Preclinical Models Of Lung Cancer Research Articles

30. RADIOSURGERY FOLLOWED BY TUMOR TREATING FIELDS FOR BRAIN METASTASES (1–10) FROM NSCLC IN THE PHASE 3 METIS TRIAL

BACKGROUNDTumor Treating Fields (TTFields) are non-invasive, loco-regional, anti-mitotic treatment modality comprising alternating electric fields. TTFields have demonstrated efficacy in preclinical non-small cell lung cancer (NSCLC) models. TTFields treatment to the brain was safe and extended overall survival in newly-diagnosed glioblastoma. The objective of the METIS study [NCT02831959] is evaluation of the efficacy and safety of TTFields in NSCLC patients with brain metastases.METHODSNSCLC patients (N=270) with 1–10 brain metastases were randomized 1:1 to stereotactic radio surgery (SRS) followed by continuous TTFields ((150 kHz, > 18 hours/day) within 7 days of SRS or supportive care. The portable device delivered TTFields to the brain using 4 transducer arrays, while patients received the best standard-of-care for systemic disease. Patients were followed every two months until second intracranial progression. Key inclusion criteria: KPS ≥70, new diagnosis of 1 inoperable or 2–10 supra- and/or infratentorial brain metastases from NSCLC amenable to SRS; and optimal therapy for extracranial disease. Prior WBRT, surgical resection of metastases, or recurrent brain metastases were exclusionary. Primary endpoint was time to 1st intracranial progression. Secondary endpoints included time to neurocognitive failure (HVLT, COWAT and TMT), overall survival, radiological response rate (RANO-BM and RECIST V1.1); quality-of-life; adverse events; time to first/second intracranial progression for patients with 1–4 and 5–10 brain metastases; bi-monthly intracranial progression rate from 2–12 months; and time to second intracranial and distant progression. The sample size (N=270) was calculated using a log-rank test (Lakatos 1988 and 2002) with 80% power at two sided alpha of 0.05 to detect a hazard ratio of 0.57. On September, 2019, an independent Data Monitoring Committee (DMC) reviewed METIS trial data collected to that point. The DMC concluded that no unexpected safety issues had emerged and recommended continuation of the METIS study as planned.

Updated Review and Perspective on 20S Proteasome Inhibitors in the Treatment of Lung Cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide. Most lung cancer patients are diagnosed at advanced stages and may benefit from pembrolizumab (anti-PD-1 antibody), cytotoxic chemotherapy and other adjuvant therapies. Despite the availability of various therapies, the response and survival rates have been low. Therefore, the study of different targets for the treatment of lung cancer has been one of the major focuses of cancer research. The ubiquitin proteasome system (UPS) is a crucial regulator of cell homeostasis and plays an essential role in the growth and development of all cells. The UPS is dysregulated in human cancer cells including lung cancer cells. Therefore, targeting UPS is potentially a selective, effective treatment for lung cancer. Bortezomib, a 20S proteasome inhibitor that is clinically approved for the management of multiple myeloma, has been studied in various preclinical and clinical models of lung cancer. Most preclinical studies have shown that a 20S proteasome inhibitor alone and its combination with other chemotherapeutic agents induce apoptosis in non-small cell lung cancer cell lines and animal models. Owing to the impressive preclinical results, many clinical trials were initiated using 20S proteasome inhibitors either as monotherapy or in combination with other conventional lung cancer therapies. Many combinational therapies of 20S PIs with conventional chemotherapy were shown to be well tolerated in clinical trials. However, there have not been any consistent data showing the beneficial effects of such proteasome inhibitor-based therapies. Low clinical efficacy of 20S PIs in lung cancer patients may be due to low drug penetration, the status of 20S proteasomes, oncogene expressions and the inherited or acquired resistance. Potential mechanisms of PI resistance or low or no clinical activity in lung cancer cells might include alteration of apoptotic proteins, overexpression or alteration of β5 subunit, or upregulation of heat shock proteins. Various cutting-edge strategies to counter this resistance or improve 20S PIs' efficacy in lung cancer cells have been reviewed which include novel combination therapies, new drug delivery systems, development of more potent PIs, and targeting different sites of the UPS. A better understanding of PI resistance mechanisms in lung cancer cells can help improve current clinical treatment strategies and clinical outcomes.

Abstract A23: Ultrarapid total abdominal FLASH irradiation in a preclinical model of ovarian cancer

Abstract Introduction: Ultrahigh dose-rate irradiation (FLASH RT) is rapidly emerging as new strategy to enhance the therapeutic index of radiotherapy by protecting normal tissues from radiation-induced toxicity while maintaining tumor control. In mice, FLASH has been shown to protect the lung, skin, and brain against radiation-induced toxicity. A recent study has utilized FLASH radiotherapy in preclinical models of lung cancer to demonstrate that FLASH RT radiotherapy achieves similar tumor control to conventional RT. While these findings identify an important role for FLASH RT in the protection of multiple tissues, the role of FLASH in enhancing the therapeutic index of abdominal and pelvic tumors located near the highly radiosensitive intestine is not known. For this purpose, we have developed an experimental platform using a clinical linear accelerator that enables total abdominal FLASH RT where rapid delivery of a single fraction of high-dose radiotherapy is performed in less than one second (200 Gy/sec). Methods: For the tumor model, ID8 cells were injected intraperitoneally into female mice. Radiation was delivered 10 days post-ID8 injection. Tumor burden was assessed at 28 days following tumor injection. Flow cytometry was used to profile infiltrating immune cells. Normal tissue toxicity was assessed through survival, total body weight, solid stool production, complete blood count, and histologic analysis. Results: In ID8 tumor-bearing mice, 14 Gy FLASH promotes GI function with a 2-fold increase in solid stool production at 5 days post-TAI. Both modalities extended median overall survival in the ID8 model by 5 days and reduced tumor burden by 50% relative to unirradiated controls. Irradiated cohorts also demonstrated an increase in the number of proliferating CD8+ and CD4+ T cells present in the ascites. Conclusion: We demonstrate that FLASH RT protects the gastrointestinal (GI) tract from lethal radiation-induced toxicity, improves epithelial integrity and GI function, enhances crypt survival, and provides efficient tumor control in a preclinical model of ovarian peritoneal metastasis. Here we present the first data supporting the potential for tumor control and GI protection using FLASH-TAI in a syngeneic orthotopic preclinical model of ovarian cancer. Citation Format: Karen Levy, Jinghui Wang, Joshua Eggold, Suchitra Natarajan, Peter Maxim, Bill Loo, Erinn Rankin. Ultrarapid total abdominal FLASH irradiation in a preclinical model of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A23.

Blood Outgrowth Endothelial Cells as a Cellular Carrier for Oncolytic Vesicular Stomatitis Virus Expressing Interferon-β in Preclinical Models of Non-Small Cell Lung Cancer

Oncolytic viruses have demonstrated efficacy in numerous tumor models including non-small cell lung cancer (NSCLC). One limitation of viral therapy for metastatic lung cancer is that systemic administration can be hindered by complement and antiviral immunity. Thus, we investigated whether ex vivo-infected blood outgrowth endothelial cells (BOECs) could be used to deliver VSV-IFNβ in preclinical models of NSCLC. BOECs were obtained from human donors or C57/Bl6 mice. VSV was engineered to produce GFP or IFNβ. Human and murine BOECs could be infected by VSV-GFP and VSV-IFNβ. Infected BOECs resulted in killing of NSCLC cells in vitro and shielded VSV-IFNβ from antibody neutralization. Mouse BOECs localized to lungs of mice bearing syngeneic LM2 lung tumors, and infected murine BOECs reduced tumor burden in this model. In an immune-deficient A549 xenograft model, mice treated with VSV-IFNβ-infected human BOECs exhibited superior antitumor activity and survival of mice (n = 10, P < .05 compared to VSV-IFNβ alone). We conclude that BOECs can be used as a carrier for delivery of oncolytic VSV-IFNβ. This may be an effective strategy for clinical translation of oncolytic virotherapy for patients with metastatic NSCLC.

Psychological stress enhances tumor growth and diminishes radiation response in preclinical model of lung cancer

Background and purposePatients with life-threatening illnesses, such as cancer, experience emotional distress. This study was to investigate the molecular and cellular mechanisms of relevant psychological stressor on tumor growth and therapeutic resistance. Materials and MethodsStress was induced in C57BL/6J mice bearing LLC lung tumors by exposure to a conspecific mice receiving inescapable foot shocks. Mice were irradiated at 7 Gy for 3 consecutive days. Behaviors were monitored by open field test (OFT), elevated plus maze (EPM), sucrose preference test (SPT), and learned helplessness (LH) test. Protein expression in tissues and cultured cells were measured by Western blot. ResultsThis study in animals showed that observing a conspecific mouse receiving foot shocks induced depression like behaviors with increased plasma corticosterone and adrenaline levels which increased tumor growth and radioresistance. Stress increased Wnt1, Drosha, and vimentin expression and decreased E-cadherin expression in tumor tissues. The combination of stress and irradiation enhanced radioresistance along with the increase in vimentin expression. The in vitro study showed that a β2-adrenergic receptor (β2-AR) agonist blocked irradiation-induced cell apoptosis and decreased cell viability, while silencing β2-AR expression reduced the protective effects of β2-AR agonist. β2-AR agonist obviously increased Wnt1 and Drosha expression in LLC-1 cells. ConclusionPsychological stress increased tumor growth and enhanced radioresistance associated with the activation of epithelial-mesenchymal transition by stress hormone-stimulated adrenergic receptors.

30P Assessing the potential role of RICTOR expression as predictive factor of response to PI3K/mTOR pathway inhibitors in preclinical models of squamous cell lung cancer

30P Assessing the potential role of RICTOR expression as predictive factor of response to PI3K/mTOR pathway inhibitors in preclinical models of squamous cell lung cancer

A32 Evaluation of the Mutant KRAS-Driven NSCLC Tumor Immune Microenvironment Using Patient-Derived Cell Line Xenografts in a Humanized Mouse Preclinical Model for Development of New Immunotherapy Approaches

Tumor cell and tumor microenvironment (TME) features that influence the response to immune checkpoint blockade in lung cancer are incompletely defined. We wanted to develop preclinical models of lung cancer to explore the relationship of non-small cell lung cancer (NSCLC) molecular characteristics (oncogenotype such as mutant KRAS and LKB1/STK11, and mRNA expression profiles) to the human TME. We exploited the use of 8 (all KRAS mutant) molecularly characterized (whole-exome seq mutation and RNAseq analyses) patient-derived NSCLC lines grown as xenografts in vivo in humanized NSG-SGM3 mice in an effort to determine the effect of different NSCLCs on the immune landscape of lung cancer xenografts. The triple transgenic NSG-SGM3 (NSGS) mice express human IL3, GM-CSF, and SCF, which combine the features of the highly immunodeficient NOD scid gamma (NSG) mouse with cytokines that support the stable engraftment of human myeloid lineages and regulatory T-cell populations. Subcutaneous KRAS mutant-driven non-NSCLC xenografts grown in NSG-SGM3 mice “humanized” with CD34+ cord blood cells were subjected to immune landscape analysis through flow cytometry, multiplex immunohistochemistry, and cytokine analysis. The xenografts were also treated with various combinations of checkpoint inhibitors, radiation, and activation of the innate immune pathway with emricasan (pan caspase inhibitor). Our results show: that the 8 KRAS mutant NSCLCs each were associated with a different spectrum of human TME; 3 of the 8 xenografts had only 0.1% of immune cell infiltrates while other xenografts had 20% immune cell infiltrates; that tumor mutation burden (TMB, absolute mutation calls 233 to 2,076) is not predictive of CD8+ T-cell infiltration in KRAS mutant-driven NSCLC xenografts. In NSCLC xenografts with high CD8+ T-cell infiltrate, the CD8+ cells in the TME were not activated, resulting in limited responses to PD-1/PD-L1 therapy. However, stimulation of the cGAS-STING innate immune pathway with emricasan followed by radiation (15 cGy) resulted in dramatic antitumor response. NSCLC xenografts grown in “humanized” mice show great intertumor heterogeneity effects on the TME even within the KRAS mutant subgroup, and it is possible to demonstrate targeted therapy such as emricasan/radiation can lead to changes in improved antitumor responses.

Abstract A091: Targeting homologous recombination repair defects in lung cancer

Abstract Non-Small Cell Lung Cancer (NSCLC) is the leading cause of cancer related mortality worldwide, and accounts for 80-85% of all lung cancer diagnoses. While targeted therapies against epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) fusions have proven to be effective for the treatment of NSCLC, only a fraction of NSCLC patients (i.e. &amp;lt;20%) benefit from these targeted agents. Although immunotherapy approaches, specifically PD-1:PD-L1 blockade, appear to be more broadly efficacious, there are still a substantial proportion of patients who may not benefit from these treatments. To investigate the potential of targeting the DNA Damage Response (DDR) pathway in lung cancer, as an alternative therapeutic approach for these patients we sought to identify whether functionally relevant HRR (Homologous Recombination Repair)-defects could be synthetically lethal with niraparib monotherapy in NSCLC PDX models. Approximately 3% of lung cancers harbor mutations in ATM (i.e. 3.5% in lung adenocarcinomas &amp; 1.4% in lung squamous cell carcinoma), which is involved in HRR as well as multiple other DNA repair and checkpoint functions, but the functional consequences of this alteration remain to be established. Studies in the past few years have demonstrated the potential of targeting the DDR pathway as a promising therapeutic strategy for lung cancer, i.e. in small cell lung cancer (SCLC) where overall response rates to immune checkpoint blockade remain poor. In addition, inhibitors targeting DDR proteins have shown promise in preclinical models of lung cancer, and are under clinical investigation as single agents and in combination with other targeted and immunotherapy agents. In this study, we have evaluated niraparib sensitivity in 57 NSCLC PDX models containing both BRCA and non-BRCA HRR mutations (n=17) as well as HRR WT models (n=40). This analysis demonstrated that niraparib sensitive models include both HRR mutant and HRR WT lung tumors (TGI &amp;gt; 70%). Amongst the PDX models containing a biallelic HRR mutation (ATM, BAP1, RAD51D &amp; XRCC2), only ATM biallelic mutant models were sensitive to niraparib (2 out of 6). Furthermore, none of the of nine monoallelic mutant models (ATM, ATR, BRCA1/2, RAD50 &amp; RAD52) were sensitive to niraparib. Surprisingly, 7.5% (3 out of 40) of the HRR WT PDX models were sensitive to niraparib. Further analysis is required to elucidate the molecular mechanisms driving niraparib sensitivity in HRR WT NSCLC. Citation Format: Asli Muvaffak, Yinghui Zhou, Bin Feng, Sarah Wang, Jing Ju Wang, Sridhar Ramaswamy, Kevin G Coleman. Targeting homologous recombination repair defects in lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A091. doi:10.1158/1535-7163.TARG-19-A091

Abstract CN08-01: IL-8 in tumor progression

Abstract Multiple cytokines, chemokines, and growth factors play critical roles as mediators of paracrine signals between the tumor and cellular components of the tumor microenvironment (TME), ultimately leading to tumor growth, survival, and progression. One of the soluble factors that has a multifaceted role in cancer progression is the chemokine IL-8 (CXCL8), which signals through the receptors CXCR1 and CXCR2.Abnormally high expression of IL-8 is found in multiple cancer types, including in triple negative breast, colorectal, lung, and head and neck tumors, among others. IL-8 can foster tumor progression by various means, including (a) promoting the trafficking of immune-suppressive cells to the tumor microenvironment, mainly granulocytic myeloid-suppressor cells (G-MDSCs), which exert a suppressive role on anti-tumor immunity; (b) promoting phenotypic plasticity in carcinoma cells via changes from an epithelial into a mesenchymal-like state; and (c) increasing the survival of cancer stem-like cell populations in the tumor. Tumor phenotypic plasticity has been linked to advanced tumor stage, presence of metastases, poor prognosis in numerous cancer types, and tumor resistance to anti-cancer therapies, including chemotherapy and small molecule-targeted therapies. Recent work has also shown that upregulation of IL-8 and enhanced mesenchymal tumor features are enriched in tumors that are resistant to checkpoint inhibitor therapy. In agreement, our laboratory has demonstrated a role for IL-8 expression in tumor resistance to lysis by immune effector cells. The multifaceted roles of IL-8 in cancer make it a promising therapeutic target. Blockade of IL-8 signaling may be achieved by direct neutralization of IL-8 via a monoclonal antibody or via inhibition of its receptors. Our laboratory has now demonstrated a role for IL-8 in the context of acquired resistance to EGFR inhibition and in the context of acquired resistance to chemotherapies in preclinical models of lung cancer. Alleviation of IL-8 signaling via a neutralizing antibody was shown to reduce mesenchymal features of the resistant cells, and markedly enhanced their sensitivity to therapies. Our group has also shown that neutralization of IL-8 via a monoclonal anti-IL8 antibody is able to revert mesenchymal features in claudin-low, triple negative tumor models, resulting in enhanced immune-mediated lysis with NK and antigen-specific T cells in vitro and reduced tumor infiltration with G-MDSC and improved responses to chemotherapy in vivo. Current work in our laboratory is evaluating inhibition of the CXCR1/2 receptors via the use of the small molecule inhibitor SX-682, as an alternative approach to abrogate signaling mediated not only by IL-8 but also by all other chemokines of the same family that bind to these receptors. In addition to IL-8, the pleiotropic cytokine TGF-β is a driver of phenotypic plasticity and cancer progression and has been implicated as a driver of T-cell exclusion from tumors. Our current experiments are exploring the hypothesis that by reducing mesenchymal features in the tumor via inhibition of IL-8 and/or TGF-β signaling we could enhance the anti-tumor efficacy of the PD-1/PD-L1 axis blockade. Citation Format: Lucas A Horn, Hanne Lind, Heidi Hempel, Kristen Fousek, Claudia Palena. IL-8 in tumor progression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN08-01. doi:10.1158/1535-7163.TARG-19-CN08-01

Abstract A095: Targeting protein-DNA interactions in the DNA damage response: Lead identification and optimization for novel inhibitors of RPA and Ku

Abstract Recent advances in our understanding of the mechanisms of the cellular response to DNA damage (DDR) have opened up an exciting array of opportunities to treat human cancer. The majority of drug discovery efforts have focused on the DDR kinases, ATM, ATR and DNA-PK, each of which is activated in response to different types of DNA damage or genomic perturbations. We have taken the novel approach of drugging this pathway by targeting the DNA binding proteins Replication protein A (RPA) and Ku, which are required to activate the ATR and DNA-PK kinases, respectively. Chemical inhibition of the DNA binding activity functionally abrogates kinase activity and provides a novel mechanism by which DDR kinases can be blocked. The identification of a potent inhibitor of the Ku-DNA interactions was gleaned from a specificity screen and was found to effectively block the Ku-DNA interaction and this inhibits DNA-PK phosphorylation activity. We identified a series of moieties and optimized pharmacophore for potent and specific inhibition of Ku and demonstrate cellular inhibition of Ku DNA binding and DNA-PK activity. Third generation Ku inhibitors were developed to optimize for cellular bioavailability and are able to sensitize cells to DNA double strand break inducing agents, consistent with the role of DNA-PK in the non-homologous end joining repair pathway. We initially identified a series of RPA-DNA interaction inhibitors from an in vitro high throughput screen. Extensive SAR was conducted to optimize for potency, specificity and physicochemical properties and led to the promotion of a lead candidate. We have determined the mechanism of action for single agent anti-cancer activity and combination activity involves chemical exhaustion of RPA. In vivo analysis of the lead candidate in pre-clinical models of lung and ovarian cancer has led to the advancement of a clinical candidate and represent a first in class RPA inhibitor poised for clinical advancement. Together, these data demonstrate the ability to chemically target structure specific protein-DNA interactions. The molecules synthesized to inhibit these interactions share some common features, but can be optimized to obtain high potency and specificity. In addition, chemical modifications can be employed to allow cellular and in vivo bioavailability while retaining potent inhibitory activity. These data reveal the potential to target a new range of macromolecular interactions to expand the anticancer armamentarium to protein-DNA interactions. Citation Format: John Turchi, Katherine S Pawelczak, Navnath Gavande, Pamela S VanderVere-Carozza. Targeting protein-DNA interactions in the DNA damage response: Lead identification and optimization for novel inhibitors of RPA and Ku [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A095. doi:10.1158/1535-7163.TARG-19-A095

Peritumoral administration of DRibbles-pulsed antigen-presenting cells enhances the antitumor efficacy of anti-GITR and anti-PD-1 antibodies via an antigen presenting independent mechanism

BackgroundTNF receptor family agonists and checkpoint blockade combination therapies lead to minimal tumor clearance of poorly immunogenic tumors. Therefore, a need to enhance the efficacy of this combination therapy arises. Antigen-presenting cells (APCs) present antigen to T cells and steer the immune response through chemokine and cytokine secretion. DRibbles (DR) are tumor-derived autophagosomes containing tumor antigens and innate inflammatory adjuvants.MethodsUsing preclinical murine lung and pancreatic cancer models, we assessed the triple combination therapy of GITR agonist and PD-1 blocking antibodies with peritumoral injections of DRibbles-pulsed-bone marrow cells (BMCs), which consisted mainly of APCs, or CD103+ cross-presenting dendritic cells (DCs). Immune responses were assessed by flow cytometry. FTY720 was used to prevent T-cell egress from lymph nodes to assess lymph node involvement, and MHC-mismatched-BMCs were used to assess the necessity of antigen presentation by the peritumorally-injected DR-APCs.ResultsTritherapy increased survival and cures in tumor-bearing mice compared to combined antibody therapy or peritumoral DR-BMCs alone. Peritumorally-injected BMCs remained within the tumor for at least 14 days and tritherapy efficacy was dependent on both CD4+ and CD8+ T cells. Although the overall percent of tumor-infiltrating T cells remained similar, tritherapy increased the ratio of effector CD4+ T cells-to-regulatory T cells, CD4+ T-cell cytokine production and proliferation, and CD8+ T-cell cytolytic activity in the tumor. Despite tritherapy-induced T-cell activation and cytolytic activity in lymph nodes, this T-cell activation was not required for tumor regression and enhanced survival. Replacement of DR-BMCs with DR-pulsed-DCs in the tritherapy led to similar antitumor effects, whereas replacement with DRibbles was less effective but delayed tumor growth. Interestingly, peritumoral administration of DR-pulsed MHC-mismatched-APCs in the tritherapy led to similar antitumor effects as MHC-matched-APCs, indicating that the observed enhanced antitumor effect was mediated independently of antigen presentation by the administered APCs.ConclusionsOverall, these results demonstrate that peritumoral DR-pulsed-BMC/DC administration synergizes with GITR agonist and PD-1 blockade to locally modulate and sustain tumor effector T-cell responses independently of T cell priming and perhaps through innate inflammatory modulations mediated by the DRibbles adjuvant. We offer a unique approach to modify the tumor microenvironment to benefit T-cell-targeted immunotherapies.

RBTT-02. RADIOSURGERY FOLLOWED BY TUMOR TREATING FIELDS FOR BRAIN METASTASES (1–10) FROM NSCLC IN THE PHASE 3 METIS TRIAL

Abstract BACKGROUND: Tumor Treating Fields (TTFields) are non-invasive, loco-regional, anti-mitotic treatment modality comprising alternating electric fields. TTFields have demonstrated efficacy in preclinical non-small cell lung cancer (NSCLC) models. TTFields treatment to the brain was safe and extended overall survival in newly-diagnosed glioblastoma. The METIS study [NCT02831959] investigates the efficacy and safety of TTFields in NSCLC patients with brain metastases. Study Design: NSCLC patients (N=270) with 1–10 brain metastases are randomized 1:1 to stereotactic radio surgery (SRS) followed by continuous TTFields ((150 kHz, &gt; 18 hours/day) within 7 days of SRS or supportive care. The portable device delivers TTFields to the brain using 4 transducer arrays, while patients receive the best standard-of-care for systemic disease. Patients are followed every two months until second intracranial progression. Key inclusion criteria: KPS ≥70, new diagnosis of 1 inoperable or 2–10 supra- and/or infratentorial brain metastases from NSCLC amenable to SRS; and optimal therapy for extracranial disease. Prior WBRT or surgical resection of metastases, a single resectable lesion or recurrent brain metastases were exclusionary. Primary endpoint was time to 1st intracranial progression. Secondary endpoints included time to neurocognitive failure (HVLT, COWAT and TMT), overall survival, radiological response rate (RANO-BM and RECIST V1.1); quality-of-life; adverse events; time to first/second intracranial progression for patients with 1–4 and 5–10 brain metastases; bi-monthly intracranial progression rate from 2–12 months; and time to second intracranial and distant progression. The sample size (N=270) was calculated using a log-rank test (Lakatos 1988 and 2002) with 80% power at two sided alpha of 0.05 to detect a hazard ratio of 0.57. In August 2018, an independent Data Monitoring Committee (DMC) performed a review of the METIS trial data collected to that point. The DMC concluded that no unexpected safety issues had emerged and recommended continuation of the METIS study as planned.

58P - IKS01, a next generation antibody drug conjugate (ADC) designed to be efficacious in tumors with low and moderate levels of folate receptor expression

BackgroundFrequent over-expression of FRA in ovarian and non-small-cell lung cancer (NSCLC), and relative lack of expression in normal tissue, make it an attractive therapeutic target. Ovarian cancer is the ninth most common cancer in women and the leading cause of death in gynaecological cancer. Lung cancer remains a leading cause of cancer-related death with NSCLC accounting for 80 % of cases. ADCs are the focus of intense interest as a means to provide selective tumor killing with increased efficacy and less off-target toxicity than standard of care chemotherapies. Clinical evaluation of mirvetuximab soravtansine in solid tumor indications demonstrated that FRA can be successfully targeted by an ADC. However, anti-tumor activity appears to be generally limited to patients whose tumors express high levels of FRA. IKS01 is an ADC comprised of an FRA-targeting antibody conjugated via Iksuda’s PermaLink conjugation technology to Femtogenix’s highly potent FGX2-62 payload. FGX2-62 is a pyrridinobenzodiazepine DNA mono-alkylating agent with pM potency in a range of tumor cell lines. IKS01 has been designed to be sufficiently potent to have target-dependent efficacy even in tumors with low or moderate expression of the target antigen. MethodsIKS01 was generated with a drug to antibody ratio of 2. Efficacy was evaluated in human cell line derived xenograft models with a range of FRA expression, including the choriocarcinoma Jeg-3 model (high expression), the ovarian adenocarcinoma OV-90 model (low expression) and the lung adenocarcinoma NCI-H2110 model (moderate expression). An FRA-targeting benchmark ADC with a format that has shown clinical efficacy was included for comparison. ResultsIKS01 is highly effective in causing tumor regressions in FRA-expressing tumor models at doses that are well tolerated. IKS01 was significantly more active than benchmark ADC in the high FRA-expressing Jeg-3 model and caused complete or near complete regressions in low/moderate FRA-expressing xenograft models where the benchmark ADC showed limited or no activity. ConclusionsIKS01 shows marked anti-tumor efficacy in pre-clinical models of ovarian and lung cancer, even in models with low or moderate FRA expression. Legal entity responsible for the studyIksuda Therapeutics Ltd. FundingIksuda Therapeutics Ltd. DisclosureJ. Thirlway: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. A. Lodge: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. A. Pelava: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. D.J. Williamson: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. D. Carta: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. M. Al Nakeeb: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. J. Mysliwy: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. P.J.M. Jackson: Shareholder/Stockholder/Stock options: Femtogenix Ltd. D.E. Thurston: Shareholder/Stockholder/Stock options: Femtogenix Ltd. R.J. Lutz: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd.

OA08.01 Organoid Cultures as Novel Preclinical Models of Non-Small Cell Lung Cancer

OA08.01 Organoid Cultures as Novel Preclinical Models of Non-Small Cell Lung Cancer

1594TiP - Radiosurgery followed by tumour treating fields (TTFields) for brain metastases (1-10) from NSCLC in the phase III METIS trial

BackgroundTumor Treating Fields (TTFields) are non-invasive, loco-regional, anti-mitotic treatment modality comprising alternating electric fields. TTFields have demonstrated efficacy in preclinical non-small cell lung cancer (NSCLC) models. TTFields treatment to the brain was safe and extended overall survival in newly-diagnosed glioblastoma. The METIS study [NCT02831959] investigates the efficacy and safety of TTFields in NSCLC patients with brain metastases. Trial designNSCLC patients (N=270) with 1-10 brain metastases are randomized 1:1 to stereotactic radio surgery (SRS) followed by continuous TTFields ((150kHz, > 18hours/day) within 7 days of SRS or supportive care. The TTFields portable device delivers TTFields to the brain using 4 transducer arrays, while patients receive the best standard-of-care for their systemic disease. Patients are followed every two months until second intracranial progression. Key inclusion criteria: KPS ≥70, new diagnosis of 1 inoperable or 2–10 supra- and/or infratentorial brain metastases from NSCLC amenable to SRS; KPS ≥70; and optimal therapy for extracranial disease. Prior WBRT or surgical resection of metastases, a single resectable lesion or recurrent brain metastases were exclusionary. Primary endpoint was time to 1st intracranial progression. Secondary endpoints included time to neurocognitive failure (HVLT, COWAT and TMT), overall survival, radiological response rate (RANO-BM and RECIST V1.1); quality-of-life; adverse events; time to first/second intracranial progression for patients with 1–4 and 5–10 brain metastases; bi-monthly intracranial progression rate from 2–12 months; and time to second intracranial and distant progression. The sample size (N=270) was calculated using a log-rank test (Lakatos 1988 and 2002) with 80% power at a two sided alpha of 0.05 to detect a hazard ratio of 0.57. In August 2018, an independent Data Monitoring Committee (DMC) performed a review of the METIS trial data collected to that point. The DMC concluded that no unexpected safety issues have emerged on the study, and recommended to continue the METIS study as planned. Clinical trial identificationNCT02831959. Legal entity responsible for the studyNovocure. FundingNovocure. DisclosureM. Mehta: Advisory / Consultancy: Varian Medical Systems ; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Celgene; Advisory / Consultancy: Tocagen; Advisory / Consultancy: AbbVie; Leadership role: Oncoceutics ; Shareholder / Stockholder / Stock options, Officer / Board of Directors: Pharmacyclics; Advisory / Consultancy: Cavion, Novocure, Varian, Agenus, Insys, Remedy, IBA. M. Ahluwalia: Advisory / Consultancy: Monteris Medical ; Advisory / Consultancy: AstraZeneca ; Advisory / Consultancy: Bristol-Myers Squibb ; Advisory / Consultancy: AbbVie; Advisory / Consultancy: CBT Pharmaceuticals; Advisory / Consultancy: Kadmon ; Advisory / Consultancy: VBI Vaccines ; Shareholder / Stockholder / Stock options: MimiVax ; Honoraria (self): Prime Oncology ; Research grant / Funding (institution): Novartis ; Research grant / Funding (institution): Novocure; Research grant / Funding (institution): Boehringer Ingelheim ; Research grant / Funding (institution): Aztra zeneca; Research grant / Funding (institution): Lilly/ImClone . P. Brown: Advisory / Consultancy: Novella Clinical. All other authors have declared no conflicts of interest.

Primary tumors induce neutrophil extracellular traps with targetable metastasis promoting effects.

Targeting the dynamic tumor immune microenvironment (TIME) can provide effective therapeutic strategies for cancer. Neutrophils are the predominant leukocyte population in mice and humans, and mounting evidence implicates these cells during tumor growth and metastasis. Neutrophil extracellular traps (NETs) are networks of extracellular neutrophil DNA fibers that are capable of binding tumor cells to support metastatic progression. Here we demonstrate for the first time that circulating NET levels are elevated in advanced esophageal, gastric and lung cancer patients compared to healthy controls. Using pre-clinical murine models of lung and colon cancer in combination with intravital video microscopy, we show that NETs functionally regulate disease progression and that blocking NETosis through multiple strategies significantly inhibits spontaneous metastasis to the lung and liver. Further, we visualize how inhibiting tumor-induced NETs decreases cancer cell adhesion to liver sinusoids following intrasplenic injection - a mechanism previously thought to be driven primarily by exogenous stimuli. Thus, in addition to neutrophil abundance, the functional contribution of NETosis within the TIME has critical translational relevance and represents a promising target to impede metastatic dissemination.

TRLS-01. RADIOSURGERY FOLLOWED BY TUMOR TREATING FIELDS (TTFIELDS) FOR BRAIN METASTASES (1–10) FROM NSCLC IN THE PHASE 3 METIS TRIAL

Tumor Treating Fields (TTFields) are non-invasive, loco-regional, anti-mitotic treatment comprising alternating electric fields that have demonstrated efficacy in preclinical non-small cell lung cancer (NSCLC) models. TTFields to the brain was safe and extended overall survival in newly-diagnosed glioblastoma. The METIS study [NCT02831959] investigates the efficacy and safety of TTFields in NSCLC patients with brain metastases. NSCLC patients (N=270) with 1–10 brain metastases are randomized 1:1 to stereotactic radio surgery (SRS) followed by continuous TTFields ((150 kHz, > 18 hours/day) within 7 days of SRS or supportive care. The TTFields portable device delivers TTFields to the brain using 4 transducer arrays, while patients receive the best standard-of-care for their systemic disease. Patients are followed every two months until second intracranial progression. Key inclusion criteria: KPS ≥70, new diagnosis of 1 inoperable or 2–10 supra- and/or infratentorial brain metastases from NSCLC amenable to SRS; KPS ≥70; and optimal therapy for extracranial disease. Prior WBRT or surgical resection of metastases, a single resectable lesion or recurrent brain metastases were exclusionary. Primary endpoint was time to 1st intracranial progression. Secondary endpoints included time to neurocognitive failure (HVLT, COWAT and TMT), overall survival, radiological response rate (RANO-BM and RECIST V1.1); quality-of-life; adverse events; time to first/second intracranial progression for patients with 1–4 and 5–10 brain metastases; bi-monthly intracranial progression rate from 2–12 months; and time to second intracranial and distant progression. The sample size (N=270) was calculated using a log-rank test (Lakatos 1988 and 2002) with 80% power at a two sided alpha of 0.05 to detect a hazard ratio of 0.57. In August 2018, an independent Data Monitoring Committee (DMC) performed a review of the METIS trial data collected to that point. The DMC concluded that no unexpected safety issues have emerged on the study, and recommended to continue the METIS study as planned.

NOTCH signaling promotes the survival of irradiated basal airway stem cells.

Radiation-induced lung injury to normal airway epithelium is a frequent side-effect and dose-limiting factor in radiotherapy of tumors in the thoracic cavity. NOTCH signaling plays key roles in self-renewal and differentiation of upper airway basal lung stem cells during development, and the NOTCH pathway is frequently deregulated in lung cancer. In preclinical lung cancer models, NOTCH inhibition was shown to improve the radiotherapy response by targeting tumor stem cells, but the effects in combination with irradiation on normal lung stem cells are unknown. NOTCH/γ-secretase inhibitors are potent clinical candidates to block NOTCH function in tumors, but their clinical implementation has been hampered by normal tissue side-effects. Here we show that NOTCH signaling is active in primary human- and murine-derived airway epithelial stem cell models and when combined with radiation NOTCH inhibition provokes a decrease in S-phase and increase in G1-phase arrest. We show that NOTCH inhibition in irradiated lung basal stem cells leads to a more potent activation of the DNA damage checkpoint kinases pATM and pCHK2 and results in an increased level of residual 53BP1 foci in irradiated lung basal stem cells reducing their capacity for self-renewal. The effects are recapitulated in ex vivo cultured lung basal stem cells after in vivo whole thorax irradiation and NOTCH inhibition. These results highlight the importance of studying normal tissue effects that may counteract the therapeutic benefit in the use of NOTCH/γ-secretase inhibitors in combination with radiation for antitumor treatment.

Abstract 4853: M6123 is an ADCC-enhanced and selective monovalent antagonist of FGFR1 showing antitumor activity in preclinical lung cancer models

Abstract Despite the considerable progress made with immunotherapies and targeted therapies, lung cancer remains a devastating disease with a high medical need for more effective and safer therapeutic options. Targeted therapies have delivered meaningful benefit to subsets of lung adenocarcinoma patients harboring specific oncogenic driver alterations but have not yet been extensively explored in other lung cancer subtypes. In the case of lung squamous carcinoma and small-cell lung cancer, amplification and overexpression of FGFR1 have been described as potential oncogenic drivers.Several small molecule tyrosine kinase inhibitors of the FGFR family are in clinical development but initial reports have suggested limited therapeutic benefit of these agents when applied as monotherapy in FGFR1-amplified lung cancer patients. To overcome toxicities associated with pan-FGFR inhibition, such as dysregulation of FGF23-mediated phosphate homeostasis, M6123 was developed as an FGFR1-selective biological antagonist. An immunization campaign in OmniRats® led to the identification of the fully human, potent and highly selective FGFR1 antibody A08 which was converted into the monovalent FGFR1 antagonist M6123 based on the proprietary SEED platform. Furthermore, M6123 was engineered for enhanced ADCC effector function. In contrast to bivalent FGFR1 antibodies, M6123 was well tolerated in mice, with no signs of body weight loss. Of note, M6123 did not interfere with hormonal FGF23 signaling in mice, a severe side effect of FGFR kinase inhibitors. Concentration-dependent inhibition of FGFR1 auto-phosphorylation was demonstrated in FGFR1-overexpressing lung cancer cells both in vitro and in vivo. Moreover, M6123 monotherapy had strong antitumor activity in specific FGFR1-expressing cell line- and patient-derived xenograft models, whilst combination with chemotherapy significantly enhanced efficacy. In summary, M6123 is a unique and clearly differentiated selective FGFR1 antagonist which warrants further testing in preclinical models to refine a predictive biomarker strategy for selection of potentially sensitive lung cancer patients. Citation Format: Christina Esdar, Christel Iffland, Astrid Zimmermann, Angela Lim, Michael Sanderson, Andree Blaukat. M6123 is an ADCC-enhanced and selective monovalent antagonist of FGFR1 showing antitumor activity in preclinical lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4853.

Abstract 4415: CDK4/6 inhibition with lerociclib (G1T38) delays acquired resistance to targeted therapies in preclinical models of non-small cell lung cancer

Abstract Tyrosine kinase inhibitors (TKIs) targeting oncogenes such as EGFR, ALK, or RET have dramatically improved anti-tumor efficacy in patients with non-small cell lung cancer (NSCLC). Despite the significant clinical benefit offered by these agents, tumor responses to single-agent TKIs nevertheless remain limited in their magnitude and duration. Here we investigate combination therapy approaches with lerociclib (G1T38), a selective, oral, and potential best-in-class inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6). Consistent with previous studies reporting frequent cell-cycle gene alterations in NSCLC, we show that lerociclib synergizes with TKIs targeting EGFR, ALK, and RET in lung cancer cell lines and enhances the efficacy of osimertinib and crizotinib in PDX models harboring EGFR or ALK mutations, respectively. Moreover, in a well-characterized murine xenograft model of EGFR-mutant NSCLC, seven days of treatment with a clinically-relevant dose of osimertinib (25 mg/kg p.o. QDx7) resulted in 60% complete tumor responses, versus 100% complete tumor responses in the osimertinib plus lerociclib (25 mg/kg osi + 100 mg/kg lero, p.o. QDx7) group. Strikingly, complete tumor responses in the combination group persisted through an additional 35 days (or more) of observation, by which time all tumors in the osimertinib monotherapy group had re-emerged - suggesting a role for lerociclib in extending the duration of antitumor response. Based on this result, we assessed the ability of lerociclib to delay the emergence of resistance by chronically treating NSCLC cell lines with TKI +/- lerociclib. These experiments revealed that combination treatment with lerociclib significantly delayed acquired TKI resistance in multiple NSCLC cell lines. The same result was achieved in vivo in an EGFR-mutant NSCLC xenograft model known to develop resistance to EGFR TKI therapy, wherein combination treatment with lerociclib delayed the outgrowth of osimertinib-resistant tumors. Studies to characterize the mechanism by which lerociclib delays TKI resistance in NSCLC are ongoing. Finally, we addressed whether combination therapy with lerociclib can treat TKI resistance after it develops. In EGFR-mutant NSCLC xenografts harboring MET amplification - one of the more common mechanisms of resistance observed to EGFR TKIs in the clinic - the combination of lerociclib plus osimertinib caused significant tumor growth inhibition (90%) compared to osimertinib monotherapy (34%) after 46 days of continuous daily dosing. Collectively, our results suggest a compelling rationale for utilizing lerociclib as a backbone therapy for multiple combination regimens in NSCLC. A phase 1b/2 clinical trial evaluating lerociclib plus osimertinib in EGFR-mutant NSCLC is ongoing (NCT03455829). Citation Format: Daniel M. Freed, Claire R. Hall, Jay C. Strum, Patrick J. Roberts. CDK4/6 inhibition with lerociclib (G1T38) delays acquired resistance to targeted therapies in preclinical models of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4415.