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Hongbin Ji
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MA17 - Molecular Mechanisms and Therapies (ID 143)
- Event: WCLC 2019
- Type: Mini Oral Session
- Track: Biology
- Presentations: 12
- Now Available
- Moderators:Eloisa Jantus-Lewintre, Hongbin Ji
- Coordinates: 9/09/2019, 15:45 - 17:15, Melbourne (1991)
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MA17.01 - Cell Lineage and Chromatin Landscape of Lung Cancer Are Controlled by GATA6 (Now Available) (ID 59)
15:45 - 17:15 | Presenting Author(s): Anna Arnal Estapé | Author(s): Wesley L. Cai, Alexandra E. Albert, Francesc López-Giráldez, Minghui Zhao, Laura E. Stevens, Kiran D. Patel, Don X. Nguyen
- Abstract
- Presentation
Background
Thoracic malignancies are histologically and biologically heterogeneous. The underlying causes of this heterogeneity are believed to be linked to the complex cellular ontogenies of lung cancers and their relationship to pulmonary development. Lineage selective transcription factors (TFs) are critical determinants of airway cell differentiation and homeostasis, but their biological requirements are often conditional. Analogously, several developmental TFs can paradoxically enhance or inhibit lung cancer progression, depending on cellular and epigenetic contexts which remain largely undefined. In this study, we report a novel function for the endodermal and pulmonary specifying TF GATA6 in lung cancer.
Method
To understand the role of GATA6 in lung tumorigenesis we used a genetically engineer mouse model (GEMMs) harboring Kras p53 mutations using progenitor cell specific gene targeting. We combined GEMMs biology with integrated analysis of the transcriptome and the chromatin landscape of lung cells derived from GATA6 deficient tumors.
Result
In this study, we uncover a conditional role for the endodermal and pulmonary specifying TF GATA6 during the initiation and progression of Kras mutant lung adenocarcinoma (LUAD). Inhibition of Gata6 in genetically engineered mouse models dampens the proliferation and increases the differentiation of LUAD tumors. These effects are influenced by the epithelial cell type that is targeted for transformation, demonstrating that GATA6 expression is an important molecular determinant of the cell of origin in Kras mutant lung cancer. In LUAD cells derived from surfactant protein C expressing progenitors, we identify multiple genomic loci that are bound by GATA6. Moreover, suppression of Gata6 in these cells significantly alters chromatin accessibility, particularly at distal enhancer elements. Analogous to its paradoxical activity in the developing lungs, GATA6 expression fluctuates during different stages of LUAD progression and can epigenetically control diverse lineage programs associated with cell proliferation, alveolar specification, BMP signaling, and epithelial plasticity.
In summary:
1) GATA6 expression varies during different stages of disease progression in the lung adenocarcinoma (LUAD) subtype
2) Suppression of Gata6 can diminish the proliferation and progression of LUAD in a manner that is influenced by the transforming progenitor of origin
3) GATA6 differentially modulates chromatin accessibility across the genome of LUAD cells
4) This epigenomic mechanism results in the activation of different lineage specific programs, including the BMP signaling pathway.
Conclusion
These findings reveal how GATA6 can modulate the chromatin landscape of lung cancer cells to control their divergent lineage dependencies during tumor progression.
D.X.N. has received research funding from AstraZeneca, Inc.
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MA17.02 - Identify Vulnerable Pathways and Improve Treatment Outcomes in LKB1-Deficient Lung Tumors (Now Available) (ID 2067)
15:45 - 17:15 | Presenting Author(s): Meng Xu Welliver | Author(s): Feng Jin, Joseph M Amann, Michael Koenig, Xiaokui Mo, David P Carbone
- Abstract
- Presentation
Background
The LKB1 tumor suppressor is inactivated in about 20% of non-small cell lung cancers (NSCLC) by mutations. Cancer cells with LKB1 deficiency exert complex effects on signal transduction and transcriptional regulation, which may cause these cells more susceptible to certain therapies comparing to cells with intact LKB1 function. Phenformin, an antidiabetic medicine from the biguanides class, has shown activities against NSCLC. Phenformin as a single agent has been shown to reduce tumor burden and prolonged survival in Kras;Lkb1 compound mutant mice but not Kras;p53 mice, suggesting specific activities in tumor with LKB1 deficiency. Currently patients with unresectable locally advanced NSCLC are treated standardly with concurrent chemoradiotherapy followed by checkpoint inhibitor, durvalumab. In this project, we test treatment sensitivity to radiotherapy and/or phenformin in lung cancer cells with intact or deficient LKB1.
Method
Human lung cancer cell lines described below were used in (1) clonogenic survival assays as well as (2) generating tumor xenograft on nude mice for tumor growth delay experiments. A549, HCC15 and Calu-1 cell lines obtained from ATCC were cultured in RPMI1640 containing 5% FBS, without antibiotics. A549 cells (LKB1 deficient, TP53 WT and KRAS mutated) or HCC15 (LKB1 deficient, TP53 mutated and KRAS WT) were transfected with empty vector or WT LKB1 or LKB1-K78I plasmids; Calu-1 (LKB1 WT, KRAS mutated and p53 deleted) transfected with empty vector or LKB1 CRIPR KO were generated as described previously.
Result
A549 cells with transfected WT-LKB1 were significantly more resistant to ionizing radiation (IR) induced cell kill (8.7% survival at 8 Gy) comparing to cells transfected with empty vector (3.7%) or kinase-dead LKB1 genes (4.2%). Similarly, HCC15 cells with transfected WT-LKB1 are significantly more resistant to IR induced cell kill (7.5%) comparing to cells transfected with empty vector (4.1%) or kinase-dead LKB1 genes (3.4%). Calu-1 cells harbor WT LKB1, and it is significantly more resistant to IR induced cell kill (8.3%) comparing to their counterpart with LKB1 KO (Calu-1 transfected with LKB1 CRIPR KO) (4.1%). When A549 cells were pretreated with 30 μM phenformin prior to, during and after IR, there was no change in survival in cells transfected with WT LKB1; however there was significant further reduction in survival in cells transfected with empty vector (LKB1 deficient). Xenograft tumors were generated in nude mice with A549 cells with the above genetic alterations. There was significant further tumor growth delay in those with A549 with deficient LKB1 comparing to those with A549 with WT LKB1 gene add-back. This tumor growth delay was further enhanced when these mice were treated with oral phenformin prior to, during and after IR treatment, confirming the in vitro experimental results.
Conclusion
Human lung cancer with deficient LKB1 are more sensitive to ionizing radiation in vitro and in vivo. This was regardless of the TP53 or KRAS mutation status. A549 cells with deficient LKB1 were also more sensitive to phenformin treatment. Phenformin treatment further sensitized LKB1 deficient lung cancer cells to IR. This suggested that LKB1 can serve as a predictive biomarker to triage patient treatments.
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MA17.03 - Importance of Cullin4 Ubiquitin Ligase in Malignant Pleural Mesothelioma (Now Available) (ID 2349)
15:45 - 17:15 | Presenting Author(s): Mayura Meerang | Author(s): Jessica Kreienbühl, Vanessa Orlowski, Michaela B Kirschner, Walter Weder, Isabelle Opitz
- Abstract
- Presentation
Background
Loss of the tumor suppressor NF2 is frequent in malignant pleural mesothelioma (MPM). NF2 suppresses tumorigenesis in part by inhibiting Cullin4 ubiquitin ligase (CUL4) complex. Here we aimed to evaluate an importance of CUL4 in MPM.
Method
We evaluated the expression of CUL4A and CUL4B in tissue microarrays using immunohistochemistry. We tested the efficacy of cullin inhibition by pevonedistat, a small molecule inhibiting cullin neddylation, in 13 cell lines and 3 primary cells in 2D and 3D culture. Four groups of SCID mice haboring intraperitoneal (ip.) pevonedistat sensitive (MSTO211H) or resistant (ACC-Meso1) cell lines were treated with pevonedistat (50 mg/kg; ip.) on a 5day on/5day off schedule for 3 cycles. Treatment efficacy was assessed by means of overall survival.
Result
CUL4B expression was associated with clinical outcomes (figure 1). Five MPM cell lines (38%) were highly sensitive to pevonedistat (IC50<500 nM). This remained true in 3D spheroid culture. The treatment induced S/G2 cell cycle arrest and accumulation of cells undergoing DNA re-replication (containing >4N DNA content) known to be mediated by p21 and CDT1 accumulation. Indeed the accumulation of p21 and CDT1 was more pronounced in pevonedistat sensitive cell lines after the treatment. Two of primary cells (67%) were sensitive to pevonedistat and also showed higher CDT1 accumulation following the treatment compared to the resistant cells. In vivo, pevonedistat treatment significantly prolonged survival of mice bearing both sensitive and resistant MPM tumors. Pevonedistat treatment reduced growth (phosphorylated histoneH3 positive) in pevonedistat sensitive tumor but increased apoptosis (cleaved–caspase3 positive) in pevonedistat resistant tumor.
Conclusion
High CUL4B expression may play a role in MPM progression. Inhibition of cullins by pevonedistat induced growth arrest and DNA re-replication strongly in a subset of MPM. The major mechanism seems to be mediated by p21 and CDT1 accumulation in vitro. Investigation of mechanisms in vivo is ongoing.
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MA17.04 - Discussant - MA17.01, MA17.02, MA17.03 (Now Available) (ID 3786)
15:45 - 17:15 | Presenting Author(s): Julian Carretero
- Abstract
- Presentation
Abstract not provided
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MA17.05 - DNA-Binding and Gene Expression Profiles in Max Deficient Small Cell Lung Cancer (Now Available) (ID 377)
15:45 - 17:15 | Presenting Author(s): Paula Llabata | Author(s): Manuel Torres-Diz, Antonio Gomez, Montse Sanchez-Cespedes
- Abstract
- Presentation
Background
The MYC pathway is frequently altered in cancer, mostly by gene activation of the MYC-family of oncogenes (fMYC) but also by genetic inactivation of MAX, the obligate partner of MYC. While the oncogenic properties of fMYC have been extensively studied, the tumour suppressor role of MAX and the function of fMYC in MAX-mutant cells remain unclear. Further, inactivating mutations in MGA, a gene that codes for another MAX-binding partner, have been found in lung cancer. MGA is a component of the non-canonical polycomb repressive complex 1 (ncPRC1) but its precise role in lung cancer development is unknown.
Method
RNA-sequencing, chromatin immunoprecipitation and proteomic analysis were performed to identify and compare the DNA binding and gene expression profiles of MYC, MGA and MAX in MAX-restituted human small cell lung cancer (SCLC)-derived cell lines.
Result
SCLC is a high-grade neuroendocrine type of lung cancer with recurrent inactivating mutations in MAX. Recent findings have described two major SCLC subtypes based on the high expression of either ASCL1 or NEUROD1 transcription factors. According to this, ASCL1 and NEUROD1 control the expression of different set of genes which defines the two subgroups of SCLC. Here, we found that MAX-mutant SCLC cells belong to the ASCL1-transcription factor dependent group of SCLCs. In the absence of MAX, even after ectopic overexpression of MYC, there was no recruitment of MYC to the DNA. The DNA binding profile of MAX in MAX-restituted cells remained unaltered after co-overexpression of MYC, despite opposed effects in gene expression. Moreover, restitution of MAX significantly shifted the DNA occupancy of MGA, from E2F6 consensus binding sites to MYC-consensus binding sites (E-boxes). Our observations also demonstrated that ncPRC1 complex is formed regardless of the presence or absence of MAX.
Conclusion
Our data supports that MYC lacks transactivation capabilities in the absence of MAX and that the tumour suppressor role of MAX relies on its capability to counteract the gene expression triggered by its partnering with fMYC. Further, we conclude that the tumor suppressor role of MGA may be related, in part, to the regulation of E2F6 promoters.
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MA17.06 - Plakophilin 1 Enhances MYC Expression, Promoting Squamous Cell Lung Cancer (Now Available) (ID 823)
15:45 - 17:15 | Presenting Author(s): Pedro Pablo Medina | Author(s): Joel Martin-Padron, Laura Boyero, María Isabel Rodríguez, Alvaro Andrades, Inés Díaz-Cano, Paola Peinado, Carlos Baliñas, Juan Carlos Alvarez Álvarez Pérez, Isabel Fernández Coira, María Esther Fárez-Vidal
- Abstract
- Presentation
Background
Plakophilin 1 (PKP1) is a member of the arm-repeat (armadillo) and plakophilin gene families, being an important component of the desmosome. Although desmosomes loss-of-function has been associated with increased cell migration and pro-oncogenic activity, we have observed consistent PKP1 overexpression in patient samples of squamous cell lung cancer (SqCLC) in comparison with lung adenocarcinoma (LUAD) and non-tumoral controls from two datasets achieved by our group, and also from three additional independent datasets.
Method
In order to explore this paradox, we developed in vitro and in vivo PKP1 gain/loss functional models in SqCLC cell lines and also we challenged our hypothesis in some LUAD cell lines.
Result
Greater cell dissemination but reduced cell proliferation was observed in CRISPR-Cas9 induced, PKP1-knockout clones. Furthermore, PKP1 expression promoted cell proliferation, cell survival, and in vivo xenograft engraftment.
Interestingly, we demonstrated through several functional experiments (chromatin immunoprecipitation, RNA immunoprecipitation, direct mutagenesis combined with luciferase assays, Western blot, qPCR... among others), and in 7 cell lines from different lung cancer subtypes (5 SqCLC and 2 LUAD cell lines), and different contexts (with and without PKP1 basal expression in order to set up gain and loss expression assays), that these pro-oncogenic activities were mediated by the functional direct relationship between PKP1 and the oncogene MYC. Specifically, PKP1 enhances MYC translation, and MYC increases PKP1 transcription, linking both proteins in a positive feedforward loop.
Conclusion
These observations provide a new molecular mechanism of cancer development, revealing PKP1 as a novel oncogene in SqCLC, and as an effective post-transcriptional regulator of MYC, which has been described as overexpressed in around 70% of NSCLC tumors.
Moreover, PKP1 unveiled as a valuable diagnostic biomarker and a potential therapeutic target for SqCLC. Importantly, PKP1 inhibition may open up the possibility of indirectly targeting MYC, not only in NSCLC (where, as mentioned before, is frequently overexpressed), but also in other tumors. This is of particular interest, because MYC is an oncogene that is dysregulated in most human cancers and is acknowledged as a “most wanted” target for cancer therapy.
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MA17.07 - Identification of AHR as a Novel Regulator of Lung Cancer Metastasis (Now Available) (ID 2331)
15:45 - 17:15 | Presenting Author(s): Silke Nothdurft | Author(s): Frank Breitenbuecher, Ross A Okimoto, Trever G Bivona, Barbara M Gruener, Michael Hoelzel, Jan Forster, Sophie Kalmbach, Alexander Schramm, Martin Schuler
- Abstract
- Presentation
Background
Curative treatment of early stage and locally advanced non-small cell lung cancer (NSCLC) relies on surgery and radiotherapy. Adjuvant or simultaneous platin-based chemotherapy is used for risk reduction in patients with large tumors and/or lymph node metastases. Still a large fraction of curatively treated patients dies from metastatic relapse. A better mechanistic understanding of lung cancer metastasis is expected to guide the development of novel rational interventions from prevention, early detection and treatment.
Method
Using a barcoded shRNA library we performed a functional in vivo screen in an orthotopic NSCLC mouse model to find target genes involved in metastatic processes. Barcoded shRNAs with significantly different representation between primary tumors and metastases were identified by next generation sequencing. Prioritized hits were functionally validated by targeted suppression in NCI-H1975 cells. Mechanistic studies were conducted in several NSCLC models in vivo and in vitro.
Result
We identified AHR, a ligand-activated transcription factor involved in regulation of biological responses to planar aromatic hydrocarbons, as potential modulator of lung cancer metastasis. Suppression of endogenous AHR by shRNA enhanced the migratory and invasive capacity of NSCLC cells in vitro. Importantly, NCI-H1975 with targeted suppression of AHR showed increased metastasis formation in an orthotopic model in vivo. High RNA expression of AHR correlates with lower likelihood of progression and superior overall survival in patients with stage I NSCLC. Mechanistically, AHR impacts matrix remodeling genes (MMP19, MMP24) as well as asparagine synthetase (ASNS), all of which have been implied in metastatic progression.
Conclusion
AHR is a novel metastasis-modulating factor in NSCLC. Its mechanism of action provides rational targets for diagnostic and therapeutic interventions.
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MA17.08 - Discussant - MA17.05, MA17.06, MA17.07 (Now Available) (ID 3787)
15:45 - 17:15 | Presenting Author(s): Paul Paik
- Abstract
- Presentation
Abstract not provided
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MA17.09 - 5-Azacytidine Inhaled Dry Powder Formulation Profoundly Improves Pharmacokinetics and Efficacy for Lung Cancer Therapy (Now Available) (ID 172)
15:45 - 17:15 | Presenting Author(s): Steven Belinsky | Author(s): Philip J Kuehl, Carmen S Tellez, Marcie J Grimes, Michael Burke, Aaron Badenoch, Devon Dubose
- Abstract
- Presentation
Background
Epigenetic therapy through its ability to activate hundreds of genes silenced by promoter hypermethylation in lung cancer could produce durable and sustained tumor regression. The demethylating agent 5-azacytidine (5AZA) is unstable in aqueous solution and subject to hydrolysis and catabolism by cytidine deaminase (CDA) in liver, thereby reducing drug concentration after systemic administration prior to reaching the lung. Delivering 5AZA by inhalation could mitigate these barriers.
Method
A stable, respirable (3.5 µM) dry powder formulation of 5AZA was generated. Pharmacokinetic (PK) studies in rats compared systemic dosing to inhaled delivery of a dry powder or aqueous formulation of 5AZA in the presence and absence of the CDA inhibitor tetrahyroduridine (THU). An orthotopic nude rat lung cancer model compared efficacy of inhaled dry powder versus aqueous 5AZA for treatment of engrafted human adenocarcinoma (Calu6, Calu3), adenocarcinoma in situ (H358), and squamous cell (RH2) tumor lines. Three weeks following engraftment (lungs contain multiple tumors), rats were treated 4 times weekly for 4 weeks, then sacrificed to assess tumor burden and genome-wide effects on the methylome in vehicle and treated tumors using the Illumina EPIC array.
Result
Plasma PK showed a ~10-fold increase in area under the curve (AUC) and a 1.5 and 5-fold increase in maximum concentration (Cmax) comparing inhaled dry powder (0.6 mg/kg) to systemic (2 mg/kg, equivalent to the human injectable dose of 75 mg/m2) and inhaled aqueous (0.6 mg/kg) 5AZA that was augmented by THU. Inhaled dry powder and aqueous 5AZA PK in lung were similar and greatly exceeded systemic (30-fold Cmax; 47-fold AUC). PK in liver and brain were superior for dry powder with 7- and 26-fold increase in AUC and 7- and 3.3-fold increase in Cmax compared to systemic or aqueous dosing. The efficacy study comparing inhaled delivery of equivalent doses (0.6 mg/kg lung dose) showed dry powder was significantly better than aqueous with a 70–80% compared to 33–50% reduction in tumor burden for Calu6, Calu3, and RH2 and equally effective in largely curing H358 tumors. A significantly increased median number of genes (175–320 versus 25–270) exhibiting ≥30% demethylation of CpGs across their promoter region was seen for Calu6, Calu3, and H358 tumors exposed to dry powder versus aqueous 5AZA, with equivalent numbers of genes demethylated for RH2.
Conclusion
Delivery of a dry powder formulation of 5AZA via an inhaler could be used to treat local and metastatic lung cancer (Support–CA196590).
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MA17.10 - Lactate Transporter Blockade as a Strategy to Overcome VEGF Inhibitor-Resistance in LKB1-Deficient NSCLC (Now Available) (ID 2647)
15:45 - 17:15 | Presenting Author(s): John Victor Heymach | Author(s): Irene Guijarro, Alissa Poteete, Sungnam Cho, Teng Zhou, You-Hong Fan, Emily Roarty, Monique Nilsson, Edwin Parra, Barbara Mino, Ignacio Wistuba, Jing Wang, Ferdinandos Skoulidis
- Abstract
- Presentation
Background
STK11/LKB1 alterations are found in 20-30% of NSCLC and used to co-occur with KRAS mutations. Because LKB1 activates AMPK, many of the best known functions of LKB1 are attributed to its ability to control metabolic alterations in cells. Our laboratory have previously reported that loss of LKB1 promotes enhanced glycolysis and elevated lactate production and more recently we demonstrated that STK11/LKB1 mutations are the strongest predictors of de novo resistance to immunotherapy in NSCLC. Prior studies have revealed an association between alterations in the LKB1/AMPK pathway and worse clinical outcomes in NSCLC and in patients treated with chemotherapy and bevacizumab. Given the roles of LKB1 in the regulation of cell metabolism and resistance to immunotherapy, it is feasible that LKB1 also impacts on the response to anti-angiogenic therapies.
Method
Xenograft mouse models were established by subcutaneous injection of H460 cells (LKB1-deficient) and H460 LKB1-expressing in nude mice and LKR10 (KRASG12D) LKB1 wild-type (K) or LKB1- knockout (KL) into 129Svmice. Mice were randomized to vehicle or B20-4.1.1 anti-VEGF antibody. Glycolytic activity of LKB1-intact and -deficient NSCLC cells was measured by Seahorse assay. We analyzed gene expression of SLC16A3 (MCT4) by qPCR and Western blot. Genetic disruption of MCT4 in the K and KL cell lines was done using CRISPR-Cas9 and mouse models were established by subcutaneous injection into mice.
Result
Mice bearing LKB1-expressing H460 xenografts treated with anti-VEGF antibody showed a significant decrease in tumor volume (p<0.05) compared with their vehicle-treated counterparts. However, mice bearing LKB1-deficient H460 xenografts showed markedly reduced efficacy of anti-VEGF therapy compared with that in LKB1-expressing xenografts. Anti-VEGF therapy significantly reduced growth of LKR10 K tumors (p<0.001) but not in LKR10 KL tumors. Microvascular density was not increased in KL tumors following anti-VEGF treatment compared to K. Human isogenic LKB1-deficient cells showed a significantly increased rate of glycolysis and lactate secretion compared with cells expressing LKB1. Human and murine LKB1-deficient cells also had increased MCT4 expression compared to K cells. Immunofluorescence and RPPA analysis of tumor samples from the K and KL mouse models showed that KL tumors upregulated MCT4 protein expression compared with K tumors (p<0.0001). The genetic disruption of MCT4 KL tumors significantly improved tumor volume reduction to anti-VEGF therapies in vivo (p<0.001).
Conclusion
LKB1 loss is associated with increased lactate secretion and resistance to VEGF inhibition in NSCLC. The targeting of the lactate transporter MCT4 enhance the sensitivity of LKB1-deficient NSCLC to anti-VEGF therapy.
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MA17.11 - High Sensitivity to PD-1 Blockade Therapy After Ld1 Depletion in KRAS-Driven Lung Cancer Through CD8+/CD3+ Tumor Infiltration and PD-L1 Induction (Now Available) (ID 2562)
15:45 - 17:15 | Presenting Author(s): Iosune Baraibar | Author(s): Marta Román, Inés López-Erdozain, Ana Oliver, Anna Vilalta, Daniel Ajona, Silvestre Vicent, Carlos De Andrea, Ruben Pio, Juan José Lasarte, Alfonso Calvo, Ignacio Gil-Bazo
- Abstract
- Presentation
Background
PD-1/PDL-1 inhibitors are approved for non-small cell lung cancer (NSCLC). However, many patients do not benefit and therapeutic combinations are under investigation. We have previously described Id1, involved in proliferation, angiogenesis and immunosuppression, as a prognostic factor in lung adenocarcinoma (LUAD) (Ponz-Sarvise, Clin Cancer Res 2011), Id1’s role in lung cancer metastasis (Castanon, Cancer Letters 2017) and more recently shown that Id1 sustains mutant KRAS-driven progression and metastasis in NSCLC (Roman, Cancer Res 2019).
In a previous syngeneic murine lung cancer model with depleted levels of Id1 using Id1-/- and Id1 wildtype C57BL/6 mice inoculated with Lewis Lung Carcinoma (LLC), we tested a combined therapeutic strategy targeting PD-1 and Id1, showing impaired tumor growth and increased survival (Gil-Bazo, presented at WCLC 2018).
Here we study a combined strategy targeting PD-1 and Id1 in a KRAS-mutant murine LUAD model and the immune-related mechanisms involved.
Method
First, a correlation between Id1 and PD-L1 mRNA expression was studied in mutant and wild-type KRAS LUAD cohorts from The Cancer Genome Atlas data set (TCGA).
Secondly, a syngeneic tumor model using Balb/c mice through subcutaneous injection of KRAS-mutant LUAD (Lacun3) cells and Id1-silenced Lacun3 (Id1sh) cells. In vitro, proliferation was measured in both cell lines through MTS assays. IFNg-induced PD-L1 expression in both cell lines and flow cytometry was used to evaluate its mechanistic effects on the immune response.
After tumor cells injection, mice were treated with an anti-PD-1 (RMP-1-14) monoclonal antibody or PBS, i.p. Tumor volumes according to Id1 status in tumor cells and the treatment administered were quantified. Vectra 3.0™ multispectral microscopy was used to characterize the tumor associated immune cells in paraffin-embedded tissues from our previous syngeneic murine lung cancer model using Id1-/- and Id1 wildtype C57BL/6 mice inoculated with LLC in which the combined blockade had been reported as effective. Immune marker antibodies were used to study expression of CD3, CD4 and CD8.
Result
An inverse, moderate and statistically significant correlation between Id1 and PD-L1 expression in mutant and wild-type KRAS LUAD cohorts from TCGA was found in both cohorts (-0.367 and -0.351, respectively, p<0.001), indicating that Id1 depletion may lead to PD-L1 expression induction.
In vitro assays showed that Id1 silencing reduced Lacun3 cells proliferation (p<0.001). Up-regulation of surface PD-L1 expression occurred in Id1sh cells, but not in Lacun3 cells, after receiving IFNg (p=0.0022). Mechanistically, in the syngeneic murine model, Id1 inhibition in the injected cells, combined with anti-PD-1 treatment, significantly induced a tumor growth impairment (p<0.001). An intense CD8+ and CD3+ immune cell infiltration was observed in LLC Id1-/- C57BL/6 mice treated with anti-PD1 (p<0.05 for CD3+ TILS), compared the control groups, possibly explaining the dramatic tumor growth impairment previously shown on the treated animals.
Conclusion
Id1 silencing may induce PD-L1 overexpression according to in silico and in vitro results. Id1 and PD-1 combined blockade in our KRAS-mutant syngeneic murine LUAD model significantly impaired tumor growth, compared to each strategy alone. A significantly increased CD3+ and CD8+ tumor infiltration and IFNg-induced PD-L1 tumor expression after the combined blockade may explain these findings.
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MA17.12 - Discussant - MA17.09, MA17.10, MA17.11 (Now Available) (ID 3788)
15:45 - 17:15 | Presenting Author(s): Shantanu Banerji
- Abstract
- Presentation
Abstract not provided
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Author of
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ES11 - Lung Cancer Plasticity and Drug Resistance (ID 14)
- Event: WCLC 2019
- Type: Educational Session
- Track: Biology
- Presentations: 1
- Now Available
- Moderators:Luca Roz, Nagahiro Saijo
- Coordinates: 9/08/2019, 15:15 - 16:45, Colorado Springs (1994)
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ES11.01 - Lung Adenocarcinoma to Squamous Cell Carcinoma Transdifferentiation and Drug Resistance (Now Available) (ID 3211)
15:15 - 16:45 | Presenting Author(s): Hongbin Ji
- Abstract
- Presentation
Abstract
Lung cancer is notorious for high heterogeneity and strong plasticity, which might contribute to the development of drug resistance. Lineage transition from lung adenocarcinoma (ADC) to squamous cell carcinoma (SCC), as implicated by clinical observation of mixed ADC and SCC pathologies in adenosquamous cell carcinoma (Ad-SCC), reflects strong cancer plasticity and potentially links to drug resistance. Using Genetically Engineered Murine Model (GEMM), we have provided in vivo evidence in supporting the ADC to SCC transdifferentiation (AST): Lkb1-deficient mouse lung ADC transdifferentiates into SCC progressively via pathologically mixed Ad-SCC. Mechanistically, we find that down-regulation of reactive oxygen species (ROS) level through N-acetyl cysteine (NAC) treatment or NRF2 expression inhibits this transition, highlighting the functional importance of ROS in regulating cancer plasticity. Pentose phosphate pathway deregulation and impaired fatty acid oxidation collectively contribute to the redox imbalance and functionally affect the AST process. Importantly, similar tumor and redox heterogeneity are also found in human LKB1-inactivated lung cancer. In preclinical trials toward metabolic stress, Lkb1-inactivated ADC can develop drug resistance through squamous transdifferentiation. Recent observations in clinic further suggest that such pathological transition might be responsible for resistance to tyrosine kinase inhibitor (TKI) therapy and chemotherapy in relapsed EGFR-mutant lung ADC patients. These findings demonstrate that lung cancer plasticity potentially affects therapeutic response and precision medicine through histological transition.
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