Synthetic lethality of drug-induced polyploidy and BCL-2 inhibition in lymphoma.

Spontaneous whole genome duplication and the adaptive mutations that disrupt genome integrity checkpoints are infrequent events in B cell lymphomas. This suggests that lymphomas might be vulnerable to therapeutics that acutely trigger genomic instability and polyploidy. Here, we report a therapeutic combination of inhibitors of the Polo-like kinase 4 and BCL-2 that trigger genomic instability and cell death in aggressive lymphomas. The synthetic lethality is selective for tumor cells and spares vital organs. Mechanistically, inhibitors of Polo-like kinase 4 impair centrosome duplication and cause genomic instability. The elimination of polyploid cells largely depends on the pro-apoptotic BAX protein. Consequently, the combination of drugs that induce polyploidy with the BCL-2 inhibitor Venetoclax is highly synergistic and safe against xenograft and PDX models. We show that B cell lymphomas are ill-equipped for acute, therapy-induced polyploidy and that BCL-2 inhibition further enhances the removal of polyploid lymphoma cells.

ASN007 is a selective ERK1/2 inhibitor with preferential activity against RAS-and RAF-mutant tumors.

Inhibition of the extracellular signal-regulated kinases ERK1 and ERK2 (ERK1/2) offers a promising therapeutic strategy in cancers harboring activated RAS/RAF/MEK/ERK signaling pathways. Here, we describe an orally bioavailable and selective ERK1/2 inhibitor, ASN007, currently in clinical development for the treatment of cancer. In preclinical studies, ASN007 shows strong antiproliferative activity in tumors harboring mutations in BRAF and RAS (KRAS, NRAS, and HRAS). ASN007 demonstrates activity in a BRAFV600E mutant melanoma tumor model that is resistant to BRAF and MEK inhibitors. The PI3K inhibitor copanlisib enhances the antiproliferative activity of ASN007 both in vitro and in vivo due to dual inhibition of RAS/MAPK and PI3K survival pathways. Our data provide a rationale for evaluating ASN007 in RAS/RAF-driven tumors as well as a mechanistic basis for combining ASN007 with PI3K inhibitors.

BET Inhibition-Induced GSK3ß Feedback Enhances Lymphoma Vulnerability to PI3K Inhibitors.

The phosphatidylinositol 3 kinase (PI3K)-glycogen synthase kinase ß (GSK3ß) axis plays a central role in MYC-driven lymphomagenesis, and MYC targeting with bromodomain and extraterminal protein family inhibitors (BETi) is a promising treatment strategy in lymphoma. In a high-throughput combinatorial drug screening experiment, BETi enhance the antiproliferative effects of PI3K inhibitors in a panel of diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma cell lines. BETi or MYC silencing upregulates several PI3K pathway genes and induces GSK3ß S9 inhibitory phosphorylation, resulting in increased ß-catenin protein abundance. Furthermore, BETi or MYC silencing increases GSK3ß S9 phosphorylation levels and ß-catenin protein abundance through downregulating the E2 ubiquitin conjugating enzymes UBE2C and UBE2T. In a mouse xenograft DLBCL model, BETi decrease MYC, UBE2C, and UBE2T and increase phospho-GSK3ß S9 levels, enhancing the anti-proliferative effect of PI3K inhibitors. Our study reveals prosurvival feedbacks induced by BETi involving GSK3ß regulation, providing a mechanistic rationale for combination strategies.

Arl13b and the non-muscle myosin heavy chain IIA are required for circular dorsal ruffle formation and cell migration.

The Arf-like protein Arl13b has been implicated in ciliogenesis and Sonic hedgehog signaling. Furthermore, we have previously shown that it regulates endocytic recycling traffic and interacts with actin. Herein, we report that the non-muscle myosin heavy chain IIA, also known as Myh9, is an Arl13b effector. Moreover, we found that both proteins localized to circular dorsal ruffles (CDRs) induced by platelet-derived growth factor stimulation and are required for their formation. CDRs are ring-shaped actin-dependent structures formed on the dorsal cell surface and are involved in diverse processes, such as macropinocytosis, integrin recycling, internalization of receptor tyrosine kinases and cell migration. We found that Arl13b or Myh9 silencing impaired cell migration, suggesting that Arl13b is required for this function through the interaction with Myh9. Moreover, Arl13b silencing impaired neural crest cell migration in zebrafish embryos. Furthermore, we showed that Arl13b is required for the formation of CDRs in migrating cells. Thus, our results indicate a new role for Arl13b in actin cytoskeleton remodeling through the interaction with Myh9, by driving the formation of CDRs necessary for cell migration.

Evidence for a purifying selection acting on the ß-lactamase locus in epidemic clones of methicillin-resistant Staphylococcus aureus.

BACKGROUND: The ß-lactamase (bla) locus, which confers resistance to penicillins only, may control the transcription of mecA, the central element of methicillin resistance, which is embedded in a polymorphic heterelogous chromosomal cassette (the SCCmec element). In order to assess the eventual correlation between bla allotypes and genetic lineages, SCCmec types and/or ß-lactam resistance phenotypes, the allelic variation on the bla locus was evaluated in a representative collection of 54 international epidemic methicillin-resistant Staphylococcus aureus (MRSA) clinical strains and, for comparative purposes, also in 24 diverse methicillin-susceptible S. aureus (MSSA) strains. RESULTS: Internal fragments of blaZ (the ß-lactamase structural gene) were sequenced for all strains. A subset of strains, representative of blaZ allotypes, was further characterized by sequencing of internal fragments of the blaZ transcriptional regulators, blaI and blaR1. Thirteen allotypes for blaZ, nine for blaI and 12 for blaR1 were found. In a total of 121 unique single-nucleotide polymorphisms (SNP) detected, no frameshift mutations were identified and only one nonsense mutation within blaZ was found in a MRSA strain. On average, blaZ alleles were more polymorphic among MSSA than in MRSA (14.7 vs 11.4 SNP/allele). Overall, blaR1 was the most polymorphic gene with an average of 24.8 SNP/allele. No correlation could be established between bla allotypes and genetic lineages, SCCmec types and/or ß-lactam resistance phenotypes. In order to estimate the selection pressure acting on the bla locus, the average dN/dS values were computed. In the three genes and in both collections dN/dS ratios were significantly below 1. CONCLUSIONS: The data strongly suggests the existence of a purifying selection to maintain the bla locus fully functional even on MRSA strains. Although, this is in agreement with the notion that in most clinical MRSA strains mecA gene is under the control of the bla regulatory genes, these findings also suggest that the apparently redundant function of blaZ gene for the MRSA resistant phenotype is still important for these strains. In addition, the data shows that the sensor-inducer blaR1 is the primary target for the accumulation of mutations in the bla locus, presumably to modulate the response to the presence of ß-lactam antibiotic.

Lipid levels including oxidized LDL in women with history of preeclampsia.

Preeclampsia (PE), a leading cause of maternal and perinatal morbidity and mortality worldwide, is a hypertensive disorder of unknown aetiology characterized by proteinuria, coagulation abnormalities and different systemic manifestations. Since there are no studies regarding the evaluation of oxidized LDL (oxLDL) in women with a history of PE, we focused on the evaluation of lipid profile and oxLDL plasma concentration several years after pregnancy to see if these women have any modifications in these parameters that may be linked to the risk of cardiovascular disease (CVD) in the future. Ninety women with a history of PE and 60 controls in a median interval of 6 years after pregnancy were recruited. Plasma oxLDL levels were measured using a two-site enzyme immunoassay. Concentration of cholesterol, triglycerides (TG), HDL-cholesterol (HDLc) and LDL-cholesterol (LDLc) were measured by automated enzymatic assays. To evaluate apoA and apoB levels automated immunoturbidimetric assays were used. In the group of women with a history of PE, gestational age at delivery was significantly earlier in comparison with the control group, whereas birth weight was significantly lower and there were more caesarean sections. Systolic and diastolic blood pressures were significantly higher in women with a history of PE than in the control group. Significantly higher obesity anthropometric markers (BMI and waist-to-hip ratio) were found in women with a history of PE. As consistent with other authors' findings, blood pressure was higher in these women, but lipid profile did not seem to play a role in the increased risk of cardiovascular disease.

Altered alanine aminotransferase and gamma-glutamyl transpeptidase in women with history of preeclampsia: association with waist-to-hip ratio and body mass index.

OBJECTIVE: Preeclampsia (PE), mostly when associated with HELLP syndrome, together with acute fatty liver of pregnancy, are the main causes of severe hepatic failure in pregnancy. Despite the number of studies in pregnancies complicated with PE, there are a few studies that focused on the evaluation of the hepatic function of these women several years after delivery. In this way, we evaluated circulating levels of AST, ALT, gammaGT and CRP several years after preeclamptic pregnancy to verify if these parameters are altered. METHODS: Eighty-nine women with previous PE and 60 women without medical complications were invited to the research centers. After the physical examination, blood was drawn for biochemical measurements. Plasma CRP levels and serum concentration of AST, ALT, gammaGT were measured by automated enzymatic assays. RESULTS: Systolic and diastolic blood pressures were significantly higher in women with history of PE than in control group as well as BMI and waist-to-hip ratio. ALT and gammaGT were significantly higher in women with previous history of PE, whereas AST and CRP presented similar levels between the two groups. Data revealed statistically significant positive correlations between ALT and gammaGT with waist-to-hip ratio and BMI. Positive correlations were also found between BMI and AST and CRP. CONCLUSION: It is possible that the increase in ALT and gammaGT levels is due to being overweight or through accumulation of visceral fat. Unaltered values of CRP suggest that the higher ALT and gammaGT values found in women with history of PE are not associated with inflammation.

Haemostatic factors in women with history of preeclampsia.

OBJECTIVE: Evaluation of haemostatic parameters--Plasma tissue plasminogen activator (t-PA), plasminogen activator inhibitor type 1 (PAI-1) and fibrin fragment D-dimer several years after the end of pregnancy to investigate if they are modified in women with history of preeclampsia (PE). STUDY DESIGN: 65 healthy women with history of PE and 54 control women with previous normal pregnancy were enrolled in this study. Groups were matched for age, time period since delivery, smoking status and alcohol consumption. t-PA, PAI-1 and fibrin fragment D-dimer antigen levels were quantified using standards commercial ELISA methods. Plasma fibrinogen was measured using automated capillary zone electrophoresis. RESULTS: Systolic and diastolic blood pressures were higher in women with history of PE. Levels of t-PA, PAI-1 and fibrinogen were similar between groups as well as the t-PA/PAI-1 ratio. A significant increase in D-dimer levels was observed in women with history of PE. CONCLUSION: The increase in D-dimer level suggests an abnormal haemostatic potential namely increased intravascular coagulation. This, together with the increased blood pressure, can reflect a tendency for an increased risk of cardiovascular/thrombotic events later in life.

Adhesion molecules (VCAM-1 and ICAM-1) and C-reactive protein in women with history of preeclampsia.

Several studies suggest that women with previous preeclampsia (PE) are at increased risk of cardiovascular disease (CVD). We examined circulating concentrations of adhesion molecules and C-reactive protein (CRP), markers for endothelial and inflammatory reactions, in addition to blood pressure and anthropometric measurements in 58 women with a history of PE and 49 control women with no pathology associated to pregnancy. Soluble adhesion molecules were measured by standard commercial ELISA methods and plasma CRP levels by automated enzymatic assays. Systolic and diastolic blood pressures and waist-to-hip ratios were significantly higher in women with history of PE than in control group. There were no significant differences in circulating levels of sICAM-1, sVCAM-1 and CRP in the study population. Women with a history of PE do not have a persistent inflammatory state that could induce overexpression of these molecules, which was supported by normal levels of CRP. The study supports the existence of common risk factors for PE and CVD, namely obesity and hypertension.