A Synergistic Interaction between Chk1- and MK2 Inhibitors in KRAS-Mutant Cancer.

KRAS is one of the most frequently mutated oncogenes in human cancer. Despite substantial efforts, no clinically applicable strategy has yet been developed to effectively treat KRAS-mutant tumors. Here, we perform a cell-line-based screen and identify strong synergistic interactions between cell-cycle checkpoint-abrogating Chk1- and MK2 inhibitors, specifically in KRAS- and BRAF-driven cells. Mechanistically, we show that KRAS-mutant cancer displays intrinsic genotoxic stress, leading to tonic Chk1- and MK2 activity. We demonstrate that simultaneous Chk1- and MK2 inhibition leads to mitotic catastrophe in KRAS-mutant cells. This actionable synergistic interaction is validated using xenograft models, as well as distinct Kras- or Braf-driven autochthonous murine cancer models. Lastly, we show that combined checkpoint inhibition induces apoptotic cell death in KRAS- or BRAF-mutant tumor cells directly isolated from patients. These results strongly recommend simultaneous Chk1- and MK2 inhibition as a therapeutic strategy for the treatment of KRAS- or BRAF-driven cancers.

The association between proinflammatory cytokine polymorphisms and cerebral palsy in very preterm infants.

Cerebral palsy (CP) is a nonprogressive motor disorder caused by white matter damage in the developing brain and is often accompanied with cognitive and sensory disabilities. The risk of CP is higher among infants born preterm than in more mature infants. Intrauterine infection/inflammation, activation of the cytokine network and elevated levels of proinflammatory cytokines in neonatal blood or in amniotic fluid to which the preterm infant is exposed, has been identified as the most common cause of preterm delivery, periventricular leukomalacia (PVL) and CP. The aim of our study was to evaluate the possible association of four TNFα promoter single nucleotide polymorphisms (SNPs) (-1031 T/C, -857 C/T, -308 G/A and -238 G/A), two IL1ß SNPs (-511 C/T and +3954 C/T) and one IL6 (-174 C/G) polymorphism with susceptibility to CP in very preterm infants. Statistically significant association between TNFα -1031 T/C high expression genotypes (TC and CC) (OR, 2.339; p=0.016) as well as between TNFα -1031 C high expression allele (OR, 2.065; p=0.013) and risk of CP was observed. In addition, statistically significant association was found between TNFα TC, CC, GG, GG -1031/-857/-308/-238 genotypes combination (OR, 3.286; p=0.034) and risk of CP. Statistically significant association between IL1ß TT, CC -511/+3954 genotypes combination and risk of CP (OR, 4.000; p=0.027) was also found. In CP patients with cystic PVL (cPVL) statistically significant association was found between TNFα -1031 T/C high expression genotypes (TC and CC) (OR, 2.361; p=0.038), IL1ß -511 C/T high expression genotype TT (OR, 3.215; p=0.030) as well as IL1ß -511 T high expression allele (OR, 1.956; p=0.019) and risk of CP. Statistically significant association was also found in patients with cPVL between TNFα TC, CC, GG, GG -1031/-857/-308/-238 genotypes combination (OR, 4.107; p=0.024), as well as IL1ß TT, CC -511/+3954 genotypes combination (OR, 7.333; p=0.005) and risk of CP. Our results suggest the role of TNFα and IL1ß polymorphisms which have previously been associated with higher circulating levels of these cytokines in genetic susceptibility to white matter damage and consequently CP in very preterm infants.

PD-L1 Dependent Immunogenic Landscape in Hot Lung Adenocarcinomas Identified by Transcriptome Analysis.

BACKGROUND: Lung cancer is the most frequent cause of cancer-related deaths worldwide. The clinical development of immune checkpoint blockade has dramatically changed the treatment paradigm for patients with lung cancer. Yet, an improved understanding of PD-1/PD-L1 checkpoint blockade-responsive biology is warranted. METHODS: We aimed to identify the landscape of immune cell infiltration in primary lung adenocarcinoma (LUAD) in the context of tumoral PD-L1 expression and the extent of immune infiltration ("hot" vs. "cold" phenotype). The study comprises LUAD cases (n = 138) with "hot" (≥150 lymphocytes/HPF) and "cold" (<150 lymphocytes/HPF) tumor immune phenotype and positive (>50%) and negative (<1%) tumor PD-L1 expression, respectively. Tumor samples were immunohistochemically analyzed for expression of PD-L1, CD4, and CD8, and further investigated by transcriptome analysis. RESULTS: Gene set enrichment analysis defined complement, IL-JAK-STAT signaling, KRAS signaling, inflammatory response, TNF-alpha signaling, interferon-gamma response, interferon-alpha response, and allograft rejection as significantly upregulated pathways in the PD-L1-positive hot subgroup. Additionally, we demonstrated that STAT1 is upregulated in the PD-L1-positive hot subgroup and KIT in the PD-L1-negative hot subgroup. CONCLUSION: The presented study illustrates novel aspects of PD-L1 regulation, with potential biological relevance, as well as relevance for immunotherapy response stratification.

Monitoring treatment effects in lung cancer-bearing mice: clinical CT and clinical MRI compared to micro-CT.

BACKGROUND: Compared to histology-based methods, imaging can reduce animal usage in preclinical studies. However, availability of dedicated scanners is limited. We evaluated clinical computed tomography (CT) and magnetic resonance imaging (MRI) in comparison to dedicated CT (micro-CT) for assessing therapy effects in lung cancer-bearing mice. METHODS: Animals received cisplatin (n = 10), sham (n = 12), or no treatment (n = 9). All were examined via micro-CT, CT, and MRI before and after treatment. Semiautomated tumour burden (TB) calculation was performed. The Bland-Altman, receiver operating characteristic (ROC), and Spearman statistics were used. RESULTS: All modalities always allowed localising and measuring TB. At all modalities, mice treated with cisplatin showed a TB reduction (p ≤ 0.012) while sham-treated and untreated individuals presented tumour growth (p < 0.001). Mean relative difference (limits of agreement) between TB on micro-CT and clinical scanners was 24.7% (21.7-27.7%) for CT and 2.9% (-4.0-9.8%) for MRI. Relative TB changes before/after treatment were not different between micro-CT and CT (p = 0.074) or MRI (p = 0.241). Mice with cisplatin treatment were discriminated from those with sham or no treatment at all modalities (p ≤ 0.001). Using micro-CT as reference standard, ROC areas under the curves were 0.988-1.000 for CT and 0.946-0.957 for MRI. TB changes were highly correlated across modalities (r ≥ 0.900, p < 0.001). CONCLUSIONS: Clinical CT and MRI are suitable for treatment response evaluation in lung cancer-bearing mice. When dedicated scanners are unavailable, they should be preferred to improve animal welfare.

Immune Cell Infiltration of the Primary Tumor, Not PD-L1 Status, Is Associated With Improved Response to Checkpoint Inhibition in Metastatic Melanoma.

Immune checkpoint inhibition has resulted in dramatic improvements in overall and relapse-free survival in patients with metastatic melanoma. The most commonly used immune checkpoint inhibitors are monoclonal antibodies targeting programmed cell death protein 1 and cytotoxic T-lymphocyte-associated protein 4. Unfortunately, a significant subset of patients fail to respond to these therapies, which has resulted in intense research efforts to identify the factors which are associated with treatment response. To this end, we investigated immune cell infiltration in primary melanomas and melanoma metastases, in addition to tumor cell PD-L1 expression, to determine whether these factors are associated with an improved outcome after immune checkpoint inhibition. Indeed, the extent of the immune cell infiltration in the primary melanoma, measured by the Immunoscore, was associated with a significantly improved response to immune checkpoint inhibition in terms of increased overall survival. However, the Immunoscore did not predict which patients would respond to treatment. The Immunoscore was significantly reduced in metastases when compared to primary melanomas. In contrast, PD-L1 expression, exhaustively tested using four commercially available anti-PD-L1 clones, did not differ significantly between primary tumors and melanoma metastases and was not associated treatment response. Whilst replication in larger, prospective studies is required, our data demonstrates the relevance of immune cell infiltration in the primary melanoma as a novel marker of improved overall survival in response to immune checkpoint inhibition.

Ethnic differences in the serotonin transporter polymorphism (5-HTTLPR) in several European populations.

The serotonin transporter (5-HTT) is a protein that has a major role in divergent psychiatric disorders, personality traits and behaviors, by regulating serotonergic synaptic function. Transcriptional activity of the 5-HTT gene (5-HTT or SLC6A4) is modulated by a polymorphic repetitive element (5-HTT gene-linked polymorphic region, 5-HTTLPR), which consists of a 44-base pairs insertion-deletion in the promoter region, creating a short (S) allele and a long (L) allele. Ethnic differences in the allele frequencies of the 5-HTTLPR exist between Caucasian and Asian populations. This study investigated ethnic differences in 5-HTTLPR in 1804 healthy Caucasian subjects from several European populations living in Croatia and the Russian Federation. The genotype and allele frequency of the 5-HTTLPR differed significantly (P<0.001) between male and female Croats, Russians, Tatars and Bashkirs, due to the lower frequency of the S allele (38% and 37%) and S/S genotype (14% and 15%) in Croat men and women compared to other studied groups. When male and female data were collapsed, Russians had marginally different allele and genotype distribution compared to Bashkirs and Tatars. Bashkirs and Tatars had similar allele and genotype frequency. The higher frequency of the S/S genotype was found in Tatars and Bashkirs compared to Croats and Russians. Gender related differences occurred only in the allele distribution within Bashkir population. These ethnic differences might be responsible for the inconsistent findings in the studies of the association between various psychiatric disorders, personality traits, behaviors and 5-HTTLPR across different ethnicities, and should be controlled to enable the generalization of results across various population groups.

AATF suppresses apoptosis, promotes proliferation and is critical for Kras-driven lung cancer.

A fundamental principle in malignant tranformation is the ability of cancer cells to escape the naturally occurring cell-intrinsic responses to DNA damage. Tumors progress despite the accumulation of DNA lesions. However, the underlying mechanisms of this tolerance to genotoxic stress are still poorly characterized. Here, we show that replication stress occurs in Kras-driven murine lung adenocarcinomas, as well as in proliferating murine embryonic and adult tissues. We identify the transcriptional regulator AATF/CHE-1 as a key molecule to sustain proliferative tissues and tumor progression in parts by inhibiting p53-driven apoptosis in vivo. In an autochthonous Kras-driven lung adenocarcinoma model, deletion of Aatf delayed lung cancer formation predominantly in a p53-dependent manner. Moreover, targeting Aatf in existing tumors through a dual recombinase strategy caused a halt in tumor progression. Taken together, these data suggest that AATF may serve as a drug target to treat KRAS-driven malignancies.

Loss of heterozygosity testing using real-time PCR analysis of single nucleotide polymorphisms.

PURPOSE: Colon cancer is a genetic disease, caused by mutations in different oncogenes and tumor-suppressor genes. The aim of this study is to evaluate the usefulness of real-time PCR SNP analysis as a new technique in the loss of heterozygosity (LOH) analysis at the E-cadherin gene locus in sporadic colon cancer. METHODS: One-hundred cases of human sporadic colon cancer and corresponding normal tissue samples were analyzed using two flanking polymorphic markers commonly used in the LOH analysis at the E-cadherin gene locus by conventional VNTR-LOH analysis. Two intragenic E-cadherin SNP markers were analyzed using real-time PCR SNP analysis. RESULTS: LOH (17.6%) was detected using flanking markers, however, no LOH was detected when the intragenic E-cadherin SNP markers were introduced into our study. Since these markers are intragenic they more accurately represent the status of the E-cadherin gene than the previously used flanking markers. CONCLUSION: In conclusion, real-time PCR SNP analysis was found to be more accurate, faster, simpler, and a more high-throughput method than the conventional VNTR-LOH analysis.

Ercc1 Deficiency Promotes Tumorigenesis and Increases Cisplatin Sensitivity in a Tp53 Context-Specific Manner.

KRAS-mutant lung adenocarcinoma is among the most common cancer entities and, in advanced stages, typically displays poor prognosis due to acquired resistance against chemotherapy, which is still largely based on cisplatin-containing combination regimens. Mechanisms of cisplatin resistance have been extensively investigated, and ERCC1 has emerged as a key player due to its central role in the repair of cisplatin-induced DNA lesions. However, clinical data have not unequivocally confirmed ERCC1 status as a predictor of the response to cisplatin treatment. Therefore, we employed an autochthonous mouse model of Kras-driven lung adenocarcinoma resembling human lung adenocarcinoma to investigate the role of Ercc1 in the response to cisplatin treatment. Our data show that Ercc1 deficiency in Tp53-deficient murine lung adenocarcinoma induces a more aggressive tumor phenotype that displays enhanced sensitivity to cisplatin treatment. Furthermore, tumors that relapsed after cisplatin treatment in our model develop a robust etoposide sensitivity that is independent of the Ercc1 status and depends solely on previous cisplatin exposure. Our results provide a solid rationale for further investigation of the possibility of preselection of lung adenocarcinoma patients according to the functional ERCC1- and mutational TP53 status, where functionally ERCC1-incompetent patients might benefit from sequential cisplatin and etoposide chemotherapy. IMPLICATIONS: This study provides a solid rationale for the stratification of lung adenocarcinoma patients according to the functional ERCC1- and mutational TP53 status, where functionally ERCC1-incompetent patients could benefit from sequential cisplatin and etoposide chemotherapy. Mol Cancer Res; 14(11); 1110-23. ©2016 AACR.

Traditionally produced sauerkraut as source of autochthonous functional starter cultures.

Spontaneous sauerkraut fermentation was performed at industrial scale in "Prehrana Inc.", Varazdin, in order to select autochthonous lactic acid bacteria (LAB) which were evaluated according probiotic criteria and tested for their capacity as probiotic starter cultures. At the end of the spontaneous sauerkraut fermentation, total LAB counts reached 9.0×10(5) CFU/ml. This underlines that the need for addition of the well characterised probiotic cultures, in appropriate viable cell counts, would be valuable in probiotic sauerkraut production. Phenotypic characterisation through API 50 CHL and SDS-PAGE of cell protein patterns revealed that Lactobacillus plantarum is predominant LAB strain in homofermentative phase of fermentation. Autochthonous LAB isolates SF1, SF2, SF4, SF9 and SF15 were selected based on the survival in in vitro gastrointestinal tract conditions. RAPD fingerprints indicated that the selected autochthonous LAB were distinct from one another. All of the strains efficiently inhibited the growth of indicator strains and satisfied technological properties such as acidification rate, tolerance to NaCl and viability during freeze-drying. Strains Lb. paraplantarum SF9 and Lb. brevis SF15, identified by AFLP DNA fingerprints, have shown the best properties to be applied as probiotic starter cultures, because of their highest adhesion to Caco-2 cells and expression of specific, protective S-layer proteins of 45 kDa in size. With addition of these strains, probiotic attribute of the sauerkraut will be achieved, including health promoting, nutritional, technological and economic advantages in large scale industrial sauerkraut production.

A functional cancer genomics screen identifies a druggable synthetic lethal interaction between MSH3 and PRKDC.

Here, we use a large-scale cell line-based approach to identify cancer cell-specific mutations that are associated with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) dependence. For this purpose, we profiled the mutational landscape across 1,319 cancer-associated genes of 67 distinct cell lines and identified numerous genes involved in homologous recombination-mediated DNA repair, including BRCA1, BRCA2, ATM, PAXIP, and RAD50, as being associated with non-oncogene addiction to DNA-PKcs. Mutations in the mismatch repair gene MSH3, which have been reported to occur recurrently in numerous human cancer entities, emerged as the most significant predictors of DNA-PKcs addiction. Concordantly, DNA-PKcs inhibition robustly induced apoptosis in MSH3-mutant cell lines in vitro and displayed remarkable single-agent efficacy against MSH3-mutant tumors in vivo. Thus, we here identify a therapeutically actionable synthetic lethal interaction between MSH3 and the non-homologous end joining kinase DNA-PKcs. Our observations recommend DNA-PKcs inhibition as a therapeutic concept for the treatment of human cancers displaying homologous recombination defects.

Posttranscriptional regulation of gene expression-adding another layer of complexity to the DNA damage response.

In response to DNA damage, cells activate a complex, kinase-based signaling network to arrest the cell cycle and allow time for DNA repair, or, if the extend of damage is beyond repair capacity, induce apoptosis. This signaling network, which is collectively referred to as the DNA damage response (DDR), is primarily thought to consist of two components-a rapid phosphorylation-driven signaling cascade that results in immediate inhibition of Cdk/cyclin complexes and a delayed transcriptional response that promotes a prolonged cell cycle arrest through the induction of Cdk inhibitors, such as p21. In recent years a third layer of complexity has emerged that involves potent posttranscriptional regulatory mechanisms that control the cellular response to DNA damage. Although much has been written on the relevance of the DDR in cancer and on the post-transcriptional role of microRNAs (miRs) in cancer, the post-transcriptional regulation of the DDR by non-coding RNAs and RNA-binding proteins (RBPs) still remains elusive in large parts. Here, we review the recent developments in this exciting new area of research in the cellular response to genotoxic stress. We put specific emphasis on the role of RBPs and the control of their function through DNA damage-activated protein kinases.

Association of MTHFR, MTR, MTRR, RFC1, and DHFR gene polymorphisms with susceptibility to sporadic colon cancer.

Altered folate levels may play an important role in colon carcinogenesis. The aim of this study was to investigate the association of polymorphisms in key folate-metabolizing genes with susceptibility to sporadic colon cancer. Six common polymorphisms (two in MTHFR and one each in MTR, MTRR, RFC1, and DHFR genes) were genotyped in 300 healthy subjects and 300 colon cancer patients from Croatia. Obtained results indicate possible protective role of MTRR 66 AA in sporadic colon cancer (OR=0.655; 95% CI=0.441-0.973; p=0.04). Maximum-likelihood analysis of haplotypes revealed a linkage disequilibrium (LD) between the two investigated polymorphisms of the MTHFR gene (C677T and A1298C), both in the control and patient groups (p<0.01 for both). LD was also detected between MTRR A66G and MTHFR A1298C polymorphisms but only in a group of patients (p<0.01). A haplotype of A66G and A1298C polymorphisms, A/A, proved to be protective (OR=0.775; 95% CI=0.603-0.996; p=0.04), whereas haplotype A/G was a risk factor for colon cancer (OR=1.270; 95% CI=1.007-1.602; p=0.04). Contrary to some previous studies, single-locus analyses identified no polymorphisms associated with risk for colon cancer, but demonstrated a possible protective effect of MTRR 66 AA genotype. The detected significant LD between two loci (MTHFR A1298C and MTRR A66G) located on different chromosomes indicates a strong selective force as a mechanism for the maintenance of their linkage. Specific combinations of alleles of these two polymorphisms showed a protective but also a risk effect on colon cancer susceptibility.

Interleukin-6-174 G/C polymorphism is not associated with IL-6 expression and susceptibility to sporadic colon cancer.

Interleukin-6 (IL-6) has been implicated in tumorigenesis; however, its role is still far from being clearly defined. Regulation of IL-6 expression is highly complex, and additional complexity is introduced by single-nucleotide polymorphisms in the IL-6 gene. These single-nucleotide polymorphisms might influence mRNA transcription, which might in turn result in increased susceptibility to certain tumors. The aim of this study was to analyze IL-6 mRNA and protein expressions in sporadic colon cancer. Influence of IL-6-174 G/C polymorphism on IL-6 mRNA expression and sporadic colon cancer susceptibility was evaluated as well. The frequency of IL-6-174 G/C was analyzed by polymerase chain reaction-restriction fragment length polymorphism analysis. IL-6 mRNA and protein expressions were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. No statistically significant difference in IL-6 mRNA expression in tumor tissue compared with the corresponding normal tissue was observed (p = 0.116). No correlation was found between IL-6 mRNA and protein expressions and clinicopathological features of sporadic colon tumors. There was no association of IL-6-174 G/C genotypes with IL-6 mRNA expression in colon tumors and corresponding normal mucous tissue (p = 0.355; p = 0.152). Finally, there was no association of IL-6-174 G/C with susceptibility to sporadic colon cancer.

IL-2 -330 T/G SNP and serum values-potential new tumor markers in neuroendocrine tumors of the gastrointestinal tract and pancreas (GEP-NETs).

Cytokines participate in tumorigenesis of gastroenteropancreatic neuroendocrine tumors (GEP-NETs). Single nucleotide polymorphisms (SNPs) in cytokine genes influence expression of proteins and are evaluated in cancer susceptibility. The aim of this study was to evaluate IL-2 -330 T/G SNP and susceptibility to GEP-NETs, and analyze the correlation between G-allele and IL-2 serum values in GEP-NET patients. Moreover we assessed the value of IL-2 as a tumor serum marker. IL-2 -330 T/G SNP was examined in 101 patients and 150 healthy volunteers and IL-2 serum levels in patients and 20 controls. Patients' IL-2 serum levels were compared to IL-2 -330 T/G genotypes and tumor functional status and finally with known markers such as chromogranin A (CgA) and 5-hydroxyindolacetic acid (5-HIAA). There was a significant difference in genotype distribution of the IL-2 -330 polymorphisms between GEP-NET and control group (p = 0.0006) as well as in the frequency of G-allele (p = 0.010). G-allele correlated with higher IL-2 serum levels (p = 0.028) and elevated in all patients, being highest in patients with functional tumors (p = 0.039). Compared to CgA and 5-HIAA, IL-2 was more specific in detecting GEP-NET patients (p < 0.0001 and p < 0.0001, respectively). Our results indicate importance of IL-2 in GEP-NET development and biochemical diagnosis.

Association of PTPN22 C1858T and CTLA-4 A49G polymorphisms with Type 1 Diabetes in Croatians.

In this case-control study the association between the PTPN22 1858T and CTLA-4 49G gene variants and T1D in Croatian population was examined. We found that distribution of PTPN22 C1858T and CTLA-4 A49G genotypes between T1D patient (n=102) and control (n=193) groups differ significantly (p<0.0001 and p=0.012, respectively). Moreover, although the risk alleles of both SNPs are distributed more frequently in patients, the significant difference is observed only for PTPN22 1858T allele (p<0.0001). This is therefore the first evidence that analyzed gene variants contribute to T1D pathogenesis in Croatian population.

IL-6-174 C/G polymorphism in the gastroenteropancreatic neuroendocrine tumors (GEP-NETs).

IL-6 is a pleiotropic cytokine with still controversial role in tumorigenesis of different cancer types. Its promoter SNP-174 C/G is associated with increased IL-6 transcription and in some tumor types with elevated IL-6 serum levels. The role of IL-6 polymorphisms and IL-6 serum values and their correlation in the gastroenteropancreatic neuroendocrine tumors is lacking. We investigated for the first time frequencies of IL-6-174 genotypes in 80 GEP-NET patients and 162 age- and sex-matched healthy controls, serum values of IL-6 in GEP-NET patients and their correlation with IL-6-174 genotypes. To analyze IL6-174 C/G polymorphism PCR-NlaIII RFLP method was used, and serum levels were measured on Immulite analyzer by enzymatic solid-phase chemiluminescent immunometric method. Serum IL-6 values were elevated (>5.9 pg/ml) in 36.8% GEP-NET patients. Differences in genotypes distribution between patients and healthy controls as well as between patients with gastrointestinal and pancreatic neuroendocrine tumors (PETs) and functioning and nonfunctioning PETs were tested by chi(2) test and Fisher's Exact test. Analysis of variance (ANOVA with proc GLM in SAS/Stat) was performed for the group comparison. Level of significance was alpha=0.05. Patients with nonfunctioning PETs had only high expression IL-6-174 CG and GG genotypes and according to genotypes differed significantly (p=0.0289) from functioning PETs. High serum IL-6 values in all GEP-NET patients correlated significantly with GG IL-6-174 genotype (p=0.0139). Nonfunctioning PET patients had significantly (p=0.000777) higher IL-6 serum values in comparison to patients with functioning PETs and gastrointestinal NETs. Serum IL-6 values correlated significantly with IL-6-174 genotypes in nonfunctioning PETs and gastrointestinal NETs (p<0.05), but not in functioning PETs.