Long-Term Treatment of Gaucher Disease with Velaglucerase Alfa in ERT-Naïve Patients from the Gaucher Outcome Survey (GOS) Registry.

Background: Gaucher disease (GD) is a rare, autosomal, recessive condition characterized by hepatosplenomegaly, thrombocytopenia, anemia, and bone abnormalities, often requiring life-long treatment. Velaglucerase alfa has improved hematologic and visceral parameters in clinical trials; however, limited long-term efficacy and safety data are available. Methods: The Gaucher Outcome Survey (GOS), a structured and validated international registry for patients with confirmed GD, provides an opportunity to evaluate long-term data from patients receiving velaglucerase alfa. Results: This analysis included 376 treatment-naïve children and adults with GD enrolled in GOS, including 20 with type 3 GD, who initiated velaglucerase alfa through participation in clinical trials or as part of their clinical management and continued treatment for a mean (range) time of 6.6 (0.003-18.6) years. Initial improvements in hematologic and visceral parameters and the biomarkers glucosylsphingosine (lyso-GL1) and chitotriosidase were observed after one year of treatment and were maintained throughout the follow-up period. Of 129 (34.3%) patients who developed adverse events during the follow-up period, events were considered related to treatment in 33 (8.8%). None led to treatment discontinuation. There were 21 deaths overall, none of which were considered related to treatment. Conclusions: This analysis of data from the GOS registry supports the safety and efficacy of velaglucerase alfa in patients with GD.

Reply to Mistry et al. The Two Substrate Reduction Therapies for Type 1 Gaucher Disease Are Not Equivalent. Comment on "Hughes et al. Switching between Enzyme Replacement Therapies and Substrate Reduction Therapies in Patients with Gaucher Disease: Data from the Gaucher Outcome Survey (GOS). J. Clin. Med. 2022, 11, 5158".

Thank you for allowing us the opportunity to respond to the commentary from Mistry and colleagues (Comment: The two substrate reduction therapies for type 1 Gaucher disease are not equivalent) [...].

Current challenges and future opportunities in patient-focused management of hereditary angioedema: A narrative review.

Patients with hereditary angioedema (HAE) experience a high burden of disease due to unpredictable, painful, disfiguring, and potentially life-threatening HAE attacks. Multiple HAE-specific medications for the on-demand treatment, short-term and long-term prophylaxis of HAE attacks have entered the market in recent years; however, the availability and access to these medications may vary between different countries. For this review, PubMed and EMBASE databases were searched for guidelines, consensus statements, and other publications on HAE management as well as publications on quality of life in patients with HAE. The current guidelines and recent literature on HAE management in specific countries are summarized with the aim to highlight the similarities and differences between guideline recommendations and the country-specific clinical practice. Improvement in quality of life, which is a key goal in HAE management, is also discussed and the country-specific trends are highlighted. Finally, the ways to achieve a more patient-centric approach to HAE management within the framework set by the clinical management guidelines are examined.

Switching between Enzyme Replacement Therapies and Substrate Reduction Therapies in Patients with Gaucher Disease: Data from the Gaucher Outcome Survey (GOS).

Switching between enzyme replacement therapies (ERT) and substrate reduction therapies (SRT) in patients with type 1 Gaucher disease (GD1) is not uncommon; however, the reasons for switchng treatments have not been explored in detail. Data from the Gaucher Outcome Survey (GOS), an international registry for patients with confirmed GD, were used to evaluate the reasons for, and consequences of, switching between these treatment types. Of the 1843 patients enrolled in GOS on 25 February 2020, 245 had undergone a treatment switch: 222 from initial ERT to SRT (of whom 88 later switched back to ERT) and 23 from initial SRT to ERT. The most common reasons for ERT-SRT switching were duration of infusion (25.4%), drug shortage (22.0%), and adverse events (AEs; 11.9%), and for SRT-ERT switching, AEs (63.6%), lack of beneficial effect (16.4%), and participation in a clinical trial (9.1%). Bodyweight and hematologic parameters largely remained stable before and after switching between ERT and SRT, although with substantial variation between patients. These findings contribute to understanding why treatment switching occurs in patients with GD, and may help physicians recognize the real-world impact of treatment switching between ERT and SRT for patients with GD.

Urinary albumin excretion with sitagliptin compared to sulfonylurea as add on to metformin in type 2 diabetes patients with albuminuria: A real-world evidence study.

AIM: To compare the change in urinary albumin to creatinine ratio (UACR) in type 2 diabetes (T2DM) patients with albuminuria who initiate sitagliptin to those who initiate a sulfonylurea (SU) as add-on to metformin monotherapy. METHOD: A cohort of T2DM patients with albuminuria (UACR >30mg/g) who initiated sitagliptin or SU as add-on dual therapy to metformin between 2008 and 2014 was extracted from the computerized medical records of a large managed care organization in Israel. Patients with albuminuria and UACR measurements available at treatment initiation and 120-365days afterwards were included. Propensity scores were calculated based on 17 factors, including demography, comorbidities, baseline levels of HbA1c, UACR, BMI, eGFR, and ACE/ARB use, and patients were matched in a 1:1 ratio. Changes in UACR were compared between the matched pairs using generalized estimating equations. RESULTS: A total of 282 eligible pairs (sitagliptin:SU) were identified. During a mean follow-up of 9months, median UACR changes were -35% (IQR=-73% to 5%) and -31% (IQR=-72% to 21%) in the sitagliptin and SU groups, respectively. Mean absolute HbA1c reductions among sitagliptin and SU groups were 0.9% and 1.0%, respectively. The magnitude of UACR reduction generally increased with greater magnitude of HbA1c reduction in both treatment groups. However, after controlling for HbA1c reduction and the interaction between HbA1c reduction and UACR reduction, sitagliptin users demonstrated a trend toward an increased likelihood of UACR reduction compared to SU users (odds ratio=1.20; 95% confidence interval: 0.99-1.47, P=0.063). CONCLUSION: Our results suggest that both sitagliptin and SU reduce albuminuria as an add-on therapy to metformin, but that sitagliptin may provide greater reductions in albuminuria independent of glycemic control when compared to SU. Larger population studies are required to further explore this.

A genomic integration method for the simultaneous visualization of endogenous mRNAs and their translation products in living yeast.

Protein localization within cells can be achieved by the targeting and localized translation of mRNA. Yet, our understanding of the dynamics of mRNA targeting and protein localization, and of how general this phenomenon is, is not clear. Plasmid-based expression systems have been used to visualize exogenously expressed mRNAs and proteins; however, these methods typically produce them at levels greater than endogenous and can result in mislocalization. Hence, a method that allows for the simultaneous visualization of endogenous mRNAs and their translation products in living cells is needed. We previously developed a method (m-TAG) to localize endogenously expressed mRNAs in yeast by chromosomal insertion of the MS2 aptamer sequence between the open-reading frame (ORF) and 3' UTR of any gene. Upon coexpression with the MS2 RNA-binding coat protein (MS2-CP) fused with GFP, the aptamer-tagged mRNAs bearing their 3' UTRs are localized using fluorescence microscopy. Here we describe an advanced method (mp-TAG) that allows for the simultaneous visualization of both endogenously expressed mRNAs and their translation products in living yeast for the first time. Homologous recombination is used to insert the mCherry gene and MS2-CP binding sites downstream from any ORF, in order to localize protein and mRNA, respectively. As proof of the concept, we tagged ATP2 as a representative gene and demonstrated that endogenous ATP2 mRNA and protein localize to mitochondria, as shown previously. In addition, we demonstrate that tagged proteins like Hhf2, Vph1, and Yef3 localize to their expected subcellular location, while the localization of their mRNAs is revealed for the first time.

Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae.

Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with mitochondria in vivo. This supports earlier cell fractionation and microarray-based studies that proposed mMP association with the mitochondrial fraction. Interestingly, a number of mMPs showed a dependency on the mitochondrial Puf3 RNA-binding protein, as well as nonessential proteins of the translocase of the outer membrane (TOM) complex import machinery, for normal colocalization with mitochondria. We examined the specific determinants of ATP2 and OXA1 mRNA localization and found a mutual dependency on the 3' UTR, Puf3, Tom7, and Tom70, but not Tom20, for localization. Tom6 may facilitate the localization of specific mRNAs as OXA1, but not ATP2, mRNA was mislocalized in tom6Δ cells. Interestingly, a substantial fraction of OXA1 and ATP2 RNA granules colocalized with the endoplasmic reticulum (ER) and a deletion in MDM10, which mediates mitochondria-ER tethering, resulted in a significant loss of OXA1 mRNA localization with ER. Finally, neither ATP2 nor OXA1 mRNA targeting was affected by a block in translation initiation, indicating that translation may not be essential for mRNA anchoring. Thus, endogenously expressed mRNAs are targeted to the mitochondria in vivo, and multiple factors contribute to mMP localization.

Phospholipase D-mTOR requirement for the Warburg effect in human cancer cells.

A characteristic of cancer cells is the generation of lactate from glucose in spite of adequate oxygen for oxidative phosphorylation. This property - known as the "Warburg effect" or aerobic glycolysis - contrasts with anaerobic glycolysis, which is triggered in hypoxic normal cells. The Warburg effect is thought to provide a means for cancer cells to survive under conditions where oxygen is limited and to generate metabolites necessary for cell growth. The shift from oxidative phosphorylation to glycolysis in response to hypoxia is mediated by the production of hypoxia-inducible factor (HIF) - a transcription factor family that stimulates the expression of proteins involved in glucose uptake and glycolysis. We reported previously that elevated phospholipase D (PLD) activity in renal and breast cancer cells is required for the expression of the α subunits of HIF1 and HIF2. We report here that the aerobic glycolysis observed in human breast and renal cancer cells is dependent on the elevated PLD activity. Intriguingly, the effect of PLD on the Warburg phenotype was dependent on the mammalian target of rapamycin complex 1 (mTORC1) in the breast cancer cells and on mTORC2 in the renal cancer cells. These data indicate that elevated PLD-mTOR signaling, which is common in human cancer cells, is critical for the metabolic shift to aerobic glycolysis.

Localization of mRNAs coding for peroxisomal proteins in the yeast, Saccharomyces cerevisiae.

Targeted mRNA trafficking and local translation may play a significant role in controlling protein localization. Here we examined for the first time the localization of all ( approximately 50) mRNAs encoding peroxisomal proteins (mPPs) involved in peroxisome biogenesis and function. By using the bacteriophage MS2-CP RNA-binding protein (RBP) fused to multiple copies of GFP, we demonstrated that >40 endogenously expressed mPPs tagged with the MS2 aptamer form fluorescent RNA granules in vivo. The use of different RFP-tagged organellar markers revealed 3 basic patterns of mPP granule localization: to peroxisomes, to the endoplasmic reticulum (ER), and nonperoxisomal. Twelve mPPs (i.e., PEX1, PEX5, PEX8, PEX11-15, DCI1, NPY1, PCS60, and POX1) had a high percentage (52%-80%) of mRNA colocalization with peroxisomes. Thirteen mPPs (i.e., AAT2, PEX6, MDH3, PEX28, etc.) showed a low percentage (30%-42%) of colocalization, and 1 mPP (PEX3) preferentially localized to the ER. The mPPs of the nonperoxisomal pattern (i.e., GPD1, PCD1, PEX7) showed <<30% colocalization. mPP association with the peroxisome or ER was verified using cell fractionation and RT-PCR analysis. A model mPP, PEX14 mRNA, was found to be in close association with peroxisomes throughout the cell cycle, with its localization depending in part on the 3'-UTR, initiation of translation, and the Puf5 RBP. The different patterns of mPP localization observed suggest that multiple mechanisms involved in mRNA localization and translation may play roles in the importation of protein into peroxisomes.

Regulation of mTORC1 and mTORC2 complex assembly by phosphatidic acid: competition with rapamycin.

mTOR, the mammalian target of rapamycin, is a critical node for control of cell growth and survival and has widely been implicated in cancer survival signals. mTOR exists in two complexes: mTORC1 and mTORC2. Phospholipase D (PLD) and its metabolite phosphatidic acid (PA) have been implicated in the regulation of mTOR; however, their role has been controversial. We report here that suppression of PLD prevents phosphorylation of the mTORC1 substrate S6 kinase (S6K) at Thr389 and the mTORC2 substrate Akt at Ser473. Suppression of PLD also blocked insulin-stimulated Akt phosphorylation at Ser473 and the mTORC2-dependent phosphorylation of PRAS40. Importantly, PA was required for the association of mTOR with Raptor to form mTORC1 and that of mTOR with Rictor to form mTORC2. The effect of PA was competitive with rapamycin-with much higher concentrations of rapamycin needed to compete with the PA-mTORC2 interaction than with PA-mTORC1. Suppressing PA production substantially increased the sensitivity of mTORC2 to rapamycin. Data provided here demonstrate a PA requirement for the stabilization of both mTORC1 and mTORC2 complexes and reveal a mechanism for the inhibitory effect of rapamycin on mTOR. This study also suggests that by suppressing PLD activity, mTORC2 could be targeted therapeutically with rapamycin.

Differential dependence of hypoxia-inducible factors 1 alpha and 2 alpha on mTORC1 and mTORC2.

Constitutive expression of hypoxia-inducible factor (HIF) has been implicated in several proliferative disorders. Constitutive expression of HIF1 alpha and HIF2 alpha has been linked to a number of human cancers, especially renal cell carcinoma (RCC), in which HIF2 alpha expression is the more important contributor. Expression of HIF1 alpha is dependent on the mammalian target of rapamycin (mTOR) and is sensitive to rapamycin. In contrast, there have been no reports linking HIF2 alpha expression with mTOR. mTOR exists in two complexes, mTORC1 and mTORC2, which are differentially sensitive to rapamycin. We report here that although there are clear differences in the sensitivity of HIF1 alpha and HIF2 alpha to rapamycin, both HIF1 alpha and HIF2 alpha expression is dependent on mTOR. HIF1 alpha expression was dependent on both Raptor (a constituent of mTORC1) and Rictor (a constitutive of mTORC2). In contrast, HIF2 alpha was dependent only on the mTORC2 constituent Rictor. These data indicate that although HIF1 alpha is dependent on both mTORC1 and mTORC2, HIF2 alpha is dependent only on mTORC2. We also examined the dependence of HIF alpha expression on the mTORC2 substrate Akt, which exists as three different isoforms, Akt1, Akt2, and Akt3. Interestingly, the expression of HIF2 alpha was dependent on Akt2, whereas that of HIF1 alpha was dependent on Akt3. Because HIF2 alpha is apparently more critical in RCC, this study underscores the importance of targeting mTORC2 and perhaps Akt2 signaling in RCC and other proliferative disorders in which HIF2 alpha has been implicated.

[Can defective TGF-Beta signaling be an Achilles heel in human cancer?].

Survival signals in cancer cells activate mTOR-the mammalian target of rapamycin. mTOR suppresses TGF-beta signals that arrest cell cycle progression in late G1-thus activated mTOR prevents cell cycle arrest at a checkpoint mediated by TGF-beta. Rapamycin treatment resurrects TGF-beta signals causing G1 arrest. Defects in TGF-beta signaling are common in human cancer, and ironically, cancer cells with defective TGF-beta signaling that do not arrest in G1, instead undergo apoptosis when treated with rapamycin. Thus, defective TGF-beta signaling may represent an Achilles heel for rational therapeutic targeting of cancer cells using rapamycin-based strategies.

Suppression of TGF-beta signaling by phospholipase D.

MDA-MB-231 human breast cancer cells have a survival signal generated by phospholipase D (PLD) that involves the activation of mTOR and MAP kinase. TGF-beta signals that block cell cycle progression in G(1) are suppressed in MDA-MB-231 cells. We report here that the elevated PLD activity in MDA-MB-231 cells suppresses TGF-beta signaling. Suppression of PLD activity or PLD expression resulted in increased phosphorylation of Smad2 and Smad3 on Ser 465/467-sites on Smads that get phosphorylated by the TGF-beta receptor and positively regulate TGF-beta signaling. The effect of PLD suppression on Smad2/3 phosphorylation was dependent on the presence of TGF-beta. Suppression of PLD also suppressed phosphorylation of Smad2 on Ser 245/250/255-sites that are phosphorylated by MAP kinase and negatively regulate TGF-beta signaling. Suppression of PLD also led to increased expression of the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1, the expression of which is stimulated in response to TGF-beta. Consistent with the elevated expression of CDK inhibitors, suppression of PLD also suppressed phosphorylation of the CDK substrate pRb. Similar effects were also seen in PANC-1 human pancreatic cancer cells. The data presented here indicate that the suppressed TGF-beta signaling in MDA-MB-231 and perhaps many other human cancer cells is due to elevated PLD activity and mediated by mTOR and MAP kinase. These results indicate that the survival signals generated by PLD involve the suppression TGF-beta signals that promote G(1) arrest.