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Inhibition of immune checkpoint proteins like programmed death 1 (PD-1) is a promising therapeutic approach for several cancers, including non-small cell lung cancer (NSCLC). Although PD-1 ligand (PD-L1) expression is used to predict anti-PD-1 therapy responses in NSCLC, its accuracy is relatively less. Therefore, we sought to identify a more accurate predictive blood biomarker for evaluating anti-PD-1 response. We evaluated the frequencies of T cells, B cells, natural killer (NK) cells, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), mononuclear myeloid-derived suppressor cells (M-MDSCs), and Lox-1+ PMN-MDSCs in peripheral blood samples of 62 NSCLC patients before and after nivolumab treatment. Correlation of immune-cell population frequencies with treatment response, progression-free survival, and overall survival was also determined. After the first treatment, the median NK cell percentage was significantly higher in responders than in non-responders, while the median Lox-1+ PMN-MDSC percentage showed the opposite trend. NK cell frequencies significantly increased in responders but not in non-responders. NK cell frequency inversely correlated with that of Lox-1+ PMN-MDSCs after the first treatment cycle. The NK cell-to-Lox-1+ PMN-MDSC ratio (NMR) was significantly higher in responders than in non-responders. Patients with NMRs ≥ 5.75 after the first cycle had significantly higher objective response rates and longer progression-free and overall survival than those with NMRs

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.

The present study investigated the effect of peanut sprout extract (PSE) as a natural resveratrol supplement on chronic bacterial prostatitis (CBP) and estradiol- induced benign prostatic hyperplasia (BPH). PSE contained a high level of resveratrol (148.51 ± 3.05 μg/g), and was tested on the mouse models of CBP . (induced by Escherichia coli 292 infection) and BPH (induced by treatment with β-estradiol and dihydrotestosterdne). PSE toxicity was assessed on the basis of changes in body weight, alanine aminotransferase activity (an indicator of hepatotoxicity), and expression of the kidney injury marker KIM-1. The effects of PSE on the histopathology of prostate tissue, the proportion of neutrophils, and immune cell profiles in the blood and spleen were examined. PSE administration did not result in any toxicity but reduced the bacterial burden and histopathological changes in the prostate. In addition, lymphocytes (CD4⁺, CD8⁺, and CD 19⁺) in the spleen were significantly increased after PSE administration in CBP mice, suggesting immune enhancement. PSE treatment of bone Snarrow-derived macrophages increased the expression of CD40, which is related to the pro-inflammatory function and host defense against pathogens. It is concluded that PSE would be a good supplement for the mitigation of prostate hyperplasia and prostatitis.

Toxoplasma gondii is an intracellular protozoan parasite that causes a Th1 cellular immunity. Our previous study showed that T. gondii lysate antigen (TLA) treatment in S180 tumor-bearing mice resulted in tumor reduction by suppressing CD31 expression, a marker of angiogenesis. In the present study, to investigate tumor suppressive effect of TLA under the absence of T lymphocytes, athymic nude mice were compared with euthymic mice in the anti-tumorigenic effect triggered by TLA in CT26 tumors. According to the results, intratumorally injected TLA reduced tumor growth and TIMP-1 level, a metastatic marker, in both euthymic and athymic mice. TLA treatment led to a sharp increase in IL-12 expression in serum cytokine profiling of athymic mice, and increased MyD88 signals in macrophages derived from the bone marrow, implying the activation of innate immunity. The selective induction of IL-12 by TLA treatment had an anti-tumorigenic effect.

Introduction: Aberrant AXL expression plays a critical role in cancer cell migration, metastasis and drug resistance. Researchers have revealed that AXL signaling is also over expressed on cells associated with tumor microenvironment. These findings highlight AXL as an attractive drug candidate for targeting tumor evasion and metastasis. Here we present SKI-G-801, a small molecule inhibitor that targets phosphorylation of AXL (IC50 = 20 nM) and its downstream signals. Methods: Inhibitory effects of SKI-G-801 on cancer viability (MTT and colony formation assay), invasion and migration (trans-well invasion assay) were examined in AXL high-expressing lung cancer cells in vitro. LLC2 lung and 4T1 breast cancer bearing mouse models were established. in addition, C57BL/6 mice were injected intravenously with B16F10 melanoma cells to establish lung metastasis model. Mice were administrated with 30 mg/kg of SKI-G-801 orally before (metastasis model) or after (syngeneic model) tumor injection. To elucidate the involvement of AXL inhibitor on tumor microenvironment, the population of T cells and myeloid cells was analyzed by flow cytometry from the LLC2 tumor. Results: Treatment of SKI-G-801 showed strong inhibition of cancer migration and invasion, when its direct killing effect on cancer cells was modest. These results were reproduced in vivo test that pretreatment of SKI-G-801 significantly reduced metastatic burden in B16F10 model (p < 0.05). LLC2 and 4T1 tumors were decreased in SKI-G-801 treatment group (p < 0.05), but not in anthemic nude mice. CD3+CD8+ T cell population and memory Tc cells were increased in SKI- G-801 treatment group (p < 0.05). Especially, granzyme B+ Tc and gp70+ tumor specific Tc were increased (p < 0.05). SKI-G-801 increase the helper T cell population; CD3+CD4+ (p < 0.05), and CD44+ memory Th cells (p < 0.01). Conclusion: SKI-G-801 demonstrates great potential in anti-cancer activity though immune responses. The anti-cancer effects lead to a reversal of the metastatic phenotype in animal model. Our results suggest that SKI-G-801 is a promising drug for prevention against metastatic cancer. Citation Format: Chun-Feng Xin, Sung Eun Kim, Kyoung-Ho Pyo, Ha Ni Jo, Jae Seok Cho, Jae Hwan Kim, Wongeun Lee, Hee Kyu Lee, Jung-Ho Kim, Ho-Juhn Song, Jong Sung Koh, Byoung Chul Cho. SKI-G-801, an AXL kinase inhibitor, blocks metastasis and induces anti-tumor immune responses in various syngeneic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2010.

Cancer immunotherapy with anti-PD-1 therapy (αPD-1) has greatly improved the survival outcome of patients with non-small cell lung cancer (NSCLC). However, the response rates of αPD-1 are around 20-30% based on clinical trials. The presence of PD-L1, tumor infiltrating lymphocyte or tumor mutation burden may be used as indicators of response, but are still limited to predict αPD-1 response. In this study, we established the machine learning based clinical decision support system (CDSS) to predict the αPD-1 response by comprehensively combining clinical information and blood-based data which are easily assessable in routine practice.We enrolled 126 patients with NSCLC treated with the αPD-1 at Yonsei cancer center. Clinical data including patient characteristics, mutation, treatment outcomes, and adverse events were collected and analyzed. Forty patients additionally had blood-based immune data by flow cytometry. There were two data sets; clinical data set (n=126) with 15 variables, and immune data set (n=40) with 37 variables. We found that 27 variables out of 52 variables are selected by recursive feature elimination. The responders are defined as PR or SD ≥6 months and the non-responders are defined as the others. Supervised learning algorithms such as the LASSO, Ridge, Elastic Net, SVM, ANN, and RF were applied to each data set for predicting the αPD-1 response. The performances of each model were evaluated according to the ROC curve or cross-validation errors. Variable importance was measured by using the random forest and gradient boosting.Patient characteristics included male (69.8%), age ≥ 60 years (66.2%), ECOG 0/1 (77.7%), adenocarcinoma (69.8%), EGFR mutations (15.1%), and PD-L1 positive (61.2%). We classified the patients into responders (38%) and non-responders (62%) in total 126 patients. A result from the clinical data set of 126 patients demonstrated that the Ridge regression model (AUC: 0.78) can more accurately predict the αPD-1 response than others. Of 15 clinical variables, some are considered to be important in the following order; tumor burden,age, PD-L1, ECOG PS, and irAE based on the random forest. When we performed the machine learning process with clinical and immune data, The Ridge regression model (AUC:0.82) showed the good performance to predict αPD-1 response, compared to the single clinical model. The machine learning based CDSS for aPD-1 to NSCLC patients has benefit for predicting aPD-1 responses. Our prediction model could be easily accessible and fast processed in routine practice. The supervised machine learning based non-invasive predictive score (NIPS) demonstrates the rate of aPD-1 response on NSCLC patients. We will validate NIPS in independent patient cohort and currently are establishing the NIPS as web-based software. Citation Format: Kyoung-Ho Pyo, Beung-Chul Ahn, Chun-Feng Xin, Dongmin Jung, Chang Gon Kim, Min Hee Hong, Byoung Chul Cho, Hye Ryun Kim. A machine learning based clinical decision support system (CDSS) for anti-PD-1 therapy using non-invasive blood marker and clinical information for lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 683.

Introduction: MERTK is thought to play a role in cancer immune evasion mechanism based on M2 type macrophage-driven immunosuppression in tumor microenvironment (TME). Thus, MERTK is a highly promising target for anti-cancer therapy under high tumor associated macrophages (TAMs) condition. We evaluated the anti-tumor effects and immune reaction of combination or single MERTK inhibitor in two lung squamous patients derived xenograft (PDX) tumor engrafted Hu-CD34-NSG models. Methods: The aPD-1 sensitive (YHIM2004) and aPD-1 non-sensitive (YHIM2009) squamous cell carcinoma PDX models were selected from pre-test with pembrolizumab. The selected two models were composed four groups; control, pembrolizumab, MERTK inhibitor, and combination. We applied pembrolizumab as aPD-1 blockade (10 mpk, q2w, i.p. inj.). The dose of MERTK inhibitor was 50 mpk, q1d by orally. The treatment was started when tumor size reached 100 mm3. Whole exome sequencing (WES), microarray (mRNA), flow cytometry, and multispectral image analysis were performed on the tumors. To elucidate the involvement of MERTK inhibitor in tumor, the tumor mutation burden, tumor microenvironment and immune cell phenotype were deconvoluted. Results: The TMB of YHIM2004 and YHIM2009 was 3.23 and 2.64 (mutations/Mb) respectively. The PD-L1 and TIL level of YHIM2004 were relatively higher than YHIM2009. Both two models were observed high macrophage in tumor. The two PDX-models, YHIM2004 (aPD-1 sensitive), YHIM2009 (aPD-1 non-sensitive) were observed anti-tumor effects in MERTK inhibitor single treatment groups (TGI = 44.23%, 67.40% respectively, p < 0.05, t-test). Especially, YHIM2004 tumor was reduced after combination therapy more than single treatment. Of note, the NOG-PDX model demonstrated no anti-tumor effect in MERTK inhibitor monotherapy, suggesting that this drug involves anti-cancer immune responses. Flow cytometry data showed increased PD-1+ and granzyme B+ T cells (p Conclusion: We concluded that MERTK signaling involves regulation of anti-cancer immune responses. Our results suggest an anti-cancer therapeutics of MERTK selective inhibitor on aPD-1 sensitive or non-sensitive tumors under high TAM tumor microenvironment. Citation Format: Kyoung-Ho Pyo, Ha-Ni Cho, Chun-Feng Xin, Jae Seok Cho, Han Na Kang, Jiyeon Yun, Hye Ryun Kim, Byoung Chul Cho. Efficacy of MERTK inhibitor in combination with pembrolizumab in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4081.

Introduction: PI3K signaling plays a pivotal role in balancing of immune responses. This study is to elucidate the role of PI3K-AKT signaling in regulating T cell metabolic fitness. Methods: PI3K/AKT inhibitors including BKM120 were used to evaluate the contribution of PI3K-AKT signaling on the metabolic fitness of T cell. Glucose metabolism and T cell activity were measured by flow cytometry, IHC, RT-PCR. Oxygen consumption rate (OCR) and extra cellular acidification rate (ECAR) were examined by seahorse. In addition, anti-tumor effects of T cells were evaluated by adaptive transfer in LLC2 tumor bearing mouse. Results: Compare to vehicle, BKM120-treated antigen-specific CD8+ T cells co-cultured with LLC2 cancer cells maintained cytotoxic effects under hypoglycemic and hypoxic conditions. Treatment of BKM120 significantly reduced tumor growth, compared to vehicle in syngeneic models (LLC2, KLN205), whereas it did not affect tumor growth in nude mice, which suggests role of activation of antitumor immunity, augmented by BKM120. Compared to vehicle, BKM120 significantly induced higher infiltration of CD8+ T cells and reduction of Foxp3+ regulatory T cell and CD11b+/Gr-1+ myeloid derived suppressor cells. Intriguingly, increased glucose uptake through overexpression of glucose transporter (GLUT1) was observed upon BKM120 treatment. Glut1 overexpression occurred through the release of feedback inhibition of mTORC2 followed by rebound AKT activation. In addition, glycolysis-related genes (Glut1, Hk, Pgam, Pdk and Pgm) and OCR/ECAR were significantly augmented in BKM120/selective PI3K-delta inhibitor (Idelalisib)-treated group. The enhanced glycolysis via PI3K signaling inhibition was significantly more prominent by Idelalisib, compared to BKM120 or selective PI3K-alpha inhibitor (BYL-719). The antigen-specific T cells pretreated with BKM120 ex vivo exhibited substantially more potent antitumor effects, compared to untreated T cells, in LLC2 tumor-bearing nude mice. Conclusion: PI3K inhibitor, specifically selective PI3K-delta inhibitor, promotes AKT-dependent metabolic fitness of T cells which sustain anti-cancer effects under nutrient/oxygen-deprived tumor microenvironment. These data support a rationale for combination of selective PI3K-delta inhibitor with checkpoint inhibitors anti-PD1 for patient with Lung cancer. Citation Format: Sung Eum Kim, Kyoung-Ho Pyo, Sang Bin Lim, Mi Ran Yun, Jae Hwan Kim, Ji Min Lee, Ha Ni Jo, Chun-Feng Xin, Jae Seok Cho, Byoung Chul Cho. Enhanced AKT-dependent metabolic fitness and antitumor effect of T lymphocytes in lung cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1098.

Introduction: EML4-ALK is a distinct molecular entity that is highly sensitive to ALK tyrosine kinase inhibitors (TKIs). Although the role of oncogenic and native ALK in modulating the immune responses has been suggested, immune checkpoint inhibitors (ICIs) have not proved efficacy in ALK-positive non-small cell lung cancer (NSCLC) so far. In this study, we evaluated comprehensive immunomodulatory effect of ALK TKI and aimed to suggest optimal method of incorporating ICIs in ALK-positive NSCLC using an EML4-ALK transgenic mice model. Methods: Ceritinib or anti-PD-1 was treated in EML4-ALK transgenic mice, and tumor response was evaluated using magnetic resonance imaging. Progression-free survival (PFS) and overall-survival (OS) were measured to compare the efficacy in mice. To examine the dynamics of immune response, flow cytometry of tumor region, cytokine-ELISA of bronchoalveolar lavage (BAL) fluid were performed at baseline and at the time of disease progression. Sequencing of ALK kinase domain was performed to identify acquired ALK mutations. Results: Upfront ceritinib was clearly more efficacious than anti-PD-1 upfront, as shown by the median PFS was 139 days vs.13 days (P < 0.05). Use of ceritinib in the first line resulted in a prolonged OS compared to the use in the second line (OS 203 days vs. 135 days, P< 0.05). The efficacy of ceritinib and anti-PD-1 combination was not more effective than ceritinib alone in the first line. Of note, we identified 1 mouse which showed a complete response to anti-PD-1 after progression on 1st line ceritinib. Tumors which progressed on ceritinib showed increased proportion of CD8+ tumor infiltrating T cells among total CD3+ T cells, and increased expression of IFN-γ in BAL fluid, representing Th1-dominant immune phenotype. In tumors which acquired resistance to ceritinib, we identified known ALK kinase domain mutations such as G1202R with a concomitant increased tumor mutational burden compared to ceritinib-naïve tumors. Ceritinib-resistant tumors also had significantly increased memory cytotoxic T cells and PD-1-expressing helper T cells in total CD3+ population. On the contrary, we noted decreased expressions of central memory T cells, effector memory T cells, IFN-γ in tumors which progressed after ceritinib and anti-PD-1 combination therapy. Conclusion: Our study confirmed that combination of ALK inhibitor and ICI is not efficacious, as evidenced by low infiltrating T cells and memory T cells. We concluded that tumor progressing on ceritinib could be sensitive to anti-PD-1 due to increased neoantigens derived from acquired ALK mutations, increased tumor mutational burden and increased number of memory T cells and PD-1-expressing helper T cells. Citation Format: Kyoung-Ho Pyo, Sun Min Lim, Ha Ni Jo, Jae Seok Cho, Jae Hwan Kim, Ji Min Lee, Chun-Feng Xin, Sung Eun Kim, Chae Won Park, Wongeun Lee, Hye Ryun Kim, Byoung Chul Cho. Finding the optimal strategy of incorporating immune checkpoint inhibitor inALK-positive non-small-cell lung cancer usingEML4-ALKtransgenic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4076.

Introduction: EML4-ALK is a distinct molecular entity that is highly sensitive to ALK tyrosine kinase inhibitors (TKIs). While many trials have showed the superiority of ALK TKIs over cytotoxic chemotherapy, the optimal sequencing of ALK TKIs is still obscure. Acquired resistance to ALK TKIs remains a key challenge, and unknown mechanisms of resistance need to be resolved. In this study, we aimed to identify optimal treatment sequence and acquired resistance mechanisms of ALK TKIs using EML4-ALK transgenic mice model that recapitulates human EML4-ALK lung adenocarcinoma. Methods: The tumorigenesis of EML4-ALK transgenic mice was based on Cre-ERT2/Lox system, and intraperitoneal injection with tamoxifen induced lung tumors. When tumor nodules were observed, EML4-ALK transgenic mice were treated with either crizotinib (150mg/kg) followed by ceritinib (75mg/kg) upon progression (N=13) or with ceritinib followed by crizotinib (N=12) upon progression. Tumor response was evaluated weekly using magnetic resonance imaging. Progression-free survival (PFS) was measured to compare the efficacy between two ALK TKIs. For the analysis of acquired resistance mechanism, whole-exome sequencing, RNA sequencing, and targeted sequencing of ALK gene were performed. Results: Head-to-head comparison of crizotinib and ceritinib was performed in the first-line setting. A total of 13 mice were treated with upfront crizotinib, and mice that showed progressive disease (PD) were crossed over to ceritinib (n=9). A total of 12 mice were treated with upfront ceritinib, and mice that showed PD (n=8) were crossed over to crizotinib. Four mice in each group were sacrificed for analysis of resistance mechanism at the time of PD. The PFS of ceritinib was significantly longer than that of crizotinib [24 weeks (95% CI, 21.4-26.5) vs. 8 weeks (95% CI, 6.5-9.0), P < 0.001]. Subsequent treatment with ceritinib following crizotinib resulted in significantly longer PFS than crizotinib following ceritinib [7 weeks (95% CI, 4.1-9.9) vs 3 weeks (95% CI, 2.5-3.5), P < 0.001]. Altogether, treatment with ceritinib followed by crizotinib showed a prolongation of PFS, compared to crizotinib followed by ceritinib (27 weeks vs. 15 weeks, P < 0.001). We noted that ALK-TKI resistant tumors harbored several copy number variations (CNVs), and nonsynonymous oncogenic mutations, but found no previously reported resistant mechanisms including secondary mutations, or CNVs. Wnt signaling related gene set was increased in both crizotinib- and ceritinib-resistant tumors, and Hippo signaling related gene set was increased in ceritinib-resistant tumors on KEGG pathway-based analysis. Conclusion: Our findings suggest that ceritinib is superior to crizotinib when used in the first-line setting. Novel mechanisms of acquired resistance such as increased Wnt and Hippo signaling need further validation. Citation Format: Kyoung-Ho Pyo, Lim Sun Min, Jae Hwan Kim, Ji Min Lee, Ha Ni Jo, Jae Soek Cho, Mi-Ran Yun, Sung Eun Kim, Hye Ryun Kim, Chun-Feng Xin, Tae-Min Kim, Byoung Chul Cho. Identification of optimal treatment sequence and acquired resistance mechanisms of ALK inhibitors using EML4-ALK transgenic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2855.

The present study investigated the effect of peanut sprout extract (PSE) as a natural resveratrol supplement on chronic bacterial prostatitis (CBP) and estradiol-induced benign prostatic hyperplasia (BPH). PSE contained a high level of resveratrol (148.51 ± 3.05 μg/g), and was tested on the mouse models of CBP (induced by Escherichia coli 292 infection) and BPH (induced by treatment with β-estradiol and dihydrotestosterone). PSE toxicity was assessed on the basis of changes in body weight, alanine aminotransferase activity (an indicator of hepatotoxicity), and expression of the kidney injury marker KIM-1. The effects of PSE on the histopathology of prostate tissue, the proportion of neutrophils, and immune cell profiles in the blood and spleen were examined. PSE administration did not result in any toxicity but reduced the bacterial burden and histopathological changes in the prostate. In addition, lymphocytes (CD4+, CD8+, and CD19+) in the spleen were significantly increased after PSE administration in CBP mice, suggesting immune enhancement. PSE treatment of bone marrow–derived macrophages increased the expression of CD40, which is related to the pro-inflammatory function and host defense against pathogens. It is concluded that PSE would be a good supplement for the mitigation of prostate hyperplasia and prostatitis.