Opioid drugs are widely used analgesics that activate the G protein-coupled µ-opioid receptor, whose endogenous neuropeptide agonists, endorphins and enkephalins, are potent pain relievers. The ...therapeutic utility of opioid drugs is hindered by development of tolerance to the analgesic effects, requiring dose escalation for persistent pain control and leading to overdose and fatal respiratory distress. The prevailing hypothesis is that the intended analgesic effects of opioid drugs are mediated by µ-opioid receptor signaling to G protein, while the side-effects of respiratory depression and analgesic tolerance are caused by engagement of the receptor with the arrestin-3 protein. Consequently, opioid drug development has focused exclusively on identifying agonists devoid of arrestin-3 engagement. Here, we challenge the prevailing hypothesis with a panel of six clinically relevant opioid drugs and mice of three distinct genotypes with varying abilities to promote morphine-mediated arrestin-3 engagement. With this genetic and pharmacological approach, we demonstrate that arrestin-3 recruitment does not impact respiratory depression, and effective arrestin-3 engagement reduces, rather than exacerbates, the development of analgesic tolerance. These studies suggest that future development of safer opioids should focus on identifying opioid ligands that recruit both G protein and arrestin-3, thereby mimicking the signaling profile of most endogenous µ-opioid receptor agonists.
Resistance to immunomodulatory drugs (IMiDs
®
) is a major cause of treatment failure, disease relapse and ultimately poorer outcomes in multiple myeloma (MM). In order to optimally deploy IMiDs and ...their newer derivates CRBN E3 ligase modulators (CELMoDs
®
) into future myeloma therapeutic regimens, it is imperative to understand the mechanisms behind the inevitable emergence of IMiD resistance. IMiDs bind and modulate Cereblon (CRBN), the substrate receptor of the CUL4
CRBN
E3 ubiquitin ligase, to target novel substrate proteins for ubiquitination and degradation. Most important of these are IKZF1 and IKZF3, key MM survival transcription factors which sustain the expression of myeloma oncogenes IRF4 and MYC. IMiDs directly target MM cell proliferation, but also stimulate T/NK cell activation by their CRBN-mediated effects, and therefore enhance anti-MM immunity. Thus, their benefits in myeloma are directed against tumor and immune microenvironment – and in considering the mechanisms by which IMiD resistance emerges, both these effects must be appraised. CRBN-dependent mechanisms of IMiD resistance, including
CRBN
genetic aberrations, CRBN protein loss and CRBN-substrate binding defects, are beginning to be understood. However, only a proportion of IMiD-resistant cases are related to CRBN and therefore additional mechanisms, which are currently less well described, need to be sought. These include resistance within the immune microenvironment. Here we review the existing evidence on both tumor and immune microenvironment mechanisms of resistance to IMiDs, pose important questions for future study, and consider how knowledge regarding resistance mechanism may be utilized to guide treatment decision making in the clinic.
Hepcidin regulates systemic iron homeostasis. Suppression of hepcidin expression occurs physiologically in iron deficiency and increased erythropoiesis but is pathologic in thalassemia and ...hemochromatosis. Here we show that epigenetic events govern hepcidin expression. Erythropoiesis and iron deficiency suppress hepcidin via erythroferrone-dependent and -independent mechanisms, respectively, in vivo, but both involve reversible loss of H3K9ac and H3K4me3 at the hepcidin locus. In vitro, pan-histone deacetylase inhibition elevates hepcidin expression, and in vivo maintains H3K9ac at hepcidin-associated chromatin and abrogates hepcidin suppression by erythropoietin, iron deficiency, thalassemia, and hemochromatosis. Histone deacetylase 3 and its cofactor NCOR1 regulate hepcidin; histone deacetylase 3 binds chromatin at the hepcidin locus, and histone deacetylase 3 knockdown counteracts hepcidin suppression induced either by erythroferrone or by inhibiting bone morphogenetic protein signaling. In iron deficient mice, the histone deacetylase 3 inhibitor RGFP966 increases hepcidin, and RNA sequencing confirms hepcidin is one of the genes most differentially regulated by this drug in vivo. We conclude that suppression of hepcidin expression involves epigenetic regulation by histone deacetylase 3.Hepcidin controls systemic iron levels by inhibiting intestinal iron absorption and iron recycling. Here, Pasricha et al. demonstrate that the hepcidin-chromatin locus displays HDAC3-mediated reversible epigenetic modifications during both erythropoiesis and iron deficiency.
IntroductionRadiogenomic analysis of patients being considered for liver resection is seldom performed in the clinic despite recent evidence indicating that quantitative MRI could improve ...posthepatectomy outcomes. Meanwhile, the increasingly accessible results from whole genome sequencing reporting on clinically actionable genetic biomarkers are yet to be fully integrated into the clinical care pathway.Methods and analysisA prospective observational cohort study of up to 200 participants is planned, recruiting adults with primary or secondary liver cancer being considered for liver resection at Hampshire Hospitals NHS Foundation Trust. The data will be evaluated to address the primary endpoint to calculate the proportion of participants in which the results from whole genome sequencing would have resulted in a change in clinical management. Participants will be offered an additional non-invasive quantitative MRI scan prior to the operation and the impact of the imaging results on treatment decision-making will be evaluated.Ethics and disseminationThis study was reviewed by the NHS Health Research Authority and given favourable opinion by the Brighton and Sussex Research Ethics Committee (REC reference: 20/PR/0222). Research findings will be discussed with a patient and public involvement and engagement group, presented at relevant scientific conferences and published in open access journals.Trial registration numberNCT04597710
Immunomodulatory drugs (IMiDs), including lenalidomide and pomalidomide, are used in the routine treatment for multiple myeloma (MM) patients. Cereblon (CRBN) is the direct molecular target of IMiDs. ...While CRBN is not an essential gene for MM cell proliferation, the frequency of CRBN genetic aberrations, including mutation, copy number loss, and exon-10 (which includes a portion of the IMiD-binding domain) splicing, have been reported to incrementally increase in later-line patients. CRBN exon-10 splicing has also been shown to be associated with decreased progression-free survival in both newly diagnosed and relapsed refractory MM patients. Although we did not find significant general splicing defects among patients with CRBN exon-10 splice variant when compared to those expressing the full-length transcript, we identified upregulated TNFA signaling via NFKB, inflammatory response, and IL-10 signaling pathways in patients with exon-10 splice variant across various data sets-all potentially promoting tumor growth via chronic growth signals. We examined master regulators that mediate transcriptional programs in CRBN exon-10 splice variant patients and identified BATF, EZH2, and IKZF1 as the key candidates across the four data sets. Upregulated downstream targets of BATF, EZH2, and IKZF1 are components of TNFA signaling via NFKB, IL2/STAT5 signaling pathways, and IFNG response pathways. Previously, BATF-mediated transcriptional regulation was associated with venetoclax sensitivity in MM. Interestingly, we found that an EZH2 sensitivity gene expression signature also correlated with high BATF or venetoclax sensitivity scores in these tumors. Together, these data provide a rationale for investigating EZH2 inhibitors or venetoclax in combination with the next generation CRBN-targeting agents, such as CELMoDs, for patients overexpressing the CRBN exon-10 splice variant.
To compare methods for homogenizing the mouse whole eye or retina for RNA extraction.
We tested five homogenization techniques for the whole eye and the retina. Two established shearing techniques ...were a version of the Potter-Elvehjem homogenizer, which uses a plastic pellet pestle in a microfuge tube, and a Dounce homogenizer. Two modern bead-beating methods used commercially manufactured devices, the Next Advance Bullet Blender and the Qiagen TissueLyser LT. The last method involved vortex mixing multiple samples simultaneously in a buffer containing a stainless-steel set screw, a novel approach. RNA was extracted from the tissue after each technique was used. Degradation of RNA was measured with the RNA integrity number (RIN score) after electrophoresis on an Agilent BioAnalyzer RNA LabChip. Nucleic acid yields were measured with ultraviolet (UV) spectroscopy in a BioTek Synergy H1 Hybrid plate reader. The purity of the nucleic acids was assessed with the mean absorbance ratio (A
/A
). The preparation time per sample was measured with a digital stopwatch. Costs of necessary consumables were calculated per ten samples.
The RIN scores for all homogenization methods and both tissue types ranged from 7.75±0.64 to 8.78±0.18; none were statistically significantly different. The total RNA yield per whole eye from the bead-based methods ranged from 7,700 to 9,800 ng and from 3,000 to 4,600 ng for the pellet pestle and Dounce shearing methods, respectively. The total RNA yield per retina from the bead-based methods ranged from 4,600 to 8,400 ng and from 2,200 to 7,400 ng for the pellet pestle and Dounce shearing methods, respectively. Homogenization was faster using the bead-based methods (about 15 min for ten samples) because multiple samples could be run simultaneously compared to the shearing methods that require samples be homogenized individually (about 45-60 min per ten samples). The costs in consumables for the methods tested ranged from $2.60 to $14.70 per ten samples. The major differences in overall costs come in the form of one-time equipment purchases, which can range from one hundred to thousands of dollars. The bead-based methods required less technician involvement and had less potential for sample contamination than the shearing methods.
The purity and quality of RNA were similar across all methods for both tissue types. The novel set screw method and the two bead-based methods (bullet blender and TissueLyser) outperformed the two shearing methods (the pellet pestle and Dounce techniques) in total RNA yields for the whole eye. Although the bullet blender, TissueLyser, and set screw methods produced comparable levels of RNA yield, purity, and quality, the set screw method was less expensive. Researchers seeking the efficiency of sophisticated bead homogenization equipment without the high equipment costs might consider this novel method.
Interactions between multiple myeloma (MM) and bone marrow (BM) are well documented to support tumour growth, yet the cellular mechanisms underlying pain in MM are poorly understood. We have used in ...vivo murine models of MM to show significant induction of nerve growth factor (NGF) by the tumour-bearing bone microenvironment, alongside other known pain-related characteristics such as spinal glial cell activation and reduced locomotion. NGF was not expressed by MM cells, yet bone stromal cells such as osteoblasts expressed and upregulated NGF when cultured with MM cells, or MM-related factors such as TNF-α. Adiponectin is a known MM-suppressive BM-derived factor, and we show that TNF-α-mediated NGF induction is suppressed by adiponectin-directed therapeutics such as AdipoRON and L-4F, as well as NF-κB signalling inhibitor BMS-345541. Our study reveals a further mechanism by which cellular interactions within the tumour-bone microenvironment contribute to disease, by promoting pain-related properties, and suggests a novel direction for analgesic development.
Multiple myeloma (MM) is a plasma cell malignancy characterised by aberrant production of immunoglobulins requiring survival mechanisms to adapt to proteotoxic stress. We here show that ...glutamyl-prolyl-tRNA synthetase (GluProRS) inhibition constitutes a novel therapeutic target. Genomic data suggest that GluProRS promotes disease progression and is associated with poor prognosis, while downregulation in MM cells triggers apoptosis. We developed NCP26, a novel ATP-competitive ProRS inhibitor that demonstrates significant anti-tumour activity in multiple in vitro and in vivo systems and overcomes metabolic adaptation observed with other inhibitor chemotypes. We demonstrate a complex phenotypic response involving protein quality control mechanisms that centers around the ribosome as an integrating hub. Using systems approaches, we identified multiple downregulated proline-rich motif-containing proteins as downstream effectors. These include CD138, transcription factors such as MYC, and transcription factor 3 (TCF3), which we establish as a novel determinant in MM pathobiology through functional and genomic validation. Our preclinical data therefore provide evidence that blockade of prolyl-aminoacylation evokes a complex pro-apoptotic response beyond the canonical integrated stress response and establish a framework for its evaluation in a clinical setting.
Novel biomarkers for tumour burden and bone disease are required to guide clinical management of plasma cell dyscrasias. Recently, bone turnover markers (BTMs) and Diffusion-Weighted Magnetic ...Resonance Imaging (DW-MRI) have been explored, although their role in the prospective assessment of multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) is unclear. Here, we conducted a pilot observational cohort feasibility study combining serum BTMs and DW-MRI in addition to standard clinical assessment. Fifty-five patients were recruited (14 MGUS, 15 smouldering MM, 14 new MM and 12 relapsed MM) and had DW-MRI and serum biomarkers (P1NP, CTX-1, ALP, DKK1, sclerostin, RANKL:OPG and BCMA) measured at baseline and 6-month follow-up. Serum sclerostin positively correlated with bone mineral density (
= 0.40-0.54). At baseline, serum BCMA correlated with serum paraprotein (
= 0.42) and serum DKK1 correlated with serum free light chains (
= 0.67); the longitudinal change in both biomarkers differed between International Myeloma Working Group (IMWG)-defined responders and non-responders. Myeloma Response Assessment and Diagnosis System (MY-RADS) scoring of serial DW-MRI correlated with conventional IMWG response criteria for measuring longitudinal changes in tumour burden. Overall, our pilot study suggests candidate radiological and serum biomarkers of tumour burden and bone loss in MM/MGUS, which warrant further exploration in larger cohorts to validate the findings and to better understand their clinical utility.
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