The MYC-Regulated RNA-Binding Proteins hnRNPC and LARP1 Are Drivers of Multiple Myeloma Cell Growth and Disease Progression and Negatively Predict Patient Survival.

Multiple myeloma (MM) is a malignant plasma cell disorder in which the MYC oncogene is frequently dysregulated. Due to its central role, MYC has been proposed as a drug target; however, the development of a clinically applicable molecule modulating MYC activity remains an unmet challenge. Consequently, an alternative is the development of therapeutic options targeting proteins located downstream of MYC. Therefore, we aimed to identify undescribed MYC-target proteins in MM cells using Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and mass spectrometry. We revealed a cluster of proteins associated with the regulation of translation initiation. Herein, the RNA-binding proteins Heterogeneous Nuclear Ribonucleoprotein C (hnRNPC) and La Ribonucleoprotein 1 (LARP1) were predominantly downregulated upon MYC depletion. CRISPR-mediated knockout of either hnRNPC or LARP1 in conjunction with redundant LARP family proteins resulted in a proliferative disadvantage for MM cells. Moreover, high expression levels of these proteins correlate with high MYC expression and with poor survival and disease progression in MM patients. In conclusion, our study provides valuable insights into MYC's role in translation initiation by identifying hnRNPC and LARP1 as proliferation drivers of MM cells and as both predictive factors for survival and disease progression in MM patients.

Translatome proteomics identifies autophagy as a resistance mechanism to on-target FLT3 inhibitors in acute myeloid leukemia.

Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in 25 % of acute myeloid leukemia (AML) patients, drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and occurs in the absence of identifiable genetic causes. This suggests that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. Here, we systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD + AML using recently developed functional translatome proteomics (measuring changes in the nascent proteome) with phosphoproteomics. This pinpointed AKT-mTORC1-ULK1-dependent autophagy as a dominant resistance mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD + cells in vitro and in primary human AML cells ex vivo. Pharmacological or genetic inhibition of autophagy increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In mice xenografted with FLT3-ITD + AML cells, co-treatment with oral FLT3 and autophagy inhibitors synergistically impaired leukemia progression and extended overall survival. Our findings identify a molecular mechanism responsible for primary FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction.

Identification of the Cysteine Protease Legumain as a Potential Chronic Hypoxia-Specific Multiple Myeloma Target Gene.

Multiple myeloma (MM) is the second most common hematologic malignancy, which is characterized by clonal proliferation of neoplastic plasma cells in the bone marrow. This microenvironment is characterized by low oxygen levels (1-6% O2), known as hypoxia. For MM cells, hypoxia is a physiologic feature that has been described to promote an aggressive phenotype and to confer drug resistance. However, studies on hypoxia are scarce and show little conformity. Here, we analyzed the mRNA expression of previously determined hypoxia markers to define the temporal adaptation of MM cells to chronic hypoxia. Subsequent analyses of the global proteome in MM cells and the stromal cell line HS-5 revealed hypoxia-dependent regulation of proteins, which directly or indirectly upregulate glycolysis. In addition, chronic hypoxia led to MM-specific regulation of nine distinct proteins. One of these proteins is the cysteine protease legumain (LGMN), the depletion of which led to a significant growth disadvantage of MM cell lines that is enhanced under hypoxia. Thus, herein, we report a methodologic strategy to examine MM cells under physiologic hypoxic conditions in vitro and to decipher and study previously masked hypoxia-specific therapeutic targets such as the cysteine protease LGMN.

Role of LTBP4 in alveolarization, angiogenesis, and fibrosis in lungs.

Deficiency of the extracellular matrix protein latent transforming growth factor-ß (TGF-ß)-binding protein-4 (LTBP4) results in lack of intact elastic fibers, which leads to disturbed pulmonary development and lack of normal alveolarization in humans and mice. Formation of alveoli and alveolar septation in pulmonary development requires the concerted interaction of extracellular matrix proteins, growth factors such as TGF-ß, fibroblasts, and myofibroblasts to promote elastogenesis as well as vascular formation in the alveolar septae. To investigate the role of LTBP4 in this context, lungs of LTBP4-deficient (Ltbp4-/-) mice were analyzed in close detail. We elucidate the role of LTBP4 in pulmonary alveolarization and show that three different, interacting mechanisms might contribute to alveolar septation defects in Ltbp4-/- lungs: 1) absence of an intact elastic fiber network, 2) reduced angiogenesis, and 3) upregulation of TGF-ß activity resulting in profibrotic processes in the lung.

Ltbp4 regulates Pdgfrß expression via TGFß-dependent modulation of Nrf2 transcription factor function.

Latent transforming growth factor beta binding protein 4 (LTBP4) belongs to the fibrillin/LTBP family of proteins and plays an important role as a structural component of extracellular matrix (ECM) and local regulator of TGFß signaling. We have previously reported that Ltbp4S knock out mice (Ltbp4S-/-) develop centrilobular emphysema reminiscent of late stage COPD, which could be partially rescued by inactivating the antioxidant protein Sestrin 2 (Sesn2). More recent studies showed that Sesn2 knock out mice upregulate Pdgfrß-controlled alveolar maintenance programs that protect against cigarette smoke induced pulmonary emphysema. Based on this, we hypothesized that the emphysema of Ltbp4S-/- mice is primarily caused by defective Pdgfrß signaling. Here we show that LTBP4 induces Pdgfrß signaling by inhibiting the antioxidant Nrf2/Keap1 pathway in a TGFß-dependent manner. Overall, our data identified Ltbp4 as a major player in lung remodeling and injury repair.

Cytochrome P450 enzymes but not NADPH oxidases are the source of the NADPH-dependent lucigenin chemiluminescence in membrane assays.

Measuring NADPH oxidase (Nox)-derived reactive oxygen species (ROS) in living tissues and cells is a constant challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized detection techniques. In search for a presumably easy, versatile, sensitive and specific technique, numerous studies have used NADPH-stimulated assays in membrane fractions which have been suggested to reflect Nox activity. However, we previously found an unaltered activity with these assays in triple Nox knockout mouse (Nox1-Nox2-Nox4-/-) tissue and cells compared to wild type. Moreover, the high ROS production of intact cells overexpressing Nox enzymes could not be recapitulated in NADPH-stimulated membrane assays. Thus, the signal obtained in these assays has to derive from a source other than NADPH oxidases. Using a combination of native protein electrophoresis, NADPH-stimulated assays and mass spectrometry, mitochondrial proteins and cytochrome P450 were identified as possible source of the assay signal. Cells lacking functional mitochondrial complexes, however, displayed a normal activity in NADPH-stimulated membrane assays suggesting that mitochondrial oxidoreductases are unlikely sources of the signal. Microsomes overexpressing P450 reductase, cytochromes b5 and P450 generated a NADPH-dependent signal in assays utilizing lucigenin, L-012 and dihydroethidium (DHE). Knockout of the cytochrome P450 reductase by CRISPR/Cas9 technology (POR-/-) in HEK293 cells overexpressing Nox4 or Nox5 did not interfere with ROS production in intact cells. However, POR-/- abolished the signal in NADPH-stimulated assays using membrane fractions from the very same cells. Moreover, membranes of rat smooth muscle cells treated with angiotensin II showed an increased NADPH-dependent signal with lucigenin which was abolished by the knockout of POR but not by knockout of p22phox. IN CONCLUSION: the cytochrome P450 system accounts for the majority of the signal of Nox activity chemiluminescence based assays.

Sestrin-2, a repressor of PDGFRß signalling, promotes cigarette-smoke-induced pulmonary emphysema in mice and is upregulated in individuals with COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. COPD is caused by chronic exposure to cigarette smoke and/or other environmental pollutants that are believed to induce reactive oxygen species (ROS) that gradually disrupt signalling pathways responsible for maintaining lung integrity. Here we identify the antioxidant protein sestrin-2 (SESN2) as a repressor of PDGFRß signalling, and PDGFRß signalling as an upstream regulator of alveolar maintenance programmes. In mice, the mutational inactivation of Sesn2 prevents the development of cigarette-smoke-induced pulmonary emphysema by upregulating PDGFRß expression via a selective accumulation of intracellular superoxide anions (O2(-)). We also show that SESN2 is overexpressed and PDGFRß downregulated in the emphysematous lungs of individuals with COPD and to a lesser extent in human lungs of habitual smokers without COPD, implicating a negative SESN2-PDGFRß interrelationship in the pathogenesis of COPD. Taken together, our results imply that SESN2 could serve as both a biomarker and as a drug target in the clinical management of COPD.

Inactivation of sestrin 2 induces TGF-beta signaling and partially rescues pulmonary emphysema in a mouse model of COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Cigarette smoking has been identified as one of the major risk factors and several predisposing genetic factors have been implicated in the pathogenesis of COPD, including a single nucleotide polymorphism (SNP) in the latent transforming growth factor (TGF)-beta binding protein 4 (Ltbp4)-encoding gene. Consistent with this finding, mice with a null mutation of the short splice variant of Ltbp4 (Ltbp4S) develop pulmonary emphysema that is reminiscent of COPD. Here, we report that the mutational inactivation of the antioxidant protein sestrin 2 (sesn2) partially rescues the emphysema phenotype of Ltbp4S mice and is associated with activation of the TGF-beta and mammalian target of rapamycin (mTOR) signal transduction pathways. The results suggest that sesn2 could be clinically relevant to patients with COPD who might benefit from antagonists of sestrin function.

Transcription factor PROX1 induces colon cancer progression by promoting the transition from benign to highly dysplastic phenotype.

The Drosophila transcription factor Prospero functions as a tumor suppressor, and it has been suggested that the human counterpart of Prospero, PROX1, acts similarly in human cancers. However, we show here that PROX1 promotes dysplasia in colonic adenomas and colorectal cancer progression. PROX1 expression marks the transition from benign colon adenoma to carcinoma in situ, and its loss inhibits growth of human colorectal tumor xenografts and intestinal adenomas in Apc(min/+) mice, while its transgenic overexpression promotes colorectal tumorigenesis. Furthermore, in intestinal tumors PROX1 is a direct and dose-dependent target of the beta-catenin/TCF signaling pathway, responsible for the neoplastic transformation. Our data underscore the complexity of cancer pathogenesis and implicate PROX1 in malignant tumor progression through the regulation of cell polarity and adhesion.

Disruption of LTBP-4 function reduces TGF-beta activation and enhances BMP-4 signaling in the lung.

Disruption of latent TGF-beta binding protein (LTBP)-4 expression in the mouse leads to abnormal lung development and colorectal cancer. Lung fibroblasts from these mice produced decreased amounts of active TGF-beta, whereas secretion of latent TGF-beta was significantly increased. Expression and secretion of TGF-beta2 and -beta3 increased considerably. These results suggested that TGF-beta activation but not secretion would be severely impaired in LTBP-4 -/- fibroblasts. Microarrays revealed increased expression of bone morphogenic protein (BMP)-4 and decreased expression of its inhibitor gremlin. This finding was accompanied by enhanced expression of BMP-4 target genes, inhibitors of differentiation 1 and 2, and increased deposition of fibronectin-rich extracellular matrix. Accordingly, increased expression of BMP-4 and decreased expression of gremlin were observed in mouse lung. Transfection of LTBP-4 rescued the -/- fibroblast phenotype, while LTBP-1 was inefficient. Treatment with active TGF-beta1 rescued BMP-4 and gremlin expression to wild-type levels. Our results indicate that the lack of LTBP-4-mediated targeting and activation of TGF-beta1 leads to enhanced BMP-4 signaling in mouse lung.

Disruption of the gene encoding the latent transforming growth factor-beta binding protein 4 (LTBP-4) causes abnormal lung development, cardiomyopathy, and colorectal cancer.

Transforming growth factor-betas (TGF-betas) are multifunctional growth factors that are secreted as inactive (latent) precursors in large protein complexes. These complexes include the latency-associated propeptide (LAP) and a latent transforming growth factor-beta binding protein (LTBP). Four isoforms of LTBPs (LTBP-1-LTBP-4) have been cloned and are believed to be structural components of connective tissue microfibrils and local regulators of TGF-beta tissue deposition and signaling. By using a gene trap strategy that selects for integrations into genes induced transiently during early mouse development, we have disrupted the mouse homolog of the human LTBP-4 gene. Mice homozygous for the disrupted allele develop severe pulmonary emphysema, cardiomyopathy, and colorectal cancer. These highly tissue-specific abnormalities are associated with profound defects in the elastic fiber structure and with a reduced deposition of TGF-beta in the extracellular space. As a consequence, epithelial cells have reduced levels of phosphorylated Smad2 proteins, overexpress c-myc, and undergo uncontrolled proliferation. This phenotype supports the predicted dual role of LTBP-4 as a structural component of the extracellular matrix and as a local regulator of TGF-beta tissue deposition and signaling.