The mTOR pathway controls phosphorylation of BRAF at T401.

BRAF serves as a gatekeeper of the RAS/RAF/MEK/ERK pathway, which plays a crucial role in homeostasis. Since aberrant signalling of this axis contributes to cancer and other diseases, it is tightly regulated by crosstalk with the PI3K/AKT/mTOR pathway and ERK mediated feedback loops. For example, ERK limits BRAF signalling through phosphorylation of multiple residues. One of these, T401, is widely considered as an ERK substrate following acute pathway activation by growth factors. Here, we demonstrate that prominent T401 phosphorylation (pT401) of endogenous BRAF is already observed in the absence of acute stimulation in various cell lines of murine and human origin. Importantly, the BRAF/RAF1 inhibitor naporafenib, the MEK inhibitor trametinib and the ERK inhibitor ulixertinib failed to reduce pT401 levels in these settings, supporting an alternative ERK-independent pathway to T401 phosphorylation. In contrast, the mTOR inhibitor torin1 and the dual-specific PI3K/mTOR inhibitor dactolisib significantly suppressed pT401 levels in all investigated cell types, in both a time and concentration dependent manner. Conversely, genetic mTOR pathway activation by oncogenic RHEB (Q64L) and mTOR (S2215Y and R2505P) mutants substantially increased pT401, an effect that was reverted by dactolisib and torin1 but not by trametinib. We also show that shRNAmir mediated depletion of the mTORC1 complex subunit Raptor significantly enhanced the suppression of T401 phosphorylation by a low torin1 dose, while knockdown of the mTORC2 complex subunit Rictor was less effective. Using mass spectrometry, we provide further evidence that torin1 suppresses the phosphorylation of T401, S405 and S409 but not of other important regulatory phosphorylation sites such as S446, S729 and S750. In summary, our data identify the mTOR axis and its inhibitors of (pre)clinical relevance as novel modulators of BRAF phosphorylation at T401.

6-Thioguanosine Monophosphate Prodrugs Display Enhanced Performance against Thiopurine-Resistant Leukemia and Breast Cancer Cells.

Thiopurines are in widespread clinical use for the treatment of immunological disorders and certain cancers. However, treatment failure due to resistance or adverse drug reactions are common, asking for new therapeutic strategies. We investigated the potential of 6-thioguanosine monophosphate (6sGMP) prodrugs to overcome resistance to 6-thioguanine. We successfully developed synthetic routes toward diverse 6sGMP prodrugs, tested their proliferation inhibitory potential in different cell lines, and examined their mode of action. Our results show that 4-acetyloxybenzyl- and cycloSaligenyl-derivatized 6sGMP prodrugs are effective antiproliferative compounds in cells that are resistant to thiopurines. We find that resistance is related to the expression of salvage pathway enzyme HGPRT. Using TUC-seq DUAL, we demonstrate the intracellular conversion of 6sGMP prodrugs into bioactive 6sGTPs. Thus, our study offers a promising strategy for thiopurine therapy by using 6sGMP prodrugs, and it suggests TUC-seq DUAL as a simple and fast method to measure the success of thiopurine therapy.

Towards a comprehensive understanding of RNA deamination: synthesis and properties of xanthosine-modified RNA.

Nucleobase deamination, such as A-to-I editing, represents an important posttranscriptional modification of RNA. When deamination affects guanosines, a xanthosine (X) containing RNA is generated. However, the biological significance and chemical consequences on RNA are poorly understood. We present a comprehensive study on the preparation and biophysical properties of X-modified RNA. Thermodynamic analyses revealed that base pairing strength is reduced to a level similar to that observed for a G•U replacement. Applying NMR spectroscopy and X-ray crystallography, we demonstrate that X can form distinct wobble geometries with uridine depending on the sequence context. In contrast, X pairing with cytidine occurs either through wobble geometry involving protonated C or in Watson-Crick-like arrangement. This indicates that the different pairing modes are of comparable stability separated by low energetic barriers for switching. Furthermore, we demonstrate that the flexible pairing properties directly affect the recognition of X-modified RNA by reverse transcription enzymes. Primer extension assays and PCR-based sequencing analysis reveal that X is preferentially read as G or A and that the ratio depends on the type of reverse transcriptase. Taken together, our results elucidate important properties of X-modified RNA paving the way for future studies on its biological significance.

Synthesis of 4-thiouridines with prodrug functionalization for RNA metabolic labeling.

Metabolic labeling has emerged as a powerful tool to endow RNA with reactive handles allowing for subsequent chemical derivatization and processing. Recently, thiolated nucleosides, such as 4-thiouridine (4sU), have attracted great interest in metabolic labeling-based RNA sequencing approaches (TUC-seq, SLAM-seq, TimeLapse-seq) to study cellular RNA expression and decay dynamics. For these and other applications (e.g. PAR-CLIP), thus far only the naked nucleoside 4sU has been applied. Here we examined the concept of derivatizing 4sU into a 5'-monophosphate prodrug that would allow for cell permeation and potentially improve labeling efficiency by bypassing the rate-limiting first step of 5' phosphorylation of the nucleoside into the ultimately bioactive 4sU triphosphate (4sUTP). To this end, we developed robust synthetic routes towards diverse 4sU monophosphate prodrugs. Using metabolic labeling assays, we found that most of the newly introduced 4sU prodrugs were well tolerated by the cells. One derivative, the bis(4-acetyloxybenzyl) 5'-monophosphate of 4sU, was also efficiently incorporated into nascent RNA.

CHD1 controls H3.3 incorporation in adult brain chromatin to maintain metabolic homeostasis and normal lifespan.

The ATP-dependent chromatin remodeling factor CHD1 is essential for the assembly of variant histone H3.3 into paternal chromatin during sperm chromatin remodeling in fertilized eggs. It remains unclear, however, if CHD1 has a similar role in normal diploid cells. Using a specifically tailored quantitative mass spectrometry approach, we show that Chd1 disruption results in reduced H3.3 levels in heads of Chd1 mutant flies. Chd1 deletion perturbs brain chromatin structure in a similar way as H3.3 deletion and leads to global de-repression of transcription. The physiological consequences are reduced food intake, metabolic alterations, and shortened lifespan. Notably, brain-specific CHD1 expression rescues these phenotypes. We further demonstrate a strong genetic interaction between Chd1 and H3.3 chaperone Hira. Thus, our findings establish CHD1 as a factor required for the assembly of H3.3-containing chromatin in adult cells and suggest a crucial role for CHD1 in the brain as a regulator of organismal health and longevity.

Efficiency and predictive parameters of outcome of a multimodal pain management concept with spinal injections in patients with low back pain: a retrospective study of 445 patients.

Low back pain is one of the most common diseases of modern civilization. Multimodal pain management (MPM) represents a central approach to avoiding surgery. Short-term results are published rarely and often incomparable because of different treatment concepts. This study compared the subjective and objective parameters as well as the anamnestic and clinical parameters of 445 patients with low back pain before and after inpatient MPM to investigate the influence of this type of therapy on short-term outcome. The majority of patients were very satisfied (39%) or satisfied (58%) with the treatment outcome. The median pain reduction for back pain was 3.0 (IQR 2.88) (numeric rating scale, NRS), thus 66% and 2.75 (IQR 3.38, 62%) for leg pain. The main pain reduction occurred within the first 10 days of treatment and was clinically significant from day 5 onwards. The outcome for patients with hospitalization of more than 10 days was significantly worse. The parameters female sex, BMI of > 30, local pain, and pain duration of 3-24 months had a significantly better outcome. In contrast, age, treatment cause, depression, anxiety, and other diseases had no statistically significant influence on outcome. MPM therapy for more than 5 days seems to be an efficient short-term approach to treating low back pain. Knowledge of some of the outcome predictors helps to early identify patients who require more intensive individual care. In the case of no clear indication for surgery, MPM can be an appropriate treatment option.

Effectiveness of a multimodal pain management concept for patients with cervical radiculopathy with focus on cervical epidural injections.

Cervical radiculopathy has become an increasing problem worldwide. Conservative treatment options have been recommended in many reviews on cervical radiculopathy, ranging from different types of physiotherapy to waiting for remission by natural history. No multimodal pain management concept (MPM) on an inpatient basis has been evaluated. This study aimed at showing the positive short-term effects of an inpatient multimodal pain management concept with focus on cervical translaminar epidural steroid injection for patients with cervical radiculopathy. 54 patients who had undergone inpatient MPM for 10 days were evaluated before and after 10-days treatment. The NRS (0-10) value for arm pain could be reduced from 6.0 (IQR 5.7-6.8) to 2.25 (IQR 2.0-3.1) and from 5.9 (IQR 4.8-6.0) to 2.0 (IQR 1.7-2.6) for neck pain. Neck pain was reduced by 57.4% and arm pain by 62.5%. 2 days after epidural steroid injection, pain was reduced by 40.1% in the neck and by 43.4% in the arms. MPM seems to be an efficient short-term approach to treating cervical radiculopathy. Cervical translaminar epidural steroid injection is an important part of this concept. In the absence of a clear indication for surgery, MPM represents a treatment option.