Non-canonical transcriptional regulation of the poor prognostic factor UGT2B17 in chronic lymphocytic leukemic and normal B cells.

BACKGROUND: High expression of the glycosyltransferase UGT2B17 represents an independent adverse prognostic marker in chronic lymphocytic leukemia (CLL). It also constitutes a predictive marker for therapeutic response and a drug resistance mechanism. The key determinants driving expression of the UGT2B17 gene in normal and leukemic B-cells remain undefined. The UGT2B17 transcriptome is complex and is comprised of at least 10 alternative transcripts, identified by previous RNA-sequencing of liver and intestine. We hypothesized that the transcriptional program regulating UGT2B17 in B-lymphocytes is distinct from the canonical expression previously characterized in the liver. RESULTS: RNA-sequencing and genomics data revealed a specific genomic landscape at the UGT2B17 locus in normal and leukemic B-cells. RNA-sequencing and quantitative PCR data indicated that the UGT2B17 enzyme is solely encoded by alternative transcripts expressed in CLL patient cells and not by the canonical transcript widely expressed in the liver and intestine. Chromatin accessible regions (ATAC-Seq) in CLL cells mapped with alternative promoters and non-coding exons, which may be derived from endogenous retrotransposon elements. By luciferase reporter assays, we identified key cis-regulatory STAT3, RELA and interferon regulatory factor (IRF) binding sequences driving the expression of UGT2B17 in lymphoblastoid and leukemic B-cells. Electrophoretic mobility shift assays and pharmacological inhibition demonstrated key roles for the CLL prosurvival transcription factors STAT3 and NF-κB in the leukemic expression of UGT2B17. CONCLUSIONS: UGT2B17 expression in B-CLL is driven by key regulators of CLL progression. Our data suggest that a NF-κB/STAT3/IRF/UGT2B17 axis may represent a novel B-cell pathway promoting disease progression and drug resistance.

Functional platelet-derived mitochondria induce the release of human neutrophil microvesicles.

Inflammation is an essential process of host defense against infections, illness, or tissue damage. Polymorphonuclear neutrophils (PMN) are among the first immune cells involved in acute inflammatory responses and are on the front line in the fight against bacterial infections. In the presence of bacterial fragments, PMN release inflammatory mediators, enzymes, and microvesicles in the extracellular milieu to recruit additional immune cells required to eliminate the pathogens. Recent evidence shows that platelets (PLTs), initially described for their role in coagulation, are involved in inflammatory responses. Furthermore, upon activation, PLT also release functional mitochondria (freeMitos) within their extracellular milieu. Mitochondria share characteristics with bacterial and mitochondrial damage-associated molecular patterns, which are important contributors in sterile inflammation processes. Deep sequencing transcriptome analysis demonstrates that freeMitos increase the mitochondrial gene expression in PMN. However, freeMitos do not affect the mitochondrial-dependent increase in oxygen consumption in PMN. Interestingly, freeMitos significantly induce the release of PMN-derived microvesicles. This study provides new insight into the role of freeMitos in the context of sterile inflammation.

Dietary omega-3 fatty acids modulate the production of platelet-derived microvesicles in an in vivo inflammatory arthritis model.

PURPOSE: The aim of this study was to investigate the effects of different ω-3 polyunsaturated fatty acid (PUFA) enriched diets, including a novel renewable plant source of ω-3 fatty acids (Buglossoides arvensis), on the development and progression of rheumatoid arthritis (RA). METHODS: RA was induced in mice consuming experimental diets using the K/BxN model. The experimental diets consisted of either a western control diet (control), diets containing B. arvensis oil or fish oil. The effects of the diets on platelets, platelet microvesicles (PMVs), and inflammatory markers such as clinical index, ankle thickness and cytokine/chemokine release were measured. RESULTS: While ω-3 PUFA-enriched diets did not prevent the development of arthritis in the K/BxN model, a significant decrease in ankle swelling was observed compared to the control group. Platelets isolated from mice consuming either low content of B. arvensis oil or fish oil diets exhibited significantly decreased PMVs production compared to mice consuming the control diet. CONCLUSION: Our study provides insight into the contribution of ω-3 PUFA supplementation in modulating the pro-inflammatory phenotype of platelets in RA pathology. Furthermore, our study suggests that low concentrations of dietary B. arvensis oil may have similar anti-inflammatory potential seen with dietary fish oil supplementation.

A novel CHD3 variant in a patient with central precocious puberty: Expanded phenotype of Snijders Blok-Campeau syndrome?

Snijders Blok-Campeau syndrome is an autosomal dominant genetic disorder first described in 2018, mostly associated with de novo variants in the CHD3 gene that affects chromatin remodeling. This syndrome is characterized by developmental delay, speech delay, and intellectual disability, but only about 60 affected individuals have been reported to date. We report a de novo likely pathogenic CHD3 variant (c.5609G > A; p. (Arg1870Gln)) in a young female presenting with features of Snijders Blok-Campeau syndrome including speech delay, autism spectrum disorder, learning difficulties, characteristic facial dysmorphisms, and a feature not previously described in this syndrome, idiopathic central precocious puberty. Her puberty was controlled with monthly injections of a GnRH analogue. Targeted exome sequencing was negative for genes known to be responsible for central precocious puberty. Our case raises the possibility that variants in CHD3 gene may also result in central precocious puberty. Strengthening this association could expand the phenotypic spectrum of the Snijders Blok-Campeau syndrome and should be included in multigene panels for precocious puberty.

Expanded phenotype of primary ciliary dyskinesia related to DRC1 pathogenic variant with dysmorphisms and vascular anomalies.

We present a case of a female diagnosed with primary ciliary dyskinesia (PCD) type 21 with non-previously reported extrapulmonary symptoms, including facial features and congenital vascular anomalies. Whole genome sequencing in our patient revealed a homozygous pathogenic variant in the DRC1 gene and no other notable structural nor punctual variants. This case demonstrates a unique clinical manifestation of PCD, which is possibly associated with the presence of a homozygous pathogenic DRC1 variant. Therefore, we suggest that analysis of DRC1 be considered with PCD type 21 when such features are present.

Emerging roles for UDP-glucuronosyltransferases in drug resistance and cancer progression.

The best-known role of UDP-glucuronosyltransferase enzymes (UGTs) in cancer is the metabolic inactivation of drug therapies. By conjugating glucuronic acid to lipophilic drugs, UGTs impair the biological activity and enhance the water solubility of these agents, driving their elimination. Multiple clinical observations support an expanding role for UGTs as modulators of the drug response and in mediating drug resistance in numerous cancer types. However, accumulating evidence also suggests an influence of the UGT pathway on cancer progression. Dysregulation of the expression and activity of UGTs has been associated with the progression of several cancers, arguing for UGTs as possible mediators of oncogenic pathways and/or disease accelerators in a drug-naive context. The consequences of altered UGT activity on tumour biology are incompletely understood. They might be associated with perturbed levels of bioactive endogenous metabolites such as steroids and bioactive lipids that are inactivated by UGTs or through non-enzymatic mechanisms, thereby eliciting oncogenic signalling cascades. This review highlights the evidence supporting dual roles for the UGT pathway, affecting cancer progression and drug resistance. Pharmacogenomic testing of UGT profiles in patients and the development of therapeutic options that impair UGT actions could provide useful prognostic and predictive biomarkers and enhance the efficacy of anti-cancer drugs.

UGT2B17 modifies drug response in chronic lymphocytic leukaemia.

BACKGROUND: High UGT2B17 is associated with poor prognosis in untreated chronic lymphocytic leukaemia (CLL) patients and its expression is induced in non-responders to fludarabine-containing regimens. We examined whether UGT2B17, the predominant lymphoid glucuronosyltransferase, affects leukaemic drug response and is involved in the metabolic inactivation of anti-leukaemic agents. METHODS: Functional enzymatic assays and patients' plasma samples were analysed by mass-spectrometry to evaluate drug inactivation by UGT2B17. Cytotoxicity assays and RNA sequencing were used to assess drug response and transcriptome changes associated with high UGT2B17 levels. RESULTS: High UGT2B17 in B-cell models led to reduced sensitivity to fludarabine, ibrutinib and idelalisib. UGT2B17 expression in leukaemic cells involved a non-canonical promoter and was induced by short-term treatment with these anti-leukaemics. Glucuronides of both fludarabine and ibrutinib were detected in CLL patients on respective treatment, however UGT2B17 conjugated fludarabine but not ibrutinib. AMP-activated protein kinase emerges as a pathway associated with high UGT2B17 in fludarabine-treated patients and drug-treated cell models. The expression changes linked to UGT2B17 exposed nuclear factor kappa B as a key regulatory hub. CONCLUSIONS: Data imply that UGT2B17 represents a mechanism altering drug response in CLL through direct inactivation but would also involve additional mechanisms for drugs not inactivated by UGT2B17.

Alternative promoters control UGT2B17-dependent androgen catabolism in prostate cancer and its influence on progression.

BACKGROUND: Perturbation of the major UGT2B17-dependent androgen catabolism pathway has the potential to affect prostate cancer (PCa) progression. The objective was to evaluate UGT2B17 protein expression in primary tumours in relation to hormone levels, disease characteristics and cancer evolution. METHODS: We conducted an analysis of a high-density prostate tumour tissue microarray consisting of 239 localised PCa cases treated by radical prostatectomy (RP). Cox proportional hazard ratio analysis was used to evaluate biochemical recurrence (BCR), and a linear regression model evaluated variations in circulating hormone levels measured by mass spectrometry. The transcriptome of UGT2B17 in PCa was established by using RNA-sequencing data. RESULTS: UGT2B17 expression in primary tumours was associated with node-positive disease at RP and linked to circulating levels of 3α-diol-17 glucuronide, a major circulating DHT metabolite produced by the UGT2B17 pathway. UGT2B17 was an independent prognostic factor linked to BCR after RP, and its overexpression was associated with development of metastasis. Finally, we demonstrated that distinctive alternative promoters dictate UGT2B17-dependent androgen catabolism in localised and metastatic PCa. CONCLUSIONS: The androgen-inactivating gene UGT2B17 is controlled by overlooked regulatory regions in PCa. UGT2B17 expression in primary tumours influences the steroidome, and is associated with relevant clinical outcomes, such as BCR and metastasis.

Post-transcriptional Regulation of UGT2B10 Hepatic Expression and Activity by Alternative Splicing.

The detoxification enzyme UDP-glucuronosyltransferase UGT2B10 is specialized in the N-linked glucuronidation of many drugs and xenobiotics. Preferred substrates possess tertiary aliphatic amines and heterocyclic amines, such as tobacco carcinogens and several antidepressants and antipsychotics. We hypothesized that alternative splicing (AS) constitutes a means to regulate steady-state levels of UGT2B10 and enzyme activity. We established the transcriptome of UGT2B10 in normal and tumoral tissues of multiple individuals. The highest expression was in the liver, where 10 AS transcripts represented 50% of the UGT2B10 transcriptome in 50 normal livers and 44 hepatocellular carcinomas. One abundant class of transcripts involves a novel exonic sequence and leads to two alternative (alt.) variants with novel in-frame C termini of 10 or 65 amino acids. Their hepatic expression was highly variable among individuals, correlated with canonical transcript levels, and was 3.5-fold higher in tumors. Evidence for their translation in liver tissues was acquired by mass spectrometry. In cell models, they colocalized with the enzyme and influenced the conjugation of amitriptyline and levomedetomidine by repressing or activating the enzyme (40%-70%; P < 0.01) in a cell context-specific manner. A high turnover rate for the alt. proteins, regulated by the proteasome, was observed in contrast to the more stable UGT2B10 enzyme. Moreover, a drug-induced remodeling of UGT2B10 splicing was demonstrated in the HepaRG hepatic cell model, which favored alt. variants expression over the canonical transcript. Our findings support a significant contribution of AS in the regulation of UGT2B10 expression in the liver with an impact on enzyme activity.

Sex-dependent association of circulating sex steroids and pituitary hormones with treatment-free survival in chronic lymphocytic leukemia patients.

Chronic lymphocytic leukemia (CLL) is not considered a hormone-regulated cancer although sex is a recognized risk factor with men more frequently diagnosed and developing progressive disease. We hypothesized that variable hormonal exposure may have a sexually dimorphic influence on treatment-free survival (TFS). In 156 CLL cases, we quantitatively profiled 29 circulating steroids (progesterone, adrenal precursors, androgens, estrogens, and catechol estrogens) as well as luteinizing hormone (LH) and follicle-stimulating hormone. Median TFS was shorter for men than that for women (80.7 vs. 135.0 months, P = 0.033). Circulating hormone profiles in CLL patients were significantly different from those of healthy donors. In male CLL cases, higher LH levels were associated with shorter TFS (adjusted hazard ratio (HRadj) 2.11; P = 0.004). In female CLL cases, high levels of the potent androgens testosterone and dihydrotestosterone and the sum of methoxy estrogens were associated with an improved TFS with HRadj values of 0.24 (P = 0.007), 0.54 (P = 0.023), and 0.31 (P = 0.034), respectively. Reduced TFS was observed for women with CLL exhibiting high expression of the steroid-inactivating UGT2B17 enzyme. This study is the first to establish a link between the outcome of CLL patients, sex steroids, and pituitary hormones, revealing a sex-specific hormonal imbalance associated with disease progression.

A Non-Canonical Role for the Glycosyltransferase Enzyme UGT2B17 as a Novel Constituent of the B Cell Receptor Signalosome.

In chronic lymphocytic leukemia (CLL), an elevated glycosyltransferase UGT2B17 expression (UGT2B17HI) identifies a subgroup of patients with shorter survival and poor drug response. We uncovered a mechanism, possibly independent of its enzymatic function, characterized by an enhanced expression and signaling of the proximal effectors of the pro-survival B cell receptor (BCR) pathway and elevated Bruton tyrosine kinase (BTK) phosphorylation in B-CLL cells from UGT2B17HI patients. A prominent feature of B-CLL cells is the strong correlation of UGT2B17 expression with the adverse marker ZAP70 encoding a tyrosine kinase that promotes B-CLL cell survival. Their combined high expression levels in the treatment of naïve patients further defined a prognostic group with the highest risk of poor survival. In leukemic cells, UGT2B17 knockout and repression of ZAP70 reduced proliferation, suggesting that the function of UGT2B17 might involve ZAP70. Mechanistically, UGT2B17 interacted with several kinases of the BCR pathway, including ZAP70, SYK, and BTK, revealing a potential therapeutic vulnerability. The dual SYK and JAK/STAT6 inhibitor cerdulatinib most effectively compromised the proliferative advantage conferred by UGT2B17 compared to the selective BTK inhibitor ibrutinib. Findings point to an oncogenic role for UGT2B17 as a novel constituent of BCR signalosome also connected with microenvironmental signaling.

Application of annotation-agnostic RNA sequencing data analysis tools for biomarker discovery in liquid biopsy.

RNA sequencing analysis is an important field in the study of extracellular vesicles (EVs), as these particles contain a variety of RNA species that may have diagnostic, prognostic and predictive value. Many of the bioinformatics tools currently used to analyze EV cargo rely on third-party annotations. Recently, analysis of unannotated expressed RNAs has become of interest, since these may provide complementary information to traditional annotated biomarkers or may help refine biological signatures used in machine learning by including unknown regions. Here we perform a comparative analysis of annotation-free and classical read-summarization tools for the analysis of RNA sequencing data generated for EVs isolated from persons with amyotrophic lateral sclerosis (ALS) and healthy donors. Differential expression analysis and digital-droplet PCR validation of unannotated RNAs also confirmed their existence and demonstrates the usefulness of including such potential biomarkers in transcriptome analysis. We show that find-then-annotate methods perform similarly to standard tools for the analysis of known features, and can also identify unannotated expressed RNAs, two of which were validated as overexpressed in ALS samples. We demonstrate that these tools can therefore be used for a stand-alone analysis or easily integrated into current workflows and may be useful for re-analysis as annotations can be integrated post hoc.

Pathogenic variants carrier screening in New Brunswick: Acadians reveal high carrier frequency for multiple genetic disorders.

BACKGROUND: Founder populations that have recently undergone important genetic bottlenecks such as French-Canadians and Ashkenazi Jews can harbor some pathogenic variants at a higher carrier rate than the general population, putting them at a higher risk for certain genetic diseases. In these populations, there can be considerable benefit to performing ethnic-based or expanded preconception carrier screening, which can help in the prevention or early diagnosis and management of some genetic diseases. Acadians are descendants of French immigrants who settled in the Atlantic Coast of Canada in the seventeenth century. Yet, the Acadian population has never been investigated for the prevalence/frequency of disease-causing genetic variants. METHODS: An exome sequencing panel for 312 autosomal recessive and 30 X-linked diseases was designed and specimens from 60 healthy participants were sequenced to assess carrier frequency for the targeted diseases. RESULTS: In this study, we show that a sample population of Acadians in South-East New Brunswick harbor variants for 28 autosomal recessive and 1 X-linked diseases, some of which are significantly more frequent in comparison to reference populations. CONCLUSION: Results from this pilot study suggests a need for further investigation of genomic variation in this population and possibly implementation of targeted carrier and neonatal screening programs.

KLF5 and NFYA factors as novel regulators of prostate cancer cell metabolism.

Prostate cancer (PCa) cells rely on the androgen receptor (AR) signaling axis to reprogram metabolism to sustain aberrant proliferation. Whether additional transcription factors participate to this reprogramming remains mostly unknown. To identify such factors, DNA motif analyses were performed in the promoter and regulatory regions of genes sensitive to androgens in PCa cells. These analyses identified two transcription factors, KLF5 and NFYA, as possibly associated with PCa cell metabolism. In clinical datasets, KLF5 and NFYA expression levels were associated with disease aggressiveness, being significantly decreased and increased, respectively, during PCa progression. Their expression was next investigated by qPCR and Western blot in human PCa cell models, revealing a positive regulation of KLF5 by androgens and a correlation between NFYA and AR protein expression status. siRNA-mediated knockdown of KLF5 increased human PCa cell proliferation rate in AR-positive cell models, suggesting a tumor suppressor function. Live-cell metabolic assays showed that knockdown of KLF5 promoted mitochondrial respiration, a key metabolic pathway associated with PCa progression. The opposite was observed for knockdown of NFYA regarding proliferation and respiration. RNA-seq analyses following the knockdown of either KLF5 and NFYA confirmed that both factors regulated distinct metabolic gene signatures, as well as other gene signatures, explaining their differential impact on PCa cell proliferation and metabolism. Overall, our findings identify KLF5 and NFYA as novel regulators of PCa cell metabolism.

Inactivation of Prostaglandin E2 as a Mechanism for UGT2B17-Mediated Adverse Effects in Chronic Lymphocytic Leukemia.

High expression of the metabolic enzyme UDP-glucuronosyltransferase UGT2B17 in chronic lymphocytic leukemia (CLL) cells was associated with poor prognosis in two independent studies. However, the underlying mechanism remains unknown. We hypothesized that UGT2B17 impacts intracellular levels of hormone-like signaling molecules involved in the regulation of gene expression in leukemic cells. We initially confirmed in a third cohort of 291 CLL patients that those with high UGT2B17 displayed poor prognosis (hazard ratio of 2.31, P = 0.015). Consistent with the unfavorable prognostic significance of elevated UGT2B17 expression in CLL patients, high UGT2B17 expression was associated with enhanced proliferation of MEC1 and JVM2 malignant B-cell models. Transcriptomic analyses revealed that high UGT2B17 was linked to a significant alteration of genes related to prostaglandin E2 (PGE2) and to its precursor arachidonic acid, both in cell models and a cohort of 448 CLL patients. In functional assays, PGE2 emerged as a negative regulator of apoptosis in CLL patients and proliferation in cells models, whereas its effect was partially abrogated by high UGT2B17 expression in MEC1 and JVM2 cells. Enzymatic assays and mass-spectrometry analyses established that the UGT2B17 enzyme inactivates PGE2 by its conjugation to glucuronic acid (GlcA) leading to the formation of two glucuronide (G) derivatives. High UGT2B17 expression was further associated with a proficient inactivation of PGE2 to PGE2-G in CLL patient cells and cell models. We conclude that UGT2B17-dependent PGE2 glucuronidation impairs anti-oncogenic PGE2 effects in leukemic cells, thereby partially contributing to disease progression in high UGT2B17 CLL patients.

Germline variability and tumor expression level of ribosomal protein gene RPL28 are associated with survival of metastatic colorectal cancer patients.

This study investigated the potential of single nucleotide polymorphisms as predictors of survival in two cohorts comprising 417 metastatic colorectal cancer (mCRC) patients treated with the FOLFIRI (folinic acid, 5-fluorouracil and irinotecan) regimen. The rs4806668G > T of the ribosomal protein gene RPL28 was associated with shorter progression-free survival and overall survival by 5 and 9 months (P = 0.002), with hazard ratios of 3.36 (P < 0.001) and 3.07 (P = 0.002), respectively. The rs4806668T allele was associated with an increased RPL28 expression in transverse normal colon tissues (n = 246, P = 0.007). RPL28 expression was higher in colorectal tumors compared to paired normal tissues by up to 124% (P < 0.001) in three independent datasets. Metastatic cases with highest RPL28 tumor expression had a reduced survival in two datasets (n = 88, P = 0.009 and n = 56, P = 0.009). High RPL28 was further associated with changes in immunoglobulin and extracellular matrix pathways. Repression of RPL28 reduced proliferation by 1.4-fold to 5.6-fold (P < 0.05) in colon cancer HCT116 and HT-29 cells. Our findings suggest that the ribosomal RPL28 protein may influence mCRC outcome.

The Intracellular Localisation and Phosphorylation Profile of the Human 5-Lipoxygenase Δ13 Isoform Differs from That of Its Full Length Counterpart.

5-Lipoxygenase (5-LO) catalyzes leukotriene (LT) biosynthesis by a mechanism that involves interactions with 5-lipoxygenase activating protein (FLAP) and coactosin-like protein (CLP). 5-LO splice variants were recently identified in human myeloid and lymphoid cells, including the catalytically inactive ∆13 isoform (5-LO∆13) whose transcript lacks exon 13. 5-LO∆13 inhibits 5-LO product biosynthesis when co-expressed with active full length 5-LO (5-LO1). The objective of this study was to investigate potential mechanisms by which 5-LO∆13 interferes with 5-LO product biosynthesis in transfected HEK293 cells. When co-expressed with 5-LO1, 5-LO∆13 inhibited LT but not 5-hydroxyeicosatetraenoic acid (5-HETE) biosynthesis. This inhibition was independent of 5-LO∆13-FLAP interactions since it occurred in cells expressing FLAP or not. In cell-free assays CLP enhances 5-LO activity through interactions with tryptophan-102 of 5-LO. In the current study, the requirement for W102 was extended to whole cells, as cells expressing the 5-LO1-W102A mutant produced little 5-LO products. W102A mutants of 5-LO∆13 inhibited 5-LO product biosynthesis as effectively as 5-LO∆13 suggesting that inhibition is independent of interactions with CLP. Confocal microscopy showed that 5-LO1 was primarily in the nucleoplasm whereas W102A mutants showed a diffuse cellular expression. Despite the retention of known nuclear localisation sequences, 5-LO∆13 was cytosolic and concentrated in ER-rich perinuclear regions where its effect on LT biosynthesis may occur. W102A mutants of 5-LO∆13 showed the same pattern. Consistent with subcellular distribution patterns, 5-LO∆13 was hyper-phosphorylated on S523 and S273 compared to 5-LO1. Together, these results reveal a role for W102 in nuclear targeting of 5-LO1 suggesting that interactions with CLP are required for nuclear localization of 5-LO1, and are an initial characterisation of the 5-LO∆13 isoform whose inhibition of LT biosynthesis appears independent of interactions with CLP and FLAP. Better knowledge of the regulation and properties of alternative 5-LO isoforms will contribute to understanding the complex regulation of LT biosynthesis.