Glucocorticoid receptor and RAS: an unexpected couple in cancer.

Constitutively activated rat sarcoma (RAS) GTPases are one of the major drivers of tumor growth and are difficult drug targets. The glucocorticoid receptor (GR), a nuclear receptor primarily acting in the nucleus, is a potent modulator of inflammation and regulator of metabolism and cell growth. Emerging evidence has revealed that GR modulates RAS-dependent signaling and RAS activation. The unliganded GR decreases RAS activation, and, upon ligand binding, GR leaves RAS complexes, is translocated into the nucleus, and unleashes the activation of RAS and its downstream pathways. GR forms a complex with RAS and RAF1 and their associated proteins, such as members of the 14-3-3 family of adapter proteins. The exploration of RAS-GR complex formation and maintenance will help to develop much-needed breakthroughs in oncogenic RAS biology and thus help to alleviate tumor growth and burden.

The nature inspired peptide [T20K]-kalata B1 induces anti-tumor effects in anaplastic large cell lymphoma.

Ribosomally synthesized and post-translationally modified peptides, such as plant cyclotides, are a diverse group of natural products well known as templates in drug discovery and therapeutic lead development. The cyclotide kalata B1 (kB1) has previously been discovered as immunosuppressive agent on T-lymphocytes, and a synthetic version of this peptide, [T20K]kB1 (T20K), has been effective in reducing clinical symptoms, such as inflammation and demyelination, in a mouse model of multiple sclerosis. Based on its T-cell modulatory impact we studied the effects of T20K and several analogs on the proliferation of anaplastic large cell lymphoma (ALCL), a heterogeneous group of clinically aggressive diseases associated with poor prognosis. T20K, as a prototype drug candidate, induces apoptosis and a proliferation arrest in human lymphoma T-cell lines (SR786, Mac-2a and the Jurkat E6.1) in a concentration dependent fashion, at least partially via increased STAT5 and p53 signaling. In contrary to its effect on IL-2 signaling in lymphocytes, the cytokine levels are not altered in lymphoma cells. In vivo mouse experiments revealed a promising activity of T20K on these cancer cells including decreased tumor weight and increased apoptosis. This study opens novel avenues for developing cyclotide-based drug candidates for therapy of patients with ALCL.

The glucocorticoid receptor associates with RAS complexes to inhibit cell proliferation and tumor growth.

Mutations that activate members of the RAS family of GTPases are associated with various cancers and drive tumor growth. The glucocorticoid receptor (GR), a member of the nuclear receptor family, has been proposed to interact with and inhibit the activation of components of the PI3K-AKT and MAPK pathways downstream of RAS. In the absence of activating ligands, we found that GR was present in cytoplasmic KRAS-containing complexes and inhibited the activation of wild-type and oncogenic KRAS in mouse embryonic fibroblasts and human lung cancer A549 cells. The DNA binding domain of GR was involved in the interaction with KRAS, but GR-dependent inhibition of RAS activation did not depend on the nuclear translocation of GR. The addition of ligand released GR-dependent inhibition of RAS, AKT, the MAPK p38, and the MAPKK MEK. CRISPR-Cas9-mediated deletion of GR in A549 cells enhanced tumor growth in xenografts in mice. Patient samples of non-small cell lung carcinomas showed lower expression of NR3C1, the gene encoding GR, compared to adjacent normal tissues and lower NR3C1 expression correlated with a worse disease outcome. These results suggest that glucocorticoids prevent the ability of GR to limit tumor growth by inhibiting RAS activation, which has potential implications for the use of glucocorticoids in patients with cancer.

Down-regulation of A20 promotes immune escape of lung adenocarcinomas.

Inflammation is a well-known driver of lung tumorigenesis. One strategy by which tumor cells escape tight homeostatic control is by decreasing the expression of the potent anti-inflammatory protein tumor necrosis factor alpha-induced protein 3 (TNFAIP3), also known as A20. We observed that tumor cell intrinsic loss of A20 markedly enhanced lung tumorigenesis and was associated with reduced CD8+ T cell-mediated immune surveillance in patients with lung cancer and in mouse models. In mice, we observed that this effect was completely dependent on increased cellular sensitivity to interferon-γ (IFN-γ) signaling by aberrant activation of TANK-binding kinase 1 (TBK1) and increased downstream expression and activation of signal transducer and activator of transcription 1 (STAT1). Interrupting this autocrine feed forward loop by knocking out IFN-α/ß receptor completely restored infiltration of cytotoxic T cells and rescued loss of A20 depending tumorigenesis. Downstream of STAT1, programmed death ligand 1 (PD-L1) was highly expressed in A20 knockout lung tumors. Accordingly, immune checkpoint blockade (ICB) treatment was highly efficient in mice harboring A20-deficient lung tumors. Furthermore, an A20 loss-of-function gene expression signature positively correlated with survival of melanoma patients treated with anti-programmed cell death protein 1. Together, we have identified A20 as a master immune checkpoint regulating the TBK1-STAT1-PD-L1 axis that may be exploited to improve ICB therapy in patients with lung adenocarcinoma.

A20/TNFAIP3 Increases ENOS Expression in an ERK5/KLF2-Dependent Manner to Support Endothelial Cell Health in the Face of Inflammation.

Rationale: Decreased expression and activity of endothelial nitric oxide synthase (eNOS) in response to inflammatory and metabolic insults is the hallmark of endothelial cell (EC) dysfunction that preludes the development of atherosclerosis and hypertension. We previously reported the atheroprotective properties of the ubiquitin-editing and anti-inflammatory protein A20, also known as TNFAIP3, in part through interrupting nuclear factor-kappa B (NF-κB) and interferon signaling in EC and protecting these cells from apoptosis. However, A20's effect on eNOS expression and function remains unknown. In this study, we evaluated the impact of A20 overexpression or knockdown on eNOS expression in EC, at baseline and after tumor necrosis factor (TNF) treatment, used to mimic inflammation. Methods and Results: A20 overexpression in human coronary artery EC (HCAEC) significantly increased basal eNOS mRNA (qPCR) and protein (western blot) levels and prevented their downregulation by TNF. Conversely, siRNA-induced A20 knockdown decreased eNOS mRNA levels, identifying A20 as a physiologic regulator of eNOS expression. By reporter assays, using deletion and point mutants of the human eNOS promoter, and knockdown of eNOS transcriptional regulators, we demonstrated that A20-mediated increase of eNOS was transcriptional and relied on increased expression of the transcription factor Krüppel-like factor (KLF2), and upstream of KLF2, on activation of extracellular signal-regulated kinase 5 (ERK5). Accordingly, ERK5 knockdown or inhibition significantly abrogated A20's ability to increase KLF2 and eNOS expression. In addition, A20 overexpression in HCAEC increased eNOS phosphorylation at Ser-1177, which is key for the function of this enzyme. Conclusions: This is the first report demonstrating that overexpression of A20 in EC increases eNOS transcription in an ERK5/KLF2-dependent manner and promotes eNOS activating phosphorylation. This effect withstands eNOS downregulation by TNF, preventing EC dysfunction in the face of inflammation. This novel function of A20 further qualifies its therapeutic promise to prevent/treat atherosclerosis.

Targeting KRAS Mutant Non-Small-Cell Lung Cancer: Past, Present and Future.

Lung cancer is the most frequent cancer with an aggressive clinical course and high mortality rates. Most cases are diagnosed at advanced stages when treatment options are limited and the efficacy of chemotherapy is poor. The disease has a complex and heterogeneous background with non-small-cell lung cancer (NSCLC) accounting for 85% of patients and lung adenocarcinoma being the most common histological subtype. Almost 30% of adenocarcinomas of the lung are driven by an activating Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation. The ability to inhibit the oncogenic KRAS has been the holy grail of cancer research and the search for inhibitors is immensely ongoing as KRAS-mutated tumors are among the most aggressive and refractory to treatment. Therapeutic strategies tailored for KRAS+ NSCLC rely on the blockage of KRAS functional output, cellular dependencies, metabolic features, KRAS membrane associations, direct targeting of KRAS and immunotherapy. In this review, we provide an update on the most recent advances in anti-KRAS therapy for lung tumors with mechanistic insights into biological diversity and potential clinical implications.

STAT3: Versatile Functions in Non-Small Cell Lung Cancer.

Signal Transducer and Activator of Transcription 3 (STAT3) activation is frequently found in non-small cell lung cancer (NSCLC) patient samples/cell lines and STAT3 inhibition in NSCLC cell lines markedly impairs their survival. STAT3 also plays a pivotal role in driving tumor-promoting inflammation and evasion of anti-tumor immunity. Consequently, targeting STAT3 either directly or by inhibition of upstream regulators such as Interleukin-6 (IL-6) or Janus kinase 1/2 (JAK1/2) is considered as a promising treatment strategy for the management of NSCLC. In contrast, some studies also report STAT3 being a tumor suppressor in a variety of solid malignancies, including lung cancer. Here, we provide a concise overview of STAT3's versatile roles in NSCLC and discuss the yins and yangs of STAT3 targeting therapies.

IDO1+ Paneth cells promote immune escape of colorectal cancer.

Tumors have evolved mechanisms to escape anti-tumor immunosurveillance. They limit humoral and cellular immune activities in the stroma and render tumors resistant to immunotherapy. Sensitizing tumor cells to immune attack is an important strategy to revert immunosuppression. However, the underlying mechanisms of immune escape are still poorly understood. Here we discover Indoleamine-2,3-dioxygenase-1 (IDO1)+ Paneth cells in the stem cell niche of intestinal crypts and tumors, which promoted immune escape of colorectal cancer (CRC). Ido1 expression in Paneth cells was strictly Stat1 dependent. Loss of IDO1+ Paneth cells in murine intestinal adenomas with tumor cell-specific Stat1 deletion had profound effects on the intratumoral immune cell composition. Patient samples and TCGA expression data suggested corresponding cells in human colorectal tumors. Thus, our data uncovered an immune escape mechanism of CRC and identify IDO1+ Paneth cells as a target for immunotherapy.

Notch inhibition overcomes resistance to tyrosine kinase inhibitors in EGFR-driven lung adenocarcinoma.

EGFR-mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFRT790M and EGFRC797S. It is generally assumed that these secondary mutations render EGFR completely unresponsive to the inhibitors, but contrary to this, we uncovered here that gefitinib and osimertinib increased STAT3 phosphorylation (p-STAT3) in EGFRT790M and EGFRC797S tumoral cells. Interestingly, we also found that concomitant Notch inhibition with gefitinib or osimertinib treatment induced a p-STAT3-dependent strong reduction in the levels of the transcriptional repressor HES1. Importantly, we showed that tyrosine kinase inhibitor-resistant tumors, with EGFRT790M and EGFRC797S mutations, were highly responsive to the combined treatment of Notch inhibitors with gefitinib or osimertinib, respectively. Finally, in patients with EGFR mutations treated with tyrosine kinase inhibitors, HES1 protein levels increased during relapse and correlated with shorter progression-free survival. Therefore, our results offer a proof of concept for an alternative treatment to chemotherapy in lung adenocarcinoma osimertinib-treated patients after disease progression.

[CHEK1 expression and inhibitors in TP53 mutant cancer]. / A checkpoint kinase 1 expressziója és gátlása TP53-mutációt hordozó rosszindulatú daganatokban.

The most frequently mutated gene in human tumors is TP53 and its mutation significantly deteriorates patients' survival. However, to date no targeted therapy is established for TP53 mutated tumors. Here, our aim was to identify druggable kinases with higher expression in TP53 mutated tumors, as well as relate these to altered prognosis. We also aimed to validate a target gene in TP53 wild type and mutant isogenic cell lines using a specific kinase inhibitor. Gene expression and mutation data were collected from 994 lung tumor samples. Samples were separated based on TP53 mutation status, and differential gene expression was compared using Mann-Whitney test between patient cohorts. Prognostic value of identified genes was validated in an array-based lung cancer dataset (n=1926). Survival analysis was performed using Cox proportional hazards regression and Kaplan-Meier survival plots. Effect of TP53 mutations on CHEK1 expression was validated in the A549 isogenic lung cancer cell line. The cell line was also treated using Chk1 protein specific kinase inhibitor to monitor cell functions. Expression of CHEK1 was elevated significantly among targetable kinases and higher expression of CHEK1 related to worse prognosis. Our results confirm the higher expression of CHEK1 kinase associated to TP53 mutations and to shorter survival.

JAK-STAT inhibition impairs K-RAS-driven lung adenocarcinoma progression.

Oncogenic K-RAS has been difficult to target and currently there is no K-RAS-based targeted therapy available for patients suffering from K-RAS-driven lung adenocarcinoma (AC). Alternatively, targeting K-RAS-downstream effectors, K-RAS-cooperating signaling pathways or cancer hallmarks, such as tumor-promoting inflammation, has been shown to be a promising therapeutic strategy. Since the JAK-STAT pathway is considered to be a central player in inflammation-mediated tumorigenesis, we investigated here the implication of JAK-STAT signaling and the therapeutic potential of JAK1/2 inhibition in K-RAS-driven lung AC. Our data showed that JAK1 and JAK2 are activated in human lung AC and that increased activation of JAK-STAT signaling correlated with disease progression and K-RAS activity in human lung AC. Accordingly, administration of the JAK1/2 selective tyrosine kinase inhibitor ruxolitinib reduced proliferation of tumor cells and effectively reduced tumor progression in immunodeficient and immunocompetent mouse models of K-RAS-driven lung AC. Notably, JAK1/2 inhibition led to the establishment of an antitumorigenic tumor microenvironment, characterized by decreased levels of tumor-promoting chemokines and cytokines and reduced numbers of infiltrating myeloid derived suppressor cells, thereby impairing tumor growth. Taken together, we identified JAK1/2 inhibition as promising therapy for K-RAS-driven lung AC.

A Mouse Model to Assess STAT3 and STAT5A/B Combined Inhibition in Health and Disease Conditions.

Genetically-engineered mouse models (GEMMs) lacking diseased-associated gene(s) globally or in a tissue-specific manner represent an attractive tool with which to assess the efficacy and toxicity of targeted pharmacological inhibitors. Stat3 and Stat5a/b transcription factors have been implicated in several pathophysiological conditions, and pharmacological inhibition of both transcription factors has been proposed to treat certain diseases, such as malignancies. To model combined inhibition of Stat3 and Stat5a/b we have developed a GEMM harboring a flox Stat3-Stat5a/b allele (Stat5/3loxP/loxP mice) and generated mice lacking Stat3 and Stat5a/b in hepatocytes (Stat5/3Δhep/Δhep). Stat5/3Δhep/Δhep mice exhibited a marked reduction of STAT3, STAT5A and STAT5B proteins in the liver and developed steatosis, a phenotype that resembles mice lacking Stat5a/b in hepatocytes. In addition, embryonic deletion of Stat3 and Stat5a/b (Stat5/3Δ/Δ mice) resulted in lethality, similar to Stat3Δ/Δ mice. This data illustrates that Stat5/3loxP/loxP mice are functional and can be used as a valuable tool to model the combined inhibition of Stat3 and Stat5a/b in tumorigenesis and other diseases.

STAT3ß is a tumor suppressor in acute myeloid leukemia.

Signal transducer and activator of transcription 3 (STAT3) exists in 2 alternatively spliced isoforms, STAT3α and STAT3ß. Although truncated STAT3ß was originally postulated to act as a dominant-negative form of STAT3α, it has been shown to have various STAT3α-independent regulatory functions. Recently, STAT3ß gained attention as a powerful antitumorigenic molecule in cancer. Deregulated STAT3 signaling is often found in acute myeloid leukemia (AML); however, the role of STAT3ß in AML remains elusive. Therefore, we analyzed the STAT3ß/α messenger RNA (mRNA) expression ratio in AML patients, where we observed that a higher STAT3ß/α mRNA ratio correlated with a favorable prognosis and increased overall survival. To gain better understanding of the function of STAT3ß in AML, we engineered a transgenic mouse allowing for balanced Stat3ß expression. Transgenic Stat3ß expression resulted in decelerated disease progression and extended survival in PTEN- and MLL-AF9-dependent AML mouse models. Our findings further suggest that the antitumorigenic function of STAT3ß depends on the tumor-intrinsic regulation of a small set of significantly up- and downregulated genes, identified via RNA sequencing. In conclusion, we demonstrate that STAT3ß plays an essential tumor-suppressive role in AML.

MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation.

The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression.

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Breaking bad family ties: Pan-ERBB blockers inhibit KRAS driven lung tumorigenesis.

Oncogenic K-RAS mutations were believed to lock the molecular switch in the ON state, independent of upstream activation. However, we demonstrate in preclinical models that activity of mutated K-RAS depends on upstream signaling events involving EGF receptor family members. This finding reveals a potential therapeutic vulnerability using pan-ERBB inhibitors to fight K-RAS mutated lung tumors.

Afatinib restrains K-RAS-driven lung tumorigenesis.

On the basis of clinical trials using first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), it became a doctrine that V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-RAS) mutations drive resistance to EGFR inhibition in non-small cell lung cancer (NSCLC). Conversely, we provide evidence that EGFR signaling is engaged in K-RAS-driven lung tumorigenesis in humans and in mice. Specifically, genetic mouse models revealed that deletion of Egfr quenches mutant K-RAS activity and transiently reduces tumor growth. However, EGFR inhibition initiates a rapid resistance mechanism involving non-EGFR ERBB family members. This tumor escape mechanism clarifies the disappointing outcome of first-generation TKIs and suggests high therapeutic potential of pan-ERBB inhibitors. On the basis of various experimental models including genetically engineered mouse models, patient-derived and cell line-derived xenografts, and in vitro experiments, we demonstrate that the U.S. Food and Drug Administration-approved pan-ERBB inhibitor afatinib effectively impairs K-RAS-driven lung tumorigenesis. Our data support reconsidering the use of pan-ERBB inhibition in clinical trials to treat K-RAS-mutated NSCLC.

AKT3 drives adenoid cystic carcinoma development in salivary glands.

Salivary gland cancer is an aggressive and painful cancer, but a rare tumor type accounting for only ~0.5% of cancer cases. Tumors of the salivary gland exhibit heterogeneous histologic and genetic features and they are subdivided into different subtypes, with adenoid cystic carcinomas (ACC) being one of the most abundant. Treatment of ACC patients is afflicted by high recurrence rates, the high potential of the tumors to metastasize, as well as the poor response of ACC to chemotherapy. A prerequisite for the development of targeted therapies is insightful genetic information for driver core cancer pathways. Here, we developed a transgenic mouse model toward establishment of a preclinical model. There is currently no available mouse model for adenoid cystic carcinomas as a rare disease entity to serve as a test system to block salivary gland tumors with targeted therapy. Based on tumor genomic data of ACC patients, a key role for the activation of the PI3K-AKT-mTOR pathway was suggested in tumors of secretory glands. Therefore, we investigated the role of Akt3 expression in tumorigenesis and report that Akt3 overexpression results in ACC of salivary glands with 100% penetrance, while abrogation of transgenic Akt3 expression could revert the phenotype. In summary, our findings validate a novel mouse model to study ACC and highlight the druggable potential of AKT3 in the treatment of salivary gland patients.

A20 Haploinsufficiency Aggravates Transplant Arteriosclerosis in Mouse Vascular Allografts: Implications for Clinical Transplantation.

BACKGROUND: Inflammation is central to the pathogenesis of transplant arteriosclerosis (TA). We questioned whether physiologic levels of anti-inflammatory A20 influence TA severity. METHODS: We performed major histocompatibility complex mismatched aorta to carotid artery interposition grafts, using wild type (WT) or A20 heterozygote (HET) C57BL/6 (H-2) donors and BALB/c (H-2) recipients, and conversely BALB/c donors and WT/HET recipients. We analyzed aortic allografts by histology, immunohistochemistry, immunofluorescence, and gene profiling (quantitative real-time reverse-transcriptase polymerase chain reaction). We validated select in vivo A20 targets in human and mouse smooth muscle cell (SMC) cultures. RESULTS: We noted significantly greater intimal hyperplasia in HET versus WT allografts, indicating aggravated TA. Inadequate upregulation of A20 in HET allografts after transplantation was associated with excessive NF-кB activation, gauged by higher levels of IkBα, p65, VCAM-1, ICAM-1, CXCL10, CCL2, TNF, and IL-6 (mostly localized to SMC). Correspondingly, cytokine-induced upregulation of TNF and IL-6 in human and mouse SMC cultures inversely correlated with A20 expression. Aggravated TA in HET versus WT allografts correlated with increased intimal SMC proliferation, and a higher number of infiltrating IFNγ and Granzyme B CD4 T cells and natural killer cells, and lower number of FoxP3 regulatory T cells. A20 haploinsufficiency in allograft recipients did not influence TA. CONCLUSIONS: A20 haploinsufficiency in vascular allografts aggravates lesions of TA by exacerbating inflammation, SMC proliferation, and infiltration of pathogenic T cells. A20 single nucleotide polymorphisms associating with lower A20 expression or function in donors of vascularized allografts may inform risk and severity of TA, highlighting the clinical implications of our findings.

Unexpected oncosuppressive role for STAT3 in KRAS-induced lung tumorigenesis.

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in the pathogenesis of several diseases and is considered a therapeutic target in solid cancers, including lung cancer. However, we recently demonstrated a tumor suppressive function of STAT3 in kirsten rat sarcoma oncogene homolog (KRAS)-driven lung cancer. Here, we discuss these findings and their consequences.