specificity to membrane type 1-matrix metalloprotease (MT1-MMP; MMP14) covalently linked through a hindered disulfide linker to the potent anti-tubulin agent DM1. MT1-MMP is involved in normal tissue remodeling and is also expressed in tumor associated stromal cells. However, overexpression is linked to increased tumor aggression and metastasis. Specifically, over-expression of MT1-MMP is also associated with poor clinical prognosis and shorter survival times in patients with NSCLC and a range of other solid tumors. The Bicycle MT1-MMP binding element within BT1718 was identified using a proprietary phage display peptide technology consisting of highly diverse phage libraries of linear amino acid sequences constrained into two loops by a central tri-functional chemical scaffold. These MT1-MMP binding Bicycles exhibit a profound affinity and specificity, more often associated with monoclonal antibodies, whilst their low molecular weight (1.5-3 kDa), akin to that of a small molecule, aids in rapid extravasation and tumor penetration. Together these attributes make Bicycles an ideal format for the targeted delivery of cytotoxic payloads. We evaluated the ability of BT1718 to bind to and kill tumor cells in vitro and in vivo in a panel of tumor cells. BT1718 demonstrated MT1-MMP target-specific binding and MT1-MMP-dependent cell killing of lung tumor cells in vitro as well as efficacy across a panel of lung tumor xenograft mouse models. For example, in the Met-amplified squamous NSCLC lung EBC-1 model, complete regressions were observed in all mice at doses as low as 5 mg/kg (iv) twice weekly and across a range of other dosing schedules, from daily to weekly. MT1-MMP-dependent activity was demonstrated by blocking target specific interactions through co-administration of an excess of unconjugated Bicycle binder, which inhibited tumour regression, or a non-binding Bicycle, which had no effect. Further evaluation in patient-derived lung xenograft (PDX) models indicates a similar activity to that seen in cell-line derived xenografts, with efficacy seen from 3mg/kg twice weekly and rapid full regression of tumors at higher doses. The molecular attributes of these Bicycles: rapid tumor penetration and specific binding, makes them ideal therapeutics for targeted delivery of toxins as Bicycle drug conjugates (BDCs). The small size of the BDC may offer a significant advantage to other targeted cytotoxic approaches such as antibody-drug conjugates due to rapid extravasation and improved tumor penetration. BT1718, a Bicycle Drug Conjugate, shows potent anti-tumor activity in human lung tumor xenograft models and IND-enabling studies are underway. Citation Format: Gavin Bennett, Robert Lutz, Peter Park, Helen Harrison, Kevin Lee. Development of BT1718, a novel Bicycle Drug Conjugate for the treatment of lung cancer [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 1167. doi:10.1158/1538-7445.AM2017-1167

Azithromycin effectively inhibits tumor angiogenesis by suppressing vascular endothelial growth factor receptor 2-mediated signaling pathways in lung cancer.

Tumor angiogenesis is essential during lung cancer development and targeting angiogenesis may possess a potential therapeutic value. The present study demonstrates that azithromycin, a Food and Drug Administration-approved antibiotic drug, is a novel tumor angiogenesis inhibitor. Azithromycin inhibits capillary network formation of human lung tumor associated-endothelial cells (HLT-ECs) in vitro and in vivo. It significantly inhibits HLT-EC adhesion and vascular endothelial growth factor (VEGF)-induced proliferation of HLT-ECs in a dose-dependent manner without affecting migration. In addition, azithromycin induces apoptosis of HLT-ECs even in the presence of VEGF. Notably, azithromycin inhibits proliferation and induces apoptosis in multiple lung cancer cell lines to a significantly reduced extent compared with in HLT-ECs, suggesting that HLT-ECs are more susceptible to azithromycin treatment. In a lung tumor xenograft model, azithromycin significantly inhibits tumor growth and its anti-tumor activities are achieved by suppressing angiogenesis. Notably, the inhibitory effects of azithromycin on angiogenesis are associated with its ability to suppress VEGF-induced activation of VEGF receptor 2 (VEGFR2), phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), focal adhesion kinase, and disruption of focal adhesion assembly and actin stress fiber formation in HLT-ECs. The present study identifies that azithromycin targets VEGFR2-mediated focal adhesion and PI3K/Akt signaling pathways in HLT-ECs, leading to the suppression of angiogenesis and lung tumor growth.

Improved antitumor activity and reduced toxicity of doxorubicin encapsulated in poly(ε-caprolactone) nanoparticles in lung and breast cancer treatment: An in vitro and in vivo study

Poly(ε-caprolactone) (PCL) nanoparticles (NPs) offer many possibilities for drug transport because of their good physicochemical properties and biocompatibility. Doxorubicin-loaded PCL NPs have been synthesized to try to reduce the toxicity of doxorubicin (DOX) for healthy tissues and enhance its antitumor effect in two tumor models, breast and lung cancer, which have a high incidence in the global population. PCL NPs were synthesized using a modified nanoprecipitation solvent evaporation method. The in vitro toxicity of PCL NPs was evaluated in breast and lung cancer cell lines from both humans and mice, as was the inhibition of cell proliferation and cell uptake of DOX-loaded PCL NPs compared to free DOX. Breast and lung cancer xenografts were used to study the in vivo antitumor effect of DOX-loaded NPs. Moreover, healthy mice were used for in vivo toxicity studies including weight loss, blood toxicity and tissue damage. The results showed good biocompatibility of PCL NPs in vitro, as well as a significant increase in the cytotoxicity and cell uptake of the drug-loaded in PCL NPs, which induced almost a 98% decrease of the IC50 (E0771 breast cancer cells). Likewise, DOX-loaded PCL NPs led to a greater reduction in tumor volume (≈36%) in studies with C57BL/6 mice compared to free DOX in both lung and breast tumor xenograft models. Nevertheless, no differences were found in terms of mouse weight. Only in the lung cancer model were significant differences in mice survival observed. In addition, DOX-loaded PCL NPs were able to reduce myocardial and blood toxicity in mice compared to free DOX. Our results showed that DOX-loaded PCL NPs were biocompatible, enhanced the antitumor effect of DOX and reduced its toxicity, suggesting that they may have an important potential application in lung and breast cancer treatments.

Oncogenic Potential of CYP24A1 in Lung Adenocarcinoma

IntroductionWe have previously demonstrated that a subset of lung cancer cells express higher CYP24A1 mRNA, a metabolizing enzyme for 1,25-D3, compared to benign tumors or surrounding normal lung and that high CYP24A1 mRNA expression is associated with poor prognosis in resected lung adenocarcinoma (AC). We hypothesized that CYP24A1 has oncogenic potential and increased CYP24A1 expression may contribute to tumor growth, whereas, CYP24A1 targeting may reduce tumor burden. MethodsTwo low CYP24A1 expressing human lung cancer cell lines (SK-LU-1 and Calu-6) were stably transfected either with an empty lentiviral vector or with the CYP24A1 expressing vector. Over-expression of mRNA and protein levels of CYP24A1 in SK-LU-1 and Calu-6 were confirmed using qRT-PCR and immunoblotting respectively. Next, effects of targeting CYP24A1 were examined in lung cancer cells (A549 and H441), which express higher basal levels of CYP24A1. Finally, we studied the effects of stable knockdown of CYP24A1 in xenograft models. ResultsOver-expression of CYP24A1 correlated with accelerated cell growth and invasion compared to control vector-transfected cells. CYP24A1 over-expression also increased RAS protein expression. Knockdown of CYP24A1 using either si- or shRNA reduced CYP24A1 mRNA and protein expression and significantly decreased cell proliferation (30-60%) and reduced mitochondrial DNA content compared to non-targeting (NT) si-/shRNA transfected/transduced cells. Transfection with CYP24A1 siRNA also decreased total RAS protein, thus reducing phosphorylated AKT. Importantly, stable knockdown of CYP24A1 in A549 and H441 lung tumor xenograft models resulted in tumor growth delay and smaller tumor size as evident from tumor bioluminescence and tumor volume measurement studies. Such observations were correlated with decreased tumor cell proliferation as evidenced by reduced Ki67 and Cyclin D staining. ConclusionsOur data suggest that CYP24A1 has oncogenic properties mediated by increasing RAS signaling, targeting of which may provide an alternate strategy to treat a subset of lung AC.

Abstract PR07: Characterization of human effectors in the tumor microenvironment of NSCLC patients and lung tumor xenograft models

Abstract Non-small cell lung cancer (NSCLC) is one the most prevalent forms of lung cancer. Lung tumors consist of a heterogeneous population of tumor cells, immune cells and stromal cells. It is critical to assess the nature and localization of immune effector cells and their expression of immune checkpoint molecules to develop more effective therapeutic strategies. In this study, we used immunohistochemistry (IHC) to assess the localization of human immune cells and expression of immune checkpoint molecules to define the microenvironment composition. Furthermore, we developed a mouse xenograft model of human lung tumor using BLT-NSG mice with a reconstituted human immune system and A549 cells. Formalin-fixed paraffin embedded NSCLC lung tumor sections (n=28 adenocarcinoma, n=16 squamous) were processed by IHC to assess expression and localization of CD4, CD8, MPO, Foxp3, CD68 and cytokeratin, as well as immune checkpoint molecules (PD-1, Tim-3, LAG-3) and ligands (PD-L1 and VISTA). For the xenograft model, BLT-NSG mice (NSG mice transplanted with fetal liver CD34+ cells and autologous thymus) at 12-16 weeks post-transplant were injected with A549-luciferase (A549-Luc) cells via transpleural or intravenous injection. Weekly bioluminescence imaging was done to assess tumor growth. Lungs and spleens were harvested and enzymatically dissociated for flow cytometric analysis of human effectors and immune checkpoint molecules. We found that significantly more human effectors are localized to the stromal compartment (cytokeratin-) than within the cytokeratin+ tumor nests. The stromal compartment contained 12-fold more CD8+ T-cells, 23-fold more CD4+ T-cells, and 11-fold more regulatory T-cells (Foxp3+). Two-fold more neutrophils (MPO+) and macrophages (CD68+) were found in stroma outside of tumor nests. There were significantly more CD8+ T-cells (p<0.05) and MPO+ neutrophils (p<0.01) localized within the cytokeratin+ areas of squamous compared to adeno tumors. PD-1, Tim-3 or LAG-3 IHC showed that 100-fold more PD-1+ cells are found within the tertiary lymphoid structures in the stromal compartment. 19-fold more Tim-3+ cells, both lymphocytes and macrophages, are found in the stromal compartment. LAG-3+ cells are lymphocytes, and localized mostly to the stromal compartment (11-fold more than in cytokeratin+ areas). PD-L1 IHC showed that 12/16 (75%) of squamous tumors and 14/28 (50%) of adeno tumors express PD-L1 on tumor cells, and H-Scores of the PD-L1 staining from squamous tumors were significantly higher than the adeno tumors (p=0.019). A majority of macrophages expressed PD-L1, but the expression level varied from sample to sample (1+ to 3+ scoring). VISTA IHC showed that lymphocytes, neutrophils and macrophages expressed VISTA, and 26-fold more VISTA+ cells are found in the stromal compartment. In our A549 xenograft model study, NSG and BLT-NSG mice with IV-injected A549 cells showed tumor growth differences by bioluminescence imaging. Flow cytometric analyses showed an increased percentage of non-naïve CD8+ (88%) and non-naïve CD4+ (97%) cells compared to spleens (44% non-naïve CD8+ cells and 74% non-naïve CD4+ cells). There was an increased expression of LAG-3 in both human CD8+ (19-90%) and human CD4+ (9-61%) non-naïve T-cells in the lungs, but LAG-3+ cells in the spleen were minimal (<6%). PD-1 expression was high in non-naïve T-cell population in both lungs and spleen (>70%). In addition, there was an up-regulation of PD-L1+(4%) and VISTA+ (12%) in the human CD14+ population in the lungs. In conclusion, the expression of various immune checkpoint molecules in effector cells may limit their localization within the stromal compartment. Furthermore, our xenograft model was able to recapitulate our observations with human patient samples, and would be a valuable in vivo mouse tool to test new therapeutic agents. Citation Format: Hyun-Jeong Ra, Gisela Brake-Silla, Evgeniy Eruslanov, Michael Sharp, Charuhas Deshpande, Michael D. Feldman, Amy Ziober, Li-Ping Wang, Manuel A. Sepulveda, Linda A. Snyder, Gwenn Danet-Desnoyers. Characterization of human effectors in the tumor microenvironment of NSCLC patients and lung tumor xenograft models. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR07.

Abstract 2142: Novel oncogenic BRaf deletions functioning as BRaf homodimer and sensitive to inhibition by LY3009120, a pan Raf and Raf dimer inhibitor

Abstract BRAF mutations, particularly the somatic hot spot BRAF V600E mutation, were discovered as major oncogenic mutations in many cancer types. It was shown that BRAF V600E is a potent oncogene that activates the MAPK pathway and functions as a BRaf monomer. BRaf selective inhibitors, vemurafenib and dabrafenib, which are effective in inhibiting the kinase activity of BRaf monomer, have demonstrated robust anti-tumor activities in BRAF mutant xenograft models and significant clinical benefit among BRAF mutant melanoma patients. In this study, we have identified and characterized novel BRaf aberrant variants, which have in-frame deletions within or adjacent to the L485-P490 region in patient samples and/or cell lines of lung, pancreatic, and ovarian cancers. Tumor cells with these endogenous BRaf deletions are resistant to BRaf monomer inhibitor vemurafenib based on inhibition of phospho-MEK and phospho-ERK, cell proliferation, and cell cycle progression. However, these cells are sensitive to LY3009120, a pan Raf and Raf dimer inhibitor. Further analysis using siRNA showed that the MEK-ERK activity in these cells is mainly dependent on BRaf, not CRaf or ARaf. Ectopical expression of the L485-P490 deleted BRaf in mouse NIH3T3 cells is able to transform the cells and form colonies comparable to BRaf V600E mutation in three-dimensional soft agar growth. More importantly, the Raf dimer disrupting mutation BRafR509H abolished the transforming activity of the L485-P490 deleted BRaf, suggesting that this BRaf deletion functions as a dimer. Further, ectopical expression of the L485-P490 deleted BRaf promotes primarily BRaf homodimerization as revealed by proximity ligation assays (PLA). It was also confirmed by PLA that BRaf homodimer is the dominant form of Raf dimers in tumor cells harboring these BRaf deletions. In lung and pancreatic tumor xenograft models developed with tumor cells with these BRaf deletions, LY3009120 treatment demonstrated significant tumor growth inhibition and regression, whereas vemurafenib treatment showed no in vivo activity. Overall, we have identified novel oncogenic BRaf deletions that function as BRaf homodimer and are sensitive to pan Raf and Raf dimer inhibitor LY3009120. Citation Format: Shih-Hsun Chen, Sean Buchanan, Youyan Zhang, Robert Van Horn, Tinggui Yin, Vipin Yadav, Swee Seong Wong, Lysiane Huber, James Henry, Ilaria Conti, James J. Starling, Gregory D. Plowman, Sheng-Bin Peng. Novel oncogenic BRaf deletions functioning as BRaf homodimer and sensitive to inhibition by LY3009120, a pan Raf and Raf dimer inhibitor. [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 2142. doi:10.1158/1538-7445.AM2015-2142

Abstract 672: ABTL0812, a new antitumor drug that inhibits the axis Akt/mTOR through a novel mechanism of action

Abstract Background: ABTL0812 is a first-in-class orally administered compound currently in Phase I/Ib First in Human Clinical Trial in patients with advanced solid tumors (NCT02201823). ABTL0812 has cytotoxic effect on a wide range of human tumor cell lines, including those which have become resistant to standard therapy. We hereby dissect the anti-tumor activity of ABTL0812, which relies on a novel mechanism of action that promotes inhibition of the Akt/mTOR axis in cancer cells. Material & methods: ABTL0812 molecular targets were identified by in silico analysis, comparing ABTL0812 chemical structure against a database including more than one million receptor-ligand interaction data. Functional relevance of the targets was confirmed biochemically and pharmacologically. ABTL0812 mechanism of action was established using human lung and pancreatic tumor cells, MEF KO cells, as well as tumor xenografts. Results: In silico screening showed that ABTL0812 binds four targets which regulate tumor progression through Akt/mTOR axis. Two of them are the transcription factors PPARα and PPARγ (Peroxisome-Proliferator Activating Receptors). In lung and pancreatic tumor cells ABTL0812 activated PPARα/γ-dependent gene transcription, while pharmacological inhibition with PPARα/γ antagonists impaired ABTL0812 cytotoxic effect. Interestingly, ABTL0812 induced transcription of the endogenous Akt inhibitor TRIB3 (tribbles homologue 3) through PPARα/γ activation. TRIB3 is a pseudokinase that inhibits Akt by direct binding and preventing its phosphorylation by mTORC2 complex. According to this, ABTL0812-induced TRIB3 overexpression resulted in inhibition of Akt phosphorylation, impaired phosphorylation of the Akt substrates TSC2 and PRAS40 and mTORC1 inhibition (pS6), which in turn promoted autophagy-mediated tumor cell death. MEF TRIB3-/- cells were resistant to ABTL0812-induced cell death, indicating that TRIB3 mediates ABTL0812 citotoxicity. Finally, Akt inhibition was observed in human lung and pancreatic tumor xenograft models treated with ABTL0812 and in human platelets incubated with ABTL0812. This supported the rational for using Akt phosphorylation as a pharmacodynamic biomarker to monitor activity of ABTL0812 in patients included in the Clinical Trial. Conclusion: ABTL0812 promotes autophagy-mediated cell death in different cancer paradigms by inhibiting the Akt/mTOR axis through a novel mechanism of action. ABTL0812 induces PPARα/γ-mediated transcription of the TRIB3 gene. Upregulated TRIB3 protein binds and inhibits Akt, leading to mTORC1 inhibition and reduction of tumor growth. These findings provide evidences that ABTL0812 may be an effective therapeutic strategy for targeting cancer. Citation Format: Tatiana Erazo, Mariana Gomez-Ferreria, Jose Alfon, Mar Lorente, Maria Salazar, Anna Lopez, Marc Cortal, Pau Munoz-Guardiola, Pedro Gascon, Guillermo Velasco, Carles Domenech, Jose M. Lizcano. ABTL0812, a new antitumor drug that inhibits the axis Akt/mTOR through a novel mechanism of action. [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 672. doi:10.1158/1538-7445.AM2015-672

A novel, soluble compound, C25, sensitizes to TRAIL-induced apoptosis through upregulation of DR5 expression.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential therapeutic agent that induces apoptosis selectively in tumor cells. However, numerous solid tumor types are resistant to TRAIL. Sensitization to TRAIL has been an area of great research interest, but has met significant challenges because of poor bioavailability, half-life, and solubility of sensitizing compounds such as curcumin. Soluble, TRAIL-sensitizing compounds were screened on the basis of similarity to the redox-active substructure of curcumin and sensitization to TRAIL-induced apoptosis. We determined the effect of the lead compound, C25, in combination with TRAIL in human cancer cell lines using MTS proliferation assays, apoptosis assays, and western blotting. Short hairpin RNA knockdown of death receptor 5 (DR5) was performed to determine whether DR5 upregulation was required for TRAIL-mediated apoptosis. In-vivo efficacy was determined using human lung tumor xenograft models. C25 helped overcome TRAIL resistance by upregulating the expression of the TRAIL receptor DR5 and apoptosis in several tumor cell lines. Blockade of DR5 expression abrogated C25 sensitization to TRAIL, demonstrating the requirement for DR5 upregulation for C25-mediated potentiation of TRAIL-mediated apoptosis. The combination of C25 and TRAIL effectively inhibited tumorigenesis in vivo. This study demonstrates the synergistic efficacy of C25 in sensitization to TRAIL-induced apoptosis in multiple tumor cell types, including highly resistant lung and ovarian tumor cell lines. Furthermore, C25 was efficacious against tumor growth in vivo. Thus, C25 may be a potential therapeutic for cancer in combination with TRAIL or DR5 agonist therapy.

Abstract 4626: Evaluating the pharmacodynamics and pharmacokinetic effects of MM-398, a nanoliposomal irinotecan (nal-IRI) in subcutaneous xenograft tumor models of human squamous cell carcinoma and small cell lung cancers

Abstract Background: MM-398 is a novel nanoliposomal encapsulation of irinotecan (nal-IRI), a topoisomerase I inhibitor. In preclinical studies, nal-IRI has been shown to greatly modify the pharmacokinetics and biodistribution of CPT-11 and its active metabolite, SN-38, thereby improving its activity. In this report, we evaluate the in vivo activity of nal-IRI in the two xenograft models of lung cancer. A pharmacodynamics (PD) study was performed to measure the drug activation and deposition parameters in these tumor models. In addition we investigated the effects of nal-IRI on tumor growth, tumor-associated macrophages (TAM's), vasculature, cell proliferation, and apoptosis. Methods: Xenograft models of subcutaneous H157 squamous cell carcinoma (SCC) and H841 small cell lung cancer (SCLC) were established in mice. A short pharmacodynamics (PD) study was performed, wherein, 24 hours after a single dose, animals were euthanized and tumors collected. PD analysis included profiling for carboxylesterase (CES) levels, vasculature (CD31), macrophage (F4/80), and metabolite (CPT-11 and SN-38) levels. For the tumor activity study, animals (5 per group) were treated by weekly i.v. injections with placebo liposome, free irinotecan at 25 mg/kg/wk, or nal-IRI at 30 and 50 mg/kg/wk for three weeks. Tumor volumes were measured with digital calipers. IHC analysis was performed for TAM content, tumor proliferation (Ki67), apoptosis, and vasculature. Results: (1) The carboxylesterase enzyme (activation) and CPT-11 (deposition) tumor levels resulted in extended intratumor SN38 duration, as predicted by the model simulation; (2) nal-IRI suppressed H157 tumor growth in a dose dependent manner, much more efficiently than free irinotecan. On day 25, nal-IRI, at 30 and 50 mg/kg/wk, inhibited tumor growth by 92.6% and 96.3%, respectively, when compared with placebo liposome. In contrast, free irinotecan inhibited tumor growth by 55.7%; (3) For the SCLC H841 xenograft, on day 35, nal-IRI at 30 and 50 mg/kg/wk, also inhibited tumor growth by 84.9% and 93.4% respectively, greater than free irinotecan by 32.7%, when compared with placebo liposome. No obvious toxicities or weight loss were noted in the nal-IRI treated groups; (4) TAM levels were significantly (p<0.05) higher in tumors treated with nal-IRI (30mg/kg and 50mg/kg), as compared to control or free irinotecan treated tumors. Conclusion: nal-IRI inhibited tumor growth in lung tumor xenograft models, suggesting the treatment of human SCC and SCLC, in which there are high unmet medical needs, as a potential target for clinical investigation. Citation Format: Daniel CF Chan, Ashish Kalra, Zhiyong Zhang, Nancy Paz, Dmitri Kirpotin, Daryl Drummond, Ulrik Nielsen, Paul A. Bunn, Jonathan Fitzgerald. Evaluating the pharmacodynamics and pharmacokinetic effects of MM-398, a nanoliposomal irinotecan (nal-IRI) in subcutaneous xenograft tumor models of human squamous cell carcinoma and small cell lung cancers. [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 4626. doi:10.1158/1538-7445.AM2014-4626

Abstract B120: Preclinical assessment of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC.

Abstract Non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations initially respond well to first generation reversible EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by acquired resistance, attributable in 60% of cases to a secondary mutation of EGFR at T790M. In addition, patients also experience skin and GI toxicity due to wild type (WT) EGFR inhibition. CO-1686 is a novel, irreversible and orally delivered kinase inhibitor that specifically targets the most frequently observed front-line and acquired resistance mutant forms of EGFR, while exhibiting minimal activity towards WT EGFR. Selectivity of CO-1686 was demonstrated using two separate biochemical assays in which CO-1686 was at least 10-fold more selective for mutant EGFR as compared to the WT receptor. In human cancer cell lines, CO-1686 potently inhibited in vitro proliferation of cells expressing mutant EGFR, while minimally affecting proliferation of WT EGFR-dependent cells. To confirm this selectivity in vivo, CO-1686 was evaluated in WT (A431), front line (HCC827, transgenic), and second line (NCI-H1975, LUM1868, transgenic) xenograft models. CO-1686 dosed at 50 mg/kg BID exhibited a minimal reduction in tumor growth in the A431 xenograft model with no significant alterations in body weight and EGFR WT pathway signaling. Comparatively, both erlotinib (75 mg/kg QD) and afatinib (20 mg/kg QD) administration resulted in profound tumor regression, but with concurrent body weight loss and EGFR WT pathway inhibition. CO-1686, when dosed as a single agent at 50 mg/kg BID and/or 100 mg/kg QD, caused tumor regressions in the HCC827 (del19), NCI-H1975 (L858R/T790M), and patient-derived lung tumor xenograft model LUM1868 (L858R/T790M). The efficacy of CO-1686 was also examined in EGFR-L858R and EGFR-L858R-T790M genetically engineered mouse (GEM) models that develop lung adenocarcinoma upon human mutant EGFR transgene induction in the lung epithelium. In both models, CO-1686 administration (50 mg/kg BID) resulted in potent anti-tumor activity as measured by MRI and histological assessment. Following a 3-week dosing period, animals treated with CO-1686 demonstrated a complete regression of established lesions. Comparatively, in the EGFR-L858R-T790M GEM model, afatinib dosed at the maximum tolerated dose (20 mg/kg QD) resulted in limited efficacy. In summary, CO-1686 exhibits potent and significant anti-tumor activity as a single agent in xenograft models driven by the most frequently observed front-line and acquired resistance mutant forms of EGFR, while exhibiting minimal activity towards WT EGFR. These data suggest that CO-1686 may have utility in both front-line and acquired resistance EGFR-mutated NSCLC, and CO-1686 is currently being evaluated in a phase I/II clinical trial in previously treated mutant EGFR NSCLC patients (ClinicalTrials.gov identifier: NCT01526928). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B120. Citation Format: Andrew D. Simmons, Henry J. Haringsma, Annette O. Walter, Robert Tjin Tham Sjin, Zoe Weaver, Andrew Allen, Thomas C. Harding. Preclinical assessment of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B120.

Efficacy of Low-Dose Oral Metronomic Dosing of the Prodrug of Gemcitabine, LY2334737, in Human Tumor Xenografts

LY2334737, an oral prodrug of gemcitabine, is cleaved in vivo, releasing gemcitabine and valproic acid. Oral dosing of mice results in absorption of intact prodrug with slow systemic hydrolysis yielding higher plasma levels of LY2334737 than gemcitabine and prolonged gemcitabine exposure. Antitumor activity was evaluated in human colon and lung tumor xenograft models. The dose response for efficacy was examined using 3 metronomic schedules, once-a-day dosing for 14 doses, every other day for 7 doses, and once a day for 7 doses, 7 days rest, followed by an additional 7 days of once-a-day dosing. These schedules gave significant antitumor activity and were well tolerated. Oral gavage of 6 mg/kg LY2334737 daily for 21 days gave equivalent activity to i.v. 240 mg/kg gemcitabine. HCl administered once a week for 3 weeks to mice bearing a patient mesothelioma tumor PXF 1118 or a non-small cell lung cancer tumor LXFE 937. The LXFE 397 tumor possessed elevated expression of the equilibrative nucleoside transporter-1 (ENT1) important for gemcitabine uptake but not prodrug uptake and responded significantly better to treatment with LY2334737 than gemcitabine (P ≤ 0.001). In 3 colon xenografts, antitumor activity of LY2334737 plus a maximally tolerated dose of capecitabine, an oral prodrug of 5-fluorouracil, was significantly greater than either monotherapy. During treatment, the expression of carboxylesterase 2 (CES2) and concentrative nucleoside transporter-3 was induced in HCT-116 tumors; both are needed for the activity of the prodrugs. Thus, metronomic oral low-dose LY2334737 is efficacious, well tolerated, and easily combined with capecitabine for improved efficacy. Elevated CES2 or ENT1 expression may enhance LY2334737 tumor response.

Stromal Platelet-Derived Growth Factor Receptor α (PDGFRα) Provides a Therapeutic Target Independent of Tumor Cell PDGFRα Expression in Lung Cancer Xenografts

In lung cancer, platelet-derived growth factor receptor α (PDGFRα) is expressed frequently by tumor-associated stromal cells and by cancer cells in a subset of tumors. We sought to determine the effect of targeting stromal PDGFRα in preclinical lung tumor xenograft models (human tumor, mouse stroma). Effects of anti-human (IMC-3G3) and anti-mouse (1E10) PDGFRα monoclonal antibodies (mAb) on proliferation and PDGFRα signaling were evaluated in lung cancer cell lines and mouse fibroblasts. Therapy studies were conducted using established PDGFRα-positive H1703 cells and PDGFRα-negative Calu-6, H1993, and A549 subcutaneous tumors in immunocompromised mice treated with vehicle, anti-PDGFRα mAbs, chemotherapy, or combination therapy. Tumors were analyzed for growth and levels of growth factors. IMC-3G3 inhibited PDGFRα activation and the growth of H1703 cells in vitro and tumor growth in vivo, but had no effect on PDGFRα-negative cell lines or mouse fibroblasts. 1E10 inhibited growth and PDGFRα activation of mouse fibroblasts, but had no effect on human cancer cell lines in vitro. In vivo, 1E10-targeted inhibition of murine PDGFRα reduced tumor growth as single-agent therapy in Calu-6 cells and enhanced the effect of chemotherapy in xenografts derived from A549 cells. We also identified that low expression cancer cell expression of VEGF-A and elevated expression of PDGF-AA were associated with response to stromal PDGFRα targeting. We conclude that stromal PDGFRα inhibition represents a means for enhancing control of lung cancer growth in some cases, independent of tumor cell PDGFRα expression.

A local effect of CYP24 inhibition on lung tumor xenograft exposure to 1,25-dihydroxyvitamin D3 is revealed using a novel LC–MS/MS assay

The vitamin D3 catabolizing enzyme, CYP24, is frequently over-expressed in tumors, where it may support proliferation by eliminating the growth suppressive effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). However, the impact of CYP24 expression in tumors or consequence of CYP24 inhibition on tumor levels of 1,25(OH)2D3in vivo has not been studied due to the lack of a suitable quantitative method. To address this need, an LC–MS/MS assay that permits absolute quantitation of 1,25(OH)2D3 in plasma and tumor was developed. We applied this assay to the H292 lung tumor xenograft model: H292 cells eliminate 1,25(OH)2D3 by a CYP24-dependent process in vitro, and 1,25(OH)2D3 rapidly induces CYP24 expression in H292 cells in vivo. In tumor-bearing mice, plasma and tumor concentrations of 1,25(OH)2D3 reached a maximum of 21.6 and 1.70ng/mL, respectively, following intraperitoneal dosing (20μg/kg 1,25(OH)2D3). When co-administered with the CYP24 selective inhibitor CTA091 (250μg/kg), 1,25(OH)2D3 plasma levels increased 1.6-fold, and tumor levels increased 2.6-fold. The tumor/plasma ratio of 1,25(OH)2D3 AUC was increased 1.7-fold by CTA091, suggesting that the inhibitor increased the tumor concentrations of 1,25(OH)2D3 independent of its effects on plasma disposition. Compartmental modeling of 1,25(OH)2D3 concentration versus time data confirmed that: 1,25(OH)2D3 was eliminated from plasma and tumor; CTA091 reduced the elimination from both compartments; and that the effect of CTA091 on tumor exposure was greater than its effect on plasma. These results provide evidence that CYP24-expressing lung tumors eliminate 1,25(OH)2D3 by a CYP24-dependent process in vivo and that CTA091 administration represents a feasible approach to increase tumor exposure to 1,25(OH)2D3.

400 Phase I study of JI-101, a novel oral tyrosine kinase inhibitor that selectively targets EphB4, VEGFR2, and PDGFRβ

Platelet-derived growth factor (PDGF) and its receptors (PDGFR) play important roles in tumorigenesis through stimulating tumor growth and promoting angiogenesis via enhancing pericyte recruitment and vessel maturation. Here we produced a neutralizing antibody, 1B3, directed against mouse PDGFRβ. 1B3 binds to PDGFRβ with high affinity (9 × 10−11 M) and blocks PDGF-BB from binding to the receptor with an IC50 of ∼1.2 nM. The antibody also blocks ligand-stimulated activation of PDGFRβ and downstream signaling molecules, including Akt and MAPK p42/44, in tumor cells. In animal studies, 1B3 significantly enhanced the antitumor and the anti-angiogenic activities of DC101, an antibody directed against mouse vascular endothelial growth factor receptor 2, in a pancreatic (BxPC-3) and a non-small cell lung (NCI-H460) tumor xenograft models. Treatment with the combination of 1B3 and DC101 in BxPC-3 xenograft-bearing mice resulted in tumor regression in 58% of mice compared to that in 18% of mice treated with DC101 alone. Taken together, these results lend great support to use PDGFRβ antagonists in combinations with other antitumor and/or anti-angiogenic agents in the treatment of a variety of cancers.