Preclinical Models Of Lung Cancer Research Articles

Abstract 4447: Deep neural network for automatic histopathologic analysis of murine lung tumors

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide, and genetically-engineered mouse models (GEMMs) of cancer provide important mechanistic and preclinical insights into this deadly disease. In particular, the “KP” model enables lung-specific inducible activation of oncogenic Kras G12D, and loss of Trp53, the two most common driver events of human non-small cell lung cancer (NSCLC). Importantly, the KP model is widely used and faithfully recapitulates molecular and histopathological features of the human disease, including progression from early hyperplasia and adenoma to invasive adenocarcinoma. However, the KP model results in multi-focal and heterogeneous tumor burden, and there is a need for improved tools to increase throughput and decrease subjectivity of tumor burden quantification and histopathological analyses. To this end, we trained a convolutional neural network (CNN) for semantic multi-class segmentation using the Aiforia(R) platform. The CNN was trained to classify and detect lung parenchyma, NSCLC tumors, and NSCLC tumor grades (grade 1-4). For supervised training, we used selected areas from 93 hematoxylin and eosin stained slides. For validation, we analyzed 34 slides completely independent of the CNN training. Tumor grades were manually annotated on the validation slides blinded to the CNN results. The overall F1 score of the CNN in grade classification was 98% and total area error 0.3%. The grade-specific F1-scores were 89%, 97%, 99%, and 98% for grades 1, 2, 3, and 4, respectively. Corresponding grade-specific total area errors were 0.4%, 0.2%, 0.4%, and 0.1%. Manual scoring independent of the training and CNN yielded similar tumor burden and grading results. In addition, the algorithm accurately recapitulates the increased tumor burden and grade seen in KP tumors harboring additional mutation of the tumor suppressor Keap1, and the delayed kinetics of KP tumors harboring a strong T cell antigen, in independent datasets. We have also extended this methodology to identification of tumors in a GEMM of small cell lung cancer, a distinct class of lung cancer with poor prognosis. In conclusion, we demonstrate that deep neural networks can be used for automated analysis and grading of preclinical models of lung cancer. We anticipate that this powerful technology will increase the throughput, sensitivity and reproducibility of hypothesis-driven studies of factors influencing tumor progression and immune response in mouse models of lung cancer. Citation Format: Peter Maxwell Kienitz Westcott, Tuomas Pitkänen, Sami Blom, Thomas Westerling, Tuomas Ropponen, Nathan Sacks, Katherine Wu, Roderick Bronson, Tuomas Tammela, Tyler Jacks. Deep neural network for automatic histopathologic analysis of murine lung tumors [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 4447.

Abstract 1301: 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 pharmacophores and moieties 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 beaks 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 J. Turchi, Pamela S. VanderVere-Carozza, Navnath S. Gavande, Katherine S. Pawelczak. 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 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 1301.

Abstract 1508: Primary tumors induce neutrophil extracellular traps with targetable metastasis promoting effects

Abstract Targeting the dynamic tumor immune microenvironment can provide effective therapeutic strategies for cancer. Neutrophils (polymorphonuclear neutrophils, PMNs) are the predominant circulating leukocyte population in humans and are vital to fight infection. Despite mounting evidence that PMNs can promote tumor progression, depleting PMNs is not a viable therapeutic option. Neutrophil extracellular traps (NETs) are networks of extracellular neutrophil DNA fibers that are capable of trapping tumor cells and promoting their growth and their metastasis. Targeting NETs can therefore be a potentially successful therapeutic option to block the tumor promoting functions of PMNs. Here we demonstrate that circulating NET levels are elevated in esophageal, gastric and lung cancer patients compared to healthy controls. This increase correlates with disease stage and NET levels are independent predictors of advanced stage. Using pre-clinical murine models of lung and colon cancer, we observe elevated NET levels in tumor bearing mice compared to non-tumor bearing mice; these levels correlated with tumor size. NET levels significantly decrease following tumor resection or treatment with DNase1, a NET degrader, or neutrophil elastase inhibitor (NEi), a NET inhibitor. NET levels do not rise following tumor inoculation in peptidyl arginine deiminase-IV knock out (PAD4-/-) mice; PAD4 being an enzyme essential for citrullination of histones, a crucial step in NET release. Moreover, PMNs from tumor bearing mice are more primed for NETosis than PMNs from non-tumor bearing mice or NEi-treated or PAD4-/- tumor bearing mice. Finally, elevated in vivo hepatic adhesion and spontaneous liver and lung metastases are observed in tumor bearing mice compared to DNase1- or NEi-treated or PAD4-/- tumor bearing mice. Therefore, inhibiting NETs represents a promising strategy to impede metastatic dissemination in several types of cancer patients. Citation Format: Roni F. Rayes, Jack G. Mouhanna, Ioana Nicolau, Phil Vourtzoumis, Carson Wong, Jules Eustache, France Bourdeau, Betty Giannias, Aya Siblini, Emma Lee, Veena Sangwan, Simon Rousseau, Daniela Quail, Logan Walsh, Nicholas Bertos, Jonathan Cools-Lartigue, Lorenzo E. Ferri, Jonathan D. Spicer. Primary tumors induce neutrophil extracellular traps with targetable metastasis promoting effects [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 1508.

Abstract 4984: Development of an improved humanized patient-derived xenograft, Hu-PDX, mouse model for evaluation of antitumor immune response in lung cancer

Abstract Current preclinical models of non-small cell lung cancer (NSCLC) do not recapitulate the human tumor microenvironment. Mice reconstituted with a human immune system and bearing human patient derived xenografts may be advantageous in evaluating human anti-tumor immune response. We developed an improved NOD scid gamma (NSG) mouse model derived from non-expanded CD34+ stem cells, without CD3+ T cell contamination, to evaluate antitumor responses to immunotherapy in NSCLC. Using fresh CD34+ from umbilical cord blood reduced humanization time significantly. Human CD45+ cell reconstitution with increased functional human lymphoid (B, T, monocytes and NK cells) and myeloid (macrophages and MDSCs) lineage repopulation, without the onset of GvHD, was achieved as early as 4 weeks post-stem cell engraftment. Published studies using expanded CD34+ derived humanization reveal compromised purity of CD34+ stem cells with an increasing number of mononuclear cells. Reconstitution of CD8+ and CD4+T cells is not achieved until 12 to 15 weeks post-engraftment at much lower levels than fresh CD34+ humanization. Single cell suspension analysis shows levels of human reconstituted T, B, NK, DC and MDSC cells at 4 weeks, which increased significantly at 6 and 9 weeks in peripheral blood, spleen and bone marrow. Human repopulated T cells were functionally active in secretion of IFN-γ by mitogenic stimuli such as PMA and IL-2 and by allogenic human cancer cells. Antigen specific CTL responses were observed when reconstituted human T cells from PDX bearing humanized mice were challenged with PDX tumor. No non-antigen specific responses were observed when T cells were co-cultured with HLA-matched human bronchial epithelial cells (HBEC). To evaluate the applicability of the humanized mouse in lung cancer translational research, we combined it with Hu-PDX or Hu-xenograft tumors and analyzed tumor growth and treatment response to the anti-PD1 checkpoint inhibitor pembrolizumab. We found that efficient engraftment of PDXs and xenograft tumors were not dependent on donor HLA-status. Similar to the clinical outcome, treatment with pembrolizumab, inhibited tumor growth significantly in both Hu-PDX, and Hu-xenograft mice regardless of donor HLA-types, increasing cytotoxic T cells and decreasing MDSC levels. Pembrolizumab had no effect on the non-humanized NSG controls. In concordance with our previous study with a syngeneic mouse tumor, the antitumor effect of check point blockade was significantly enhanced when combined with nanoparticle systemically deliveredTUSC2, a tumor suppressor and immunomodulatory gene, in a KRAS mutant lung metastasis humanized mouse model. In conclusion, fresh CD34+ are more effective than their expanded counterparts in humanizing mice, do so in much reduced time, and recapitulate the immune response to cancer. Citation Format: Ismail M. Meraz, Mourad Majidi, Feng Meng, RuPing Shao, Min Jin Ha, Shinya Neri, Bingliang Fang, Steven H. Lin, Peggy T. Tinkey, Elizabeth J. Shpall, Jeffrey Morris, Jack A. Roth. Development of an improved humanized patient-derived xenograft, Hu-PDX, mouse model for evaluation of antitumor immune response in 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 4984.

Abstract 1499: Recolonizing microbiota may impact tumor response to PD-1 inhibition following antibiotic and radiotherapy treatment in a bioluminescent orthotopic model of murine lung cancer

Abstract Mounting clinical and preclinical evidence demonstrates an important role for the intestinal microbiome in mediating efficacy of immune checkpoint inhibitor (ICI) antibody therapy across a number of tumor contexts. We sought to determine the effects of microbiome modulation on ICI therapy in a clinically recapitulative orthotopic murine lung cancer model. The microbiome of C57Bl/6NHsd mice was sterilized with antibiotic (ampicillin, streptomycin and colistin) for ten days, resulting in a 99.9% mean decrease in fecal aerobic and anaerobic bacterial load in comparison to naïve mice, handled under specific pathogen free (SPF) conditions. Luciferase-expressing murine Lewis lung carcinoma cells (LL/2-Red-FLuc) were surgically implanted into the left lung parenchyma of all animals. Animals were treated with locoregional radiotherapy (2x 9Gy fractions) targeted to the left lung. The gastrointestinal microbiome was reconstituted via oral gavage Q3D of ~1e09/dose commensal A. muciniphila (A. muc) and E. hirae (E. hir) over five doses or sterile saline as control and animals were randomized within commensal/saline treatment groups to equivalent mean tumor burden as measured by Lumina Series III In-Vivo Imaging System (IVIS). Animals were then administered anti-murine PD-1 or isotype control (0.25 mg) antibody (Ab) treatments Q3D over four doses via intraperitoneal injection. Tumor growth was monitored by IVIS over the course of the study, and ex-vivo IVIS was performed on lungs at endpoint (Day 12 post-tumor implantation). Tumor growth of microbiota non-reconstituted antibiotic-sterilized animals was only slightly inhibited by anti-PD-1 therapy. Animals administered commensal A. muc and E. hir and treatments also displayed slightly inhibited tumor growth kinetics, similar to those observed under saline/anti-PD-1 therapy. Fecal microbial sequencing and immunophenotypic analyses are ongoing. This study demonstrates the utility and ongoing development of a clinically recapitulative contextually accurate preclinical murine lung cancer model to assess the effects of specific microbiota in mediating the efficacy of anti-tumor immunotherapy. Citation Format: Benjamin G. Cuiffo, Caitlin S. Parello, Chelsea Ritchie, Nicholas Rivelli, Alexandra Kury, Sallyann Vu, Gavin Gagnon, Veronica Ritchie, Kasey Reardon, Catarina Costa, Samantha Rogers, Gregory D. Lyng, Stephen T. Sonis. Recolonizing microbiota may impact tumor response to PD-1 inhibition following antibiotic and radiotherapy treatment in a bioluminescent orthotopic model of murine 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 1499.

Host transcriptomic signatures associated with dysbiosis in a preclinical model of lung cancer.

3107 Background: Enrichment of the lower airway microbiota with oral commensals has been associated with transcriptomic changes affecting several inflammatory pathways associated with non-small cell lung cancer (NSCLC) development and progression. Using a mouse model of NSCLC, we evaluated the effects of lower airway dysbiosis on tumor progression and host transcriptomics. Methods: Preclinical model of lung cancer was constructed by introducing luminescence-tagged Kras mutated cells into C57/B6 mice, causing lung cancer to develop. Lower airway dysbiosis was induced by weekly intratracheal challenge with either PBS or Veillonella parvula in wild type and lung cancer mice. Experiments were repeated twice to evaluate for survival as well as lower airway host response using flow cytometry and RNA sequencing (HiSeq). Sequence data was processed using a validated mouse gene expression signature matrix with cibersort from https://cibersort.stanford.edu and DESeq using FDR correction. Results: In wild type mice, lower airway dysbiosis with Veillonella did not affect the survival, weight gain or airway lumen diameter. Among lung cancer mice, dysbiosis led to increased mortality, weight loss, and tumor burden. Multiple transcriptomic signatures were identified among the dysbiosis groups (both in WT and lung cancer mice). Unsupervised hierarchical clustering of immune cell profiles using cibersort on whole transcriptome showed near perfect separation between the four experimental conditions. Amongst the most differentially enriched immune cell subsets, we identified that lung dysbiosis upregulates genes annotated to Th1 and Th2 cells (p < 0.01, q < 0.2). Using flow cytometry, we identified that PD-1, IL-17, and ROR-gamma are differentially expressed in CD4+ cells in dysbiosis conditions, and these patterns are consistent in whole RNA transcriptome. Conclusions: Transcriptomic signatures reveal immune profiles associated with dysbiosis, an experimental condition associated with worse outcomes in lung cancer. This investigation provides novel insights into how disruption of the lower airway microbiome may contribute to the pathogenesis of NSCLC.

Lenvatinib (len) plus pembrolizumab (pembro) in patients (pts) with advanced melanoma previously exposed to anti–PD-1/PD-L1 agents: Phase 2 LEAP-004 study.

TPS9594 Background: Pembro, a PD-1 inhibitor, has shown effective antitumor activity, deep and durable responses, and survival benefit in treatment-naive pts and those with previously treated metastatic melanoma. Len, a potent inhibitor of VEGF receptors 1-3, FGF receptors 1-4, PDGF receptor α, RET, and KIT, combined with a PD-1 inhibitor showed antitumor activity superior to either agent alone in preclinical models of colorectal and lung cancer. Additionally, len plus pembro showed anti-tumor activity and was well-tolerated (24-wk ORR, 47.6%; TRAE: gr 3/4, 67%; gr 5, 0%) in pts with advanced melanoma previously treated with 0-2 therapies in the phase 1b/2 KEYNOTE-146 trial. The efficacy and safety of len plus pembro combination therapy will be evaluated in an open-label, phase 2 trial of pts with advanced melanoma that progressed on PD-1/PD-L1 inhibitor therapy (NCT03776136). Methods: Key inclusion criteria: age ≥18 years, histologically/cytologically confirmed unresectable stage III-IV melanoma that progressed (per iRECIST) within 12 weeks of last dose of an approved PD-1/PD-L1 inhibitor therapy (≥2 doses as monotherapy or combined with other therapies), measurable disease, ECOG PS 0/1, no active autoimmune disease, and adequate organ function. Pts must provide a baseline tumor sample. Pts will receive len 20 mg/day orally plus pembro 200 mg IV Q3W for approximately 2 years (35 doses of pembro), after which they may receive len alone until PD or unacceptable toxicity. Response will be assessed per RECIST v1.1 based on blinded independent central review (BICR) Q9W until week 54, Q12W until week 102, and Q24W thereafter. Pts with CR may discontinue treatment after ≥24 weeks of therapy; eligible pts may continue treatment beyond initial RECIST- or iRECIST-defined PD. AEs will be assessed throughout treatment and for 90 days (120 days for serious AEs) after last dose and graded per NCI CTCAE v4.0. Pts will be followed-up for survival status Q12W. The primary efficacy end point is ORR per modified RECIST v1.1 (BICR). Key secondary end points are PFS and DOR per modified RECIST v1.1 (BICR), OS, and safety; an exploratory biomarker analysis is planned. Clinical trial information: NCT03776136.

A Novel Laser Fiberscope for Simultaneous Imaging and Phototherapy of Peripheral Lung Cancer

Phototherapy is an alternative treatment for patients with localized non-small cell lung cancer who are unable to undergo surgical resection. However, phototherapy is currently limited to treatment of centrally located lung cancer, with the much larger proportion of peripheral lesions remaining inaccessible. There are also concerns over the accuracy of targeted laser treatment because of the need to exchange visualization and irradiation fibers during therapy, preventing the operator from confirming the final location of the irradiation fiber. A newly developed parallel-type ultrasmall composite optical fiberscope (Laser-eYe Ultrathin fiberscope [LYU]), which enables simultaneous white-light imaging and phototherapy, was evaluated in preclinical lung cancer models. Three models were used: human lung cancer xenografts (A549) in mice, orthotopic VX2 lung tumors in rabbits, and exvivo pig lungs into which A549 tumor tissue was transplanted. A multifunctional porphyrin-phospholipid nanoparticle (porphysome) was used as a photosensitizer to evaluate fluorescence-guided photothermal therapy. The LYU's 0.97mm diameter and hydrophilic coating allowed easy passage through the working channel of all types of bronchoscopes and controlled guidance of the LYU tip in any desired direction. The LYU could visualize the peripheral bronchus and porphysome-laden peripheral tumors. The LYU could also perform photothermal therapy with simultaneous imaging. The LYU enables simultaneous imaging and phototherapy that allows accurate irradiation of peripheral lung cancers. This new laser device may enable ultraminimally invasive transbronchial treatment of peripheral lung cancer.

Ex vivo Hsp70-Activated NK Cells in Combination With PD-1 Inhibition Significantly Increase Overall Survival in Preclinical Models of Glioblastoma and Lung Cancer.

Heat shock protein 70 (Hsp70) which is expressed on the plasma membrane of highly aggressive tumors including non-small cell lung carcinoma and glioblastoma multiforme serves as a target for Hsp70-targeting NK cells. Herein, we aimed to investigate the antitumor effects of a combined therapy consisting of ex vivo Hsp70-peptide TKD/IL-2-activated NK cells in combination with mouse/human anti-PD-1 antibody in a syngeneic glioblastoma and a xenograft lung cancer mouse model. Mice with membrane Hsp70 positive syngeneic GL261 glioblastoma or human xenograft A549 lung tumors were sham-treated with PBS or injected with ex vivo TKD/IL-2-activated mouse/human NK cells and mouse/human PD-1 antibody either as a single regimen or in combination. Tumor volume was assessed by MR scanning and tumor-infiltrating CD8+ T, NK, and PD-1+ cells were quantified by immunohistochemistry (IHC). We could show that the adoptive transfer of ex vivo TKD/IL-2-activated mouse NK cells or the inhibition of PD-1 resulted in tumor growth delay and an improved overall survival (OS) in a syngeneic glioblastoma mouse model. A combination of both therapies was well-tolerated and significantly more effective with respect to both outcome parameters than either of the single regimens. A combined treatment in a xenograft lung cancer model showed identical effects in immunodeficient mice bearing human lung cancer after adoptive transfer of TKD/IL-2-activated human effector cells and a human PD-1 antibody. Tumor control was associated with a massive infiltration with CD8+ T and NK cells in both tumor models and a decreased in PD-1 expression on immune effector cells. In summary, a combined approach consisting of activated NK cells and anti-PD-1 therapy is safe and results in a long-term tumor control which is accompanied by a massive tumor immune cell infiltration in 2 preclinical tumor models.

Advances in Preclinical Models of Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an invasive and high-grade neuroendocrine malignant tumor. It is characterized by short doubling time, high proliferation rate, and early extensive metastasis. SCLC is sensitive to radiotherapy and chemotherapy in the initial stage; however, it can easily relapse and develop drug resistance. In the past five years, there has been a resurgence of research on SCLC worldwide, including the establishment of SCLC cells, the development of related genetically engineered mouse models (GEMMs) and the establishment of patient-derived xenograft models (PDXs). These studies have identified new potential therapeutic vulnerabilities for SCLC, leading to new clinical trials. In this perspective, the establishment, application, and advantages and disadvantages of three preclinical research models of SCLC are systematically summarized.

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

Abstract The recent FDA approvals of palbociclib, ribociclib, and abemaciclib in breast cancer validate cyclin-dependent kinases 4 and 6 (CDK4/6) as key therapeutic targets, and warrant investigations in other tumor types with frequently observed alterations in the CDK4/6 pathway. Towards this end, we assessed the preclinical antitumor efficacy of G1T38, an oral, potent, and selective small-molecule CDK4/6 inhibitor in clinical development, in models of non-small cell lung cancer (NSCLC). Importantly, preclinical studies have shown that G1T38 has a differentiated and potential best-in-class profile since it can be dosed continuously without causing severe neutropenia, which could result in better tumor efficacy. Initial efficacy screening of G1T38 in 60 patient-derived NSCLC xenografts revealed significant tumor growth inhibition (TGI), primarily in lung adenocarcinomas harboring frequently observed oncogenic alterations in KRAS, EGFR, BRAF, and ALK. In vitro analyses show that CDK4/6 inhibition enhances the antiproliferative effect of inhibitors targeting these oncogenes (or their signaling pathways), suggesting a possible role for G1T38 in augmenting response and/or delaying acquired resistance to agents currently utilized in the clinic. These results were validated in vivo in mouse NSCLC xenograft models with defined oncogenic alterations, including EGFR mutations. For example, in H1975 xenografts harboring EGFR L858R/T790M (the T790M mutation is responsible for over 50% of cases of acquired resistance to first-generation EGFR inhibitors), combination of G1T38 with erlotinib resulted in 77% TGI after 18 days–a significant improvement over either therapy alone. Moreover, in H1975 xenografts, G1T38 delayed resistance to the second-generation EGFR inhibitor afatinib, and upon development of resistance to afatinib monotherapy, addition of G1T38 to afatanib treatment resulted in stabilization of tumor growth. G1T38 also augmented the response of EGFR-mutant NSCLC xenografts to the third-generation EGFR inhibitor osimertinib, significantly enhancing TGI compared to either therapy alone. As a result, a safety, pharmacokinetics, and efficacy study of G1T38 + osimertinib (G1T38-03) is scheduled for initiation in 1Q18 in patients with EGFR-mutant, T790M-positive NSCLC. Additional efforts are ongoing to investigate the combination of G1T38 with targeted therapies in NSCLC murine models harboring alterations in KRAS, ALK, and BRAF. Together, our results suggest a compelling rationale for utilizing G1T38 as a backbone for multiple targeted therapy combination regimens in NSCLC. Citation Format: Jessica A. Sorrentino, Daniel M. Freed, John E. Bisi, Jay C. Strum, Patrick J. Roberts. The CDK4/6 inhibitor G1T38 enhances response to targeted therapies in preclinical models of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1522.

The Autotaxin-Lysophosphatidic Acid Axis Promotes Lung Carcinogenesis.

Pathogenesis and progression of lung cancer are governed by complex interactions between the environment and host genetic susceptibility, which is further modulated by genetic and epigenetic changes. Autotaxin (ATX, ENPP2) is a secreted glycoprotein that catalyzes the extracellular production of lysophosphatidic acid (LPA), a growth-factor-like phospholipid that is further regulated by phospholipid phosphatases (PLPP). LPA's pleiotropic effects in almost all cell types are mediated through at least six G-protein coupled LPA receptors (LPAR) that exhibit overlapping specificities, widespread distribution, and differential expression profiles. Here we use both preclinical models of lung cancer and clinical samples (from patients and healthy controls) to investigate the expression levels, activity, and biological role of the above components of the ATX/LPA axis in lung cancer. ENPP2 was genetically altered in 8% of patients with lung cancer, whereas increased ATX staining and activity were detected in patient biopsies and sera, respectively. Moreover, PLPP3 expression was consistently downregulated in patients with lung cancer. Comparable observations were made in the two most widely used animal models of lung cancer, the carcinogen urethane-induced and the genetically engineered K-rasG12D -driven models, where genetic deletion of Enpp2 or Lpar1 resulted in disease attenuation, thus confirming a procarcinogenic role of LPA signaling in the lung. Expression profiling data analysis suggested that metabolic rewiring may be implicated in the procarcinogenic effects of the ATX/LPA axis in K-ras- G12D -driven lung cancer pathogenesis.Significance: These findings establish the role of ATX/LPA in lung carcinogenesis, thus expanding the mechanistic links between pulmonary fibrosis and cancer. Cancer Res; 78(13); 3634-44. ©2018 AACR.

Patient-derived conditionally reprogrammed cells maintain intra-tumor genetic heterogeneity

Preclinical in vitro models provide an essential tool to study cancer cell biology as well as aid in translational research, including drug target identification and drug discovery efforts. For any model to be clinically relevant, it needs to recapitulate the biology and cell heterogeneity of the primary tumor. We recently developed and described a conditional reprogramming (CR) cell technology that addresses many of these needs and avoids the deficiencies of most current cancer cell lines, which are usually clonal in origin. Here, we used the CR cell method to generate a collection of patient-derived cell cultures from non-small cell lung cancers (NSCLC). Whole exome sequencing and copy number variations are used for the first time to address the capability of CR cells to keep their tumor-derived heterogeneity. Our results indicated that these primary cultures largely maintained the molecular characteristics of the original tumors. Using a mutant-allele tumor heterogeneity (MATH) score, we showed that CR cells are able to keep and maintain most of the intra-tumoral heterogeneity, suggesting oligoclonality of these cultures. CR cultures therefore represent a pre-clinical lung cancer model for future basic and translational studies.

Combination of bemcentinib (BGB324): A first-in-class selective oral AXL inhibitor, with pembrolizumab in patients with triple negative breast cancer and adenocarcinoma of the lung.

TPS43 Background: Activation of the receptor tyrosine kinase AXL has a profound suppressive effect on the innate immune system. AXL is overexpressed on multiple cell types in the tumour immune microenvironment including dendritic cells, NK cells and tumour-associated macrophages. AXL signalling in immune cells supports tumour immune escape by downregulating dendritic cell activity, modulating efferocytosis as well as favouring an immunosuppressive chemokine profile and M-MDSC expansion. AXL is prevalent in tumours resistant to anti-PD-1 therapy (Hugo, 2016). Axl expression in tumour cells confers resistance to effector T cell cytotoxicity. Bemcentinib (BGB324) is a first-in-class, highly selective and orally bioavailable small molecule AXL inhibitor in phase II clinical development. In pre-clinical models of pancreas, breast and lung cancer, inhibition of AXL signalling with bemcentinib reversed multiple tumour immune suppressive mechanisms as evidenced by increased infiltration of cytotoxic T lymphocytes, NK and NKT cells and decreased infiltration of M-MDSCs (Wnuk-Lipinska, 2017). Bemcentinib enhanced the effect of immune checkpoint blockade via PD-1 or CTLA-4 in lung and mammary adenocarcinoma mouse models and achieved sustained tumour immunity. Methods: BGBC007 (NCT03184558) and BGBC008 (NCT03184571) are open-label, phase II studies designed to assess the anti-tumour activity of bemcentinib in combination with pembrolizumab in patients with previously treated TNBC and adenocarcinoma of the lung respectively. All patients will be treated with bemcentinib in combination with pembrolizumab continuously for up to two years. The primary endpoint is objective response rate, secondary endpoints include duration of response, progression free survival according to RECIST 1.1, pharmacokinetics, safety and tolerability. Pretreatment tumour specimens are scheduled to assess AXL expression/signalling and PD-L1 expression; the levels of circulating immune-related cytokines and soluble AXL receptor will also be measured in longitudinal patient plasma samples. Both studies are open to recruitment. Clinical trial information: NCT03184558.

Abstract A160: Combination of necitumumab with second- or third-generation EGFR tyrosine kinase inhibitors downregulates DNA repair mechanisms and induces durable tumor remissions in xenograft NSCLC models with EGFR mutations

Abstract First- , second- , and third-generation EGFR tyrosine kinase inhibitors (TKIs) demonstrate profound tumor responses in EGFR mutation-positive NSCLC. Nevertheless, most patients invariably develop acquired resistance. The objective of the present study was to evaluate the effects of dual EGFR blockade using a ligand-blocking antibody (necitumumab) and second- or third-generation TKIs (afatinib or osimertinib) in preclinical NSCLC models with EGFR mutations. Afatinib and osimertinib (AZD9291), second- and third-generation EGFR TKIs, respectively, are irreversible covalent inhibitors of the EGFR tyrosine kinase, with the latter specifically targeting a mutant form of the receptor (EGFR-T790M). We sought to investigate whether combination of necitumumab (LY3012211) with afatinib and osimertinib would provide any benefit over the monotherapies in preclinical mouse models of lung cancer. Using PC-9 and NCI-H1975 xenograft models of NSCLC that harbor EGFR mutations, we observed that dual EGFR blockade of ligand binding and receptor tyrosine kinase activity improved the antitumor efficacy and, more importantly, resulted in durable tumor remissions compared to either single-agent therapy. Additionally, we explored two dosing schedules of necitumumab/osimertinib combination treatment (concurrent versus phased). Concurrent administration of both agents for four weeks was significantly more efficacious compared to the phased schedule (e.g., administration of osimertinib for one week followed by necitumumab and osimertinib combination for additional three weeks). To provide mechanistic insights into the combinatorial activity observed, we employed high-content gene expression analysis using nCounter Pan-Cancer Pathways assay. Pathway analysis of the combination and single-agent treatment groups identified multiple shared and treatment-specific pathway alterations. Genes implicated in FGFR, PI3K pathways and extracellular remodeling appeared to be upregulated by combination and monotherapies and suggested potential common mechanisms of acquired resistance to EGFR inhibitors, whereas compromised DNA repair mechanisms were identified in PC-9 and NCI-H1975 tumors upon treatment with both combinations. In conclusion, these data demonstrate that combination of necitumumab with second- or third-generation EGFR TKIs results in an improved antitumor efficacy compared to the respective monotherapies; a more profound inhibition of EGFR pathway and compromised DNA repair mechanisms may underlie this effect. A phase I clinical trial of necitumumab and osimertinib in EGFR mutation-positive stage IV or recurrent NSCLC who have progressed on a previous EGFR TKI is currently ongoing (NCT02496663). Citation Format: Amelie Forest, Erik R. Rasmussen, Thompson N. Doman, Michael Amatulli, Rajiv Bassi, Gerald E. Hall, Jason R. Manro, Manisha Brahmachary, David Surguladze, Yung-mae M. Yao, Michael D. Kalos, Ruslan D. Novosiadly. Combination of necitumumab with second- or third-generation EGFR tyrosine kinase inhibitors downregulates DNA repair mechanisms and induces durable tumor remissions in xenograft NSCLC models with EGFR mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A160.

Harnessing innate lung anti-cancer effector functions with a novel bacterial-derived immunotherapy

ABSTRACTAcute infection is known to induce strong anti-tumor immune responses, but clinical translation has been hindered by the lack of an effective strategy to safely and consistently provoke a therapeutic response. These limitations are overcome with a novel treatment approach involving repeated subcutaneous delivery of a Klebsiella-derived investigational immunotherapeutic, QBKPN. In preclinical models of lung cancer, QBKPN administration consistently showed anti-cancer efficacy, which was dependent on Klebsiella pre-exposure, but was independent of adaptive immunity. Rather, QBKPN induced anti-tumor innate immunity that required NK cells and NKG2D engagement. QBKPN increased NK cells and macrophages in the lungs, altered macrophage polarization, and augmented the production of cytotoxic molecules. An exploratory trial in patients with non-small cell lung cancer demonstrated QBKPN was well tolerated, safe, and induced peripheral immune changes suggestive of macrophage polarization and reduction of PD-1 and PD-L1 expression on leukocytes. These data demonstrate preclinical efficacy, and clinical safety and tolerability, for this cancer immunotherapy strategy that exploits innate anti-tumor immune mechanisms.

PUMA mediates the anti-cancer effect of osimertinib in colon cancer cells.

Osimertinib, an irreversible EGFR/HER2 inhibitor, has been found to be effective in the cancer cell with EGFR gene mutations in preclinical lung cancer models. However, the effect of osimertinib in colorectal cancer (CRC) cells is unclear. In the present study, we investigated how osimertinib suppresses CRC cells growth and potentiates effects of other chemotherapeutic drugs. We found that p73-mediated osimertinib-induced p53 upregulated modulator of apoptosis (PUMA) expression irrespective of p53 status following PI3K/AKT pathway inhibition in CRC cells. Furthermore, PUMA is required for osimertinib-induced apoptosis. In addition, osimertinib also synergized with 5-FU to induce significant apoptosis via PUMA in CRC cells. These results demonstrated a critical role of PUMA in mediating the anticancer effects of osimertinib and suggest that PUMA induction can be used as an indicator of osimertinib sensitivity.

Extended topoisomerase 1 inhibition through liposomal irinotecan results in improved efficacy over topotecan and irinotecan in models of small-cell lung cancer.

Liposomal irinotecan (irinotecan liposome injection, nal-IRI), a liposomal formulation of irinotecan, is designed for extended circulation relative to irinotecan and for exploiting discontinuous tumor vasculature for enhanced drug delivery to tumors. Following tumor deposition, nal-IRI is taken up by phagocytic cells followed by irinotecan release and conversion to its active metabolite, SN-38. Sustained inhibition of topoisomerase 1 by extended SN-38 exposure as a result of delivery by nal-IRI is hypothesized to enable superior antitumor activity compared with traditional topoisomerase 1 inhibitors such as conventional irinotecan and topotecan. We evaluated the antitumor activity of nal-IRI compared with irinotecan and topotecan in preclinical models of small-cell lung cancer (SCLC) including in a model pretreated with carboplatin and etoposide, a first-line regimen used in SCLC. Nal-IRI demonstrated antitumor activity in xenograft models of SCLC at clinically relevant dose levels, and resulted in complete or partial responses in DMS-53, DMS-114, and NCI-H1048 cell line-derived models as well as in three patient-derived xenograft models. The antitumor activity of nal-IRI was superior to that of topotecan in all models tested, which generally exhibited limited control of tumor growth and was superior to irinotecan in four out of five models. Further, nal-IRI demonstrated antitumor activity in tumors that progressed following treatment with topotecan or irinotecan, and demonstrated significantly greater antitumor activity than both topotecan and irinotecan in NCI-H1048 tumors that had progressed on previous carboplatin plus etoposide treatment. These results support the clinical development of nal-IRI in patients with SCLC.

Inhibition of ataxia telangiectasia related-3 (ATR) improves therapeutic index in preclinical models of non-small cell lung cancer (NSCLC) radiotherapy

Background and purposeTo evaluate the impact of ATR inhibition using AZD6738 in combination with radiotherapy on the response of non-small cell lung cancer (NSCLC) tumour models and a murine model of radiation induced fibrosis. Materials and methodsAZD6738 was evaluated as a monotherapy and in combination with radiation in vitro and in vivo using A549 and H460 NSCLC models. Radiation induced pulmonary fibrosis was evaluated by cone beam computed tomography (CBCT) and histological staining. ResultsAZD6738 specifically inhibits ATR kinase and enhanced radiobiological response in NSCLC models but not in human bronchial epithelial cells (HBECs) in vitro. Significant tumour growth delay was observed in cell line derived xenografts (CDXs) of H460 cells (p<0.05) which were less significant in A549 cells. Combination of AZD6738 with radiotherapy showed no significant change in lung tissue density by CBCT (p>0.5) and histological scoring of radiation induced fibrosis (p>0.5). ConclusionInhibition of ATR with AZD6738 in combination with radiotherapy increases tumour growth delay without observable augmentation of late radiation induced toxicity further underpinning translation towards clinical evaluation in NSCLC.

Abstract DDT02-04: A novel PRMT5 inhibitor with potent in vitro and in vivo activity in preclinical lung cancer models

Abstract PRMT5 is a type II methyltransferase that specifically adds methyl groups to arginine as a long-lasting post-translational modification. The PRMT5/MEP50 complex regulates a plethora of cellular processes, such as epigenetics and splicing, which are notable events involved in tumorigenesis. Although not frequently mutated or amplified in tumors, elevated PRMT5 protein levels in lung and hematologic cancers are correlated with poorer survival. The PRMT5 inhibitor JNJ-64619178 has been selected as a clinical candidate based on its high selectivity and potency (subnanomolar range) under different in vitro and cellular conditions, paired with favorable pharmacokinetics and safety properties. JNJ-64619178 binds into the SAM binding pocket and reaches the substrate binding pocket to inhibit PRMT5/MEP50 function in a time-dependent manner. Broad cell line panel profiling of JNJ-64619178 revealed a wide range of sensitivity, which is indicative of a genomic dependency instead of a general cytotoxic on-target consequence of PRMT5 inhibition. Further investigations indicate a synthetic lethal correlation between PRMT5 inhibition and key cancer driver pathways. JNJ-64619178, dosed orally (10 mg/kg, every day), showed selective and efficient blockage of the methylation of SMD1/3 proteins, which are crucial components of the spliceosome and substrates of PRMT5/MEP50. JNJ-64619178 also demonstrated tumor regression in a biomarker-driven human small cell lung cancer xenograft model (NCI-H1048) and prolonged tumor growth inhibition after dosing cessation. In rodent and nonrodent toxicology studies, a tolerated dose of JNJ-64619178 has been identified, with the observed toxicity consistent with on-target activity. In summary, JNJ-64619178 has a favorable preclinical package that supports clinical testing in patients diagnosed with lung cancer and hematologic malignancies. Citation Format: Dirk Brehmer, Tongfei Wu, Geert Mannens, Lijs Beke, Petra Vinken, Dana Gaffney, Weimei Sun, Vineet Pande, Jan-Willem Thuring, Hillary Millar, Italo Poggesi, Ivan Somers, An Boeckx, Marc Parade, Erika van Heerde, Thomas Nys, Carol Yanovich, Barbara Herkert, Tinne Verhulst, Marc Du Jardin, Lieven Meerpoel, Christopher Moy, Gaston Diels, Marcel Viellevoye, Wim Schepens, Alain Poncelet, Joannes T. Linders, Edward C. Lawson, James P. Edwards, Dushen Chetty, Sylvie Laquerre, Matthew V. Lorenzi. A novel PRMT5 inhibitor with potent in vitro and in vivo activity in preclinical lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr DDT02-04. doi:10.1158/1538-7445.AM2017-DDT02-04