Non-canonical functions of cell cycle cyclins and cyclin-dependent kinases.

The roles of cyclins and their catalytic partners, the cyclin-dependent kinases (CDKs), as core components of the machinery that drives cell cycle progression are well established. Increasing evidence indicates that mammalian cyclins and CDKs also carry out important functions in other cellular processes, such as transcription, DNA damage repair, control of cell death, differentiation, the immune response and metabolism. Some of these non-canonical functions are performed by cyclins or CDKs, independently of their respective cell cycle partners, suggesting that there was a substantial divergence in the functions of these proteins during evolution.

Matched Analyses of Brain Metastases versus Primary Non-Small Cell Lung Cancer Reveal a Unique microRNA Signature.

Distant spreading of tumor cells to the central nervous system in non-small cell lung cancer (NSCLC) occurs frequently and poses major clinical issues due to limited treatment options. RNAs displaying differential expression in brain metastasis versus primary NSCLC may explain distant tumor growth and may potentially be used as therapeutic targets. In this study, we conducted systematic microRNA expression profiling from tissue biopsies of primary NSCLC and brain metastases from 25 patients. RNA analysis was performed using the nCounter Human v3 miRNA Expression Assay, NanoString technologies, followed by differential expression analysis and in silico target gene pathway analysis. We uncovered a panel of 11 microRNAs with differential expression and excellent diagnostic performance in brain metastasis versus primary NSCLC. Five microRNAs were upregulated in brain metastasis (miR-129-2-3p, miR-124-3p, miR-219a-2-3p, miR-219a-5p, and miR-9-5p) and six microRNAs were downregulated in brain metastasis (miR-142-3p, miR-150-5p, miR-199b-5p, miR-199a-3p, miR-199b-5p, and miR-199a-5p). The differentially expressed microRNAs were predicted to converge on distinct target gene networks originating from five to twelve core target genes. In conclusion, we uncovered a unique microRNA profile linked to two target gene networks. Our results highlight the potential of specific microRNAs as biomarkers for brain metastasis in NSCLC and indicate plausible mechanistic connections.

miR-100-5p confers resistance to ALK tyrosine kinase inhibitors Crizotinib and Lorlatinib in EML4-ALK positive NSCLC.

Lung cancer causes the highest number of cancer-related deaths worldwide. Resistance to therapy is a major clinical issue contributing to the poor prognosis of lung cancer. In recent years, targeted therapy has become a concept where subgroups of non-small cell lung cancer (NSCLC) with genetically altered receptor tyrosine kinases are targeted by tyrosine kinase inhibitors (TKIs). One such subgroup harbors a gene fusion of echinoderm microtubule-associated protein-like 4 (EML4) with anaplastic lymphoma kinase (ALK). Although most NSCLC patients with EML4-ALK fusions initially respond to ALK TKI-therapy they eventually develop resistance. While ALK kinase domain mutations contribute to ALK TKI-refractoriness, they are only present in a fraction of all ALK TKI-resistant tumors. In this study we sought to explore a possible involvement of microRNAs (miRNAs) in conferring resistance to ALK TKIs in ALK TKI-refractory NSCLC cell lines. We subjected our ALK TKI-refractory cancer cells along with parental cancer cells to systematic miRNA expression arrays. Furthermore, ALK TKI-refractory cancer cells were exposed to a synthetic miRNA inhibitory Locked Nucleic Acid (LNA)-library in the presence of ALK TKIs Crizotinib or Lorlatinib. The outcome of the combined approaches uncovered miR-100-5p to confer resistance to Crizotinib and Lorlatinib in EML4-ALK NSCLC cells and to be a potential therapeutic target in drug resistance.

miR-126-5p targets Malate Dehydrogenase 1 in non-small cell lung carcinomas.

Malate Dehydrogenase (MDH) 1 has recently been shown to be highly expressed and display prognostic value in non-small cell lung carcinomas (NSCLCs). However, it is not known how MDH1 expression is regulated and there is no current molecular or chemical strategy that specifically targets MDH1. This may be due to structural and enzymatic similarities with its isoenzyme, malate dehydrogenase 2 (MDH2). However, MDH1 and MDH2 are encoded by distinct genes and this opens up the possibility for modulation at the expression level. Here, we screened in silico for microRNAs (miRs) that selectively targets the 3'UTR region of MDH1. These analyses revealed that mir-126-5p has three binding sites in the 3'UTR region of MDH1. Additionally, we show that expression of miR-126-5p suppresses the enzymatic activity of MDH1, mitochondrial respiration and caused cell death in NSCLC cell lines.

The c-MYC oncoprotein, the NAMPT enzyme, the SIRT1-inhibitor DBC1, and the SIRT1 deacetylase form a positive feedback loop.

Silent information regulator 1 (SIRT1) represents an NAD(+)-dependent deacetylase that inhibits proapoptotic factors including p53. Here we determined whether SIRT1 is downstream of the prototypic c-MYC oncogene, which is activated in the majority of tumors. Elevated expression of c-MYC in human colorectal cancer correlated with increased SIRT1 protein levels. Activation of a conditional c-MYC allele induced increased levels of SIRT1 protein, NAD(+), and nicotinamide-phosphoribosyltransferase (NAMPT) mRNA in several cell types. This increase in SIRT1 required the induction of the NAMPT gene by c-MYC. NAMPT is the rate-limiting enzyme of the NAD(+) salvage pathway and enhances SIRT1 activity by increasing the amount of NAD(+). c-MYC also contributed to SIRT1 activation by sequestering the SIRT1 inhibitor deleted in breast cancer 1 (DBC1) from the SIRT1 protein. In primary human fibroblasts previously immortalized by introduction of c-MYC, down-regulation of SIRT1 induced senescence and apoptosis. In various cell lines inactivation of SIRT1 by RNA interference, chemical inhibitors, or ectopic DBC1 enhanced c-MYC-induced apoptosis. Furthermore, SIRT1 directly bound to and deacetylated c-MYC. Enforced SIRT1 expression increased and depletion/inhibition of SIRT1 reduced c-MYC stability. Depletion/inhibition of SIRT1 correlated with reduced lysine 63-linked polyubiquitination of c-Myc, which presumably destabilizes c-MYC by supporting degradative lysine 48-linked polyubiquitination. Moreover, SIRT1 enhanced the transcriptional activity of c-MYC. Taken together, these results show that c-MYC activates SIRT1, which in turn promotes c-MYC function. Furthermore, SIRT1 suppressed cellular senescence in cells with deregulated c-MYC expression and also inhibited c-MYC-induced apoptosis. Constitutive activation of this positive feedback loop may contribute to the development and maintenance of tumors in the context of deregulated c-MYC.

Phosphorylation by Cdk2 is required for Myc to repress Ras-induced senescence in cotransformation.

The MYC and RAS oncogenes are frequently activated in cancer and, together, are sufficient to transform rodent cells. The basis for this cooperativity remains unclear. We found that although Ras interfered with Myc-induced apoptosis, Myc repressed Ras-induced senescence, together abrogating two main barriers of tumorigenesis. Inhibition of cellular senescence required phosphorylation of Myc at Ser-62 by cyclin E/cyclin-dependent kinase (Cdk) 2. Cdk2 interacted with Myc at promoters, where it affected Myc-dependent regulation of genes, including Bmi-1, p16, p21, and hTERT, which encode proteins known to control senescence. Repression of senescence by Myc was abrogated by the Cdk inhibitor p27Kip1, which is induced by antiproliferative signals like IFN-gamma or by pharmacological inhibitors of Cdk2 but not by inhibitors of other Cdks. In contrast, a phospho-mimicking Myc-S62D mutant was resistant to these manipulations. Inhibition of cyclin E/Cdk2 reversed the senescence-associated gene expression pattern imposed by Myc/cyclin E/Cdk2. This indicates a role of Cdk2 as a transcriptional cofactor and activator of the antisenescence function of Myc and provides mechanistic insight into the Myc-p27Kip1 antagonism. Finally, our findings highlight that pharmacological inhibition of Cdk2 activity is a potential therapeutical principle for cancer therapy, in particular for tumors with activated Myc or Ras.

Cerebrospinal fluid as a liquid biopsy for molecular characterization of brain metastasis in patients with non-small cell lung cancer.

OBJECTIVES: Non-small cell lung cancer (NSCLC) with brain metastases (BM) is a challenging clinical issue with poor prognosis. No data exist regarding extensive genetic analysis of cerebrospinal fluid (CSF) and its correlation to associated tumor compartments. MATERIALS AND METHODS: We designed a study across multiple NSCLC patients with matched material from four compartments; primary tumor, BM, plasma and CSF. We performed enrichment-based targeted next-generation sequencing analysis of ctDNA and exosomal RNA in CSF and plasma and compared the outcome with the solid tumor compartments. RESULTS: An average of 105 million reads per sample was generated with fractions of mapped reads exceeding 99% in all samples and with a mean coverage above 10,000x. We observed a high degree of overlap in variants between primary lung tumor and BM. Variants specific for the BM/CSF compartment included in-frame deletions in AR, FGF10 and TSC1 and missense mutations in HNF1a, CD79B, BCL2, MYC, TSC2, TET2, NRG1, MSH3, NOTCH3, VHL and EGFR. CONCLUSION: Our approach of combining ctDNA and exosomal RNA analyses in CSF presents a potential surrogate for BM biopsy. The specific variants that were only observed in the CNS compartments could serve as targets for individually tailored therapies in NSCLC patients with BM.

c-Myc associates with ribosomal DNA and activates RNA polymerase I transcription.

The c-Myc oncoprotein regulates transcription of genes that are associated with cell growth, proliferation and apoptosis. c-Myc levels are modulated by ubiquitin/proteasome-mediated degradation. Proteasome inhibition leads to c-Myc accumulation within nucleoli, indicating that c-Myc might have a nucleolar function. Here we show that the proteins c-Myc and Max interact in nucleoli and are associated with ribosomal DNA. This association is increased upon activation of quiescent cells and is followed by recruitment of the Myc cofactor TRRAP, enhanced histone acetylation, recruitment of RNA polymerase I (Pol I), and activation of rDNA transcription. Using small interfering RNAs (siRNAs) against c-Myc and an inhibitor of Myc-Max interactions, we demonstrate that c-Myc is required for activating rDNA transcription in response to mitogenic signals. Furthermore, using the ligand-activated MycER (ER, oestrogen receptor) system, we show that c-Myc can activate Pol I transcription in the absence of Pol II transcription. These results suggest that c-Myc coordinates the activity of all three nuclear RNA polymerases, and thereby plays a key role in regulating ribosome biogenesis and cell growth.

Concepts and Design of Introducing Synthetic MicroRNAs into Mammalian Cells.

MicroRNAs are pleiotropic gene modulators affecting numerous cellular processes in development and disease. Due to their small size, microRNAs can easily be synthesized for the purpose of mechanistic or therapeutic studies in biological processes, including autophagy. Depending on the biological question posed, approaches of modulating microRNAs involve either microRNA mimic or inhibitory nucleic acid molecules. This protocol outlines the detailed methodological steps to introduce synthetic microRNA drugs into target cells in vitro and in vivo and how to monitor their function. In addition, it provides insights on how to control the adverse effects when ectopically expressing synthetic microRNA mimic molecules.

Development and validation of a dynamic survival nomogram for metastatic non-small cell lung cancer based on the SEER database and an external validation cohort.

Background: Limited efficacy and poor prognosis are common in patients with metastatic non-small cell lung cancer (NSCLC). An accurate and useful nomogram helps the clinician predict the prognosis of the patients. However, there has been no previous report on the nomogram specially for predicting the overall survival (OS) of metastatic NSCLC patients. Methods: A total of 18,343 patients diagnosed with metastatic NSCLC in the Surveillance, Epidemiology, and End Results (SEER) database were included and divided into the training cohort (n=12,840) and the internal validation cohort (n=5,503), and 242 patients in Renji Hospital were additionally enrolled as the external validation cohort. Demographical, clinical, and OS data were collected. A Cox proportional hazards regression model was used to develop a nomogram based on the training cohort. To validate the nomogram, we applied C-indexes, calibration curves, receiver operating characteristic (ROC) curve, decision curve analysis (DCA), and a Kaplan-Meier survival curve. Results: The multivariate Cox regression model found that there were a total of 16 independent risk factors for OS of the patients (all 16 factors showed P<0.001), which were integrated into the nomogram with a C-index of 0.702 [95% confidence interval (CI): 0.684-0.720]. The nomogram also exhibited good prognostic value in the internal validation cohort (C-index =0.699, 95% CI: 0.673-0.725) and external validation cohort (C-index =0.695, 95% CI: 0.653-0.737). The ROC and Kaplan-Meier survival curve analyses demonstrated a high discriminative ability. High-risk patients had significantly less favorable OS than low-risk patients in the SEER population and external validation cohort (both P<0.001). The DCA analysis showed that the nomogram provided better prognosis prediction than the tumor-node-metastasis (TNM) staging system. Conclusions: We constructed and validated a dynamic nomogram with 16 variables based on a large-scale population of SEER database to predict the prognosis of metastatic NSCLC patients. The nomogram is expected to provide higher predictive ability and accuracy than the TNM staging system.

Ras-Related Protein Rab-32 and Thrombospondin 1 Confer Resistance to the EGFR Tyrosine Kinase Inhibitor Osimertinib by Activating Focal Adhesion Kinase in Non-Small Cell Lung Cancer.

Treatment with the tyrosine kinase inhibitor (TKI) osimertinib is the standard of care for non-small cell lung cancer (NSCLC) patients with activating mutations in the epidermal growth factor receptor (EGFR). Osimertinib is also used in T790M-positive NSCLC that may occur de novo or be acquired following first-line treatment with other EGFR TKIs (i.e., gefitinib, erlotinib, afatinib, or dacomitinib). However, patients treated with osimertinib have a high risk of developing resistance to the treatment. A substantial fraction of the mechanisms for resistance is unknown and may involve RNA and/or protein alterations. In this study, we investigated the full transcriptome of parental and osimertinib-resistant cell lines, revealing 131 differentially expressed genes. Knockdown screening of the genes upregulated in resistant cell lines uncovered eight genes to partly confer resistance to osimertinib. Among them, we detected the expression of Ras-related protein Rab-32 (RAB32) and thrombospondin 1 (THBS1) in plasmas sampled at baseline and at disease progression from EGFR-positive NSCLC patients treated with osimertinib. Both genes were upregulated in progression samples. Moreover, we found that knockdown of RAB32 and THBS1 reduced the expression of phosphorylated focal adhesion kinase (FAK). Combination of osimertinib with a FAK inhibitor resulted in synergistic toxicity in osimertinib-resistant cells, suggesting a potential therapeutic drug combination for overcoming resistance to osimertinib in NSCLC patients.

Elevated expression of miR-494-3p is associated with resistance to osimertinib in EGFR T790M-positive non-small cell lung cancer.

Background: Non-small cell lung cancer (NSCLC) harboring activating mutations in the gene encoding epidermal growth factor receptor (EGFR) is amenable for targeted therapy with tyrosine kinase inhibitors (TKIs). Eventually, resistance to TKI-therapy occurs resulting in disease progression. A substantial fraction of resistance mechanisms is unknown and may involve alterations in the RNA or protein landscape. MicroRNAs (miRNAs) have been frequently suggested to play roles in various forms of cancer including NSCLC. However, a role of miRNAs in acquired resistance to EGFR TKIs remains elusive. In this work, we aimed to investigate the potential involvement of miRNAs in acquired resistance to the third-generation EGFR TKI osimertinib in NSCLC. Methods: We combined miRNA expression profiling with miRNA-inhibitory screening to identify miRNAs involved in conferring resistance to osimertinib. Finally, we validated our top miRNA candidate by profiling longitudinal plasma exosomal RNA from patients receiving osimertinib as second-line therapy in a clinical trial. Results: Various miRNAs displayed differential expression in parental versus osimertinib-refractory NSCLC cells. miRNA-inhibitory screening revealed miR-494-3p to partially confer resistance to osimertinib in vitro. Expression of miR-494-3p was significantly elevated in plasma sampled at disease progression compared to plasma sampled at treatment baseline in a cohort of 21 EGFR T790M-mutation positive NSCLC patients receiving osimertinib. Conclusions: Our results highlight the need for further therapeutic exploration of miR-494-3p in in vivo models of EGFR-mutant NSCLC.

Plasma RNA profiling unveils transcriptional signatures associated with resistance to osimertinib in EGFR T790M positive non-small cell lung cancer patients.

Background: Targeted therapy with tyrosine kinases inhibitors (TKIs) against epidermal growth factor receptor (EGFR) is part of routine clinical practice for EGFR mutant advanced non-small cell lung cancer (NSCLC) patients. These patients eventually develop resistance, frequently accompanied by a gatekeeper mutation, T790M. Osimertinib is a third-generation EGFR TKI displaying potency to the T790M resistance mutation. Here we aimed to analyze if exosomal RNAs, isolated from longitudinally sampled plasma of osimertinib-treated EGFR T790M NSCLC patients, could provide biomarkers of acquired resistance to osimertinib. Methods: Plasma was collected at baseline and progression of disease from 20 patients treated with osimertinib in the multicenter phase II study TKI in Relapsed EGFR-mutated non-small cell lung cancer patients (TREM). Plasma was centrifuged at 16,000 g followed by exosomal RNA extraction using Qiagen exoRNeasy kit. RNA was subjected to transcriptomics analysis with Clariom D. Results: Transcriptome profiling revealed differential expression [log2(fold-change) >0.25, false discovery rate (FDR) P<0.15, and P(interaction) >0.05] of 128 transcripts. We applied network enrichment analysis (NEA) at the pathway level in a large collection of functional gene sets. This overall enrichment analysis revealed alterations in pathways related to EGFR and PI3K as well as to syndecan and glypican pathways (NEA FDR <3×10-10). When applied to the 40 individual, sample-specific gene sets, the NEA detected 16 immune-related gene sets (FDR <0.25, P(interaction) >0.05 and NEA z-score exceeding 3 in at least one sample). Conclusions: Our study demonstrates a potential usability of plasma-derived exosomal RNAs to characterize molecular phenotypes of emerging osimertinib resistance. Furthermore, it highlights the involvement of multiple RNA species in shaping the transcriptome landscape of osimertinib-refractory NSCLC patients.

MYCMI-7: A Small MYC-Binding Compound that Inhibits MYC: MAX Interaction and Tumor Growth in a MYC-Dependent Manner.

Deregulated expression of MYC family oncogenes occurs frequently in human cancer and is often associated with aggressive disease and poor prognosis. While MYC is a highly warranted target, it has been considered "undruggable," and no specific anti-MYC drugs are available in the clinic. We recently identified molecules named MYCMIs that inhibit the interaction between MYC and its essential partner MAX. Here we show that one of these molecules, MYCMI-7, efficiently and selectively inhibits MYC:MAX and MYCN:MAX interactions in cells, binds directly to recombinant MYC, and reduces MYC-driven transcription. In addition, MYCMI-7 induces degradation of MYC and MYCN proteins. MYCMI-7 potently induces growth arrest/apoptosis in tumor cells in a MYC/MYCN-dependent manner and downregulates the MYC pathway on a global level as determined by RNA sequencing. Sensitivity to MYCMI-7 correlates with MYC expression in a panel of 60 tumor cell lines and MYCMI-7 shows high efficacy toward a collection of patient-derived primary glioblastoma and acute myeloid leukemia (AML) ex vivo cultures. Importantly, a variety of normal cells become G1 arrested without signs of apoptosis upon MYCMI-7 treatment. Finally, in mouse tumor models of MYC-driven AML, breast cancer, and MYCN-amplified neuroblastoma, treatment with MYCMI-7 downregulates MYC/MYCN, inhibits tumor growth, and prolongs survival through apoptosis with few side effects. In conclusion, MYCMI-7 is a potent and selective MYC inhibitor that is highly relevant for the development into clinically useful drugs for the treatment of MYC-driven cancer. Significance: Our findings demonstrate that the small-molecule MYCMI-7 binds MYC and inhibits interaction between MYC and MAX, thereby hampering MYC-driven tumor cell growth in culture and in vivo while sparing normal cells.

Construction of a Full-Length 3'UTR Reporter System for Identification of Cell-Cycle Regulating MicroRNAs.

Three prime untranslated region (3'UTR) reporter constructs are widely used by the scientific community to functionally link microRNAs (miRNAs) to suppression of mRNA expression. However, full-length 3'UTR vectors are rarely employed due to labor-intensive cloning work. Instead, 3'UTR fragments containing putative miRNA binding sites are commonly utilized to mechanistically validate miRNAs. Assaying truncated 3'UTRs may falsely validate miRNAs due to altered positioning of binding sites in respect to 3'UTR length and RNA secondary structure. Here we present a detailed protocol for the construction of full-length 3'UTR luciferase reporter constructs that was used to unveil miRNAs regulating multiple cell-cycle factors.

A MicroRNA Gene Panel Predicts the Vaginal Microbiota Composition.

The vaginal microbiota plays an essential role in vaginal health. The vaginas of many reproductive-age women are dominated by one of the Lactobacillus species. However, the vaginas of a large number of women are characterized by the colonization of several other anaerobes. Notably, some women with the non-Lactobacillus-dominated vaginal microbiota develop bacterial vaginosis, which has been correlated with sexually transmitted infections and other adverse outcomes. However, interactions and mechanisms linking the vaginal microbiota to host response are still under investigation. There are studies suggesting a link between human microRNAs and gut microbiota, but limited analysis has been carried out on the interplay of microRNAs and vaginal microbiota. In this study, we performed a microRNA expression array profiling on 67 vaginal samples from young Swedish women. MicroRNAs were clustered into distinct groups according to vaginal microbiota composition. Interestingly, 182 microRNAs were significantly elevated in their expression in the non-Lactobacillus-dominated community, suggesting an antagonistic relationship between Lactobacillus and microRNAs. Of the elevated microRNAs, 10 microRNAs displayed excellent diagnostic potential, visualized by receiver operating characteristics analysis. We further validated our findings in 34 independent samples where expression of top microRNA candidates strongly separated the Lactobacillus-dominated community from the non-Lactobacillus-dominated community in the vaginal microbiota. Notably, the Lactobacillus crispatus-dominated community showed the most profound differential microRNA expression compared with the non-Lactobacillus-dominated community. In conclusion, we demonstrate a strong relationship between the vaginal microbiota and numerous genital microRNAs, which may facilitate a deeper mechanistic interplay in this biological niche.IMPORTANCE Vaginal microbiota is correlated with women's health, where a non-Lactobacillus-dominated community predisposes women to a higher risk of disease, including human papillomavirus (HPV). However, the molecular relationship between the vaginal microbiota and host is largely unexplored. In this study, we investigated a link between the vaginal microbiota and host microRNAs in a group of young women. We uncovered an inverse correlation of the expression of microRNAs with the abundance of Lactobacillus species in the vaginal microbiota. Particularly, the expression of microRNA miR-23a-3p and miR-130a-3p, displaying significantly elevated levels in non-Lactobacillus-dominated communities, predicted the bacterial composition of the vaginal microbiota in an independent validation group. Since targeting of microRNAs is explored in the clinical setting, our results warrant investigation of whether microRNA modulation could be used for treating vaginosis recurrence and vaginosis-related diseases. Conversely, commensal bacteria could be used for treating diseases with microRNA aberrations.

Pharmacological inactivation of CDK2 inhibits MYC/BCL-XL-driven leukemia in vivo through induction of cellular senescence.

Deregulated expression of the MYC oncogene is a frequent event during tumorigenesis and generally correlates with aggressive disease and poor prognosis. While MYC is a potent inducer of apoptosis, it often suppresses cellular senescence, which together with apoptosis is an important barrier against tumor development. For this latter function, MYC is dependent on cyclin-dependent kinase 2 (CDK2). Here, we utilized a MYC/BCL-XL-driven mouse model of acute myeloblastic leukemia (AML) to investigate whether pharmacological inhibition of CDK2 can inhibit MYC-driven tumorigenesis through induction of senescence. Purified mouse hematopoietic stem cells transduced with MYC and BCL-XL were transplanted into lethally irradiated mice, leading to the development of massive leukemia and subsequent death 15-17 days after transplantation. Upon disease onset, mice were treated with the selective CDK2 inhibitor CVT2584 or vehicle either by daily intraperitoneal injections or continuous delivery via mini-pumps. CVT2584 treatment delayed disease onset and moderately but significantly improved survival of mice. Flow cytometry revealed a significant decrease in tumor load in the spleen, liver and bone marrow of CVT2584-treated compared to vehicle-treated mice. This was correlated with induced senescence evidenced by reduced cell proliferation, increased senescence-associated ß-galactosidase activity and heterochromatin foci, expression of p19ARF and p21CIP1, and reduced phosphorylation (activation) of pRb, while very few apoptotic cells were observed. In addition, phosphorylation of MYC at Ser-62 was decreased. In summary, inhibition of CDK2 delayed MYC/BCL-XL-driven AML linked to senescence induction. Our results suggest that CDK2 is a promising target for pro-senescence cancer therapy, in particular for MYC-driven tumors, including leukemia.

Multiplex immune protein profiling of fine-needle aspirates from patients with non-small-cell lung cancer reveals signatures associated with PD-L1 expression and tumor stage.

Biomarker signatures identified through minimally invasive procedures already at diagnosis of non-small-cell lung cancer (NSCLC) could help to guide treatment with immune checkpoint inhibitors (ICI). Here, we performed multiplex profiling of immune-related proteins in fine-needle aspirate (FNA) samples of thoracic lesions from patients with NSCLC to assess PD-L1 expression and identify related protein signatures. Transthoracic FNA samples from 14 patients were subjected to multiplex antibody-based profiling by proximity extension assay (PEA). PEA profiling employed protein panels relevant to immune and tumor signaling and was followed by Qlucore® Omics Explorer analysis. All lesions analyzed were NSCLC adenocarcinomas, and PEA profiles could be used to monitor 163 proteins in all but one sample. Multiple key immune signaling components (including CD73, granzyme A, and chemokines CCL3 and CCL23) were identified and expression of several of these proteins (e.g., CCL3 and CCL23) correlated to PD-L1 expression. We also found EphA2, a marker previously linked to inferior NSCLC prognosis, to correlate to PD-L1 expression. Our identified protein signatures related to stage included, among others, CXCL10 and IL12RB1. We conclude that transthoracic FNA allows for extensive immune and tumor protein profiling with assessment of putative biomarkers of important for ICI treatment selection in NSCLC.

TGF-beta enforces senescence in Myc-transformed hematopoietic tumor cells through induction of Mad1 and repression of Myc activity.

Inhibition of tumor growth factor (TGF)-beta-mediated cell cycle exit is considered an important tumorigenic function of Myc oncoproteins. Here we found that TGF-beta1 enforced G(1) cell cycle arrest and cellular senescence in human U-937 myeloid tumor cells ectopically expressing v-Myc, which contains a stabilizing mutation frequently found in lymphomas. This correlated with induced expression of the Myc antagonist Mad1, resulting in replacement of Myc for Mad1 at target promoters, reduced histone acetylation and strong repression of Myc-driven transcription. The latter was partially reversed by histone deacetylase (HDAC) inhibitors, consistent with involvement of Mad1. Importantly, knockdown of MAD1 expression prevented TGF-beta1-induced senescence, underscoring that Mad1 is a crucial component of this process. Enforced Mad1 expression sensitized U-937-myc cells to TGF-beta and restored phorbol ester-induced cell cycle exit, but could not alone induce G(1) arrest, suggesting that Mad1 is required but not sufficient for cellular senescence. Our results thus demonstrate that TGF-beta can override Myc activity despite a stabilizing cancer mutation and induce senescence in myeloid tumor cells, at least in part by induction of Mad1. TGF-beta-induced senescence, or signals mimicking this pathway, could therefore potentially be explored as a therapeutic principle for treating hematopoietic and other tumors with deregulated MYC expression.

An immune gene expression signature distinguishes central nervous system metastases from primary tumours in non-small-cell lung cancer.

BACKGROUND: Dissemination of non-small-cell lung cancer (NSCLC) in the central nervous system is a frequent and challenging clinical problem. Systemic or local therapies rarely prolong survival and have modest activity regarding local control. Alterations in gene expression in brain metastasis versus primary tumour may increase aggressiveness and impair therapeutic efforts. METHODS: We identified 25 patients with surgically removed NSCLC brain metastases in two different patient cohorts. For 13 of these patients, primary tumour samples were available. Gene expression analysis using the nCounter® PanCancer Immune Profiling gene expression panel (nanoString technologies Inc.) was performed in brain metastases and primary tumour samples. Identification of differentially expressed genes was conducted on normalized data using the nSolver analysis software. RESULTS: We compared gene expression patterns in brain metastases with primary tumours. Brain metastasis samples displayed a distinct clustering pattern compared to primary tumour samples with a statistically significant downregulation of genes related to immune response and immune cell activation. Results from KEGG term analysis on differentially expressed genes revealed a concomitant enrichment of multiple KEGG terms associated with the immune system. We identified a 12-gene immune signature that clearly separated brain metastases from primary tumours. CONCLUSIONS: We identified a unique gene downregulation pattern in brain metastases compared with primary tumours. This finding may explain the lower intracranial efficacy of systemic therapy, especially immunotherapy, in brain metastasis of patients with NSCLC.