LKB1 Research Articles

Abstract 793: Association of serine/threonine kinase 11 mutations and response to programmed cell death 1 inhibitors in metastatic gastric cancer

Abstract Background: Programmed cell death 1 (PD-1) inhibitors have shown therapeutic efficacy in metastatic gastric cancer (mGC). However, only a small portion the patients derive clinical benefits from PD-1 inhibitors, and no predictive biomarkers have been established in mGC. Inactivating mutations in serine/threonine kinase 11 (STK11), also known as liver kinase B1, are associated with immune suppression in the tumor microenvironment and poor response to PD-1 inhibitors in KRAS-mutant lung adenocarcinoma. Therefore, we hypothesized that STK11 inactivating mutations would be associated with inferior clinical response to PD-1 inhibitors in mGC. Methods: We analyzed 59 patients with mGC who had been treated with PD-1 inhibitors and whose tumors had been analyzed by targeted high-throughput sequencing. Clinicopathological characteristics, including Epstein-Barr virus positivity (EBV+), programmed death-ligand 1 (PD-L1) positivity, high microsatellite instability (MSI-H), response to PD-1 inhibitors, and survival were retrospectively evaluated. Result: STK11 mutations were identified in 30 (50.8%) patients, and were all missense mutations. Three patients (5.1%) had simultaneous STK11 gene amplification and mutation. STK11 mutations were not significantly associated with MSI-H, the number of tumor mutations, PD-L1+, EBV+, or the response to PD-1 inhibitors. Patients with STK11 mutations had prolonged overall survival (median: 19.0 vs 11.6 months, p=0.15), and progression-free survival (4.2 vs 1.9 months, p=0.06) when treated with PD-1 inhibitors, but these differences were not statistically significant. In addition, the distribution of STK11 mutations was not associated with the response to PD-1 inhibitor treatment. Patients with STK11 inactivating mutations without STK11 gene amplification had significantly prolonged progression-free survival compared to patients with intact wild type STK11 or STK11 gene amplification (4.8 vs 1.0 months, p=0.04). However, in multivariate Cox regression analysis with MSI-H, PD-L1+, EBV+, the number of mutations, and type of PD-1 inhibitor used (pembrolizumab vs nivolumab), only MSI-H and PD-L1+ were significantly associated with longer progression-free survival. Conclusions: In mGC, the presence of STK11 mutation was not predictive of the response to PD-1 inhibitors. Instead, patients with MSI-H or PD-L1+ tumors displayed superior clinical responses to PD-1 inhibitors. Citation Format: Minsuk Kwon, Kyung Kim, Suhye Choi, Jung Yong Hong, Seung Tae Kim, Se Hoon Park, Joon Oh Park, Young Suk Park, Ho Yeong Lim, Kyoung-Mee Kim, Jeeyun Lee. Association of serine/threonine kinase 11 mutations and response to programmed cell death 1 inhibitors in metastatic gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 793.

Abstract 2487: Mutation of the STK11 gene predicts recurrence of breast cancer

Abstract Introduction: Breast cancer is the most common cancer in women, and some patients develop recurrence after standard therapy. Effective predictors are urgently needed to detect recurrence earlier. Methods: We conducted a comprehensive study via an experimental and bioinformatics approach to detect mutated genes in breast cancer. Twenty-seven breast cancer patients who developed recurrence within 24 months postoperatively and 22 control cancer patients without recurrence were enrolled from National Cheng Kung University Hospital in Taiwan. Targeted deep sequencing was performed to assess the mutations among individuals with breast cancer using a panel of 143 cancer-associated genes. Bioinformatics and public databases were used to predict the protein functions of the mutated genes. Results: Mutations were identified in 49 breast cancer specimens, and the most frequently mutated genes were BRCA2, TP53, APC, ATM, BRCA1, NOTCH1, TET2, NF1, TSC2, PIK3CA, TSC1, PTEN, MSH2, PTCH1, PIK3R1, STK11, RB1, BAP1, CDH1 and FBXW7. Mutation of these genes was correlated with protein phosphorylation and autophosphorylation. Among these highly mutated genes, mutations of STK11 were associated with poor prognosis and increased recurrence of breast cancer. Knockdown of STK11 in triple negative breast cancer cell lines increases transcription of cytokines and modulates immune response. Conclusion: Our findings suggest that mutation of STK11 is correlated with early recurrence of breast cancer patients and it will become a powerful prognostic marker for recurrence of breast cancer. Suppression of STK11 signaling by gene mutation may contribute to immune escape. Citation Format: Chih-Yang Wang, Yung-Chieh Chang, Yao-Lung Kuo, Kuo-Ting Lee, Pai-Sheng Chen, Chun Hei Antonio Cheung, Che-Hung Shen, Chih-Peng Chang, Ming-Derg Lai, Meng-Ru Shen, Hui-Ping Hsu. Mutation of the STK11 gene predicts recurrence of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2487.

Abstract PR03: YAP/TAZ requirement in mesenchyme-originated intestinal hamartomatous polyposis

Abstract Hamartomatous polyposis syndromes (HPS) are rare genetic syndromes, including Peutz-Jeghers syndrome (PJS) and PTEN hamartoma tumor syndrome (Cowden and Bannayan-Riley-Ruvalcaba syndromes), which are caused by germline mutations of STK11 (LKB1) and PTEN genes, respectively. HPS is characterized by the development of hamartomatous polyps in the GI tract; however, the cellular origins and molecular mechanism underlying hamartomatous polyposis remain poorly understood. Here we demonstrate that specific disruption or deletion of Stk11 or Pten in mouse GI mesenchyme, but not in the epithelium, generates hamartomatous polyps histologically resembling GI polyps observed in PJS and Cowden syndrome patients. Genetically, we show that mTOR activity is essential for Pten but not Stk11 deletion-driven hamartomatous polyposis. Furthermore, we show that AMPK1/2 deletion is not able to initiate PJS polyp formation. However, we find significant upregulation of mesenchymal YAP/TAZ activity in PJS polyps and demonstrate that YAP/TAZ in gut mesenchyme are required for Stk11 deletion-induced PJS hamartomatous polyposis. Our studies suggest a mesenchymal origin of GI polyps in HPS and reveal the distinct roles of YAP/TAZ and mTOR in gut mesenchyme during hamartomatous polyposis, which likely have important therapeutic implication for treating these HPSs. This abstract is also being presented as Poster A06. Citation Format: Jennifer L. Cotton, Kyvan Dang, Qi Li, Randy L. Johnson, Y. Tony Ip, Xu Wu, Junhao Mao. YAP/TAZ requirement in mesenchyme-originated intestinal hamartomatous polyposis [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr PR03.

Jejunal Polyps that Lead to Small Bowel Intussusception in Adults: A Rare Case Report

Small intestine polyps are very rare in adults. Incidental finding of those polyps; sending them for histopathology that comes out to be Peutz-Jeghers syndrome (PJS) is extremely rare. PJS is an inherited disease characterized with typical hamartomas polyps in intestine along with distinctive skin pattern and mucocutaneous pigmentation. Gene mutation is responsible for 93% of PJS cases, among which majority of cases occur due to mutation in STK11 gene. PJS patients have 15-fold higher risk of developing cancer, including intestinal, extra-intestinal, breast, lung, pancreatic and colorectal cancer. Although PJS incidence is very low but its early recognition is very important for lowering morbidity and mortality. In this study, we have reported a very rare case of PJS in adults that was operated for small bowel obstruction secondary to intussusception. These cases are very rare in literature.

Two cases of somatic STK11 mosaicism in Danish patients with Peutz–Jeghers syndrome

Peutz-Jeghers syndrome (PJS) is a hereditary polyposis syndrome characterized by hamartomatous Peutz-Jeghers polyps in the gastrointestinal tract, mucocutaneous pigmentations, and increased risk for intestinal and extraintestinal cancer. In more than two-third of patients it is possible to detect pathogenic variants in the serine/threonine kinase 11 (STK11) gene, but so far is knowledge about genetic causes in the remaining part of patients limited. Reports of STK11 mosaicism are rare but may be an explanation in some patients without initial findings of pathogenic variants in STK11. We report two Danish patients with STK11 mosaicism detected in blood when using Next-Generation Sequencing. This is only the sixth and seventh patient reported in the literature, and we compare phenotypes of the reported cases. The results indicate that STK11 mosaicism is more frequent than anticipated and highlight that mosaicism should be considered in patients with clinical suspicion of PJS or patients fulfilling the diagnostic criteria.

Serine/threonine kinase 11 (STK11) mutations and immunotherapy resistance in patients with non-small cell lung cancer.

e15055 Background: There is immune evasion and resistance to checkpoint inhibitors (CPI). Programmed death-ligand 1 (PD-L1) and Tumor mutational burden (TMB) might help us predict response, but we have not yet validated biomarkers that can predict resistance. Mutations (mut) in STK11 can induce epigenetic changes that confer proliferative advantages to cancer cells and preliminary reports have suggested that they can confer resistance to CPI. We investigated the role of STK11 and KRAS mut as markers of poor response to CPI in patients (pts) with non-small cell lung cancer (NSCLC). Methods: Clinical outcomes of 127 pts with stage IIIB-IV NSCLC who were tested for KRAS and STK11 mut and received CPI were evaluated for progression free survival (PFS) and overall survival (OS). For statistical analysis log-rank tests were used to compare OS and PFS, chi-squared tests were used to compare 1-year survivals and proportions among different variables, and Kaplan-Meier survival curves were used to report OS and PFS. Results: Of the 127 pts: 31 had STK11 mut, 14 had STK11+KRAS mut (SKM group), and 10 pts were in the SKMP group (STK11+KRAS mut+PD-L1 (-)). Median age was 65y (27-88y). Males were 54% of the total and 30 pts (24%) were Hispanic (H). STK11 mut patients had an inferior PFS and OS as shown in table. Pts in SKM and SKMP groups had worse outcomes; however, not all the P values were significant. The difference in OS and 1-year OS were very impressive and significant when compared between the SKMP group (4m and 30%) and the wild type group (15m and 73%). There were no significant differences in clinical outcomes for H vs. non-H White pts. Conclusions: The presence of STK11 mut were associated with shorter OS/PFS in NSCLC pts treated with CPIs, proving its utility as a negative predictive marker. This can be enhanced combining STK11 and KRAS mut and possibly adding PD-L1 (-). These findings are consistent with recent studies that have reported STK11 mut as a genomic driver of primary resistance. Due to the small sample size further studies will be needed to validate these findings. [Table: see text]

Modulating tumor immune microenvironment by the STK11/LKB1 signaling in breast cancer.

e15185 Background: Standard treatment for breast cancer patients includes surgery, chemotherapy, radiotherapy, target and endocrine therapy. Immune checkpoint inhibitors are newly developing medications. The theoretical basis of immunotherapy is the interaction between cancer cells and tumor-infiltrating immune cells. Cancer cells secrete cytokines and create a specific tumor immune microenvironment (TIME) to attract or modulate immune cells. Further, genetic mutations or copy-number variations in cancer cells contribute to immunosuppression. Liver kinase B1 (LKB1) protein ( STK11 gene) is the upstream of AMP activated Protein Kinase (AMPK)/mammalian Target of Rapamycin Complex 1 (mTORC1) signaling pathway. STK11/LKB1 is one of the possible pathways modulating TIME. Methods: Twenty-seven breast cancer patients who developed recurrence within postoperative 24 months and 22 control cancer patients without recurrence were enrolled from National Cheng Kung University Hospital in Taiwan. Targeted deep sequencing was performed to assess the mutations among individuals with breast cancer using a panel of 143 cancer-associated genes. Bioinformatics and public databases were used to predict the protein functions of the STK11 genes. Immunohistochemical staining of LKB1 protein was performed in clinical specimens. Immune-related molecules were analyzed by RNA sequencing and cytokine array after suppression of STK11. Results: Mutations of STK11 gene were detected in recurrent patients and associated with poor prognosis of patients. From immunohistochemical study, the patients with low LKB1 expression had a worse survival. We utilized CRISPER/Cas9 system to transfect sgRNA into three mouse cell lines, including M158, NF639 and PY8119. RNA sequencing was performed in parental and Stk11-sgRNA cells. Immune-related pathways were ranked in the top 10 networks. Increased mRNA expression of Csf3 (protein G-CSF), Cxcl5, and Ccl2 was detected. The results are confirmed by cytokine array. The expression of G-CSF (gene Csf3) and CXCL5 (gene Cxcl5) proteins was increased in Stk11-sgRNA cells. The results were similar with RNA sequencing. Conclusions: Our findings suggest that suppression of STK11/LKB1 is correlated with early recurrence of breast cancer patients and contributes to modulate TIME. The STK11/LKB1 and downstream AMPK/mTORC1 pathways may be potential targets for immunotherapy.

Molecular subtyping reveals stage-specific mutational landscape and prognostic predicting factors of stage IV NSCLC.

e21607 Background: The mutational profile of non-small cell lung cancer (NSCLC) has been widely studied. However, the correlation between mutational profile and the prognosis of various molecular subtypes of NSCLC has not been fully investigated. Methods: We established the mutational landscape from 244 stage I-IV NSCLC patients using a 605-gene next generation sequencing panel. Molecular subtyping was performance based on the mutational status of EGFR, TP53, KRAS and STK11 genes according to reported methods. Sequencing data were analyzed with R packages and statistics analysis was performed with Graphpad PRISM 5.0 software. P ≤ 0.05 was regarded as statistically significant. Results: Differential SNV/INDEL mutation frequency across stage I-IV was found in EGFR, TP53, ATM, CDKN2A and NF1 (P < 0.05), differential CNV frequency was found in EGFR, RB1, PIK3CA, TERT, KRAS, FGFR1 and ERBB2 (P < 0.05), and differential SV frequency was found in ROS1 (P < 0.05). TMB of stage III-IV patients was higher than those of stage I (P < 0.05). TMB was higher in patients with TP53 but without EGFR mutations (P < 0.05). The association between prognosis and mutational status was further investigated in stage IV NSCLC. Patients with EGFR SNV/INDEL mutations exhibited better PFS and OS time, while patients with KRAS and/or STK11 SNV/INDEL mutations, or without mutations in EGFR, TP53, KRAS and STK11 exhibited worse PFS and OS time. However, no significant difference was observed in PFS and OS between patients with or without TP53 SNV/INDEL, EGFR CNV, ERBB2 CNV or TERT CNV variations. Further analysis showed that male or age≥62 exhibited worse OS time (P < 0.05), while smoking history, palliative surgery and palliative radiotherapy did not affect the PFS and OS time of stage IV patients. Conclusions: We found differential frequency in SNV/INDEL, CNV and SV variations across stage I-IV NSCLC. EGFR SNV/INDEL, KRAS SNV/INDEL, STK11 SNV/INDEL mutation, gender and age were found to be prognostic predicting factors for stage IV NSCLC.

Association of a STK11/KEAP1-mutation gene expression signature in lung adenocarcinoma with immune desertion in squamous cell carcinomas and mediation by NFE2L2 deregulation.

3082 Background: KEAP1 and STK11 mutations are associated with resistance to immune checkpoint inhibition (ICI) in non-small cell lung cancer (NSCLC). Mechanisms are currently unknown. Methods: We examined mutation, methylation, copy number and gene expression data from the cancer genome atlas (TCGA) lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LSCC), head and neck squamous cell carcinoma (HNSCC) and cervical carcinoma (CESC) data sets as well as public single cell gene expression data from a HNSCC cohort. Pathway annotations were performed using gene set enrichment analysis. A previously published cohort of NSCLC patients treated with ICI was analyzed for the predictive value of NFE2L2 mutations on PFS. Results: Annotation of STK11 and KEAP1 mutant LUAD revealed identical gene set enrichment for mitochondrial metabolism and downregulation of the STING-pathway, immune checkpoints, and interferon signaling. A STK11/KEAP1-mutation derived gene expression signature was established in LUAD and found to be driven by NFE2L2-regulated genes. This gene expression signature was independently predictive of immune desertion in LSCC, CESC and HNSCC and associated with STING-pathway downregulation in single cell sequencing analyses in HNSCC. KEAP1 and STK11 mutations were less frequent in LSCC, CESC and HNSCC but NFE2L2 mutations were identified in 15, 6 and 5%, respectively. NFE2L2 mutant SCC exhibited upregulation of the 15-gene- signature as well as immune desertion. In NSCLC, NFE2L2 mutations were associated with significantly worse PFS with ICI. Conclusions: Alterations of KEAP1, STK11, NFE2L2 and other related genes are linked to NFE2L2 target gene upregulation and immune desertion in LUAD, CESC, LSCC and HNSCC alike. The NFE2L2 pathway should be investigated clinically as a putative negative predictive biomarker for ICI and a potential therapeutic target.

LKB1/STK11 Expression in Lung Adenocarcinoma and Associations With Patterns of Recurrence

Mutations in the serine/threonine kinase 11 (STK11)/liver kinase B1 (LKB1) have been implicated in mediating resistance to checkpoint blockade among patients with advanced lung adenocarcinoma. We sought to examine the associations between clinicopathologic characteristics, tumor LKB1 expression, features of the immune microenvironment, and postoperative prognosis among patients with early stage lung adenocarcinoma undergoing surgical therapy. Formalin-fixed, paraffin-embedded specimens of patients undergoing resection of stage I to III, chemotherapy-naïve adenocarcinomas (1997 to 2008) were analyzed using tissue microarray sectioning. Sublobar resections were excluded. Intratumoral LKB1/STK11 expression was quantified as H-score. In a subset, tumor-associated immune cell populations were quantified using whole tumor sections in peritumoral and intratumoral compartments. In all, 104 patients met inclusion criteria. Expression of LKB1/STK11 (median H-score 102.9) was higher in women (median 123.3) than in men (100, P= .004) and in never-smokers (median 145) than in former/current smokers (100, P= .002). Expression of LKB1/STK11 was positively correlated with intratumoral infiltration of cluster of differentiation (CD) 3+ (r= 0.351, P= .005), CD4+ (r= 0.436, P < .001), and CD8+ (r= 0.263, P= .049) cells. Patients with extrathoracic recurrence had lower tumor expression of LKB1/STK11 than did other patients with recurrent disease. On multivariate analysis, low LKB1/STK11 expression remained independently associated with poor disease-free survival and distant disease-free survival. Low LKB1/STK11 expression is associated with specific patient characteristics and poor postoperative prognosis in chemotherapy-naïve lung adenocarcinoma. Further investigation is warranted to delineate its clinical significance in the context of evaluating novel therapeutic agents in patients with resectable disease.

Clinical and Genetic Analyses of 38 Chinese Patients with Peutz-Jeghers Syndrome.

Background Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant inherited disease caused by a germline mutation in the STK11 gene. It is characterized by mucocutaneous pigmentation, gastrointestinal hamartomatous polyps, and cancer predisposition. Aims We aimed to summarize the main clinical and genetic features of Chinese PJS patients and assessed the genotype-phenotype correlations. Methods Thirty-eight patients clinically diagnosed with Peutz-Jeghers syndrome were included in this study from 2016 to 2019. Combined direct sequencing and multiplex ligation-dependent probe amplification tests were used to detect germline heterogeneous STK11 mutations. RNA sequencing was performed in polyps of PJS patients and control groups to evaluate the difference in expression of STK11. The genotype-phenotype correlations were calculated by Kaplan-Meier analyses. Results All 26 probands and 12 affected relatives had germline heterogeneous STK11 mutations among which 8 variants were novel. Individuals with missense mutations had their first surgery and other symptoms significantly later than individuals with null mutations. Conclusion This study expanded the spectrum of STK11 gene mutations and further elucidated individuals with null mutations of STK11 typically had an earlier onset of PJS symptoms and needed earlier management.

Genetic Profile of Young Chinese Patients with Lung Adenocarcinoma

背景与目的全球肺癌的发病率正呈逐年上升趋势，其中腺癌所占的百分比日益升高。据统计，全球的肺癌平均初诊年龄在70岁左右，虽然肺癌仍以老年患者居多，但发病年龄的年轻化趋势愈加明显。结合现有研究数据，我们已知在非小细胞肺癌中，年轻患者疾病的发生有其独特的生物学特点。但年轻肺腺癌患者的基因组学特性和临床特征仍有待确定。本研究采用高通量测序（next-generation sequencing technology, NGS）技术对中国年轻肺腺癌患者的基因突变状态进行了研究。方法共收集了89例年龄≤45岁的肺腺癌患者组织标本，所有患者均知情同意。使用NGS检测用于确定癌组织中驱动基因突变。此外，对同期行NGS检测的95例 > 45岁肺腺癌患者的基因组和临床病理特征进行回顾性分析。结果根据年龄分类对184例肺腺癌患者的驱动基因突变频率进行了分析，揭示了年龄≤45岁的年轻组患者的独特基因特征。其中间变淋巴瘤激酶（anaplastic lymphoma kinase, ALK）融合基因和人表皮生长因子受体-2（human epidermal growth factor receptor 2, HER2）基因的突变频率较高。而鼠类肉瘤病毒癌基因（kirsten rat sarcoma viral oncogene, KRAS）、丝氨酸/苏氨酸蛋白激酶11（serine/threonine kinase 11, STK11）和表皮生长因子受体（epidermal growth factor receptor, EGFR）20外显子突变的趋势则相反，这些突变在年龄 > 45岁的老年组中更为常见。此外，年轻组EGFR基因突变同时伴有肿瘤蛋白p53（tumor protein p53, TP53）基因突变较老年组更为普遍（81.6% vs 44.9%），这可能使其应用EGFR酪氨酸激酶抑制剂（EGFR-tyrosine kinase inhibitor, EGFR-TKI）后疗效较差。结论NGS分析显示年轻腺癌患者具有独特的基因突变特点。在年轻患者中发现EGFR/TP53共突变的频率较高，这些独特的基因组学特征对临床治疗有重要的指导意义。

Association of serine/threonine kinase 11 mutations and response to programmed cell death 1 inhibitors in metastatic gastric cancer

Programmed cell death 1 (PD-1) inhibitors have shown therapeutic efficacy in metastatic gastric cancer (mGC). However, no predictive biomarkers have been established in mGC. Inactivating mutations in serine/threonine kinase 11 (STK11) are associated with poor response to PD-1 inhibitors in KRAS-mutant lung adenocarcinoma. Therefore, we hypothesized that STK11 inactivating mutations would be associated with inferior clinical response to PD-1 inhibitors in mGC. We analyzed 59 mGC patients who had been treated with PD-1 inhibitors and whose tumors had been analyzed by targeted high-throughput sequencing. STK11 mutations were identified in 30 (50.8%) patients, and were all missense mutations. Three patients (5.1%) had STK11 gene amplification and mutation, simultaneously. Patients with STK11 mutations had prolonged overall survival (median: 19.0 vs 11.6 months, p = 0.15), and progression-free survival (4.2 vs 1.9 months, p = 0.06) when treated with PD-1 inhibitors, but these differences were not statistically significant. Patients with STK11 inactivating mutations without STK11 gene amplification had significantly prolonged progression-free survival compared to patients with wild type STK11 or STK11 gene amplification (4.8 vs 1.0 months, p = 0.04). However, in multivariate Cox regression analysis with high microsatellite instability (MSI-H), the number of tumor mutations, PD Ligand-1 (PD-L1)+, Epstein-Barr virus positivity (EBV)+, and type of PD-1 inhibitor used (pembrolizumab vs nivolumab), only MSI-H and PD-L1+ were significantly associated with longer progression-free survival. In mGC, the presence of STK11 mutation was not predictive of the response to PD-1 inhibitors. Instead, patients with MSI-H or PD-L1+ tumors displayed superior clinical responses to PD-1 inhibitors.

AMPK-Independent LKB1 Activity Is Required for Efficient Epithelial Ovarian Cancer Metastasis.

Epithelial ovarian cancer (EOC) spreads by direct dissemination of malignant cells and multicellular clusters, known as spheroids, into the peritoneum followed by implantation and growth on abdominal surfaces. Using a spheroid model system of EOC metastasis, we discovered that Liver kinase B1 (LKB1), encoded by the STK11 gene, and its canonical substrate AMP-activated protein kinase (AMPK) are activated in EOC spheroids, yet only LKB1 is required for cell survival. We have now generated STK11-knockout cell lines using normal human FT190 cells and three EOC cell lines, OVCAR8, HeyA8, and iOvCa147. STK11KO did not affect growth and viability in adherent culture, but it decreased anchorage-independent growth of EOC cells. EOC spheroids lacking LKB1 had markedly impaired growth and viability, whereas there was no difference in normal FT190 spheroids. To test whether LKB1 loss affects EOC metastasis, we performed intraperitoneal injections of OVCAR8-, HeyA8-, and iOvCa147-STK11KO cells, and respective controls. LKB1 loss exhibited a dramatic reduction on tumor burden and metastatic potential; in particular, OVCAR8-STK11KO tumors had evidence of extensive necrosis, apoptosis, and hypoxia. Interestingly, LKB1 loss did not affect AMPKα phosphorylation in EOC spheroids and tumor xenografts, indicating that LKB1 signaling to support EOC cell survival in spheroids and metastatic tumor growth occurs via other downstream mediators. We identified the dual-specificity phosphatase DUSP4 as a commonly upregulated protein due to LKB1 loss; indeed, DUSP4 knockdown in HeyA8-STK11KO cells partially restored spheroid formation and viability. IMPLICATIONS: LKB1 possesses key tumor-promoting activity independent of downstream AMPK signaling during EOC metastasis.

STING Pathway Expression Identifies NSCLC With an Immune-Responsive Phenotype.

Although the combination of anti-programmed cell death-1 or anti-programmed cell death ligand-1 (PD-L1) with platinum chemotherapy is a standard of care for NSCLC, clinical responses vary. Even though predictive biomarkers (which include PD-L1 expression, tumor mutational burden, and inflamed immune microenvironment) are validated for immunotherapy, their relevance to chemoimmunotherapy combinations is less clear. We have recently reported that activation of the stimulator of interferon genes (STING) innate immune pathway enhances immunotherapy response in SCLC. Here, we hypothesize that STING pathway activation may predict and underlie predictive correlates of antitumor immunity in NSCLC. We analyzed transcriptomic and proteomic profiles in two NSCLC cohorts from our institution (treatment-naive patients in the Profiling of Resistance Patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax study and relapsed patients in the Biomarker-Integrated Approaches of Targeted Therapy for Lung CancerElimination study) and The Cancer Genome Atlas (N= 1320). Tumors were stratified by STING activation on the basis of protein or mRNA expression of cyclic GMP-AMPsynthase, phospho-STING, and STING-mediated chemokines (chemokine ligand 5 [CCL5] and C-X-C motif chemokine 10 [CXCL10]). STING activation in patient tumors and in platinum-treated preclinical NSCLC models was correlated with biomarkers of immunotherapy response. STING activation is associated with higher levels ofintrinsic DNA damage, targetable immune checkpoints, and chemokines in treatment-naive and relapsed lung adenocarcinoma. We observed that tumors with lower STING and immune gene expression show higher frequency of serine-threonine kinase 11 (STK11) mutations; however, we identified a subset of these tumors that are TP53 comutated and display high immune- and STING-related gene expression. Treatment with cisplatin increases STING pathway activation and PD-L1 expression in multiple NSCLC preclinical models, including adeno- and squamous cell carcinoma. STING pathway activation in NSCLC predicts features of immunotherapy response and is enhanced by cisplatin treatment. This suggests a possible predictive biomarker and mechanism for improved response to chemoimmunotherapy combinations.

DNA-dependent protein kinase regulates lysosomal AMP-dependent protein kinase activation and autophagy

ABSTRACT Macroautophagy/autophagy is a central component of the cytoprotective cellular stress response. To enlighten stress-induced autophagy signaling, we screened a human kinome siRNA library for regulators of autophagic flux in MCF7 human breast carcinoma cells and identified the catalytic subunit of DNA-dependent protein kinase PRKDC/DNA-PKcs as a positive regulator of basal and DNA damage-induced autophagy. Analysis of autophagy-regulating signaling cascades placed PRKDC upstream of the AMP-dependent protein kinase (AMPK) complex and ULK1 kinase. In normal culture conditions, PRKDC interacted with the AMPK complex and phosphorylated its nucleotide-sensing γ1 subunit PRKAG1/AMPKγ1 at Ser192 and Thr284, both events being significantly reduced upon the activation of the AMPK complex. Alanine substitutions of PRKDC phosphorylation sites in PRKAG1 reduced AMPK complex activation without affecting its nucleotide sensing capacity. Instead, the disturbance of PRKDC-mediated phosphorylation of PRKAG1 inhibited the lysosomal localization of the AMPK complex and its starvation-induced association with STK11 (serine/threonine kinase 11). Taken together, our data suggest that PRKDC-mediated phosphorylation of PRKAG1 primes AMPK complex to the lysosomal activation by STK11 in cancer cells thereby linking DNA damage response to autophagy and cellular metabolism. Abbreviations: AXIN1: axin 1; 3-MA: 3-methyladenine; 5-FU: 5-fluorouracil; AA mutant: double alanine mutant (S192A, T284A) of PRKAG1; ACACA: acetyl-CoA carboxylase alpha; AICAR: 5-Aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; ATG: autophagy-related; ATM: ataxia telangiectasia mutated; ATR: ATM serine/threonine kinase; AV: autophagic vacuole; AVd: degradative autophagic vacuole; AVi: initial autophagic vacuole; BECN1: beclin 1; BSA: bovine serum albumin; CBS: cystathionine beta-synthase; CDK7: cyclin dependent kinase 7; CDKN1A: cyclin dependent kinase inhibitor 1A; EGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione S transferase; H2AX/H2AFX: H2A.X variant histone; HBSS: Hanks balanced salt solution; IP: immunopurification; IR: ionizing radiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K9: mitogen-activated protein kinase kinase kinase 9; mRFP: monomeric red fluorescent protein; mCh: mCherry; MCM7: minichromosome maintenance complex component 7; MTORC1: mechanistic target of rapamycin kinase complex 1; NHEJ: non-homologous end joining; NRBP2: nuclear receptor binding protein 2; NTC: non-targeting control; NUAK1: NUAK family kinase 1; PBS: phosphate-buffered saline; PIK3AP1: phosphoinositide-3-kinase adaptor protein 1; PIK3CA: phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha; PIKK: phosphatidylinositol 3-kinase-related kinase; PRKAA: protein kinase AMP-activated catalytic subunit alpha; PRKAB: protein kinase AMP-activated non-catalytic subunit beta; PRKAG: protein kinase AMP-activated non-catalytic subunit gamma; PRKDC: protein kinase, DNA-activated, catalytic subunit; RLuc: Renilla luciferase; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TP53: tumor protein p53; TSKS: testis specific serine kinase substrate; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type.

Radiographic Surveillance of Patients with Non-BRCA1/2 Pathogenic Variants.

The National Comprehensive Cancer Network (NCCN) developed clinical practice guidelines for germline pathogenic variants in highly penetrant genes, such asTP53andPTEN, and in moderately penetrant genes, such asCHEK2, ATM andPALB2. Whether the practice of radiographic surveillance of patients with pathogenic variants in genes other than BRCA1/2 complies with current NCCN guidelines remains unclear. Retrospective review of patients identified with pathogenic variants in genes other than BRCA1/2 from 2007 through 2017 to determine if radiographic surveillance was in accordance with NCCN guidelines for mammography and consideration of magnetic resonance imaging (MRI). Exclusions included variants of unknown significance, pathogenic variants not associated with an increased risk of breast cancer, and previous breast cancer diagnosis. After exclusions, 35 patients with pathogenic variants in ATM, CDH1, CHEK2, NBN, PALB2, PTEN, and STK11 genes were reviewed to assess whether radiographic surveillance was in accordance with NCCN guidelines. Guidelines for those with variants in ATM, CHEK2 and NBN includes annual mammography with tomosynthesis and consideration of breast MRI at age 40, variants in CDH1 and PALB2 at age 30, variants in PTEN at age 30-35 or 5-10 years before the earliest family breast cancer, and variants in STK11 at age 25. Of these 35 patients, 11 (31%) received mammography only; 11 (31%) received mammography and MRI, and 13 (37%) received no radiographic surveillance. Two of the 35 (6%) patients who received radiographic surveillance were diagnosed with ductal carcinoma in situ or invasive breast cancer. Thirty-one percent of patients with pathogenic variants in genes other than BRCA1/2 received both mammography and MRI. Thirty-seven percent of patients with these highly penetrant and moderately penetrant genes received no radiographic follow-up, clearly demonstrating an opportunity for improvement.

Dietary Lactic Acid Bacteria Modulate Yolk Components and Cholesterol Metabolism by Hmgr Pathway in Laying Hens

ABSTRACT This study aimed to investigate the effect of dietary lactic acid bacteria (LAB) on egg production, yolk components, cholesterol metabolism, and enterohepatic circulation of bile acids in hens. Four treatment diets included a control and LAB added at 3 × 105 (low), 3 × 107 (medium), or 3 × 109 (high) cfu/kg. The treatment LAB contained equal amounts of Lactobacillus acidophilus, Lactobacillus plantarum, and Enterococcus faecium. Results showed that high LAB increased (p<0.05) laying rate, egg mass, and yolk phospholipid, but decreased (p<0.05) yolk triglyceride and phosvitin. Diets with LAB decreased (p<0.05) yolk and serum cholesterol content, and serum bile acid by 9.3 to 39.9%. In liver, high LAB downregulated (p<0.05) mRNA expression of serine/threonine kinase 11 (STK11), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), AMP-activated protein kinase catalytic subunit (PRKAA1, 2), and protein phosphatase catalytic subunits (PPP2CA, PPP2CB and PPP3CA) by 49.5 to 175.4%. In mucosa, high LAB downregulated (p<0.05) PRKAA1 and HMGR by 68.2 and 69.6%, respectively; but upregulated (p<0.05) PPP2CA and PPP2CB by 51.2 and 45%, respectively. Linear decreasing (p≤0.035) responses to LAB doses were found on cholesterol, phosvitin, bile acid, and hepatic gene expressions, and quadratic (p≤0.006) effects on yolk cholesterol and hepatic STK11. It is concluded that probiotic LAB can improve yolk components and decrease hepatic cholesterol synthesis by regulating HMGR pathway in hens.

Role of the Serine/Threonine Kinase 11 (STK11) or Liver Kinase B1 (LKB1) Gene in Peutz-Jeghers Syndrome.

Peutz-Jeghers syndrome (PJS) is a well-described inherited syndrome, characterized by the development of gastrointestinal polyps and characteristic mucocutaneous freckling. PJS is an autosomal prevailing disease, due to genetic mutation on chromosome 19p, manifested by restricted mucocutaneous melanosis in association with gastrointestinal (GI) polyposis. The gene for PJS has recently been shown to be a serine/threonine kinase, known as LKB1 or STK11, which maps to chromosome subband 19p13.3. This gene has a putative coding region of 1302 bp, divided into nine exons, and acts as a tumor suppressor in the hamartomatous polyps of PJS patients and in the other neoplasms that develop in PJS patients. It is probable that these neoplasms develop from hamartomas, but it remains possible that the LKB1 or STK11 locus plays a role in a different genetic pathway of tumor growth in the cancers of PJS patients. This article focuses on the role of LKB1 or STK11 gene expression in PJS and related cancers.

Next generation sequencing identifies novel potential actionable mutations for grade I meningioma treatment.

Meningiomas are common brain tumors that arise from the meningeal membranes that envelope the brain and spinal cord. The World Health Organization classifies these tumors into three histopathological grades. Because of tumor recurrence, treating meningiomas may be challenging even in well-differentiated grade I (GI) neoplasms. Indeed, around 5% of completely resected GI meningiomas relapse within 5 years. Therefore, identifying driver mutations in GI meningiomas through next generation sequencing (NGS) assays is paramount. The aim of this study was to validate the use of the 50-gene AmpliSeq Hotspot Cancer Panel v2 to identify the mutational status of 23 GI meningioma, namely, 12 non recurrent and 11 recurrent. In 18 out of the 23 GI meningiomas analyzed, we identified at least one gene mutation (78.2%). The most frequently mutated genes were c-kit (39.1%), ATM (26.1%), TP53 (26.1%), EGFR (26.1%), STK11 (21.7%), NRAS (17.4%), SMAD4 (13%), FGFR3 (13%), and PTPN11 (13%); less frequent mutations were SMARCB1 (8.7%), FLT3 (8.7%), KRAS (8.7%), FBWX7 (8.7%), ABL1 (8.7%), ERBB2 (8.7%), IDH1 (8.7%), BRAF (8.7%), MET (8.7%), HRAS (4.3%), RB1 (4.3%), CTNNB1 (4.3%), PIK3CA (4.3%), VHL (4.3%), KDR (4.3%), APC (4.3%), NOTCH1 (4.3%), JAK3 (4.3%), and SRC (4.3%). To our knowledge, mutations in all of these genes, except for TP53, STK11, SMARCB1, PIK3CA, VHL, and BRAF, have never been described before in meningiomas. Hence, these findings demonstrate the viability of NGS to detect new genetic alterations in GI meningiomas. Equally important, this technology enabled us to detect possible novel actionable mutations not previously associated with GI and for which selective inhibitors already exist.