Defects in 8-oxo-guanine repair pathway cause high frequency of C > A substitutions in neuroblastoma

Neuroblastomas are childhood tumors with frequent fatal relapses after induction treatment, which is related to tumor evolution with additional genomic events. Our whole-genome sequencing data analysis revealed a high frequency of somatic cytosine > adenine (C > A) substitutions in primary neuroblastoma tumors, which was associated with poor survival. We showed that increased levels of C > A substitutions correlate with copy number loss (CNL) of OGG1 or MUTYH. Both genes encode DNA glycosylases that recognize 8-oxo-guanine (8-oxoG) lesions as a first step of 8-oxoG repair. Tumor organoid models with CNL of OGG1 or MUTYH show increased 8-oxoG levels compared to wild-type cells. We used CRISPR-Cas9 genome editing to create knockout clones of MUTYH and OGG1 in neuroblastoma cells. Whole-genome sequencing of single-cell OGG1 and MUTYH knockout clones identified an increased accumulation of C > A substitutions. Mutational signature analysis of these OGG1 and MUTYH knockout clones revealed enrichment for C > A signatures 18 and 36, respectively. Clustering analysis showed that the knockout clones group together with tumors containing OGG1 or MUTYH CNL. In conclusion, we demonstrate that defects in 8-oxoG repair cause accumulation of C > A substitutions in neuroblastoma, which contributes to mutagenesis and tumor evolution

Design of kidney-targeted drug-carrier conjugates for the inhibition of profibrotic signaling cascades

In the past decades, the worldwide number of patients with chronic kidney disease (CKD) has increased remarkably and it is expected that this increasing trend will continue during the coming years (1). At this moment, therapy is focused on the prevention of progressive CKD by treatment of the underlying causes, such as hypertension, diabetes and obesity. CKD is asymptomatic in the initial stage and is often diagnosed in a stage at which treatment of the underlying cause alone is not sufficient to stop loss of kidney function. As a consequence, CKD will gradually progress to end-stage renal disease (ESRD) and renal dialysis or kidney transplantation are then the only possibilities for patients to survive. Because of the lack of clinically available drugs that can halt the progression of CKD and the seriousness of the disease, there is a high need for novel drugs. This thesis focuses on the intracellular delivery of drugs into the proximal tubular cells of the kidneys. Tubulointerstitial fibrosis is one of the common hallmarks of progressive CKD and renal drug targeting to the proximal tubular cells can be of great value in the development of therapeutics that halt or reverse tubulointerstitial fibrosis

Renal targeting of kinase inhibitors

Activation of proximal tubular cells by fibrotic and inflammatory mediators is an important hallmark of chronic kidney disease. We have developed a novel strategy to intervene in renal fibrosis, by means of locally delivered kinase inhibitors. Such compounds will display enhanced activity within tubular cells and reduced unwanted systemic effects. In our approach kinase inhibitors are linked to the renal carrier lysozyme using a platinum-based linker that binds drugs via a coordinative linkage. Many kinase inhibitors contain aromatic nitrogen atoms able to bind to this linker without the need of prior derivatization. The resulting drug-lysozyme conjugates are rapidly filtered in the glomerulus into the tubular lumen and subsequently reabsorbed via the endocytic pathway for clearance of low-molecular weight proteins. An important property of the formed conjugates is their in vivo stability and the sustained drug release profile within target cells. This review summarizes the state-of-the-art of drug targeting to the kidney. Furthermore, we will highlight recent results obtained with kinase inhibitor-lysozyme conjugates targeted to different kinases, i.e. the transforming growth factor (TGF)-beta-receptor kinase, p38 MAPkinase and Rho-associated kinase. Both in vitro and in vivo results demonstrated their efficient tubular uptake and beneficial therapeutic effects, superior to treatment with free kinase inhibitors. These proof-of-concept studies clearly indicate the feasibility of drug targeting for improving the renal specificity of kinase inhibitors.(c) 2008 Elsevier B.V. All rights reserved

Intervention in growth factor activated signaling pathways by renally targeted kinase inhibitors

Cell-specific targeting to renal tubular cells is an interesting approach to enhance the accumulation of drugs in the kidney. Low molecular weight proteins are rapidly filtered and extensively accumulate in proximal tubular cells. We therefore have used lysozyme (LZM,14 kDa) as a tubular cell-specific carrier for the delivery of kinase inhibitors. Two different kinase inhibitors (LY364947 and erlotinib, directed to either the TGF-beta receptor kinase or the EGF receptor) were individually conjugated to LZM via a novel platinum-based linker (Universal Linkage System; ULS). The cellular handling and pharmacological efficacy of the conjugates were evaluated in cultured proximal tubular cells (HK-2 cells). Both conjugates were efficiently internalized via endocytosis. TGF-beta or EGF activated HK-2 cells showed a strong activation of the studied kinases and the conjugates inhibited these events, as was demonstrated by Western blotting of phosphorylated downstream mediators and quantitative gene expression analysis. In conclusion, we have developed tubular cell-specific kinase inhibitor-LZM conjugates via a novel linker strategy, which both showed to be effective in vitro. Future in vivo studies should show their potential for the treatment of renal diseases. (C) 2008 Elsevier B.V. All rights reserved

Intervention in growth factor activated signaling pathways by renally targeted kinase inhibitors

Cell-specific targeting to renal tubular cells is an interesting approach to enhance the accumulation of drugs in the kidney. Low molecular weight proteins are rapidly filtered and extensively accumulate in proximal tubular cells. We therefore have used lysozyme (LZM,14 kDa) as a tubular cell-specific carrier for the delivery of kinase inhibitors. Two different kinase inhibitors (LY364947 and erlotinib, directed to either the TGF-beta receptor kinase or the EGF receptor) were individually conjugated to LZM via a novel platinum-based linker (Universal Linkage System; ULS). The cellular handling and pharmacological efficacy of the conjugates were evaluated in cultured proximal tubular cells (HK-2 cells). Both conjugates were efficiently internalized via endocytosis. TGF-beta or EGF activated HK-2 cells showed a strong activation of the studied kinases and the conjugates inhibited these events, as was demonstrated by Western blotting of phosphorylated downstream mediators and quantitative gene expression analysis. In conclusion, we have developed tubular cell-specific kinase inhibitor-LZM conjugates via a novel linker strategy, which both showed to be effective in vitro. Future in vivo studies should show their potential for the treatment of renal diseases. (C) 2008 Elsevier B.V. All rights reserved

Whole genome, transcriptome and methylome profiling enhances actionable target discovery in high-risk pediatric cancer

The Zero Childhood Cancer Program is a precision medicine program to benefit children with poor-outcome, rare, relapsed or refractory cancer. Using tumor and germline whole genome sequencing (WGS) and RNA sequencing (RNAseq) across 252 tumors from high-risk pediatric patients with cancer, we identified 968 reportable molecular aberrations (39.9% in WGS and RNAseq, 35.1% in WGS only and 25.0% in RNAseq only). Of these patients, 93.7% had at least one germline or somatic aberration, 71.4% had therapeutic targets and 5.2% had a change in diagnosis. WGS identified pathogenic cancer-predisposing variants in 16.2% of patients. In 76 central nervous system tumors, methylome analysis confirmed diagnosis in 71.1% of patients and contributed to a change of diagnosis in two patients (2.6%). To date, 43 patients have received a recommended therapy, 38 of whom could be evaluated, with 31% showing objective evidence of clinical benefit. Comprehensive molecular profiling resolved the molecular basis of virtually all high-risk cancers, leading to clinical benefit in some patients.Marie Wong ... Jordan R. Hansford ... et al