MED12 and BRD4 cooperate to sustain cancer growth upon loss of mediator kinase.

Mediator kinases (CDK8/19) are transcriptional regulators broadly implicated in cancer. Despite their central role in fine-tuning gene-expression programs, we find complete loss of CDK8/19 is tolerated in colorectal cancer (CRC) cells. Using orthogonal functional genomic and pharmacological screens, we identify BET protein inhibition as a distinct vulnerability in CDK8/19-depleted cells. Combined CDK8/19 and BET inhibition led to synergistic growth retardation in human and mouse models of CRC. Strikingly, depletion of CDK8/19 in these cells led to global repression of RNA polymerase II (Pol II) promoter occupancy and transcription. Concurrently, loss of Mediator kinase led to a profound increase in MED12 and BRD4 co-occupancy at enhancer elements and increased dependence on BET proteins for the transcriptional output of cell-essential genes. In total, this work demonstrates a synthetic lethal interaction between Mediator kinase and BET proteins and exposes a therapeutic vulnerability that can be targeted using combination therapies.

Constitutive STAT3 Serine Phosphorylation Promotes Helicobacter-Mediated Gastric Disease.

Gastric cancer is associated with chronic inflammation (gastritis) triggered by persistent Helicobacter pylori (H. pylori) infection. Elevated tyrosine phosphorylation of the latent transcription factor STAT3 is a feature of gastric cancer, including H. pylori-infected tissues, and aligns with nuclear transcriptional activity. However, the transcriptional role of STAT3 serine phosphorylation, which promotes STAT3-driven mitochondrial activities, is unclear. Here, by coupling serine-phosphorylated (pS)-STAT3-deficient Stat3SA/SA mice with chronic H. felis infection, which mimics human H. pylori infection in mice, we reveal a key role for pS-STAT3 in promoting Helicobacter-induced gastric pathology. Immunohistochemical staining for infiltrating immune cells and expression analyses of inflammatory genes revealed that gastritis was markedly suppressed in infected Stat3SA/SA mice compared with wild-type mice. Stomach weight and gastric mucosal thickness were also reduced in infected Stat3SA/SA mice, which was associated with reduced proliferative potential of infected Stat3SA/SA gastric mucosa. The suppressed H. felis-induced gastric phenotype of Stat3SA/SA mice was phenocopied upon genetic ablation of signaling by the cytokine IL-11, which promotes gastric tumorigenesis via STAT3. pS-STAT3 dependency by Helicobacter coincided with transcriptional activity on STAT3-regulated genes, rather than mitochondrial and metabolic genes. In the gastric mucosa of mice and patients with gastritis, pS-STAT3 was constitutively expressed irrespective of Helicobacter infection. Collectively, these findings suggest an obligate requirement for IL-11 signaling via constitutive pS-STAT3 in Helicobacter-induced gastric carcinogenesis.

Constitutive type I interferon modulates homeostatic balance through tonic signaling.

Interferons (IFNs) were discovered as cytokines induced during and protecting from viral infection. They have been documented to play essential roles in numerous physiological processes beyond antiviral and antimicrobial defense, including immunomodulation, cell cycle regulation, cell survival, and cell differentiation. Recent data have also uncovered a potentially darker side to IFN, including roles in inflammatory diseases, such as autoimmunity and diabetes. IFN can have effects in the absence of acute infection, highlighting a physiologic role for constitutive IFN. Type I IFNs are constitutively produced at vanishingly low quantities and yet exert profound effects, mediated in part through modulation of signaling intermediates required for responses to diverse cytokines. We review evidence for a yin-yang of IFN function through its role in modulating crosstalk between multiple cytokines by both feedforward and feedback regulation of common signaling intermediates and postulate a homeostatic role for IFN through tonic signaling in the absence of acute infection.

STAT3-driven hematopoiesis and lymphopoiesis abnormalities are dependent on serine phosphorylation.

Deregulated activation of the latent transcription factor STAT3 has been implicated in the pathogenesis of myeloproliferative and lymphoproliferative hematologic disorders. The uncontrolled activation of STAT3 has traditionally been assigned to its elevated phosphorylation at tyrosine 705 (pY705) and associated nuclear transcriptional activity. By contrast, a transcriptional role for serine 727 phosphorylation (pS727) of STAT3 has recently emerged, suggesting that pS727 may account for the pathological activity of STAT3 in certain disease settings. Here, by coupling pS727-STAT3-deficient Stat3SA/SA mice with a STAT3-driven mouse model (gp130F/F) for myeloproliferative and lymphoproliferative pathologies, we reveal a key role for pS727-STAT3 in promoting multiple hematologic pathologies. The genetic blockade of pS727-STAT3 in gp130F/F:Stat3SA/SA mice ameliorated the neutrophilia, thrombocytosis, splenomegaly and lymphadenopathy that are features of gp130F/F mice. The protection against thrombocytosis in gp130F/F:Stat3SA/SA mice coincided with normalized megakaryopoiesis in both bone marrow and spleen compartments. Interestingly, pS727-STAT3-mediated abnormal lymphopoiesis in gp130F/F mice was more pronounced in lymph nodes compared to thymus, and was characterized by elevated numbers of B cells at the expense of T cells. Furthermore, pS727-STAT3 dependency for these hematologic pathologies coincided with transcriptional activity on STAT3-regulated genes, rather than its effect on mitochondrial and metabolic genes. Collectively, these findings suggest that pS727 plays a critical pathological role in modulating the transcriptional activity of STAT3 in hematologic disorders.

Toll-like receptor 2 regulates metabolic reprogramming in gastric cancer via superoxide dismutase 2.

Toll-like receptors (TLRs) play critical roles in host defense after recognition of conserved microbial- and host-derived components, and their dysregulation is a common feature of various inflammation-associated cancers, including gastric cancer (GC). Despite the recent recognition that metabolic reprogramming is a hallmark of cancer, the molecular effectors of altered metabolism during tumorigenesis remain unclear. Here, using bioenergetics function assays on human GC cells, we reveal that ligand-induced activation of TLR2, predominantly through TLR1/2 heterodimer, augments both oxidative phosphorylation (OXPHOS) and glycolysis, with a bias toward glycolytic activity. Notably, DNA microarray-based expression profiling of human cancer cells stimulated with TLR2 ligands demonstrated significant enrichment of gene-sets for oncogenic pathways previously implicated in metabolic regulation, including reactive oxygen species (ROS), p53 and Myc. Moreover, the redox gene encoding the manganese-dependent mitochondrial enzyme, superoxide dismutase (SOD)2, was strongly induced at the mRNA and protein levels by multiple signaling pathways downstream of TLR2, namely JAK-STAT3, JNK MAPK and NF-κB. Furthermore, siRNA-mediated suppression of SOD2 ameliorated the TLR2-induced metabolic shift in human GC cancer cells. Importantly, patient-derived tissue microarrays and bioinformatics interrogation of clinical datasets indicated that upregulated expression of TLR2 and SOD2 were significantly correlated in human GC, and the TLR2-SOD2 axis was associated with multiple clinical parameters of advanced stage disease, including distant metastasis, microvascular invasion and stage, as well as poor survival. Collectively, our findings reveal a novel TLR2-SOD2 axis as a potential biomarker for therapy and prognosis in cancer.

STAT3: a multifaceted oncoprotein.

Signal transducer and activator of transcription (STAT) 3 is a key signalling protein engaged by a multitude of growth factors and cytokines to elicit diverse biological outcomes including cellular growth, differentiation, and survival. The complete loss of STAT3 is not compatible with life and even partial loss of function mutations lead to debilitating pathologies like hyper IgE syndrome. Conversely, augmented STAT3 activity has been reported in as many as 50% of all human tumours. The dogma of STAT3 activity posits that it is a tyrosine phosphorylated transcription factor which modulates the expression of hundreds of genes. However, the regulation and biological consequences of STAT3 activation are far more complex. In addition to tyrosine phosphorylation, STAT3 is decorated with a plethora of post-translational modifications which regulate STAT3's nuclear function in addition to its non-genomic activities. In addition to these emerging complexities in the biochemical regulation of STAT3 activity, recent studies reveal that STAT3 is either oncogenic or a tumour suppressor. This review will explore these complexities.

A phase IIa study of HA-irinotecan, formulation of hyaluronic acid and irinotecan targeting CD44 in extensive-stage small cell lung cancer.

Preclinical studies in small cell lung cancer (SCLC) have shown that hyaluronic acid (HA) can be effectively used to deliver chemotherapy and selectively decrease CD44 expressing (stem cell-like) tumour cells. The current study aimed to replicate these findings and obtain data on safety and activity of HA-irinotecan (HA-IR). Eligible patients with extensive stage SCLC were consented. A safety cohort (n = 5) was treated with HA-IR and Carboplatin (C). Subsequently, the patients were randomised 1:1 to receive experimental (HA-IR + C) or standard (IR + C) treatment, to a maximum of 6 cycles. The second line patients were added to the study and treated with open label HA-IR + C. Tumour response was measured after every 2 cycles. Baseline tumour specimens were stained for CD44s and CD44v6 expression. Circulating tumour cells (CTCs) were enumerated before each treatment cycle. Out of 39 patients screened, 34 were evaluable for the study. The median age was 66 (range 39-83). The overall response rates were 69% and 75% for experimental and standard arms respectively. Median progression free survival was 42 and 28 weeks, respectively (p = 0.892). The treatments were well tolerated. The incidence of grade III/IV diarrhea was more common in the standard arm, while anaemia was more common in the experimental arm. IHC analysis suggested that the patients with CD44s positive tumours may gain survival benefit from HA-IR. HA-IR is well tolerated and active in ES-SCLC. The effect of HA-IR on CD44s + cancer stem-like cells provide an early hint towards a potential novel target.

Cre-dependent DNA recombination activates a STING-dependent innate immune response.

Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell-cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies.

STAT3 supports experimental K-RasG12D-induced murine myeloproliferative neoplasms dependent on serine phosphorylation.

Juvenile myelomonocytic leukemia, acute myeloid leukemia (AML), and other myeloproliferative neoplasms (MPNs) are genetically heterogeneous but frequently display activating mutations in Ras GTPases and activation of signal transducer and activator of transcription 3 (STAT3). Altered STAT3 activity is observed in up to 50% of AML correlating with poor prognosis. Activated STAT proteins, classically associated with tyrosine phosphorylation, support tumor development as transcription factors, but alternative STAT functions independent of tyrosine phosphorylation have been documented, including roles for serine-phosphorylated STAT3 in mitochondria supporting transformation by oncogenic Ras. We examined requirements for STAT3 in experimental murine K-Ras-dependent hematopoietic neoplasia. We show that STAT3 is phosphorylated on S727 but not Y705 in diseased animals. Moreover, a mouse with a point mutation abrogating STAT3 S727 phosphorylation displayed delayed onset and decreased disease severity with significantly extended survival. Activated K-Ras required STAT3 for cytokine-independent growth of myeloid progenitors in vitro, and mitochondrially restricted STAT3 and STAT3-Y705F, both transcriptionally inert mutants, supported factor-independent growth. STAT3 was dispensable for growth of normal or K-Ras-mutant myeloid progenitors in response to cytokines. However, abrogation of STAT3-S727 phosphorylation impaired factor-independent malignant growth. These data document that serine-phosphorylated mitochondrial STAT3 supports neoplastic hematopoietic cell growth induced by K-Ras.

Mitochondrial STAT3: Powering up a potent factor.

The JAK-STAT3 signaling pathway is engaged by many cytokines and growth factor stimuli to control diverse biological processes including proliferation, angiogenesis, survival, immune modulation, and metabolism. For over two decades it has been accepted that STAT3-dependent biology is due to its potency as a transcription factor capable of regulating the expression of many hundreds of genes. However, recent evidence of non-canonical and non-genomic activities of STAT3 has emerged. The most exciting of these activities is its capacity to translocate into the mitochondria where it regulates the activity of the electron transport chain and the opening of the mitochondrial permeability transition pore. These have broad consequences including cell survival and the production of reactive oxygen species and ATP in both normal tissue and under pathological conditions. Despite these fascinating observations there are many key unanswered questions about the mechanism of STAT mitochondrial activity.

Mapping the IMiD-dependent cereblon interactome using BioID-proximity labelling.

Immunomodulatory imide drugs (IMiDs) are central components of therapy for multiple myeloma (MM). IMiDs bind cereblon (CRBN), an adaptor for the CUL4-DDB1-RBX1 E3 ligase to change its substrate specificity and induce degradation of 'neosubstrate' transcription factors that are essential to MM cells. Mechanistic studies to date have largely focussed on mediators of therapeutic activity and insight into clinical IMiD toxicities is less developed. We adopted BioID2-dependent proximity labelling (BioID2-CRBN) to characterise the CRBN interactome in the presence and absence of various IMiDs and the proteasome inhibitor, bortezomib. We aimed to leverage this technology to further map CRBN interactions beyond what has been achieved by conventional proteomic techniques. In support of this approach, analysis of cells expressing BioID2-CRBN following IMiD treatment displayed biotinylation of known CRBN interactors and neosubstrates. We observed that bortezomib alone significantly modifies the CRBN interactome. Proximity labelling also suggested that IMiDs augment the interaction between CRBN and proteins that are not degraded, thus designating 'neointeractors' distinct from previously disclosed 'neosubstrates'. Here we identify Non-Muscle Myosin Heavy Chain IIA (MYH9) as a putative CRBN neointeractor that may contribute to the haematological toxicity of IMiDs. These studies provide proof of concept for proximity labelling technologies in the mechanistic profiling of IMiDs and related E3-ligase-modulating drugs.

Functional crosstalk between type I and II interferon through the regulated expression of STAT1.

Autocrine priming of cells by small quantities of constitutively produced type I interferon (IFN) is a well-known phenomenon. In the absence of type I IFN priming, cells display attenuated responses to other cytokines, such as anti-viral protection in response to IFNgamma. This phenomenon was proposed to be because IFNalpha/beta receptor1 (IFNAR1) is a component of the IFNgamma receptor (IFNGR), but our new data are more consistent with a previously proposed model indicating that regulated expression of STAT1 may also play a critical role in the priming process. Initially, we noticed that DNA binding activity of STAT1 was attenuated in c-Jun(-/-) fibroblasts because they expressed lower levels of STAT1 than wild-type cells. However, expression of STAT1 was rescued by culturing c-Jun(-/-) fibroblasts in media conditioned by wild-type fibroblasts suggesting they secreted a STAT1-inducing factor. The STAT1-inducing factor in fibroblast-conditioned media was IFNbeta, as it was inhibited by antibodies to IFNAR1, or when IFNbeta expression was knocked down in wild-type cells. IFNAR1(-/-) fibroblasts, which cannot respond to this priming, also expressed reduced levels of STAT1, which correlated with their poor responses to IFNgamma. The lack of priming in IFNAR1(-/-) fibroblasts was compensated by over-expression of STAT1, which rescued molecular responses to IFNgamma and restored the ability of IFNgamma to induce protective anti-viral immunity. This study provides a comprehensive description of the molecular events involved in priming by type I IFN. Adding to the previous working model that proposed an interaction between type I and II IFN receptors, our work and that of others demonstrates that type I IFN primes IFNgamma-mediated immune responses by regulating expression of STAT1. This may also explain how type I IFN can additionally prime cells to respond to a range of other cytokines that use STAT1 (e.g., IL-6, M-CSF, IL-10) and suggests a potential mechanism for the changing levels of STAT1 expression observed during viral infection.

Circulating Tumour DNA (ctDNA) as a Predictor of Clinical Outcome in Non-Small Cell Lung Cancer Undergoing Targeted Therapies: A Systematic Review and Meta-Analysis.

BACKGROUND: Liquid biopsy (LB) analysis using (ctDNA)/cell-free DNA (cfDNA) is an emerging alternative to tissue profiling in (NSCLC). LB is used to guide treatment decisions, detect resistance mechanisms, and predicts responses, and, therefore, outcomes. This systematic review and meta-analysis evaluated the impact of LB quantification on clinical outcomes in molecularly altered advanced NSCLC undergoing targeted therapies. METHODS: We searched Embase, MEDLINE, PubMed, and Cochrane Database, between 1 January 2020 and 31 August 2022. The primary outcome was progression-free survival (PFS). Secondary outcomes included overall survival (OS), objective response rate (ORR), sensitivity, and specificity. Age stratification was performed based on the mean age of the individual study population. The quality of studies was assessed using the Newcastle-Ottawa Scale (NOS). RESULTS: A total of 27 studies (3419 patients) were included in the analysis. Association of baseline ctDNA with PFS was reported in 11 studies (1359 patients), while that of dynamic changes with PFS was reported in 16 studies (1659 patients). Baseline ctDNA-negative patients had a trend towards improved PFS (pooled hazard ratio [pHR] = 1.35; 95%CI: 0.83-1.87; p < 0.001; I2 = 96%) than ctDNA-positive patients. Early reduction/clearance of ctDNA levels after treatment was related to improved PFS (pHR = 2.71; 95%CI: 1.85-3.65; I2 = 89.4%) compared to those with no reduction/persistence in ctDNA levels. The sensitivity analysis based on study quality (NOS) demonstrated improved PFS only for good [pHR = 1.95; 95%CI: 1.52-2.38] and fair [pHR = 1.99; 95%CI: 1.09-2.89] quality studies, but not for poor quality studies. There was, however, a high level of heterogeneity (I2 = 89.4%) along with significant publication bias in our analysis. CONCLUSIONS: This large systematic review, despite heterogeneity, found that baseline negative ctDNA levels and early reduction in ctDNA following treatment could be strong prognostic markers for PFS and OS in patients undergoing targeted therapies for advanced NSCLC. Future randomised clinical trials should incorporate serial ctDNA monitoring to further establish the clinical utility in advanced NSCLC management.

Potential Avenues for Exosomal Isolation and Detection Methods to Enhance Small-Cell Lung Cancer Analysis.

Around the world, lung cancer has long been the main factor in cancer-related deaths, with small-cell lung cancer (SCLC) being the deadliest form of lung cancer. Cancer cell-derived exosomes and exosomal miRNAs are considered promising biomarkers for diagnosing and prognosis of various diseases, including SCLC. Due to the rapidity of SCLC metastasis, early detection and diagnosis can offer better diagnosis and prognosis and therefore increase the patient's chances of survival. Over the past several years, many methodologies have been developed for analyzing non-SCLC-derived exosomes. However, minimal advances have been made in SCLC-derived exosome analysis methodologies. This Review discusses the epidemiology and prominent biomarkers of SCLC. Followed by a discussion about the effective strategies for isolating and detecting SCLC-derived exosomes and exosomal miRNA, highlighting the critical challenges and limitations of current methodologies. Finally, an overview is provided detailing future perspectives for exosome-based SCLC research.

MYC drives platinum resistant SCLC that is overcome by the dual PI3K-HDAC inhibitor fimepinostat.

BACKGROUND: Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer with an appalling overall survival of less than 5% (Zimmerman et al. J Thor Oncol 14:768-83, 2019). Patients typically respond to front line platinum-based doublet chemotherapy, but almost universally relapse with drug resistant disease. Elevated MYC expression is common in SCLC and has been associated with platinum resistance. This study evaluates the capacity of MYC to drive platinum resistance and through screening identifies a drug capable of reducing MYC expression and overcoming resistance. METHODS: Elevated MYC expression following the acquisition of platinum resistance in vitro and in vivo was assessed. Moreover, the capacity of enforced MYC expression to drive platinum resistance was defined in SCLC cell lines and in a genetically engineered mouse model that expresses MYC specifically in lung tumors. High throughput drug screening was used to identify drugs able to kill MYC-expressing, platinum resistant cell lines. The capacity of this drug to treat SCLC was defined in vivo in both transplant models using cell lines and patient derived xenografts and in combination with platinum and etoposide chemotherapy in an autochthonous mouse model of platinum resistant SCLC. RESULTS: MYC expression is elevated following the acquisition of platinum resistance and constitutively high MYC expression drives platinum resistance in vitro and in vivo. We show that fimepinostat decreases MYC expression and that it is an effective single agent treatment for SCLC in vitro and in vivo. Indeed, fimepinostat is as effective as platinum-etoposide treatment in vivo. Importantly, when combined with platinum and etoposide, fimepinostat achieves a significant increase in survival. CONCLUSIONS: MYC is a potent driver of platinum resistance in SCLC that is effectively treated with fimepinostat.

A STAT3 protein complex required for mitochondrial mRNA stability and cancer.

Signal transducer and activator of transcription 3 (STAT3) is a potent transcription factor necessary for life whose activity is corrupted in diverse diseases, including cancer. STAT3 biology was presumed to be entirely dependent on its activity as a transcription factor until the discovery of a mitochondrial pool of STAT3, which is necessary for normal tissue function and tumorigenesis. However, the mechanism of this mitochondrial activity remained elusive. This study uses immunoprecipitation and mass spectrometry to identify a complex containing STAT3, leucine-rich pentatricopeptide repeat containing (LRPPRC), and SRA stem-loop-interacting RNA-binding protein (SLIRP) that is required for the stability of mature mitochondrially encoded mRNAs and transport to the mitochondrial ribosome. Moreover, we show that this complex is enriched in patients with lung adenocarcinoma and that its deletion inhibits the growth of lung cancer in vivo, providing therapeutic opportunities through the specific targeting of the mitochondrial activity of STAT3.

Targeting Menin disrupts the KMT2A/B and polycomb balance to paradoxically activate bivalent genes.

Precise control of activating H3K4me3 and repressive H3K27me3 histone modifications at bivalent promoters is essential for normal development and frequently corrupted in cancer. By coupling a cell surface readout of bivalent MHC class I gene expression with whole-genome CRISPR-Cas9 screens, we identify specific roles for MTF2-PRC2.1, PCGF1-PRC1.1 and Menin-KMT2A/B complexes in maintaining bivalency. Genetic loss or pharmacological inhibition of Menin unexpectedly phenocopies the effects of polycomb disruption, resulting in derepression of bivalent genes in both cancer cells and pluripotent stem cells. While Menin and KMT2A/B contribute to H3K4me3 at active genes, a separate Menin-independent function of KMT2A/B maintains H3K4me3 and opposes polycomb-mediated repression at bivalent genes. Release of KMT2A from active genes following Menin targeting alters the balance of polycomb and KMT2A at bivalent genes, facilitating gene activation. This functional partitioning of Menin-KMT2A/B complex components reveals therapeutic opportunities that can be leveraged through inhibition of Menin.

Ligand-dependent hedgehog signaling maintains an undifferentiated, malignant osteosarcoma phenotype.

TP53 and RB1 loss-of-function mutations are common in osteosarcoma. During development, combined loss of TP53 and RB1 function leads to downregulation of autophagy and the aberrant formation of primary cilia, cellular organelles essential for the transmission of canonical Hedgehog (Hh) signaling. Excess cilia formation then leads to hypersensitivity to Hedgehog (Hh) ligand signaling. In mouse and human models, we now show that osteosarcomas with mutations in TP53 and RB1 exhibit enhanced ligand-dependent Hh pathway activation through Smoothened (SMO), a transmembrane signaling molecule required for activation of the canonical Hh pathway. This dependence is mediated by hypersensitivity to Hh ligand and is accompanied by impaired autophagy and increased primary cilia formation and expression of Hh ligand in vivo. Using a conditional genetic mouse model of Trp53 and Rb1 inactivation in osteoblast progenitors, we further show that deletion of Smo converts the highly malignant osteosarcoma phenotype to benign, well differentiated bone tumors. Conversely, conditional overexpression of SHH ligand, or a gain-of-function SMO mutant in committed osteoblast progenitors during development blocks terminal bone differentiation. Finally, we demonstrate that the SMO antagonist sonidegib (LDE225) induces growth arrest and terminal differentiation in vivo in osteosarcomas that express primary cilia and Hh ligand combined with mutations in TP53. These results provide a mechanistic framework for aberrant Hh signaling in osteosarcoma based on defining mutations in the tumor suppressor, TP53.

Nongenomic STAT5-dependent effects on Golgi apparatus and endoplasmic reticulum structure and function.

We report unexpected nongenomic functions of signal transducer and activator of transcription (STAT) 5 species in the cytoplasm aimed at preserving the structure and function of the Golgi apparatus and rough endoplasmic reticulum (ER) in vascular cells. Immunoimaging and green fluorescent protein-tagged-STAT5a protein localization studies showed the constitutive association of nonphosphorylated STAT5a, and to a lesser extent STAT5b, with the Golgi apparatus and of STAT5a with centrosomes in human pulmonary arterial endothelial and smooth muscle cells. Acute knockdown of STAT5a/b species using small interfering RNAs (siRNAs), including in the presence of an mRNA synthesis inhibitor (5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole), produced a dramatic phenotype within 1 day, consisting of dilatation and fragmentation of Golgi cisternae, a marked tubule-to-cyst change in the ER, increased accumulation of reticulon-4 (RTN4)/Nogo-B and atlastin-3 (ATL3) at cyst-zone boundaries, cystic separation of the outer and inner nuclear membranes, accompanied by scalloped/lunate distortion of the nucleus, with accumulation of RTN4 on convex sides of distorted nuclei. These cells showed inhibition of vesicular stomatitis virus G protein glycoprotein trafficking, mitochondrial fragmentation, and reduced mitochondrial function. STAT5a/b(-/-) mouse embryo fibroblasts also showed altered ER/Golgi dynamics. RTN4 knockdown using siRNA did not affect development of the cystic phenotype; ATL3 siRNA led to effacement of cyst-zone boundaries. In magnetic-bead cross-immunopanning assays, ATL3 bound both STAT5a and STAT5b. Remarkably, this novel cystic ER/lunate nucleus phenotype was characteristic of vascular cells in arterial lesions of idiopathic pulmonary hypertension, an unrelentingly fatal human disease. These data provide evidence of a STAT-family protein regulating the structure of a cytoplasmic organelle and implicate this mechanism in the pathogenesis of a human disease.

Lineage-restricted neoplasia driven by Myc defaults to small cell lung cancer when combined with loss of p53 and Rb in the airway epithelium.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by loss of function TP53 and RB1 mutations in addition to mutations in other oncogenes including MYC. Overexpression of MYC together with Trp53 and Rb1 loss in pulmonary neuroendocrine cells of the mouse lung drives an aggressive neuroendocrine low variant subtype of SCLC. However, the transforming potential of MYC amplification alone on airway epithelium is unclear. Therefore, we selectively and conditionally overexpressed MYC stochastically throughout the airway or specifically in neuroendocrine, club, or alveolar type II cells in the adult mouse lung. We observed that MYC overexpression induced carcinoma in situ which did not progress to invasive disease. The formation of adenoma or SCLC carcinoma in situ was dependent on the cell of origin. In contrast, MYC overexpression combined with conditional deletion of both Trp53 and Rb1 exclusively gave rise to SCLC, irrespective of the cell lineage of origin. However, cell of origin influenced disease latency, metastatic potential, and the transcriptional profile of the SCLC phenotype. Together this reveals that MYC overexpression alone provides a proliferative advantage but when combined with deletion of Trp53 and Rb1 it facilitates the formation of aggressive SCLC from multiple cell lineages.