CDK12 is hyperactivated and a synthetic-lethal target in BRAF-mutated melanoma.

Melanoma is the deadliest form of skin cancer and considered intrinsically resistant to chemotherapy. Nearly all melanomas harbor mutations that activate the RAS/mitogen-activated protein kinase (MAPK) pathway, which contributes to drug resistance via poorly described mechanisms. Herein we show that the RAS/MAPK pathway regulates the activity of cyclin-dependent kinase 12 (CDK12), which is a transcriptional CDK required for genomic stability. We find that melanoma cells harbor constitutively high CDK12 activity, and that its inhibition decreases the expression of long genes containing multiple exons, including many genes involved in DNA repair. Conversely, our results show that CDK12 inhibition promotes the expression of short genes with few exons, including many growth-promoting genes regulated by the AP-1 and NF-κB transcription factors. Inhibition of these pathways strongly synergize with CDK12 inhibitors to suppress melanoma growth, suggesting promising drug combinations for more effective melanoma treatment.

Peak plasma concentration of total and free bupivacaine after erector spinae plane and pectointercostal fascial plane blocks.

PURPOSE: Erector spinae plane blocks (ESPB) and pectointercostal fascial (PIFB) plane blocks are novel interfascial blocks for which local anesthetic (LA) doses and concentrations necessary to achieve safe and effective analgesia are unknown. The goal of this prospective observational study was to provide the timing (Tmax) and concentration (Cmax) of maximum total and free plasma bupivacaine after ESPB in breast surgery and after PIFB in cardiac surgery patients. METHODS: Erector spinae plane blocks or PIFBs (18 patients per block; total, 36 patients) were performed with 2 mg⋅kg-1 of bupivacaine with epinephrine 5 µg⋅mL-1. Our principal outcomes were the mean or median Cmax of total and free plasma bupivacaine measured 10, 20, 30, 45, 60, 90, 180, and 240 min after LA injection using liquid chromatography with tandem mass spectrometry. RESULTS: For ESPB, the mean (standard deviation [SD]) total bupivacaine Cmax was 0.37 (0.12) µg⋅mL-1 (range, 0.19 to 0.64), and the median [interquartile range (IQR)] Tmax was 30 [50] min (range, 10-180). For ESPB, the mean (SD) free bupivacaine Cmax was 0.015 (0.017) µg⋅mL-1 (range, 0.003-0.067), and the median [IQR] Tmax was 30 [20] min (range, 10-120). After PIFB, mean plasma concentrations plateaued at 60-240 min. For PIFB, the mean (SD) total bupivacaine Cmax was 0.32 (0.21) µg⋅mL-1 (range, 0.14-0.95), with a median [IQR] Tmax of 120 [150] min (range, 30-240). For PIFB, the mean (SD) free bupivacaine Cmax was 0.019 (0.010) µg⋅mL-1 (range, 0.005-0.048), and the median [IQR] Tmax was 180 [120] min (range, 30-240). For both ESPB and PIFB, we observed no correlations between pharmacokinetic and demographic parameters. CONCLUSION: Total and free bupivacaine Cmax observed after ESPB and PIFB with 2 mg⋅kg-1 of bupivacaine with epinephrine 5 µg⋅mL-1 were five to twenty times lower than levels considered toxic in the literature.

RéSUMé: OBJECTIF: Les blocs des muscles érecteurs du rachis (ESP) et les blocs des plans fasciaux pecto-intercostaux (PIFB) sont de nouveaux blocs interfasciaux pour lesquels les doses et les concentrations d'anesthésique local (AL) nécessaires à obtenir une analgésie sécuritaire et efficace sont inconnues. L'objectif de cette étude observationnelle prospective était de déterminer le moment d'administration (Tmax) et la concentration (Cmax) de bupivacaïne plasmatique totale et plasmatique libre maximale après un bloc ESP pour chirurgie mammaire et après un PIFB chez les patients en chirurgie cardiaque. MéTHODE: Des blocs ESP ou PIFB (18 patients par bloc; total, 36 patients) ont été réalisés avec 2 mg⋅kg-1 de bupivacaïne et 5 µg⋅mL-1 d'épinéphrine. Nos principaux critères d'évaluation étaient la Cmax moyenne ou médiane de bupivacaïne plasmatique totale et libre mesurée 10, 20, 30, 45, 60, 90, 180 et 240 min après l'injection d'AL par chromatographie liquide avec spectrométrie de masse en tandem. RéSULTATS: Pour le bloc ESP, la Cmax de bupivacaïne totale moyenne (écart type [ET]) était de 0,37 (0,12) µg⋅mL-1 (plage, 0,19 à 0,64), et le Tmax médian [écart interquartile (ÉIQ)] était de 30 [50] min (intervalle, 10­180). Pour le bloc ESP, la Cmax de bupivacaïne libre moyenne (ET) était de 0,015 (0,017) µg⋅mL-1 (plage, 0,003­0,067), et le Tmax médian [ÉIQ] était de 30 [20] min (intervalle, 10­120). Après un PIFB, les concentrations plasmatiques moyennes ont plafonné à 60­240 min. Pour le bloc PIFB, la Cmax de bupivacaïne totale moyenne (ET) était de 0,32 (0,21) µg⋅mL-1 (plage, 0,14­0,95), et le Tmax médian [ÉIQ] était de 120 [150] min (intervalle, 30­240). Pour le bloc PIFB, la Cmax de bupivacaïne libre moyenne (ET) était de 0,019 (0,010) µg⋅mL-1 (plage, 0,005­0,048), et le Tmax médian [ÉIQ] était de 180 [120] min (intervalle, 30­240). Pour le bloc ESP et le PIFB, nous n'avons observé aucune corrélation entre les paramètres pharmacocinétiques et démographiques. CONCLUSION:  : Les Cmax de bupivacaïne totale et libre observées après un bloc ESP et PIFB avec 2 mg⋅kg-1 de bupivacaïne avec 5 µg⋅mL-1 d'épinéphrine étaient cinq à vingt fois plus faibles que les niveaux considérés comme toxiques dans la littérature.

Mesenchymal stromal cell ciliogenesis is abrogated in response to tumor necrosis factor-α and requires NF-κB signaling.

The primary cilium is a cell surface-anchored sensory organelle which expression is lost in hypoxic cancer cells and during mesenchymal stromal cells (MSC) adaptation to low oxygen levels. Since pro-inflammatory cues are among the early events which promote tumor angiogenesis, we tested the inflammatory cytokine tumor necrosis factor (TNF)-α and found that it triggered a dose-dependent loss of the primary cilia in MSC. This loss was independent of IFT88 expression, was abrogated by progranulin, an antagonist of the TNF receptor and required the NF-κB signaling intermediates IκB kinase α, ß, and γ, as well as NF-κB p65. These findings strengthen the concept that the primary cilium may serve as a biomarker reflecting the tumor-supporting potential of MSC and their capacity to adapt to hypoxic and pro-inflammatory cues.

MT1-MMP expression level status dictates the in vitro action of lupeol on inflammatory biomarkers MMP-9 and COX-2 in medulloblastoma cells.

Local inflammation-induced extracellular matrix structural changes are a prerequisite to neoplastic invasion by pediatric intracranial tumors. Accordingly, increased expression of matrix metalloproteinases MMP-2 and MMP-9, two inflammation-induced matrix metalloproteinases (MMPs), may further aid the transformed cells either to infiltrate adjacent tissues or to enter the peripheral circulation. In the context of neuroinflammation, MMP-9 has been linked to processes such as blood-brain barrier opening and invasion of neural tissue by blood-derived immune cells. Given its reported anti-inflammatory and anticancer properties, we investigated the in vitro pharmacological effects of lupeol, a diet-derived triterpenoid, on MMP-9 and cyclooxygenase (COX)-2 expressions in a pediatric medulloblastoma DAOY cell line model. Lupeol was unable to inhibit the increased MMP-9 and COX-2 expression in phorbol 12-myristate 13-acetate (PMA)-treated cells, but was rather found to synergize with PMA to induce both biomarkers' expression. A contribution of the membrane type-1 (MT1)-MMP was also revealed, since lupeol/PMA treatments triggered proMMP-2 activation, and that MT1-MMP gene silencing reversed the combined effects of lupeol/PMA on both MMP-9 and COX-2. The mRNA stabilizing factor HuR was also found increased in the combined lupeol/PMA treatment, suggesting stabilization processes of the MMP-9 and COX-2 transcripts. We postulate that lupeol's anti-inflammatory properties may exert better pharmacological action within low MT1-MMP expressing tumors. Furthermore, these evidences add up to the new pleiotropic molecular mechanisms of action of MT1-MMP, and prompt for evaluating the future in vitro pharmacological properties of lupeol under pro-inflammatory experimental set-up.

Pharmacological targeting of ß-adrenergic receptor functions abrogates NF-κB signaling and MMP-9 secretion in medulloblastoma cells.

Targeting of the vascular endothelium compartment explains, in part, the therapeutic efficacy of the nonselective ß-adrenergic antagonist propranolol against common endothelial tumors such as hemangiomas. In vitro, the antiangiogenic biological activity of propranolol was shown to inhibit human brain microvascular endothelial cell tubulogenesis. However, possible interference of propranolol with cell signaling associated with the tumoral compartment remains unexplored. We therefore assessed the potency of propranolol against a pediatric brain tumor- derived DAOY medulloblastoma cell model. Gene expression of ß(1)-, ß(2)-, and ß(3)-adrenergic receptors was confirmed in DAOY cells by semiquantitative RT-PCR. We next found that propranolol dose-dependently inhibited induction of the key extracellular matrix-degrading and blood-brain barrier disrupting enzyme matrix metalloproteinase- 9 (MMP-9) by phorbol 12-myristate 13-acetate (PMA). Propranolol not only inhibited PMA- induced phosphorylation of the extracellular signal-regulated kinase (Erk), but also that of IkappaB (IκB), preventing the IκB phosphorylation which is a prerequisite for IκB degradation. Propranolol inhibition of IκB phosphorylation was shown to occur with optimal efficacy at 30 µM. Although propranolol, at up to 100 µM, did not affect cell viability, it potentiated PMA- mediated signaling that ultimately led to diminished phosphorylation of Akt. The anti-Erk and anti-Akt phosphorylation effects are both suggestive of antiproliferative and antisurvival signaling, respectively. Our data are therefore indicative of a pharmacological role for propranolol against ß-adrenergic receptor signaling functions involving the nuclear factor-kappaB-mediated regulation of MMP-9.