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Supplementary Table 2. siRNA sequences

Supplementary Figure 2. Significant weight loss in LC-2/ad xenograft mice treated with vandetanib Mice bearing LC-2/ad cells were treated with vehicle only (Vehicle), dovitinib (DO 30mg/kg), dovitinib (DO 60mg/kg), or vandetanib (VA 50mg/kg) for 32days. Average percent change in body weight relative to initial body weight is shown. Error bars represent SEM **P < 0.001 for vehicle vs vandetanib 50mg/kg treatment group.

Supplementary Figure 1. Supplementary Figure S1. Dovitinib inhibits RET kinase and its downstream signaling in lung cancer cells harboring RET rearrangement A, Western blot analysis of indicated markers in LC-2/ad cells after treated with indicated dose of dovitinib for 4hr. B. Western blot analysis to measure the levels of total FGFR3, FLT3, RET, and KIT upon siRNA treatment in LC-2/ad cell line. Cells were transfected with indicated siRNA for 48hr.

RET rearrangement is a newly identified oncogenic mutation in lung adenocarcinoma (LADC). Activity of dovitinib (TKI258), a potent inhibitor of FGFR, VEGFR, and PDGFR, in RET-rearranged LADC has not been reported. The aims of the study are to explore antitumor effects and mechanisms of acquired resistance of dovitinib in RET-rearranged LADC. Using structural modeling and in vitro analysis, we demonstrated that dovitinib induced cell-cycle arrest at G0–G1 phase and apoptosis by selective inhibition of RET kinase activity and ERK1/2 signaling in RET-rearranged LC-2/ad cells. Strong antitumor effect of dovitinib was observed in an LC-2/ad tumor xenograft model. To identify the acquired resistance mechanisms to dovitinib, LC-2/ad cells were exposed to increasing concentrations of dovitinib to generate LC-2/ad DR cells. Gene-set enrichment analysis of gene expression and phosphor-kinase revealed that Src, a central gene in focal adhesion, was activated in LC-2/ad DR cells. Saracatinib, an src kinase inhibitor, suppressed ERK1/2 phosphorylation and growth of LC-2/ad DR cells. Taken together, these findings suggest that dovitinib can be a potential therapeutic option for RET-rearranged LADC, in which acquired resistance to dovitinib can be overcome by targeting Src. Mol Cancer Ther; 14(10); 2238–48. ©2015 AACR.

Supplementary Table 1. Docking score of dovitinib, vandetanib, and sunitinib against RET kinase.

Supplementary Figure 3A. Inhibition of FAK activity in LC-2/ad cells and LC-2/ad DR cells. A, MTT assays of LC-2/ad and LC-2/ad DR cells. Cells were treated with indicated dose of dovitinib, PF-52,271, or both for 72hr, and cell viability was determined using MTT. Means are derived from three replicate (n=3). Error bars indicate mean {plus minus} SE of three replicates.

Supplementary Figure 3B and C. Inhibition of FAK activity in LC-2/ad cells and LC-2/ad DR cells B, Western blot analysis of indicated markers in LC-2/ad and LC-2/ad DR cells after treated with 1mM of dovitinib (DO), dasatinib (DA), saracatinib (SA), or PF562,271 (PF) for 4hr. C, Western blot analysis of indicated markers in LC-/2ad and LC-2/ad DR cells after treated with indicated dose of dovitinib (DO) for 4hr.

Gene expression levels in H3122, LR, and panobinostat treated LR cells and the promoter or enhancer H3K27 ac gain or loss

Supplementary Figure S1. Viability of H3122-LR and H2228-LR cells Supplementary Figure S2. Gene set enrichment analysis and western blot Supplementary Figure S3. Effects of AXL inhibition on EMT gene expression in LR cells, related to Fig. 1E. Supplementary Figure S4. Mice with established H3122-derived tumors were treated with four doses (50, 75, 87.5, or 100 mg/kg) of ceritinib to derive ceritinib-resistant tumors Supplementary Figure S5. Enhancer remodeling during ceritinib resistance, related to Fig. 2. Supplementary Figure S6. Genes with H3K27ac loss or gain in their promoters during ceritinib resistance, related to Fig.2.

Supplementary Materials and Methods that contain detailed experimental procedures

Supplementary Figure legend

Anaplastic lymphoma kinase (ALK) inhibitors are highly effective in patients with ALK fusion–positive lung cancer, but acquired resistance invariably emerges. Identification of secondary mutations has received considerable attention, but most cases cannot be explained by genetic causes alone, raising the possibility of epigenetic mechanisms in acquired drug resistance. Here, we investigated the dynamic changes in the transcriptome and enhancer landscape during development of acquired resistance to ALK inhibitors. Histone H3 lysine 27 acetylation (H3K27ac) was profoundly altered during acquisition of resistance, and enhancer remodeling induced expression changes in both miRNAs and mRNAs. Decreased H3K27ac levels and reduced miR-34a expression associated with the activation of target genes such as AXL. Panobinostat, a pan-histone deacetylase inhibitor, altered the H3K27ac profile and activated tumor-suppressor miRNAs such as miR-449, another member of the miR-34 family, and synergistically induced antiproliferative effects with ALK inhibitors on resistant cells, xenografts, and EML4-ALK transgenic mice. Paired analysis of patient samples before and after treatment with ALK inhibitors revealed that repression of miR-34a or miR-449a and activation of AXL were mutually exclusive of secondary mutations in ALK. Our findings indicate that enhancer remodeling and altered expression of miRNAs play key roles in cancer drug resistance and suggest that strategies targeting epigenetic pathways represent a potentially effective method for overcoming acquired resistance to cancer therapy.Significance: Epigenetic deregulation drives acquired resistance to ALK inhibitors in ALK-positive lung cancer. Cancer Res; 78(12); 3350–62. ©2018 AACR.

Supplementary Figure S1,S2,and S3

Supplementary figure legends S1 to S12

Prostaglandin E2 (PGE2) is widely recognized as one of the major bioactive lipids that, with the striking regenerative potential, promote drug-resistance in cancer cells as well as immune evasion in the tumor microenvironment (TME). Primarily driven by apoptotic cell death, PGE2 is thought to elicit wound-healing responses to help provide an immunosuppressive and proliferative niche that supports cancer stem cell repopulation and thereby therapy-resistance. While COX1/2 inhibitors that attenuate PGE2 production have shown promising anti-cancer effects in various (pre-)clinical settings, the gastrointestinal- and cardiotoxicities precluded their development as anti-cancer agents. It is anticipated that specific targeting of PGE2 signaling via its cognate receptors constitutes a safer and potentially more effective approach. Of the receptor subtypes EP1-4, Gα,s-coupled EP2 and EP4 are believed to be directly involved in immunosuppressive effects of PGE2.OCT-598 is a novel, highly potent and selective EP2/EP4 dual antagonist with Ki values of 23 nM and 0.2 nM vs EP2 and EP4, respectively. PGE2 inhibited normal differentiation of human monocytes into CD1a+CD16- dendritic cells under the presence of GM-CSF and IL-4 and promoted differentiation towards CD1a-CD16+ macrophages in vitro. However, EP2/EP4 dual inhibition by OCT-598 reversed this phenomenon to a greater extent than either EP2- or EP4-specific inhibitor alone. In vivo, OCT-598 effected tumor growth inhibition in multiple syngeneic mouse models as a single agent as well as in combination with an immune checkpoint blocker (ICB). Furthermore, the addition of OCT-598 to the lung cancer standard-of-care regimen (anti-PD-1 plus chemotherapy) in TC-1 mouse lung adenocarcinoma model gave rise to complete tumor regression. In conclusion, dual blockade of EP2 and EP4 by OCT-598 is shown to be a compelling strategy to reinforce antitumor effects by thwarting PGE2-mediated therapy resistance and immune evasion.Findings from this study provide a rationale for clinical development of OCT-598 as a therapeutic option for human malignant cancers. Citation Format: Youngrae Lee, Sujeong Baek, Dong Kwon Kim, Yeri Lee, Donggeon Kim, Seongin Jo, Sang Kyun Lim, Young Sook Shin, Soonsang Kwon, Seung Min Yang, Young Taek Kim, Seong-San Kang, Chun-Bong Synn, Kwangmin Na, Mi Hyun Kim, Heekyung Han, Yu Jin Han, Sungwoo Lee, Jae Hwan Kim, Mi Ran Yun, Youngseon Byeon, Young Seob Kim, Ji Yun Lee, Jii Bum Lee, Chang Gon Kim, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho, Taeyoung Yoon. OCT-598, a novel EP2/EP4 dual antagonist, promotes anti-tumor immune responses in syngeneic mouse tumor models in combination with standard-of-care chemo- and immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3234.

Heterogeneity in resistant to immunotherapies of tumor microenvironment (TME) has been implicated in immunotherapies to cause immune evasion or drug resistance. This study was conducted to explore the heterogeneity of TME through multiplex IHC, spatial and RNA sequencing analysis. We selected a sample from a lung adenocarcinoma patient without EGFR-activating mutation and expressing 30% of PD-L1. For quantitative analysis by multiplex IHC, various markers including CD4, CD8, FoxP3, granzyme B, CD20 and pan-cytokeratin were stained with 7 different fluorescence dyes, which was imaged with Vectra Polaris (Akoya). For scRNAseq and spatial RNAseq, we used 5’ chromium library kit (10X Genomics) to make library construction. Integrated raw data was generated using Cell ranger, Seurat pipeline and Azimuth package. The tumor area was divided into 16 clusters in which we selected 2 clusters based on CD3/45 expression. There was a noticeable distinction between the two clusters which were defined as the ‘High’ region (CD45highCD3high cluster) and the ‘Low’ region (CD45lowCD3low cluster). By multiplex IHC, percentage of CD8+T cells was higher in the ‘High’ region than in the ‘Low’ region (8.5% vs. 0.8%, respectively). Subsequent analysis of two clusters using spatial and single cell RNA seq, the ‘Low’ region was characterized by increased hypoxia-associated gene expressions including HIF1A, HIF3A and VEGFA. Various immune cells were abundant in the ‘High’ region and CD45 expression level was 11-fold higher in the ‘High’ region compared to the ‘Low’ region. Cytokine/chemokine network analysis via spatial RNAseq revealed that gene expression of tumor necrosis factor (TNF) family-associated factors increased in the 'High' region compared to the ‘Low’ region (TNF, FAS, TRAIL, RANKL and CD40), which is well-known to promotes apoptosis, programmed cell death, or necrosis of certain cancer. Additionally, the ‘High’ region also had elevated levels of the PD-1/PD-L1, CD155, CD122/TIGIT, Siglec10/CD24, LAG3/LAGLS3, and CD47/CD172a axes, suggesting active immune responses. Intriguingly, combined analyses showed that ‘High’ region showed enhanced level of CD44 expression as the leading-edged gene, which suggests the metastatic potential of tumor cells. Furthermore, scRNA analysis confirmed that CD44 expression was mainly higher in macrophages, suggesting that tumor-associated macrophages partially affected tumor cell metastasis in the ‘High’ region. Our finding suggests that understanding the intratumoral immunological heterogeneity of lung adenocarcinoma can help to study the mechanism of tumor heterogeneity by integrated spatial RNAseq and scRNAseq analyses. This type of technique could be applied to understand complex networks of anti-tumor immune activities, drug resistance mechanisms and immunotherapeutic response of cancer. Citation Format: Seul Lee, Jae-Hwan Kim, Kwangmin Na, Seung Min Yang, Dong Kwon Kim, Sujeong Baek, Seong-san Kang, Yu Jin Han, Chun-Bong Synn, Mi hyun Kim, Heekyung Han, Young Taek Kim, Sungwoo Lee, Youngseon Byeon, Young Seob Kim, Ji Yun Lee, Jii Bum Lee, Chang Gon Kim, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. Characterization of immunological heterogeneity in the tumor microenvironment by integrated analyses using single cell RNAseq, spatial RNAseq and multiplex IHC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6780.

Background: The Aryl hydrocarbon receptor (AhR) is one of the most predominant regulators of cancer metabolism. The AhR exerts important immunosuppressive functions by activating Treg cells and myeloid-derived suppressor cells and repressing CD8+ effector T cells. Here, we propose that a best-in-class AhR inhibitor, DA-4505, improves anti-tumor efficacy via modulation of tumor immune surveillance compared to BAY2416964, an AHR antagonist drug candidate being studied in the clinical phase. Methods: To evaluate anti-tumor effects of DA-4505 and BAY2416964, the two AhR inhibitors were dosed at 10 mg/kg once daily alone or in combination with aPD-1 (10 mg/kg) in surgical and chemotherapy models, and a PDX model (YHIM2004). Tumor volume, relapse, and survival were evaluated, and immune profiles were analyzed with IHC, flow cytometry, and scRNAseq. Results: A significant tumor reduction appeared in the CT26 and 4T1 tumor models after the DA-4505 treatment compared to vehicle group (P Conclusion: The AhR inhibitor DA-4505 demonstrated an improvement in anti-tumor efficacy. In addition, it has shown a synergistic effect when combined with aPD-1. Discoveries from this study provide a preclinical rationale for future clinical implications in solid tumor. Citation Format: DongKwon Kim, Sujeong Baek, Seung Min Yang, Yu Jin Han, Seong-san Kang, Chun-Bong Synn, Mi Hyun Kim, Heekyung Han, Kwangmin Na, Young Taek Kim, Sungwoo Lee, Taedong Han, Hyounmie Doh, Jongho Cho, Dajeong Kim, Daewon Cha, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Mi Ran Yun, Ji Yun Lee, Jii Bum Lee, Chang Gon Kim, Min Hee Hong, Sun Min Lim, Byoung Chul Cho, Kyoung-Ho Pyo. A novel AhR inhibitor ‘DA-4505’ improved the anti-cancer efficacy of surgical and chemotherapy via synergistic anti-tumor effects of aPD-1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5107.

Introduction: EGFR mutations holds the major targets for drug in lung adenocarcinoma (LUAD). Despite the tremendous study of EGFR mutant (MT) LUAD, the comprehensive interpretation of the heterogeneous character of LUAD harboring EGFR MT remains a key challenge. Here, we investigated the heterogeneity of EGFR MT LUAD and explored the tumor microenvironment (TME) in EGFR MT LUAD. Method: We performed single-cell RNA sequencing (scRNA-seq) from 135 LUAD patients which consist of normal(n=24), EGFR wild (WT)(n=18), and MT(n=93). Also, we used whole genome sequencing and bulk-RNA sequencing to validate with scRNA-seq results. From 898,648 cells, main cell types were classified. To explore the various characteristics of MT LUAD tumor cells, we used two ways: i) We re-clustered epithelial cells populating the normal, WT, and MT. ii) We re-clustered only MT epithelial cells. In each analysis, we identified the tumor character in the clusters using differential expressed genes analysis, lineage tracing, clinical information, mutation, and trajectory analysis. Also, we extracted each main cell type except epithelial cells, and identified subtypes of main cell types. Finally, we revealed the interaction of cellular components in TME. Results: In the analysis of epithelial cells, we identified characteristics of specific EGFR MT by comparing of EGFR WT and MT tumors in clusters with similar biological features. The cluster represented by alveolar type 2 (AT2) known as initiation of LUAD was populating normal, WT, and MT. In this cluster, MT- and WT-associated pathway shared but differently significant between MT and WT in the pathway analysis. The cluster represented by proliferative is mostly comprised tumor cells and we found significantly increased the expression of MDK, CD24 in the MT of the cluster. In the analysis of only MT epithelial cells, 2 of clusters were stage-specific cluster: i) The cluster annotated as early stage cluster, ii) The cluster annotated as advanced stage cluster. Trajectory showed that there is a pseudotemporal continuum, following the stage from early stage cluster to advanced stage cluster. Also, based on the lineage tracing, 2 of clusters revealed lineage-specific clusters: i) The cluster annotated as AT2 was enriched from early stage cells, ii) The cluster annotated as basal cell known as origin of lung squamous cell carcinoma(LUSC) was enriched from advanced stage cells. Psedotemporal ordering of these cluster revealed AT2 cluster transdifferentiate into basal cell cluster which implied the possibility of LUAD to LUSC transition by drug resistance. In the interaction of MT and WT TME, the number of signaling received epithelial cells from myeloid cells, endothelial cells, and fibroblasts as sender increased compared with the interaction of normal. Conclusion: We shed light on the ecosystem of TME according to clinical and biological feature of tumor in EGFR mutant LUAD. Citation Format: You Won Lee, Eun Ji Lee, Seung Yeon Oh, Kyoung-Ho Pyo, Seong Gu Heo, YoungJoon Park, Su-Jin Choi, Kyumin Lim, Ju-hyeon Lee, Jae Hwan Kim, Jii Bum Lee, Ji Yoon Lee, Sun Min Lim, Chang Gon Kim, Min Hee Hong, Mi Ran Yun, Byoung Chul Cho. Phenotype profiling of tumor microenvironment in EGFR mutant lung adenocarcinoma with multi-omics data. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5935.

Introduction: Unmet needs exist for immunotherapy targeting PD-1/PD-L1 in head and neck squamous cell carcinoma (HNSCC) and lung squamous cell carcinoma (LUSC) due to its suboptimal response. Amivantamab, a bispecific antibody targeting epidermal growth factor receptor (EGFR) and c-Met, has been demonstrated to induce antibody-dependent cytotoxicity and trogocytosis in tumor cells. We hypothesized that combination of amivantamab with pembrolizumab may synergistically enhance antitumor immunity. In this study, we present comprehensive immunomodulatory and synergistic antitumor efficacy of amivantamab and pembrolizumab in humanized HNSCC and LUSC mice models. Methods: EGFR and MET-expressing tumors from a HNSCC and a LUSC patient were transplanted into Hu-CD34-NSG to establish humanized patient-derived xenograft (PDX) models. Tumor-bearing PDXs were treated with vehicle, pembrolizumab (10mpk, Q5D, n=10), amivantamab (10mpk, BIW, n=10), or a combination of pembrolizumab and amivantamab (n=10). Analysis of immune modulatory responses within the tumor microenvironment (TME) using multiplexed IHC, flow cytometry, and single cell RNA sequencing was performed. Results: Combination of amivantamab and pembrolizumab showed a significant reduction of tumor volume (p Conclusion: Our study demonstrated combinatorial benefits of amivantamab and pembrolizumab by effectively remodeling TME, providing a preclinical rationale to clinically combine amivantamab and PD-1 blockade treatments. Citation Format: Sun Min Lim, Chun-Bong Synn, Seong-san Kang, DongKwon Kim, Soo-Hwan Lee, Sujeong Baek, Seung Min Yang, Yu Jin Han, Mi hyun Kim, Heekyung Han, Kwangmin Na, Young Taek Kim, Sungwoo Lee, Mi Ran Yun, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Jii Bum Lee, Ji Yun Lee, Chang Gon Kim, Min Hee Hong, Kyoung-Ho Pyo, Joshua Curtin, Bharvin Patel, Isabelle Bergiers. Combinatorial activity of amivantamab and pembrolizumab in head and neck squamous cell carcinoma and lung squamous cell carcinoma expressing wild-type EGFR and MET [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5865.

Introduction: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a standard modality of the 1st-line treatments for patients with EGFR-mutated non-small-cell lung cancer (NSCLC). The FDA approved osimertinib, a third-generation EGFR-TKI, is highly selective for EGFR-activating mutations (exon 19 deletion [del19] or L858R point mutation in exon 21 [L858R]) as well as EGFR-T790M mutation. Nevertheless, resistance inevitably emerges and leads to disease progression. Acquired EGFR-C797S mutation is the most common on-target resistance mechanism to osimertinib in first- and second-line settings, and there are currently no approved targeted therapies. We aimed to evaluate the efficacy of BI-732, a fourth-generation EGFR-TKI developed to overcome the C797S mutation. Materials and Methods: We constructed several types of Ba/F3 cells expressing EGFR mutations containing the C797S mutation as follows: EGFRdel19/C797S, EGFRL858R/C797S, EGFRdel19/T790M/C797S, and EGFRL858R/T790M/C797S. We also constructed PC9 cells harboring EGFRdel19/C797S (named PC9-DC cells) using the CRISPR/Cas9 system. Furthermore, we generated two patient-derived cells, YU-1097 with EGFRdel19/T790M/C797S and YU-1182 with EGFRL858R/C797S. The in vitro efficacy of BI-732 was tested in several cell lines harboring the EGFR-C797S mutation mentioned above. We further evaluated in vivo antitumor activity of BI-732 in subcutaneous YU-1097 xenograft model and intracranial YU-1097-tumor model.Results: BI-732 effectively inhibited the viability of Ba/F3 cells expressing EGFRdel19/C797S (IC50, 6.8 nM), EGFRL858R/C797S (IC50, 213.4 nM), EGFRdel19/T790M/C797S (IC50, 3.8nM), and EGFRL858R/T790M/C797S (IC50, 15.2 nM), concomitant with marked reduction in EGFR and downstream signal phosphorylation. Moreover, BI-732 exhibited comparable nanomolar in vitro activity to osimertinib on EGFR activating mutation while sparing wild-type EGFR activity. We further confirmed the superior antiproliferative efficacy of BI-732 in PC9-DC (IC50, 24.9 nM), YU-1182 (IC50, 73 nM) and YU-1097 cells (IC50, 2.9 nM). Oral administration of BI-732 at a concentration of 25 mg/kg as a single agent resulted in significant tumor regression with 183.2% tumor growth inhibition (TGI) in the subcutaneous YU-1097 xenograft model. In a combination treatment strategy with osimertinib, BI-732 showed enhanced antitumor activity at much lower concentrations than monotherapy. BI-732 also demonstrated the ability to penetrate brain-blood barrier (BBB) in the intracranial YU-1097-tumor models.Conclusions: BI-732 is a potent, selective, and orally available fourth-generation EGFR-TKI for EGFR mutations including C797S and has efficient BBB penetration. Our findings suggest that BI-732 can be effective against EGFR mutant NSCLC, especially EGFRDel19/T790M/C797S and EGFRDel19/C797S, that have progressed with previous EGFR inhibitors. Citation Format: Eun Ji Lee, Jiyun Lee, Seung Yeon Oh, You Won Lee, Ju young Kim, Su-Jin Choi, Sewon Park, Mi Ra Yu, Jae Hwan Kim, Kyoung-Ho Pyo, Jii Bum Lee, Min Hee Hong, Sun Min Lim, Anke Baum, Lydia Woelflingseder, Harald Engelhardt, Mark Petronczki, Flavio Solca, Mi Ran Yun, Byoung Chul Cho. BI-732, a novel fourth-generation EGFR-TKI, demonstrates promising activities against the C797S-mediated EGFR-TKI resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4476.

A recently developed treatment strategy for lung cancer that combines immune checkpoint inhibitors with chemotherapy has been applied as a standard treatment for lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), and it has improved the outcomes of chemotherapy. Maintenance treatment with anti-PD-1 antibody (aPD-1) enhances the effect of immunochemical combination therapy and improves therapeutic efficacy, which contributes toward a significant improvement in patient survival rates. The AXL receptor tyrosine kinase (AXL), which is expressed in tumor cells, plays an essential role in the resistance of cancers to chemotherapy and immunotherapy, and stimulates signaling associated with epithelial-mesenchymal transition (EMT) in metastatic cancer. AXL is thus an attractive target for controlling resistance to anti-tumor therapies. In this study, we examined the effect of AXL inhibitors on immune activation and tumor growth in TC1 and C3PQ mouse tumor models, in the context of clinical immunotherapy/chemotherapy and maintenance treatment, using an aPD-1 with/without pemetrexed. To determine the optimal timing for administration of SKI-G-801, an AXL inhibitor, we investigated its anti-tumor effects based on inclusion at the immunochemotherapy and maintenance therapy stages. We also performed flow cytometry-based immune profiling of myeloid cells and lymphoid cells at different points in the treatment schedule, to investigate the immune activation and anti-tumor effects of the AXL inhibitor. The addition of SKI-G-801 to the immune checkpoint inhibitor and chemotherapy stage, as well as the maintenance therapy stage, produced the best anti-tumor results, and significant tumor growth inhibition was observed in both the TC1 and C3PQ models. Both models also exhibited increased proportion of effector memory helper T cells and increased expression of CD86+ macrophages. Especially, regulatory T cells were significantly reduced in the TC1 tumor model and there was an increase in central memory cytotoxic T cell infiltration and an increased proportion of macrophages with high CD80 expression in the C3PQ tumor model. These results suggest increased infiltration of T cells, consistent with previous studies using AXL inhibitors. It is expected that the results from this study will serve as a stepping stone for clinical research to improve the existing standard of care.

Background Recurrent and/or metastatic squamous cell carcinoma of head and neck (R/M SCCHN) is a common cancer with high recurrence and mortality. Current treatments have low response rates (RRs). Methods Fifty-three patients with R/M SCCHN received continuous oral buparlisib. In parallel, patient-derived xenografts (PDXs) were established in mice to evaluate resistance mechanisms and efficacy of buparlisib/cetuximab combination. Baseline and on-treatment tumour genomes and transcriptomes were sequenced. Based on the integrated clinical and PDX data, 11 patients with progression under buparlisib monotherapy were treated with a combination of buparlisib and cetuximab. Results For buparlisib monotherapy, disease control rate (DCR) was 49%, RR was 3% and median progression-free survival (PFS) and overall survival (OS) were 63 and 143 days, respectively. For combination therapy, DCR was 91%, RR was 18% and median PFS and OS were 111 and 206 days, respectively. Four PDX models were originated from patients enrolled in the current clinical trial. While buparlisib alone did not inhibit tumour growth, combination therapy achieved tumour inhibition in three of seven PDXs. Genes associated with apoptosis and cell-cycle arrest were expressed at higher levels with combination treatment than with buparlisib or cetuximab alone. Conclusions The buparlisib/cetuximab combination has significant promise as a treatment strategy for R/M SCCHN. Clinical Trial Registration NCT01527877.

Objectives AXL‐mediated activation of aberrant tyrosine kinase drives various oncogenic processes and facilitates an immunosuppressive microenvironment. We evaluated the anti‐tumor and anti‐metastatic activities of SKI‐G‐801, a small‐molecule inhibitor of AXL, alone and in combination with anti‐PD‐1 therapy. Methods In vitro pAXL inhibition by SKI‐G‐801 was performed in both human and mouse cancer cell lines. Immunocompetent mouse models of tumor were established to measure anti‐metastatic potential of SKI‐G‐801. Furthermore, SKI‐G‐801, anti‐PD‐1 or their combination was administered as an adjuvant or neoadjuvant in the 4T1 tumor model to assess their potential for clinical application. Results SKI‐G‐801 robustly inhibited pAXL expression in various cell lines. SKI‐G‐801 alone or in combination with anti‐PD‐1 potently inhibited metastasis in B16F10 melanoma, CT26 colon and 4T1 breast models. SKI‐G‐801 inhibited the growth of B16F10 and 4T1 tumor‐bearing mice but not immune‐deficient mice. An antibody depletion assay revealed that CD8+ T cells significantly contributed to SKI‐G‐801‐mediated survival. Anti‐PD‐1 and combination group were observed the increased CD8+Ki67+ and effector T cells and M1 macrophage and decreased M2 macrophage, and granulocytic myeloid‐derived suppressor cell (G‐MDSC) compared to the control group. The neoadjuvant combination of SKI‐G‐801 and anti‐PD‐1 therapy achieved superior survival benefits by inducing more profound T‐cell responses in the 4T1 syngeneic mouse model. Conclusion SKI‐G‐801 significantly suppressed tumor metastasis and growth by enhancing anti‐tumor immune responses. Our results suggest that SKI‐G‐801 has the potential to overcome anti‐PD‐1 therapy resistance and allow more patients to benefit from anti‐PD‐1 therapy., SKI‐G‐801, a novel AXL inhibitor, significantly suppressed tumor metastasis and growth by enhancing anti‐tumor immune responses. Our results suggest that SKI‐G‐801 has the potential to overcome resistance to anti‐PD‐1 therapy and expand the number of patients who benefit from anti‐PD‐1 therapy.

Simple Summary Despite the rapid advancement in immunotherapy and targeted agents, many patients diagnosed with cancer have poor prognosis with dismal overall survival. One of the key hallmarks of cancer is the ability of cancer cells to reprogram their energy metabolism. O-GlcNAcylation is an emerging potential mechanism for cancer cells to induce proliferation and progression of tumor cells and resistance to chemotherapy. This review summarizes the mechanism behind O-GlcNAcylation and discusses the role of O-GlcNAcylation, including its function with receptor tyrosine kinase and chemo-resistance in cancer, and immune response to cancer and as a prognostic factor. Further pre-clinical studies on O-GlcNAcylation are warranted to assess the clinical efficacy of agents targeting O-GlcNAcylation. Abstract Cancer cells are able to reprogram their glucose metabolism and retain energy via glycolysis even under aerobic conditions. They activate the hexosamine biosynthetic pathway (HBP), and the complex interplay of O-linked N-acetylglucosaminylation (O-GlcNAcylation) via deprivation of nutrients or increase in cellular stress results in the proliferation, progression, and metastasis of cancer cells. Notably, cancer is one of the emerging diseases associated with O-GlcNAcylation. In this review, we summarize studies that delineate the role of O-GlcNAcylation in cancer, including its modulation in metastasis, function with receptor tyrosine kinases, and resistance to chemotherapeutic agents, such as cisplatin. In addition, we discuss the function of O-GlcNAcylation in eliciting immune responses associated with immune surveillance in the tumor microenvironment. O-GlcNAcylation is increasingly accepted as one of the key players involved in the activation and differentiation of T cells and macrophages. Finally, we discuss the prognostic role of O-GlcNAcylation and potential therapeutic agents such as O-linked β-N-acetylglucosamine-transferase inhibitors, which may help overcome the resistance mechanism associated with the reprogramming of glucose metabolism.

Simple Summary Patients with epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer can have central nervous system (CNS) metastases during their disease course. A high unmet medical need exists especially for patients with T790M-positive NSCLC whose disease progressed after first-line EGFR-TKI. Osimertinib is a third-generation EGFR-TKI with selective activity for both sensitizing and EGFR T790M mutations and has improved CNS activity over first- and second-generation EGFR TKIs and chemotherapies. This study confirmed the clinical activity and CNS efficacy of osimertinib in an unselected real-world population. Abstract Up to 40% of patients with epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC) may develop central nervous system (CNS) metastases throughout their disease. Moreover, the first- and second-generation EGFR-tyrosine kinase inhibitors have limited efficacy because of their poor blood–brain barrier permeability. Therefore, we conducted preplanned analyses of ASTRIS, a clinical study of the third-generation EGFR-TKI osimertinib to demonstrate its potential role in intracranial response efficacies. We retrospectively examined 89 NSCLC patients with brain evaluation who were not amenable to curative surgery or radiotherapy and received osimertinib upon confirmation of the presence of the T790M mutation. We collected the information regarding patients’ baseline characteristics, baseline intracranial status, including leptomeningeal metastases (LM), and intracranial responses measured by Response Evaluation Criteria in Solid Tumors version 1.1, using independent central review. The median age was 60 years, and 69.7% of the patients were female. Sixty-five patients (73.0%) had brain metastases (BM) at baseline and nineteen patients (23.5%) had additional LM. Among patients with brain metastases, 24 (36.9%) had ≥1 measurable brain metastases and 16 were evaluated for the objective response. In the CNS evaluable for response set, the intracranial objective response rate (cORR) and disease control rate (cDCR) were 62.5% (95% confidence interval (CI), 38.3–82.6%) and 93.8% (95% CI, 74.3–99.3%), respectively. The median intracranial progression-free survival (cPFS) was 13.0 (95% CI, 7.21–18.8) months, including patients with measurable and non-measurable BM or LM. Our cORR, cDCR, and cPFS were comparable to those observed in previous clinical trials. The outcome of this study helps to demonstrate the potential role of intracranial efficacies of osimertinib 80 mg administration in T790M-positive advanced NSCLC with/without BM or LM.

Background Immune checkpoint blockade with Programmed cell death 1 (PD-1)/PD-L1 inhibitors has been effective in various malignancies and is considered as a standard treatment modality for patients with non-small-cell lung cancer (NSCLC). However, emerging evidence show that PD-1/PD-L1 blockade can lead to hyperprogressive disease (HPD), a flair-up of tumor growth linked to dismal prognosis. This study aimed to evaluate the incidence of HPD and identify the determinants associated with HPD in patients with NSCLC treated with PD-1/PD-L1 blockade. Patients and methods We enrolled patients with recurrent and/or metastatic NSCLC treated with PD-1/PD-L1 inhibitors between April 2014 and November 2018. Clinicopathologic variables, dynamics of tumor growth, and treatment outcomes were analyzed in patients with NSCLC who received PD-1/PD-L1 blockade. HPD was defined according to tumor growth kinetics (TGK), tumor growth rate (TGR), and time to treatment failure (TTF). Immunophenotyping of peripheral blood CD8+ T lymphocytes was conducted to explore the potential predictive biomarkers of HPD. Results A total of 263 patients were analyzed. HPD was observed in 55 (20.9%), 54 (20.5%), and 98 (37.3%) patients according to the TGK, TGR, and TTF. HPD meeting both TGK and TGR criteria was associated with worse progression-free survival [hazard ratio (HR) 4.619; 95% confidence interval (CI) 2.868–7.440] and overall survival (HR, 5.079; 95% CI, 3.136–8.226) than progressive disease without HPD. There were no clinicopathologic variables specific for HPD. In the exploratory biomarker analysis with peripheral blood CD8+ T lymphocytes, a lower frequency of effector/memory subsets (CCR7−CD45RA− T cells among the total CD8+ T cells) and a higher frequency of severely exhausted populations (TIGIT+ T cells among PD-1+CD8+ T cells) were associated with HPD and inferior survival rate. Conclusion HPD is common in NSCLC patients treated with PD-1/PD-L1 inhibitors. Biomarkers derived from rationally designed analysis may successfully predict HPD and worse outcomes, meriting further investigation of HPD.

Purpose: Given that osimertinib is the only approved third-generation EGFR-TKI against EGFR activating and resistant T790M mutated non–small cell lung cancer (NSCLC), additional mutant-selective inhibitors with a higher efficacy, especially for brain metastases, with favorable toxicity profile are still needed. In this study, we investigated the antitumor efficacy of YH25448, an oral, mutant-selective, irreversible third-generation EGFR-TKI in preclinical models. Experimental Design: Antitumor activity of YH25448 was investigated in vitro using mutant EGFR-expressing Ba/F3 cells and various lung cancer cell lines. In vivo antitumor efficacy, ability to penetrate the blood–brain barrier (BBB), and skin toxicity of YH25448 were examined and compared with those of osimertinib using cell lines and PDX model. Results: Compared with osimertinib, YH25448 showed a higher selectivity and potency in kinase assay and mutant EGFR-expressing Ba/F3 cells. In various cell line models harboring EGFR activating and T790M mutation, YH25448 effectively inhibited EGFR downstream signaling pathways, leading to cellular apoptosis. When compared in vivo at equimolar concentrations, YH25448 produced significantly better tumor regression than osimertinib. Importantly, YH25448 induced profound tumor regression in brain metastasis model with excellent brain/plasma and tumor/brain area under the concentration–time curve value. YH25448 rarely suppressed the levels of p-EGFR in hair follicles, leading to less keratosis than osimertinib in animal model. The potent systemic and intracranial activity of YH25448 has been shown in an ongoing phase I/II clinical trial for advanced EGFR T790M mutated NSCLC (NCT03046992). Conclusions: Our findings suggest that YH25448 is a promising third-generation EGFR inhibitor, which may be more effective and better tolerated than the currently approved osimertinib.

Background: The IDO is an enzyme responsible for catabolizing tryptophan (Trp) to kynurenine (Kyn). The kynurenine exert important immunosuppressive functions by activating Treg cells and myeloid-derived suppressor cells. YH29407 is a novel IDO1 inhibitor, improved the pharmacodynamics compared to previous IDO inhibitors. Methods: To evaluate antitumor effects and immune profiles of YH29407, YH29407 was dosed at 50 or 100 mg/kg twice daily (B.I.D.) alone or in combination with anti-PD-1 (10 mg/kg, B.I.W., i.p.) and BMS-986205 was dosed at 125 mg/kg once daily (Q.D.) alone or in combination with anti-PD-1 in a MC38 syngeneic tumor model. The antitumor effects during 11 days of administration of YH29407 alone or combinations and BMS-986205 alone or combination in the MC38 model were measured. Measurements were carried out up to day 50 for survival testing. After 3 days of treatment for each condition, immune cell profiles were evaluated by flow cytometry. Results: The YH29407 at dose of 100 mg/kg B.I.D combination treatment group showed the best effects on tumor size reduction. The group constituting >TGI:70% contained 100% (15/15) of the combination treatment group, whereas 15% (2/13) of competitor BMS-986205 combination group were contained. In addition, in the combination treatment group, most tumors showed an aspect of decrease including complete responses of 5 mice. Moreover, there was a complete response in YH29407 alone group but, not observed in BMS-986205 group. According to flow cytometry analysis, the combination treatment group showed higher CD3+ total T cells compared to the vehicle group (*p Conclusion: Taken together, we suggest that YH29407 is the best combination partner with immune checkpoint inhibitors for solid tumor. Citation Format: Kyoung-Ho Pyo, Dong Kwon Kim, Se-Woong Oh, Gyu-Jin Lee, Ho-Woong Kang, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Chun-Bong Synn, Wongeun Lee, Su Hwan Lee, Seul Lee, Seung Min Yang, Soyeon Lee, Yunjoo Joo, Eun Ji Lee, Sun Min Lim, Byoung Chul Cho. Combination therapy with anti-PD-1 and YH29407, a novel IDO1 inhibitor, enhances T cell-mediated antitumor immunity in MC38 tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5481.

Introduction: Acquired resistance to ALK-tyrosine kinase inhibitors (ALK-TKIs) treatment, particularly target-off resistance, remains a clinical challenge for ALK-rearranged non-small cell lung cancer (NSCLC). To explore novel vulnerabilities of ALK TKI-resistant cancer cells, we focused on their distinct metabolic pathways for growth and survival. Experimental Design: To investigate metabolic pathways in resistance mechanisms, we generated ALK-TKIs -acquired-resistant in vitro/vivo models. We screened metabolite mechanisms using metabolite assay kit, Seahorse Extracellular Flux Analyzer, real-time PCR, western blot, RNA-seq in resistant models. Results: Through an integrated transcriptomic and metabolic assay screening approach, we identified the enhanced reliance on glutamine metabolism in target-off ALK-TKIs-resistant cells. Specifically, resistant cells were characterized by upregulation of glutaminase 1 (GLS1), a mitochondrial enzyme hydrolyzing glutamine into glutamate, simultaneously with downregulation of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this metabolic state intensively accelerates glutaminolysis and subsequent mitochondrial glutamine-derived aspartate synthesis, resulting in TKI resistance by reinforcing antioxidant capacity with increase of NADPH and glutathione. Mechanistically, GLS1 inhibition elicited a marked reduction of cell growth with increase of reactive oxygen species (ROS) in resistant cells, which was restored by supplementation of exogenous aspartate. The antitumor activity of GLS1 inhibition against resistant tumor cells was further validated in in vivo experiments, patient-derived xenograft (PDX) and EML4-ALK transgenic mice. More importantly, glutaminase inhibitor CB-839 enhanced the therapeutic efficacy of anti-PD-L1 treatment in immune checkpoint blockade (ICB)-resistant EML4-ALK transgenic mice. Conclusion: Our findings highlight a new metabolic vulnerability of ALK-TKIs resistant tumors and provide a rationale for targeting GLS1 as a potential treatment option to overcome ALK-TKIs resistance. Citation Format: You Won Lee, Hun Mi Choi, Seung Yeon Oh, Eun Ji Lee, Kyoung-Ho Pyo, Jae Hwan Kim, Youngseon Byeon, Seong Gu Heo, Sun Min Lim, Min Hee Hong, Chang Gon Kim, Hye Ryun Kim, Mi Ran Yun, Byoung Chul Cho. Targeting adaptive metabolic program as a novel treatment approach for TKIs-failed ALK-positive NSCLCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB544.

Background: Immune checkpoint inhibitor has emerged as remarkable therapeutic that improves the anti-cancer effect of patients. However, response rate is low in a large proportion of patients. The overall efficacy of immune checkpoint inhibitor therapy remains unsatisfactory. Recently, live biotherapeutic products (LBPs) have emerged as potential therapeutics to overcome the limitation of immune checkpoint inhibitor. Here, we demonstrated the efficacy of CJRS-10671 and CJRS-10672 in tumor bearing mice models. Methods: To evaluate anti-tumor effects, 109 CFU/mouse CJRS-10671 was administered via oral gavage twice daily (BID) alone or in combination with anti-PD-1 (10 mg/kg, QOD, i.p.) for 10 days in a Lewis Lung Carcinoma (LLC1) syngeneic tumor model. Tumor growth was measured by calipers 3 times per week. We analyzed immune cell profiles by flow cytometry. In lung squamous cell carcinoma patient derived xenograft (PDX) model,109 CFU/mouse CJRS-10671 and CJRS-10672 were orally administered twice daily (BID) alone or in combination with pembrolizumab (10 mg/kg, Q5D, i.p.) for 35 days. Tumor growth was measured by calipers 3 times per week. We analyzed immune cell profiles and single cell RNA sequencing. Results: In LLC1 syngeneic tumor model, CJRS-10671 alone suppressed tumor growth comparable to that of immune checkpoint inhibitor (anti-PD-1 antibody) only treatment group. Combined administration of CJRS-10671 and immune checkpoint inhibitor significantly suppressed tumor growth (pTGI-75%).We confirmed that administration of CJRS-10671 increased population of CD8+ central memory T cells in tumor. Also, TGI (%) directly related features were mostly antigen presentation and monocyte origin cells, especially macrophage in immune-profiling analysis. Furthermore, we observed that CJRS-10671 and CJRS-10672 were associated with signaling of damage-associated molecular pattern (DAMP) and especially CJRS-10671 was related to nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling by single cell RNA sequencing analysis. Conclusion: CJRS-10671 and CJRS-10672, single administration or combination with anti-PD-1 antibody, have potential anti-tumor effect. Citation Format: Arim Min, Bo-eun Kwon, Hyunjeong Kim, Hyunkyung Park, Jiyoung Lee, Kyoung-Ho Pyo, Byoung Chul Cho. Novel bacteria strains, CJRS-10671 and CJRS-10672, enhance anti-tumor efficacy in LLC1 syngeneic model and humanized PDX mice model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3527.

Background Although cancer immunotherapy showed promising results in hematological malignancies, it has come up with relatively low tumor response for many solid tumors partly due to immune-suppressive tumor microenvironment (TME). Because of the immune-suppressive nature of TME, TME has been an active area of research and therapeutic target for restoring immune system and subsequent tumor growth inhibition. Among the many components in TME, cancer-associated fibroblasts (CAFs) are one of the key cell components of TME where one of the promising solid-tumor TME marker, fibroblast-activating protein (FAP) is highly expressed. Here we have developed an antibody-cytokine fusion protein from our TMEkine™ platform containing anti-FAP and IL-12. Our TMEkine™ (anti-FAP-IL-12) molecule induced strong anti-cancer effects in preclinical solid tumor models by immune-modulation. Methods IL-12 cytokine was mutated in TMEkine™ (anti-FAP-IL-12) to reduce systemic toxicity and its binding affinity was tested to FAP and IL-12 receptor. The anti-tumor activity of anti-FAP-IL-12 was investigated on CT26 (murine colorectal cancer) syngeneic mouse models with/without NIH-3T3 (murine fibroblast). Additionally, mice showing complete response after anti-FAP-IL-12 administration were re-injected CT26 with/without 4T1 cells for re-challenge study to monitor long-term durable response generated from the initial immune activation. Results We showed that TMEkine™ (anti-FAP-IL-12) interacts with FAP and IL-12 receptor. IL-12 activity was attenuated by our IL-12 mutants. We also showed that TMEkine™ (anti-FAP-IL-12) induced IFN-γ from primary human T cells and NK cells. TMEkine™ (anti-FAP-IL-12) administration resulted in significant reduction of the tumor burden in both CT26+NIH-3T3/FAP+ and CT26/FAP+ models. In the re-challenge experiments, CT26 tumor growth was inhibited significantly compared to 4T1 tumor suggesting memory immune response was generated in TMEkine™ (anti-FAP-IL-12) treated mice. Conclusions These findings provide evidences that the treatment of anti-FAP/IL-12 TMEkine™ induced anti-cancer effects without serious adverse effects. Anti-FAP/IL-12 has a strong potential to provide a therapeutic option for cancer-specific immunomodulator and cancer cell eradication.

Background Targeting transforming growth factor-β (TGF-β) is reported to augment the efficacy of immune checkpoint inhibitors (ICIs) through either enhanced anti-tumor immunity or the correction of tumor microenvironment (TME). Therefore, the combination of vactosertib, a highly selective TGF-β RI kinase inhibitor, and durvalumab is anticipated to improve anti-tumor activity of the ICI. A phase 1b/2a study was conducted to evaluate the combination of vactosertib and durvalumab in patients with advanced NSCLC who progressed after platinum-based chemotherapy. Methods Patients were treated with vactosertib at a dose of 200 mg twice daily (five days on and two days off) and durvalumab at a dose of 1500 mg every four weeks. Eligible patients were ≥19 years old with good performance status (ECOG 0–1) and have no prior exposure to immune checkpoint inhibitors or other TGF- β R1 kinase inhibitors. The objectives of this analysis were to evaluate the safety, antitumor activity including objective response rate (ORR), duration of response (DOR), and time to response (TTR) as well as circulating pharmacodynamic biomarkers related to TGF-β signaling. Response was assessed per RECIST (v1.1). Results By August 4 2020, twenty-six PD-L1 positive (SP263 assay) patients were analyzed. Median age was 61.5 years (range 48–83), 69.2% were male, median number of previous lines of chemotherapy was 1 (range 1–4), and all patients were PD-L1 positive (15 patients with PD-L1≥25% and 11 patients with PD-L1 1–24%). The most frequently reported treatment-related adverse events (TRAE) were itching (38.5%) and skin rash (34.6%), but no Gr≥3 itching and rash were observed. Each case of the following was reported as Grade 3 TRAEs: adrenal insufficiency, anemia, and pneumonitis; Grade 4 TRAE, CPK increase, was observed in one patient. Objective response rate was 30.8% and 40.0% in patients with PD-L1≥1% and ≥25% respectively. Circulating PAI-1 and CTGF evaluated in 15 patients decreased significantly on Cycle 1 day 5. Ongoing biomarker results will be presented. Conclusions The combination of vactosertib and durvalumab has demonstrated a manageable safety profile and encouraging anti-tumor activity as a potential therapeutic strategy in patients with advanced NSCLC. The efficacy outcomes of this combination in a larger number of patients with advanced NSCLC will be followed. Trial Registration NCT03732274 Ethics Approval The study was approved by Ethics Board of Severance Hospital (4-2018-0892), National Cancer Center (NCC2019-0057), St. Vincent’s Hospital (VC19MDDF0205), and Chungbuk National University Hospital (2019-08-015).

BackgroundEML4-ALK is a distinct molecular entity that is highly sensitive to ALK tyrosine kinase inhibitors (TKIs). Immune checkpoint inhibitors (ICIs) have not proved efficacy in ALK-positive non-small cell lung cancer so far. In this study, we performed a mouse clinical trial using EML4-ALK transgenic mice model to comprehensively investigate immunomodulatory effects of ALK TKI and to investigate the mechanisms of resistance to ICIs.MethodsEML4-ALK transgenic mice were randomized to three treatment arms (arm A: antiprogrammed death cell protein-1 (PD-1), arm B: ceritinib, arm C: anti-PD-1 and ceritinib), and tumor response was evaluated using MRI. Progression-free survival and overall survival were measured to compare the efficacy. Flow cytometry, multispectral imaging, whole exome sequencing and RNA sequencing were performed from tumors obtained before and after drug resistance.ResultsMouse clinical trial revealed that anti-PD-1 therapy was ineffective, and the efficacy of ceritinib and anti-PD-1 combination was not more effective than ceritinib alone in the first line. Dynamic changes in immune cells and cytokines were observed following each treatment, while changes in T lymphocytes were not prominent. A closer look at the tumor immune microenvironment before and after ceritinib resistance revealed increased regulatory T cells and programmed death-ligand 1 (PD-L1)-expressing cells both in the tumor and the stroma. Despite the increase of PD-L1 expression, these findings were not accompanied by increased effector T cells which mediate antitumor immune responses.ConclusionsALK-positive tumors progressing on ceritinib is not immunogenic enough to respond to immune checkpoint inhibitors.

Additional file 1 : Supplementary Table 1. Baseline characteristics of patients (N = 15). Supplementary Table 2. Detailed pathologic information of F0 and F2 generation. Supplementary Table 3. Multivariate analysis to determine the association between covariates and patients- derived xenograft establishment. Supplementary Table 4. Latency time of 15 established PDX models. Supplementary Table 5. Detailed information of established head and neck cancer patient-derived xenografts

Additional file 2 : Supplementary Figure 1. Comparison of histopathologic and immunohistochemistry findings between patients and their matched patient-derived xenografts (PDXs, F2 generation) of head and neck cancer squamous cell carcinoma (HNSCC). Left columns show hematoxylin and eosin-staining and right columns show immunohistochemical staining of p63, a marker of squamous cell carcinoma, from patients and the PDX of each model. Representative stained sections are shown (magnification: 200× in patient samples; scale bars = 100 μm).Supplementary Figure 2. P16 immunohistochemistry staining of tumor tissue between patient and PDX model. Supplementary Figure 3. Ki67 immunohistochemistry staining on tumor tissue of patient derived xenograft model. Supplementary Figure 4. Tumor growth rate to anti-cancer therapy in YHIM-3006 and 3011 models.

Purpose Agonists of the stimulator of interferon genes (STING) play a key role in activating the STING pathway by promoting the production of cytokines. In this study, we investigated the antitumor effects and activation of the systemic immune response of treatment with DMXAA (5,6-dimethylxanthenone-4-acetic acid), a STING agonist, in EML4-ALK lung cancer and CT26 colon cancer. Materials and Methods The abscopal effects of DMXAA in the treatment of metastatic skin nodules were assessed. EML4-ALK lung cancer and CT26 colon cancer models were used to evaluate these effects after DMXAA treatment. To evaluate the expression of macrophages and T cells, we sacrificed the tumor-bearing mice after DMXAA treatment and obtained the formalin-fixed paraffin-embedded (FFPE) tissue and tumor cells. Immunohistochemistry and flow cytometry were performed to analyze the expression of each FFPE and tumor cell. Results We observed that highly infiltrating immune cells downstream of the STING pathway had increased levels of chemokines after DMXAA treatment. In addition, the levels of CD80 and CD86 in antigen-presenting cells were significantly increased after STING activation. Furthermore, innate immune activation altered the systemic T cell-mediated immune responses, induced proliferation of macrophages, inhibited tumor growth, and increased numbers of cytotoxic memory T cells. Tumor-specific lymphocytes also increased in number after treatment with DMXAA. Conclusion The abscopal effect of DMXAA treatment on the skin strongly reduced the spread of EML4-ALK lung cancer and CT26 colon cancer through the STING pathway and induced the presentation of antigens.

Introduction:Osimertinib is a third-generation EGFR tyrosine kinase inhibitor (TKI) that is approved for the use of EGFR-mutant non-small cell lung cancer (NSCLC) patients. In this study, we investigated the acquired resistance mechanisms in NSCLC patients and patient-derived preclinical models.Methods:Formalin-fixed paraffin-embedded tumor samples and plasma samples from 55 NSCLC patients who were treated with osimertinib were collected at baseline and at progressive disease (PD). Next-generation sequencing was performed in tumor and plasma samples using a 600-gene hybrid capture panel designed by AstraZeneca. Osimertinib-resistant cell lines and patient-derived xenografts and cells were generated and whole exome sequencing and RNA sequencing were performed. In vitro experiments were performed to functionally study the acquired mutations identified.Results:A total of 55 patients and a total of 149 samples (57 tumor samples and 92 plasma samples) were analyzed, and among them 36 patients had matched pre- and post-treatment samples. EGFR C797S (14%) mutation was the most frequent EGFR-dependent mechanism identified in all available progression samples, followed by EGFR G824D (6%), V726M (3%), and V843I (3%). Matched pre- and post-treatment sample analysis revealed in-depth acquired mechanisms of resistance. EGFR C797S was still most frequent (11%) among EGFR-dependent mechanism, while among EGFR-independent mechanisms, PIK3CA, ALK, BRAF, EP300, KRAS, and RAF1 mutations were detected. Among Osimertinib-resistant cell lines and patient-derived models, we noted acquired mutations which were potentially targetable such as NRAS p.Q61K, in which resistance could be overcome with combination of osimertinib and trametinib. A patient-derived xenograft established from osimertinib-resistant patient revealed KRAS p.G12D mutation which could be overcome with combination of osimertinib, trametinib, and buparlisib.Conclusion:In this study, we explored the genetic profiles of osimertinib-resistant NSCLC patient samples using targeted deep sequencing. In vitro and in vivo models harboring osimertinib resistance revealed potential novel treatment strategies after osimertinib failure.