Renal transplantation for infantile and juvenile cystinosis: Two case report and review of the literature.

Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by cystine buildup in various tissues, including the kidneys. Renal involvement is the primary manifestation, leading to end-stage renal disease (ESRD) if left untreated. Kidney transplantation (KT) in patients with cystinosis has significantly improved their prognosis for the disease outcome. Detailed reports on preoperative and Long-term postoperative management in these patients remain sparse. This report discusses the outcomes of two young adult patients of Middle Eastern descent with cystinosis who underwent KT. The first patient, diagnosed with infantile nephropathic cystinosis treated by cystine-depleting therapy, was operated by KT at the age of 18. The second patient, diagnosed with juvenile cystinosis, underwent transplantation at the age of 35 after being treated with hemodialysis. Our report describes detailed pre- and postoperative managements, including laboratory results, and pharmacological interventions. Both cases highlighted the varying clinical manifestations and disease severity between infantile and juvenile cystinosis. Pre-transplant conditions included renal dysfunction, growth retardation, secondary hyperparathyroidism, anemia, and extrarenal manifestations. Following KT, both patients experienced regained renal function, resolution of extrarenal complications, and normalization of laboratory parameters. Furthermore, both patients showed excellent postoperative outcomes with no acute rejection or allograft-related complications. KT is the treatment of choice for cystinosis patients with ESRD. Long-term follow-up post-transplantation is crucial to maintain good graft function. Further studies may elucidate optimal pre- and postoperative management protocols for this rare condition.

Neurologic involvement in cystinosis: Focus on brain lesions and new evidence of four-repeat (4R-) Tau immunoreactivity.

Nephropathic cystinosis is a rare autosomal recessive storage disorder caused by CTNS gene mutations, leading to autophagy-lysosomal pathway impairment and cystine crystals accumulation. Neurologic involvement is highly variable and includes both neurodevelopmental and neurodegenerative disturbances, as well as focal neurologic deficits. By presenting longitudinal data of a 28-year-old patient with a large infratentorial lesion, we summarized the pathology, clinical and imaging features of neurological involvement in cystinosis patients. Brain damage in form of cystinosis-related cerebral lesions occurs in advanced disease phases and is characterized by the accumulation of cystine crystals, subsequent inflammation with vasculitis-like features, necrosis, and calcification. Epilepsy is a frequent comorbidity in affected individuals. Steroids might play a role in the symptomatic treatment of "stroke-like" episodes due to edematous-inflammatory lesions, but probably do not change the overall prognosis. Lifelong compliance to depleting therapy with cysteamine still represents the main therapeutic option. However, consequences of CTNS gene defects are not restricted to cystine accumulation. New evidence of four-repeat (4R-) Tau immunoreactivity suggests concurrent progressive neurodegeneration in cystinosis patients, highlighting the need of innovative therapeutic strategies, and shedding light on the crosstalk between proteinopathies and autophagy-lysosomal system defects. Eventually, emerging easily accessible biomarkers such as serum neurofilament light chains (NfL) might detect subclinical neurologic involvement in cystinosis patients.

Event-related potential (ERP) evidence for visual processing differences in children and adults with cystinosis (CTNS gene mutations).

BACKGROUND: Cystinosis, a rare lysosomal storage disease caused by mutations in the CTNS gene, is characterized by cystine crystallization and accumulation within multiple tissues, including kidney and brain. Its impact on neural function appears mild relative to its effects on other organs during early disease, but since therapeutic advances have led to substantially increased life expectancy, neurological implications are of increasing interest, necessitating deeper understanding of the impact of cystinosis on neurocognitive function. Behavioral difficulties have been reported in cystinosis in the visual domain. Very little is known, however, about how the brains of people living with cystinosis process visual information. This is especially interesting given that cystine accumulation in the cornea and posterior ocular structures is a hallmark of cystinosis. METHODS: Here, high-density scalp electrophysiology was recorded to visual stimuli (during a Go/No-Go task) to investigate visual processing in individuals with cystinosis, compared to age-matched controls. Analyses focused on early stages of cortical visual processing. RESULTS: The groups differed in their initial cortical response, with individuals with cystinosis exhibiting a significantly larger visual evoked potential (VEP) in the 130-150 ms time window. The groups also differed in the associations between neural responses and verbal abilities: While controls with higher IQ scores presented larger neural responses, that relationship was not observed in cystinosis. CONCLUSIONS: The enlarged VEP in cystinosis could be the result of cortical hyperexcitability and/or differences in attentional engagement and explain, at least partially, the visual and visual-spatial difficulties described in this population.

Cu2+-Pyropheophorbide a-Cystine Conjugate: Synergistic Photodynamic/Chemodynamic Therapy and Glutathione Depletion Improves the Antitumor Efficacy and Downregulates the Hypoxia-Inducing Factor.

Cancer immune escape, metastasis, recurrence, and multidrug resistance are all associated with hypoxia in the tumor microenvironment (TME). We synthesized a CuPPaCC conjugate for reactive oxygen species (ROS)-mediated cancer therapy. CuPPaCC continuously produced cytotoxic ROS and oxygen through a photo-chemocycloreaction, alleviated hypoxia, and inhibited the expression of a hypoxia-inducing factor (HIF-1α). CuPPaCC was synthesized from pyromania phyllophyllic acid a (PPa), cystine (CC), and copper ions, and its structure was characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The ability of CuPPaCC to produce ROS and oxygen after photodynamic therapy (PDT) in vitro and in vivo was investigated. The ability of CuPPaCC to consume glutathione was investigated. CuPPaCC toxicity (light and dark) in CT26 cells was analyzed by MTT and live/dead cell staining. The anticancer effect of CuPPaCC in vivo was investigated in CT26 Balb/c mice. When stimulated by the TME, CuPPaCC released Cu2+ and PPaCC, and the singlet oxygen yield increased from 34 to 56.5%. The dual ROS-generating mechanism via a Fenton-like reaction/photoreaction and dual glutathione depletion via Cu2+/CC multiplied the antitumor efficacy of CuPPaCC. The photo-chemocycloreaction continued to produce oxygen and maintained high ROS levels even after PDT, significantly alleviating hypoxia in the TME and downregulating the expression of HIF-1α. CuPPaCC thus showed excellent antitumor activity in vitro and in vivo. These results showed that the strategy could be effective in improving the antitumor efficacy of CuPPaCC and could be used as a synergistic regimen for cancer therapy.

The gene therapy for corneal pathology with novel nonsense cystinosis mouse lines created by CRISPR Gene Editing.

PURPOSE: Cystinosis is an autosomal recessive lysosomal storage disease (LSDs) caused by mutations in the gene encoding cystinosin (CTNS) that leads to cystine crystal accumulation in the lysosome that compromises cellular functions resulting in tissue damage and organ failure, especially in kidneys and eyes. However, the underlying molecular mechanism of its pathogenesis remains elusive. Two novel mice lines created via CRISPR are used to examine the pathogenesis of cystinosis in the kidney and cornea and the treatment efficacy of corneal pathology using self-complimentary Adeno-associated viral (scAAV-CTNS) vector. METHODS: The CRISPR technique generated two novel cystinotic mouse lines, Ctnsis1 (an insertional mutation) and Ctnsis2 (a nonsense mutation). Immune histochemistry, renal functions test and HRT2 in vivo confocal microscopy were used to evaluate the age-related renal pathogenesis and treatment efficacy of the scAAV-CTNS virus in corneal pathology. RESULTS: Both mutations lead to the production of truncated Ctns proteins. Ctnsis1 and Ctnsis 2 mice exhibit the characteristic of cystinotic corneal crystal phenotype at four-week-old. Treatment with the scAAV-CTNS viral vector decreased the corneal crystals in the treated mice cornea. Ctnsis 1 show renal abnormalities manifested by increased urine volume, reduced urine osmolality, and the loss of response to Desmopressin (dDAVP) at 22-month-old but Ctnsis2 don't manifest renal pathology up to 2 years of age. CONCLUSIONS: Both Ctnsis1 and Ctnsis2 mice exhibit phenotypes resembling human intermediate nephropathic and ocular cystinosis, respectively. scAAV-CTNS viral vectors reduce the corneal cystine crystals and have a great potential as a therapeutic strategy for treating patients suffering from cystinosis.

Cysteine depletion sensitizes prostate cancer cells to agents that enhance DNA damage and to immune checkpoint inhibition.

BACKGROUND: Prostate Cancer (PCa) represents one of the most commonly diagnosed neoplasms in men and is associated with significant morbidity and mortality. Therapy resistance and significant side effects of current treatment strategies indicate the need for more effective agents to treat both androgen-dependent and androgen-independent PCa. In earlier studies, we demonstrated that depletion of L-cysteine/cystine with an engineered human enzyme, Cyst(e)inase, increased intracellular ROS levels and inhibited PCa growth in vitro and in vivo. The current study was conducted to further explore the mechanisms and potential combinatorial approaches with Cyst(e)inase for treatment of PCa. METHODS: DNA single strand breaks and clustered oxidative DNA damage were evaluated by alkaline comet assay and pulsed field gel electrophoresis, respectively. Neutral comet assay and immunofluorescence staining was used to measure DNA double strand breaks. Cell survival and reactive oxygen species level were measured by crystal violet assay and DCFDA staining, respectively. Western blot was used to determine protein expression. FACS analyses were preformed for immune cell phenotyping. Allograft and xenograft tumor models were used for assessing effects on tumor growth. RESULTS: PCa cells treated with Cyst(e)inase lead to DNA single and double strand breaks resulted from clustered oxidative DNA damage (SSBs and DSBs). Cyst(e)inase in combination with Auranofin, a thioredoxin reductase inhibitor, further increased intracellular ROS and DNA DSBs and synergistically inhibited PCa cell growth in vitro and in vivo. A combination of Cyst(e)inase with a PARP inhibitor (Olaparib) also increased DNA DSBs and synergistically inhibited PCa cell growth in vitro and in vivo without additional ROS induction. Knockdown of BRCA2 in PCa cells increased DSBs and enhanced sensitivity to Cyst(e)inase. Finally, Cyst(e)inase treatment altered tumor immune infiltrates and PD-L1 expression and sensitized PCa cells to anti-PD-L1 treatment. CONCLUSIONS: The current results demonstrate the importance of oxidative DNA damage either alone or in combination for Cyst(e)inase-induced anticancer activity. Furthermore, cysteine/cystine depletion alters the tumor immune landscape favoring enhanced immune checkpoint inhibition targeting PD-L1. Thus, combinatorial approaches with Cyst(e)inase could lead to novel therapeutic strategies for PCa.

Cystinosis. / Cystinose.

Cystinosis is a very rare autosomal recessive lysosomal storage disorder with an incidence of 1 : 150,000 - 1 : 200,000, and is caused by mutations in the CTNS gene encoding the lysosomal membrane protein cystinosin, which transports cystine out of the lysosome into the cytoplasm. As a result, accumulation of cystine occurs in almost all cells and tissues, especially in the kidneys, leading to multiple organ involvement. Introduction of drug therapy with cysteamine in the mid 1980s, along with the availability of renal replacement therapy in childhood, have dramatically improved patient outcome. Whereas patients used to die without therapy with end-stage renal failure during the first decade of life, nowadays most patients live well into adulthood without renal replacement therapy, and several reach 40 years. There is robust evidence that early initiation and sustained lifelong therapy with cysteamine are both essential for morbidity and mortality. The rarity of the disease and the multi-organ involvement present an enormous challenge for those affected and the providers of care for this patient group.

Posterior Segment Involvement in Infantile Nephropathic Cystinosis - A Review.

Cystinosis is a rare lysosomal storage disease with a prevalence of 1 : 100 000 - 1 : 200 000 cases. It is caused by biallelic mutations in the CTNS gene, which encodes cystinosin, that transport cystine out of the lysosomes. Due to its dysfunction, cystine crystals accumulate in the lysosomes and ultimately cause apoptosis of the cell. Since cystinosin is ubiquitously present in the body, cystine crystals are deposited in every body structure and lead to the dysfunction of various organ systems in the course of time. Cystine crystals deposited in the cornea are a clinical hallmark of the disease, while there is less awareness of concomitant posterior segment alterations. Symmetrical pigment epithelial mottling and patches of depigmentation frequently start in the periphery and progress towards the posterior pole and can be encountered upon fundus biomicroscopy. Spectral-domain optical coherence tomography (SD-OCT) is an elegant tool for visualizing chorioretinal cystine crystals at the posterior pole. An SD-OCT-based clinical grading of the severity of the chorioretinal manifestation can potentially be applied as a biomarker for systemic disease status and for monitoring oral therapy adherence in the future. Along with previous histological examinations, it may also give information about the location of cystine crystals in the choroid and retina. This review aims to increase the awareness of vision-threatening retinal and choroidal changes in cystinosis and the concomitant findings in SD-OCT.

[Cystinosis: From the gene identification to the first gene therapy clinical trial]. / Cystinose - De la découverte du gène aux premiers essais de thérapie génique.

Cystinosis is an autosomal recessive metabolic disease characterized by lysosomal accumulation of cystine in all the cells of the body. Infantile cystinosis begins in infancy by a renal Fanconi syndrome and eventually leads to multi-organ failure, including the kidney, eye, thyroid, muscle, and pancreas, eventually causing premature death in early adulthood. The current treatment is the drug cysteamine that only delays the progression of the disease. We identified the gene involved, CTNS, and showed that the encoded protein, cystinosin, is a proton-driven cystine transporter. We generated a mouse model of cystinosis, the Ctns-/- mice, that recapitulates the main disease complications. The goal was next to develop a gene therapy approach for cystinosis. We used bone marrow stem cells as a vehicle to bring the healthy CTNS gene to tissues, and we showed that wild-type hematopoietic stem and progenitor cell (HSPC) transplantation led to abundant tissue integration of bone marrow-derived cells, significant decrease of tissue cystine accumulation and long-term kidney, eye and thyroid preservation. We then developed an autologous transplantation approach of HSPCs modified ex vivo using a lentiviral vector to introduce a functional CTNS cDNA, and showed its efficacy in Ctns-/- mice. We conducted the pharmacology/toxicology studies, developed the manufacturing process using human CD34+ cells, and design the clinical trial. We received Food and Drug Administration (FDA)-clearance to start a phase 1/2 clinical trial for cystinosis in December 2018. Six patients have been treated so far. In this review, we describe the path to go from the gene to a gene therapy approach for cystinosis.

Title: Cystinose - De la découverte du gène aux premiers essais de thérapie génique. Abstract: La cystinose est une maladie métabolique autosomique récessive caractérisée par une accumulation lysosomale de cystine dans toutes les cellules de l'organisme. La cystinose infantile débute dans la petite enfance par un syndrome de Fanconi et aboutit à une détérioration progressive de la fonction de la plupart des organes, y compris les reins, les yeux, la thyroïde, les muscles et le pancréas, et finit par entraîner une mort prématurée. Le traitement par la cystéamine ne permet que de retarder la progression de la maladie. Afin de développer une approche de thérapie génique pour la cystinose, un modèle murin qui présente les principales complications de la maladie a été développé grâce à l'identification du gène CTNS, dont le produit, la cystinosine, est un co-transporteur de cystine-protons. Cette revue décrit les étapes allant de la découverte du gène à la thérapie génique pour la cystinose, qui a permis de traiter six patients jusqu'à présent.

Corneal Densitometry to Assess the Corneal Cystine Deposits in Patients With Cystinosis.

PURPOSE: The purpose of this study was to assess the suitability of corneal densitometry measurements obtained with Scheimpflug imaging in estimating the corneal changes caused by cystine deposits in the cornea in patients with cystinosis. METHODS: Scheimpflug imaging (Pentacam) was performed for 14 patients with cystinosis and 16 age-matched controls. Pentacam data were used for analysis of the corneal densitometry at different zones in the cornea for patients with cystinosis and controls. Densitometry measurements were compared with the corneal crystal scores obtained from the slitlamp images for patients with cystinosis. RESULTS: There was no statistically significant difference in keratometry measurements between the 2 groups ( P > 0.05). Corneal thickness was found to be significantly higher in the control group when compared with the cystinosis group ( P = 0.0004). The mean corneal densitometry was significantly higher in patients with cystinosis when compared with controls at most of the corneal layers and zones. The corneal densitometry readings for the right and left eyes showed moderate positive correlation with the corneal crystal score with a ceiling effect being reached at the maximum corneal crystal score of 3. CONCLUSIONS: Corneal densitometry obtained through Pentacam can be used as an objective estimate of the level of cystine crystals present in patients with cystinosis. The clinical estimate of corneal crystal score, although effective at low levels of crystal deposition, does not allow for accurate estimates of change when the level of crystal deposition is high leading to limited utility when assessing treatment effects. Hence, densitometry measurements can potentially be used to assess treatment efficacy of cystinosis treatments in clinical settings.

Potent nanoreactor-mediated ferroptosis-based strategy for the reversal of cancer chemoresistance to Sorafenib.

The drug resistance of cancer cells is related to a variety of mechanisms, among which the destruction of redox homeostasis is one of the key factors. Ferroptosis, an intracellular iron-dependent form of cell death, is related to the production of oxidative stress. The accumulation of lipid peroxidation (LPO) during ferroptosis disrupts intracellular redox homeostasis, thereby affecting the sensitivity of tumor cells to drugs. In this work, we proposed a ferroptosis strategy based on LPO accumulation, reduced glutathione generation via inhibition of SLC3A2 protein and inactivated glutathione peroxidase 4 (GPX4) to reverse the chemoresistance of cancer cells. The Fenton reaction based on the ferroptosis-inducing nanoreactors (Au/Fe-GA/Sorafenib@PEG) not only generated hydroxyl radicals (·OH) under laser irradiation to realize the accumulation of LPO, but also depleted GSH to increase the accumulation of LPO. Meanwhile, the cystine uptake of cells was inhibited by Sorafenib, resulting in reduced GSH synthesis and inactivated GPX4. In vitro and in vivo experiments demonstrated AFG/SFB@PEG + Laser group could inactivate GPX4 and the enhanced ferroptosis can reverse chemo-resistance caused by continuous upregulation of GPX4 levels in cells through 'self-rescue'. The study proposed the mechanism and feasibility of ferroptosis to reverse drug resistance, providing a promising strategy for chemo-resistant cancer treatment. STATEMENT OF SIGNIFICANCE: Herein, we proposed a ferroptosis strategy based on LPO accumulation, reduced glutathione generation via inhibition of SLC3A2 protein, and inactivated glutathione peroxidase 4 (GPX4) to reverse chemoresistance of cancer cells. The Fenton reaction based on the ferroptosis-inducing nanoreactors (Au/Fe-GA/Sorafenib@PEG) not only generated hydroxyl radicals (·OH) under laser irradiation to realize the accumulation of LPO but also depleted GSH to increase the accumulation of LPO. Meanwhile, the cystine uptake of cells was inhibited by Sorafenib, resulting in reduced GSH synthesis and inactivated GPX4. In vitro and in vivo experiments demonstrated AFG/SFB@PEG + Laser group could inactivate GPX4 and the enhanced ferroptosis can reverse chemo-resistance caused by continuous upregulation of GPX4 levels in cells through 'self-rescue'.

SLC12A5 promotes hepatocellular carcinoma growth and ferroptosis resistance by inducing ER stress and cystine transport changes.

BACKGROUND: Hepatocellular carcinoma (HCC) has a poor prognosis and new effective treatments are needed. SLC12A5 plays important roles in multiple complex pathological states and is overexpressed in a variety of malignancies. However, the effects of SLC12A5 in HCC have not been determined. METHODS: SLC12A5 expression was assessed by immunostaining and western blotting. A cell viability assay was used to detect cell proliferation. Flow cytometry was used to evaluate the intracellular calcium concentration and cell cycle. Ferroptosis was detected by transmission electron microscopy, lipid peroxidation, and glutathione assays. Subcutaneous tumor formation experiments were used to validate the tumorigenic effect of SLC12A5 in vivo. RNA-seq was used to evaluate the molecular mechanisms underlying the effects of SLC12A5. The therapeutic efficacy of targeting SLC12A5 was assessed in a patient-derived xenograft (PDX) model. RESULTS: High SLC12A5 expression was strongly associated with a poor clinical prognosis and promoted HCC growth. Mechanistically, SLC12A5 promoted ER stress to enhance calcium release and upregulated PNCK expression levels. Concomitantly, PNCK was significantly activated by calcium ions released from the ER. PNCK activated and induced the phosphorylation of PI3K/AKT/mTOR pathway components. Furthermore, SLC12A5 inhibited ferroptosis in HCC by upregulating the expression of xCT, a cystine transporter. CONCLUSION: High SLC12A5 levels were correlated with a poor prognosis, promoted tumorigenesis, and inhibited ferroptosis in HCC. These findings suggested that SLC12A5 is a therapeutic target and provide insight into the link between ER stress and ferroptosis in HCC.

Genistein improves renal disease in a mouse model of nephropathic cystinosis: a comparison study with cysteamine.

Cysteamine is currently the only therapy for nephropathic cystinosis. It significantly improves life expectancy and delays progression to end-stage kidney disease; however, it cannot prevent it. Unfortunately, compliance to therapy is often weak, particularly during adolescence. Therefore, finding better treatments is a priority in the field of cystinosis. Previously, we found that genistein, an isoflavone particularly enriched in soy, can revert part of the cystinotic cellular phenotype that is not sensitive to cysteamine in vitro. To test the effects of genistein in vivo, we fed 2-month-old wild-type and Ctns-/- female mice with either a control diet, a genistein-containing diet or a cysteamine-containing diet for 14 months. Genistein (160 mg/kg/day) did not affect the growth of the mice or hepatic functionality. Compared with untreated mice at 16 months, Ctns-/- mice fed with genistein had lower cystine concentrations in their kidneys, reduced formation of cystine crystals, a smaller number of LAMP1-positive structures and an overall better-preserved parenchymal architecture. Cysteamine (400 mg/kg/day) was efficient in reverting the lysosomal phenotype and in preventing the development of renal lesions. These preclinical data indicate that genistein ameliorates kidney injury resulting from cystinosis with no side effects. Genistein therapy represents a potential treatment to improve the outcome for patients with cystinosis.

A new proof of evidence of cysteamine quantification for therapeutic drug monitoring in patients with cystinosis.

BACKGROUND: To date, measurement of intracellular cystine is used for the therapeutic monitoring of patients affected by cystinosis in treatment with cysteamine. Since this method is time and sample consuming, development of a faster method to quantify cysteamine would be extremely useful in order to help clinicians to adjust dosages of cysteamine and to define better the pharmacokinetic profile of this drug. The aim of the study was to develop a liquid chromatography tandem mass spectrometry method for the quantification of cysteamine in plasma samples and to test its applicability on plasma samples derived from patients with nephropathic infantile cystinosis in treatment with cysteamine. RESULTS: The percentage of accuracy of the developed method varied between 97.80 and 106.00% and CV% between 0.90 and 6.93%. There was no carry over. The calibration curves were built from 2.5 to 50 µM. The limit of detection and the lower limit of quantification occurred at 0.25 and 1.25 µM respectively. Cysteamine was stable up to 2 months at -20 °C. Concentrations of cysteamine and intracellular cystine of 4 patients were in line with data previously reported. CONCLUSION: The proposed method showed an appropriate selectivity, specificity, linearity, sensibility, accuracy, precision and good applicability to samples.

Therapeutic implications of mitochondrial stress-induced proteasome inhibitor resistance in multiple myeloma.

The connections between metabolic state and therapy resistance in multiple myeloma (MM) are poorly understood. We previously reported that electron transport chain (ETC) suppression promotes sensitivity to the BCL-2 antagonist venetoclax. Here, we show that ETC suppression promotes resistance to proteasome inhibitors (PIs). Interrogation of ETC-suppressed MM reveals integrated stress response-dependent suppression of protein translation and ubiquitination, leading to PI resistance. ETC and protein translation gene expression signatures from the CoMMpass trial are down-regulated in patients with poor outcome and relapse, corroborating our in vitro findings. ETC-suppressed MM exhibits up-regulation of the cystine-glutamate antiporter SLC7A11, and analysis of patient single-cell RNA-seq shows that clusters with low ETC gene expression correlate with higher SLC7A11 expression. Furthermore, erastin or venetoclax treatment diminishes mitochondrial stress-induced PI resistance. In sum, our work demonstrates that mitochondrial stress promotes PI resistance and underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic state.

Nephropathic cystinosis in Poland: a 40-year retrospective study.

INTRODUCTION: Nephropathic cystinosis (NC) is a rare, autosomal recessive disorder leading to lysosomal accumulation of cystine. It is caused by mutations in the CTNS gene encoding a cystine cotransporter cystinosin. The infantile (INC) and juvenile (JNC) forms are distinguished. The former, responsible for 95% of cases, is characterized by development of renal Fanconi syndrome, end-stage kidney disease (ESKD), and extrarenal complications. A therapy with cysteamine significantly improves outcomes. There are limited data on NC in the Central Eastern European countries. OBJECTIVES: We aimed to evaluate the prevalence, genetic background, and clinical course of NC in the Polish population. PATIENTS AND METHODS: We performed a retrospective analysis of data of all identified NC patients in Poland. RESULTS: Between 1982 and 2017, 15 patients with NC (13 ICN, 2 JCN) were identified. The most common mutations of the CTNS gene were c.18_c.21delGACT and c.681+1G>A, whereas only 2 patients carried the 57 kb deletion. The majority (11/13) of INC patients with limited access to the cysteamine therapy developed ESKD at a median age of 11 years and 9 of them received kidney transplants. Three INC patients died at a median age of 24 years. In contrast, 2 INC patients treated adequately present normal kidney function and growth at the age of 13 and 11 years. Two JNC patients presented a milder course. CONCLUSIONS: The prevalence of NC in Poland is much lower than in the Western countries and its molecular background appears to be different. The unfavorable course in the majority of INC patients was caused by a limited access to the cysteamine treatment.

Cystinosin-deficient rats recapitulate the phenotype of nephropathic cystinosis.

The lysosomal storage disease cystinosis is caused by mutations in CTNS, encoding the cystine transporter cystinosin, and in its severest form leads to proximal tubule dysfunction followed by kidney failure. Patients receive the drug-based therapy cysteamine from diagnosis. However, despite long-term treatment, cysteamine only slows the progression of end-stage renal disease. Preclinical testing in cystinotic rodents is required to evaluate new therapies; however, the current models are suboptimal. To solve this problem, we generated a new cystinotic rat model using CRISPR/Cas9-mediated gene editing to disrupt exon 3 of Ctns and measured various parameters over a 12-mo time course. Ctns-/- rats display hallmarks of cystinosis by 3-6 mo of age, as demonstrated by a failure to thrive, excessive thirst and urination, cystine accumulation in tissues, corneal cystine crystals, loss of LDL receptor-related protein 2 in proximal tubules, and immune cell infiltration. High levels of glucose, calcium, albumin, and protein were excreted at 6 mo of age, consistent with the onset of Fanconi syndrome, with a progressive diminution of urine urea and creatinine from 9 mo of age, indicative of chronic kidney disease. Kidney histology and immunohistochemistry showed proximal tubule atrophy and glomerular damage as well as classic "swan neck" lesions. Overall, Ctns-/- rats show a disease progression that more faithfully recapitulates nephropathic cystinosis than existing rodent models. The Ctns-/- rat provides an excellent new rodent model of nephropathic cystinosis that is ideally suited for conducting preclinical drug testing and is a powerful tool to advance cystinosis research.NEW & NOTEWORTHY Animal models of disease are essential to perform preclinical testing of new therapies before they can progress to clinical trials. The cystinosis field has been hampered by a lack of suitable animal models that fully recapitulate the disease. Here, we generated a rat model of cystinosis that closely models the human condition in a timeframe that makes them an excellent model for preclinical drug testing as well as being a powerful tool to advance research.

Evaluation of NACA and diNACA in human cystinosis fibroblast cell cultures as potential treatments for cystinosis.

BACKGROUND: Cystinosis is a rare autosomal recessive lysosomal storage disease, associated with high morbidity and mortality. Mutations in the CTNS gene disable a membrane protein responsible for the transport of cystine out of the lysosome. Loss of transporter function leads to intralysosomal cystine accumulation and long-term damage to various tissues and organs, including the kidneys, eyes, liver, muscles, pancreas, and brain. The only cystine-depletion therapy for treatment of cystinosis is cysteamine which requires frequent administration of high doses and often causes gastrointestinal pain as well as pungent sulfurous odor in patients. The current in vitro study evaluated antioxidants, N-acetylcysteine amide (NACA; NPI-001) and (2R,2R')-3,3'-disulfanediyl bis(2-acetamidopropanamide) (diNACA; NPI-002), as potential treatments for cystinosis. METHODS: Cytotoxicity of cysteamine, NACA and diNACA was evaluated in cultured human cystinotic fibroblasts (HCFs). HCFs were cultured in 96 well plates incubated for 0-72 h in the presence of 25, 50 or 75 µM each of either cysteamine, NACA or diNACA along with an untreated control. Media was removed and cell viability assessed. Next, cystine-depleting activities of cysteamine, NACA and diNACA were screened in HCFs cell culture utilizing an inexpensive, proven colorimetric assay. HCFs were seeded and allowed to reach approximately 80% confluence before the addition of the test articles: 50 µM of either cysteamine, NACA or diNACA in media along with an untreated control. HCFs were incubated, harvested, and cystine was reduced to cysteine, the concentration of which was then determined per quantity of protein compared to a cysteine standard. Statistically significant cystine depletion was determined by paired t-test versus untreated control (p < 0.05). RESULTS: Neither cysteamine, NACA nor diNACA at 25, 50 or 75 µM caused cytotoxicity in HCFs. Treatment with all tested concentrations (25, 50 or 75 µM) of either NACA or diNACA at 48 or 72 h resulted in statistically significant increases in cell viability, relative to untreated control, whereas the higher concentrations (50 or 75 µM) of cysteamine achieved statistical significance at both timepoints but not the lowest concentration (25 µM). All test articles depleted cystine from HCFs compared to control. NACA depletion of cystine was statistically superior to cysteamine at 6, 24 and 48 h and numerically greater at 72 h. DiNACA depletion of cystine was statistically superior to cysteamine at 6 and 48 h, slightly numerically greater at 24 h and slightly less at 72 h. CONCLUSIONS: NACA and diNACA were non cytotoxic to HCFs and significantly increased cell viability. Cystine reduction was determined as percent of control after incubation with 50 µM of NACA, diNACA or cysteamine in HCFs cell culture for 6, 24, 48 and 72 h. Of the three test articles, NACA exhibited most rapid and greatest potency in cystine reduction. Rank order potency for cystine reduction over time was observed, NACA > diNACA ≥ cysteamine. Therefore, further study of NACA and diNACA as potential treatments for cystinosis is warranted.

Ocular treatment of cystinosis with eye drop containing cysteamine / Ciszteamintartalmú szemcsepp alkalmazása cystinosisban

Cystinosis is a rare lysosomal storage disease affecting amino acid metabolism, characterized by the accumulation and crystallization of cystine in various tissues, primarily in the eye and kidney. The major ophthalmic symptom is photophobia, which is related to the corneal deposition of cystine crystals. The light sensitivity significantly impairs the quality of life of the affected patients, thus, effective ophthalmic treatment to reduce the crystal density is very importance. In the current case report, we present the characteristic ocular clinical appearance and treatment options of cystinosis by reviewing the literature. A simple aqueous solution of cysteamine, which aids in the dissolution of crystals, has been widely used in topical treatment in the past, however, its therapeutic efficacy is debatable. Recently, a new viscous formulation of cysteamine has been proposed for ophthalmic treatment. For the treatment of corneal cystine crystals in our patient, the new viscous format of cysteamine has been applied, and therapeutic effects were recorded for a year. Applying the viscous cysteamine formulation, a marked and gradual decrease in photophobia was observed in our patient in the first year of the treatment. Anterior-segment optical coherence tomography and in vivo confocal microscopy represented a continuous decrease in the density of corneal crystals even from the first month of the treatment period. The aim of our case report is to present the ophthalmic symptoms of cystinosis and the results of the first clinical application of viscous formulation of cysteamin eye drops in Hungary in a cystinosis patient.

Cystine uptake inhibition potentiates front-line therapies in acute myeloid leukemia.

By querying metabolic pathways associated with leukemic stemness and survival in multiple AML datasets, we nominated SLC7A11 encoding the xCT cystine importer as a putative AML dependency. Genetic and chemical inhibition of SLC7A11 impaired the viability and clonogenic capacity of AML cell lines in a cysteine-dependent manner. Sulfasalazine, a broadly available drug with xCT inhibitory activity, had anti-leukemic activity against primary AML samples in ex vivo cultures. Multiple metabolic pathways were impacted upon xCT inhibition, resulting in depletion of glutathione pools in leukemic cells and oxidative stress-dependent cell death, only in part through ferroptosis. Higher expression of cysteine metabolism genes and greater cystine dependency was noted in NPM1-mutated AMLs. Among eight anti-leukemic drugs, the anthracycline daunorubicin was identified as the top synergistic agent in combination with sulfasalazine in vitro. Addition of sulfasalazine at a clinically relevant concentration significantly augmented the anti-leukemic activity of a daunorubicin-cytarabine combination in a panel of 45 primary samples enriched in NPM1-mutated AML. These results were confirmed in vivo in a patient-derived xenograft model. Collectively, our results nominate cystine import as a druggable target in AML and raise the possibility to repurpose sulfasalazine for the treatment of AML, notably in combination with chemotherapy.