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Jillian W.P. Bracht



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    FP03 - Immuno-biology and Novel Immunotherapeutics (Phase I and Translational) (ID 151)

    • Event: WCLC 2020
    • Type: Posters (Featured)
    • Track: Immuno-biology and Novel Immunotherapeutics (Phase I and Translational)
    • Presentations: 1
    • Moderators:
    • Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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      FP03.04 - Selinexor can Inhibit Nuclear Export of HMGB1, a Negative Predictive Marker for Immunotherapy Response (ID 3616)

      00:00 - 00:00  |  Presenting Author(s): Jillian W.P. Bracht

      • Abstract
      • Presentation
      • Slides

      Introduction

      The high mobility group box 1 (HMGB1) normally acts as a DNA chaperone in the nucleus, but was previously shown to be involved in various inflammatory diseases and cancer. Endogenous stimuli and oxidative stress can induce the transport of HMGB1 from the nucleus to the cytoplasm through exportin 1 (XPO1/CRM1). In addition, XPO1 was shown to be responsible for nuclear transport of HIV-1. Consequent release of HMGB1 from the cell as a damage-associated molecular pattern (DAMP) was found to have a paradoxical role in cell survival and death, by either activating an immune response through TLRs and RAGE receptors, or by inhibition of the immune response through CD24 and TIM-3 receptors. Selinexor (KPT-330) is an FDA-approved XPO1 inhibitor that prevents the release of HMGB1 from the nucleus to the cytoplasm. Selinexor was previously shown to be effective in KRAS mutant lung adenocarcinoma cell lines. We hypothesized that HMGB1 and XPO1 may be important biomarkers in cancer patients that will receive immune checkpoint inhibitors (ICIs).

      Methods

      Pre-ICI-treatment FFPE tumor tissue samples from 15 HIV-infected and 30 non-HIV-infected cancer patients, mainly lung cancer, were analyzed using the nCounter NanoString platform with the Human PanCancer IO360 panel. The IO360 panel can be used to analyze the expression of 770 genes related to tumor biology, immune response and microenvironment. HMGB1 mRNA expression results were correlated with HIV status and clinical benefit (CB; objective response or stable disease of more than 24 weeks by RECIST1.1 criteria). Next, XPO1 was inhibited in lung cancer cell lines using selinexor and integrase inhibitors (INSTIs) and effects on cell viability and nuclear export of HMGB1 were evaluated. Moreover, combination therapies using selinexor and EGFR or MET inhibitors were tested and effects on cell viability were determined.

      Results

      HMGB1 mRNA expression was lower in patients that had CB from ICI treatment (p = 0.04). No significant differences were found in HMGB1 mRNA expression between patients with- and without HIV-1 infections. Kaplan Meier analysis revealed that patients with lower HMGB1 mRNA expression have better overall survival, both in our patient cohort (p = 0.004), and a TCGA lung adenocarcinoma patient cohort (p = 0.003). Inhibition of the HMGB1 transporter, using either selinexor or INSTIs, can prevent nuclear export of HMGB1 in the PC9 lung cancer cell line and inhibit cell proliferation in EGFR and KRAS mutant cell lines. Combination treatments with selinexor decreased cell viability in lung cancer cell lines when compared to single treatment.

      Conclusion

      In this study we found HMGB1 mRNA to be lower expressed in tumor tissue of pre-ICI-treated cancer patients with clinical benefit from treatment, indicating that HMGB1 expression may be used as a negative predictive marker for immunotherapy response. Further exploration should focus on validating this finding in a larger patient cohort and future in vivo studies should reveal if the combination of ICIs and XPO1 inhibitors could yield better responses to immunotherapy. Therefore, HMGB1 may be used to predict clinical benefit from ICI treatment and provides a new target for cancer treatment.

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    P60 - Tumor Biology and Systems Biology - Basic and Translational Science - Immune Bio (ID 198)

    • Event: WCLC 2020
    • Type: Posters
    • Track: Tumor Biology and Systems Biology - Basic and Translational Science
    • Presentations: 1
    • Moderators:
    • Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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      P60.12 - Baseline Tumor Immune Cell Infiltration and Activation can Predict Checkpoint Inhibitor Pneumonitis in Lung Cancer Patients (ID 3620)

      00:00 - 00:00  |  Presenting Author(s): Jillian W.P. Bracht

      • Abstract
      • Slides

      Introduction

      Treatment with anti PD-1/PD-L1 antibodies has demonstrated clinical efficacy in non-small cell lung cancer (NSCLC), increasing its use significantly. Besides anti-tumoral activity in some patients (pts), T-cell activation after immune checkpoint inhibitors (ICIs) can lead to an imbalance in the immune system and induce self-reactive T-cells in the lung tissue. This process can eventually lead to life-threatening immune-related adverse events (irAEs), like checkpoint inhibitor pneumonitis (CIP). We hypothesized that a pre-treatment state of chronic inflammation or immune system imbalance within the lung tissue could predict which pts are at higher risk of developing CIP.

      Methods

      Pre-ICI-treatment FFPE tissue samples from 18 pts that developed CIP and from 27 pts that did not, were retrospectively collected. Gene expression analysis was performed using the NanoString nCounter platform with the Human PanCancer IO360 panel, harboring 770 genes related to tumor biology, immune response and microenvironment. In addition, the IO360 panel contains gene signatures to measure immune system activity. Differential expression (DE) analysis was carried out based on the development of CIP. Finally, a classifier algorithm was created using a recursive feature selection with a leave one out cross validation method to predict which combination of genes is most effective to predict CIP development. In addition, we analyzed healthy adjacent lung tissue from 4 CIP, and 5 non-CIP pts.

      Results

      DE analysis revealed 72 differentially expressed genes (DEGs) in pre-treatment tissue of pts with- and without CIP development. Most DEGs were upregulated in pts that developed CIP. The algorithm yielded a 16-gene signature that was able to separate pts that developed CIP from patients that did not with receiver operating characteristic (ROC) areas under the curve (AUCs) of 0.80 to 0.92. Genes with an increased expression in the CIP developing cohort were found to be involved in apoptosis, MAPK pathway, antigen presentation and costimulatory signaling. Panel-incorporated immune signatures were also evaluated. Neutrophil-, NK-cell and TH1 cell populations were found to be lower in pts that developed CIP. This indicates a general lower pre-treatment immune cell population abundance and activation in CIP developing pts. Since the overall response rate (ORR) in these pts was high (52.1%), it can be hypothesized that there is a common antigen recognition for T-cells between tumor and healthy lung cells after ICI treatment. In addition, stronger activation of self-reactive T cells in the surrounding healthy lung tissue, instead of within the tumor and tumor microenvironment, could explain the occurrence of CIP in a subgroup of pts. Analysis of the healthy adjacent lung tissue revealed that CIP pts have higher macrophage, T-cell and neutrophil infiltration and activation in their healthy tissue, compared to nonCIP pts.

      Conclusion

      We have developed a 16-gene signature that can predict which pts are more likely to develop CIP. This could be exploited by extensively monitoring those pts to allow for early intervention. In addition, we found that CIP developing pts have a higher immune cell infiltration in their healthy tissue which could explain CIP development. These findings need further validation in a larger patient cohort.

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    P61 - Tumor Biology and Systems Biology - Basic and Translational Science - KRAS (ID 199)

    • Event: WCLC 2020
    • Type: Posters
    • Track: Tumor Biology and Systems Biology - Basic and Translational Science
    • Presentations: 1
    • Moderators:
    • Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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      P61.01 - Imipramine Blue (IP) plus MET Tyrosine Kinase Inhibitors (TKI) Suppress Lung Adenocarcinoma (LUAD) KRAS Mutation Tumor Growth (ID 1348)

      00:00 - 00:00  |  Author(s): Jillian W.P. Bracht

      • Abstract
      • Slides

      Introduction

      KRAS mutations in LUAD co-occur with TP53 mutations and LKB1 non-synonymous mutations (nsm), portending a poor prognosis. MET amplification has not been considered. Previously, we identified OTSSP167 as a PAK1 kinase inhibitor with significant activity in A549 (KRASG12-C and LKB1nsm). OTSSP167 plus auranofin (PKCι) shows high synergism and inhibits tumor growth in mice in the H23 cell line (KRASG12-C, p53mut and LKB1nsm) (Ito et al, Cell Comm and Signaling 2019). In addition, OTSSP167 has a potent MELK inhibition effect. We hypothesize that MET expression could be upregulated in KRAS-mutant cell lines, based on the fact that the putative signaling pathway, MELK-forkhead box M1 (FOXM1)-MET, could be present in KRAS mutant cells. In the current study, we examined the combination of MET TKIs with imipramine blue (FOXM1 inhibitor).

      Methods

      Quantitative real time PCR of MET and FOXM1 was performed in 4 KRAS-mutant cell lines (A549, H23, H460 and Calu6). LKB1 mRNA was assessed in 32 advanced KRAS-mutant LUAD patients. Cell proliferation assays were performed in A549 and H23, and in the EBC1 (MET amplified lung cancer cell line). Synergy was defined by a combination index (CI) of < 0.75 by Chou-Talalay. Cell lines were treated with IP and MET TKIs (crizotinib, savolitinib and tepotinib).

      Results

      MET mRNA was elevated in A549 and H23 (which both carry LKB1nsm) but not in H460 and Calu6. FOXM1 mRNA was overexpressed in H23. Synergy (CI<0.75) was seen with IP plus tepotinib in the A549 and H23 cell lines, but not in H460 and Calu6. Synergy was also noted with IP plus crizotinib, but not with savolitinib. The CI of IP plus MET TKIs in the EBC1 cell line (which is only MET amplified) was >1. The median overall survival for KRAS-mutant LUAD patients with low LKB1 was 1.1 months versus 19.4 for those with high LKB1 (p=<0.005).

      Conclusion

      The bona fide of MET TKIs (tepotinib) plus IP in KRAS cell lines with LKB1 nsm, encourages the determination of clinical benefit of tepotinib plus IP in KRAS mutant LUAD patients. The liaison of MET and LKB1 nsm should be further investigated. LKB1 mRNA expression, together with MET and FOXM1 mRNA expression, warrants assessment in KRAS LUAD patients.

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    P65 - Tumor Biology and Systems Biology - Basic and Translational Science - NC RNA (ID 204)

    • Event: WCLC 2020
    • Type: Posters
    • Track: Tumor Biology and Systems Biology - Basic and Translational Science
    • Presentations: 1
    • Moderators:
    • Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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      P65.04 - Tracking circRNAs in Lung Adenocarcinoma Samples as Promising Biomarkers for Cancer Detection using the NanoString nCounter®  (ID 3502)

      00:00 - 00:00  |  Author(s): Jillian W.P. Bracht

      • Abstract
      • Slides

      Introduction

      Lung cancer is positioned as the foremost cause of cancer death at global scale. The lack of reliable biomarkers for early detection seems to be the main cause underlying this high mortality rate; therefore, the discovery of new candidates allowing timely diagnosis of the disease is rather imperative.

      Circular RNAs (circRNAs) have strongly emerged as valuable tissue-specific biomarkers of different disorders, including lung cancer. The covalently linked 5´-3´ends provide them not only with a distinctive structure but also keeps them exempted from the exonuclease activity making them very stable. They have been described to be highly expressed in extracellular vesicles (EVs) when compared with mRNA; however, despite of the aforementioned properties, their potential as biomarkers has not been fully explored in lung cancer, yet very far to be implemented in the liquid biopsy settings.

      Through this study we demonstrate the feasibility of using the NanoString nCounter® Flex platform for the study of circRNAs in different fresh and formalin-fixed paraffin embedded (FFPE) lung cancer material including EVs, hence paving the way for the development of new diagnosis platforms based on this technology and the different circRNA expression patterns.

      Methods

      Lung cancer cell lines were cultured under standard laboratory conditions until harvested for RNA extraction. EVs were isolated from the cell culture medium by ultracentrifugation.

      FFPE tissue samples were retrospectively collected; samples were macro-dissected and total RNA was extracted.

      Patient blood was separated into plasma and cellular fractions by centrifugation. Plasma-derived EVs were isolated using the miRCURY Exosome Serum/Plasma Kit.

      RNA from EVs was extracted with TRI-Reagent.

      For mRNA depleted samples, a treatment with RNase R was performed.

      Gene expression analysis was carried out using the NanoString nCounter® platform with a customized panel harboring 85 circRNA related to the biology of the disease.

      Results

      First runs showed circRNA expression in lung cancer cell lines. A comparative analysis of total RNA versus mRNA-depleted RNA was performed resulting in an overall circRNA enrichment of the latter. An analogous experiment was performed in FFPE PC9 cells; however, circRNA enrichment was not achieved.

      Likewise, FFPE lung cancer tissue samples were analyzed; Consequently, circRNA expression has been evident in all samples analyzed so far.

      EV-derived RNA (exRNA) from cell lines was also tested in the nCounter®. Counts corresponding to different transcripts were evident in all samples without previous amplification step. Similar results were found in exRNA from one NSCLC patient. More patient samples are currently being collected to validate these results.

      Conclusion

      Through this project we have demonstrated the feasibility of using nCounter® for the study of circRNAs in different lung cancer materials. RNase R treatment proved to be beneficial for circRNA enrichment in fresh samples but not FFPE samples, probably due to the mechanical and chemical breakage they may experiment during purification, thus becoming susceptible to RNase R degradation.

      CircRNAs were also expressed in EVs; however, more patient samples need to be tested. Pre-amplification steps are advisable in the future to explored the sensitivity of the technology before being implemented into the liquid biopsy settings.

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