Requirement of the NF- B Subunit p65/RelA for K-Ras-Induced Lung Tumorigenesis

K-Ras-induced lung cancer is a very common disease, for which there are currently no effective therapies. Because therapy directly targeting the activity of oncogenic Ras has been unsuccessful, a different approach for novel therapy design is to identify critical Ras downstream oncogenic targets. Given that oncogenic Ras proteins activate the transcription factor NF-κB, and the importance of NF-κB in oncogenesis, we hypothesized that NF-κB would be an important K-Ras target in lung cancer. To address this hypothesis, we generated an NF-κB-EGFP reporter mouse model of K-Ras-induced lung cancer and determined that K-Ras activates NF-κB in lung tumors in situ. Furthermore, a mouse model was generated where activation of oncogenic K-Ras in lung cells was coupled with inactivation of the NF-κB subunit p65/RelA. In this model, deletion of p65/RelA reduces the number of K-Ras-induced lung tumors both in the presence and absence of the tumor suppressor p53. Lung tumors with loss of p65/RelA have higher numbers of apoptotic cells, reduced spread and lower grade. Using lung cell lines expressing oncogenic K-Ras, we show that NF-κB is activated in these cells in a K-Ras-dependent manner and that NF-κB activation by K-Ras requires IKKβ kinase activity. Taken together, these results demonstrate the importance of the NF-κB subunit p65/RelA in K-Ras induced lung transformation and identify IKKβ as a potential therapeutic target for K-Ras-induced lung cancer

IKKβ kinase promotes stemness, migration, and invasion in KRAS-driven lung adenocarcinoma cells

KRAS oncogenic mutations are widespread in lung cancer and, because direct targeting of KRAS has proven to be challenging, KRAS-driven cancers lack effective therapies. One alternative strategy for developing KRAS targeted therapies is to identify downstream targets involved in promoting important malignant features, such as the acquisition of a cancer stem-like and metastatic phenotype. Based on previous studies showing that KRAS activates nuclear factor kappa-B (NF-κB) through inhibitor of nuclear factor kappa-B kinase β (IKKβ) to promote lung tumourigenesis, we hypothesized that inhibition of IKKβ would reduce stemness, migration and invasion of KRAS-mutant human lung cancer cells. We show that KRAS-driven lung tumoursphere-derived cells exhibit stemness features and increased IKKβ kinase activity. IKKβ targeting by different approaches reduces the expression of stemness-associated genes, tumoursphere formation, and self-renewal, and preferentially impairs the proliferation of KRAS-driven lung tumoursphere-derived cells. Moreover, we show that IKKβ targeting reduces tumour cell migration and invasion, potentially by regulating both expression and activity of matrix metalloproteinase 2 (MMP2). In conclusion, our results indicate that IKKβ is an important mediator of KRAS-induced stemness and invasive features in lung cancer, and, therefore, might constitute a promising strategy to lower recurrence rates, reduce metastatic dissemination, and improve survival of lung cancer patients with KRAS-driven disease

C/EBPa controls acquisition and maintenance of adult hematopoietic stem cell quiescence

Summary In blood, transcription factor C/EBPa is essential for myeloid differentiation and has been implicated in regulating self-renewal of fetal liver (FL) hematopoietic stem cells (HSCs). However, its function in adult HSCs has remained unknown. Here, using an inducible knockout model we found that C/EBPa deficient adult HSCs underwent a pronounced expansion with enhanced proliferation, characteristics resembling FL HSCs. Consistently, transcription profiling of C/EBPa deficient HSCs revealed a gene expression programme similar to FL HSCs. Moreover we observed that age-specific C/EBPa expression correlated with its inhibitory effect on HSC cell cycle. Mechanistically we identified N-Myc as C/EBPa downstream target, and loss of C/EBPa resulted in de-repression of N-Myc. Our data establish C/EBPa as a central determinant in the switch from fetal to adult HSCs

Unsuspected role of the brain morphogenetic gene Otx1 in hematopoiesis

Otx1 belongs to the paired class of homeobox genes and plays a pivotal role in brain development. Here, we show that Otx1 is expressed in hematopoietic pluripotent and erythroid progenitor cells. Moreover, bone marrow cells from mice lacking Otx1 exhibit a cell-autonomous impairment of the erythroid compartment. In agreement with these results, molecular analysis revealed decreased levels of erythroid genes that include the SCL and GATA-1 transcription factors. Accordingly, a gain of function of SCL rescues the erythroid deficiency in Otx1-/- mice. Taken together, our findings indicate a function for Otx1 in the regulation of blood cell production. There is growing evidence suggesting that common cellular and molecular mechanisms orchestrate differentiation in various tissues. Homeobox-containing genes seem to be strong candidate genes to regulate a number of developmental processes, including neurogenesis and hematopoiesis. Members of the Otx family (Otx1, Otx2, Otx3, and Crx) are the vertebrate homologues of the Drosophila head gap gene orthodenticle and encode transcription factors containing a bicoid-like homeodomain. They are temporally and spatially regulated during development and seem to be required for proper head and sense organ patterning. Otx1, Otx2, and Otx3 show partially overlapping, but distinct expression patterns, and Otx2, the first to be activated during development, plays a major role in gastrulation and in the early specification of the anterior neural plate. In contrast, Otx1 shows a later onset and is involved in corticogenesis, sense organ development, and pituitary function. Mice bearing targeted deletion of Otx1 are affected by a permanent epileptic phenotype and show multiple brain abnormalities and morphological defects of the acoustic and visual sense organs. In addition, at the prepubescent stage, they exhibit transient dwarfism and hypogonadism because of low levels of pituitary hormones. In the present study, we have investigated whether Otx1 also plays a role in blood cell production, as several homeobox genes of different families are involved in normal and/or malignant hematopoiesis

Treatment of Chronic Myelogenous Leukemia by Blocking Cytokine Alterations Found in Normal Stem and Progenitor Cells

SummaryLeukemic cells disrupt normal patterns of blood cell formation, but little is understood about the mechanism. We investigated whether leukemic cells alter functions of normal hematopoietic stem and progenitor cells. Exposure to chronic myelogenous leukemia (CML) caused normal mouse hematopoietic progenitor cells to divide more readily, altered their differentiation, and reduced their reconstitution and self-renewal potential. Interestingly, the normal bystander cells acquired gene expression patterns resembling their malignant counterparts. Therefore, much of the leukemia signature is mediated by extrinsic factors. Indeed, IL-6 was responsible for most of these changes. Compatible results were obtained when human CML were cultured with normal human hematopoietic progenitor cells. Furthermore, neutralization of IL-6 prevented these changes and treated the disease

Acetylation of C/EBP alpha inhibits its granulopoietic function

CCAAT/enhancer-binding protein alpha (C/EBP alpha) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBP alpha at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBP alpha DNA-binding ability and modulates C/EBP alpha transcriptional activity. Acetylated C/EBP alpha is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)-mediated granulocytic differentiation of 32Dcl3 cells. C/EBP alpha mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBP alpha-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBP alpha and demonstrate the importance of C/EBP alpha acetylation in myeloid differentiation

CCAAT/Enhancer Binding Protein β Is Dispensable for Development of Lung Adenocarcinoma

Lung cancer is the leading cause of cancer death worldwide. Although disruption of normal proliferation and differentiation is a vital component of tumorigenesis, the mechanisms of this process in lung cancer are still unclear. A transcription factor, C/EBPβ is a critical regulator of proliferation and/or differentiation in multiple tissues. In lung, C/EBPβ is expressed in alveolar pneumocytes and bronchial epithelial cells; however, its roles on normal lung homeostasis and lung cancer development have not been well described. Here we investigated whether C/EBPβ is required for normal lung development and whether its aberrant expression and/or activity contribute to lung tumorigenesis. We showed that C/EBPβ was expressed in both human normal pneumocytes and lung adenocarcinoma cell lines. We found that overall lung architecture was maintained in Cebpb knockout mice. Neither overexpression of nuclear C/EBPβ nor suppression of CEBPB expression had significant effects on cell proliferation. C/EBPβ expression and activity remained unchanged upon EGF stimulation. Furthermore, deletion of Cebpb had no impact on lung tumor burden in a lung specific, conditional mutant EGFR lung cancer mouse model. Analyses of data from The Cancer Genome Atlas (TCGA) revealed that expression, promoter methylation, or copy number of CEBPB was not significantly altered in human lung adenocarcinoma. Taken together, our data suggest that C/EBPβ is dispensable for development of lung adenocarcinoma

Fatty acid synthase mediates EGFR palmitoylation in EGFR mutated non‐small cell lung cancer

Abstract Metabolic reprogramming is widely known as a hallmark of cancer cells to allow adaptation of cells to sustain survival signals. In this report, we describe a novel oncogenic signaling pathway exclusively acting in mutated epidermal growth factor receptor (EGFR) non‐small cell lung cancer (NSCLC) with acquired tyrosine kinase inhibitor (TKI) resistance. Mutated EGFR mediates TKI resistance through regulation of the fatty acid synthase (FASN), which produces 16‐C saturated fatty acid palmitate. Our work shows that the persistent signaling by mutated EGFR in TKI‐resistant tumor cells relies on EGFR palmitoylation and can be targeted by Orlistat, an FDA‐approved anti‐obesity drug. Inhibition of FASN with Orlistat induces EGFR ubiquitination and abrogates EGFR mutant signaling, and reduces tumor growths both in culture systems and in vivo. Together, our data provide compelling evidence on the functional interrelationship between mutated EGFR and FASN and that the fatty acid metabolism pathway is a candidate target for acquired TKI‐resistant EGFR mutant NSCLC patients

Identification of a targetable KRAS-mutant epithelial population in non-small cell lung cancer

Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencingin aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas