Chromosome 11q loss and MYCN amplification demonstrate synthetic lethality with checkpoint kinase 1 inhibition in neuroblastoma

Neuroblastoma is the most common extracranial solid tumor found in children and despite intense multi-modal therapeutic approaches, low overall survival rates of high-risk patients persist. Tumors with heterozygous loss of chromosome 11q and MYCN amplification are two genetically distinct subsets of neuroblastoma that are associated with poor patient outcome. Using an isogenic 11q deleted model system and high-throughput drug screening, we identify checkpoint kinase 1 (CHK1) as a potential therapeutic target for 11q deleted neuroblastoma. Further investigation reveals MYCN amplification as a possible additional biomarker for CHK1 inhibition, independent of 11q loss. Overall, our study highlights the potential power of studying chromosomal aberrations to guide preclinical development of novel drug targets and combinations. Additionally, our study builds on the growing evidence that DNA damage repair and replication stress response pathways offer therapeutic vulnerabilities for the treatment of neuroblastoma

Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4-8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions

Mutations in ALK signaling pathways conferring resistance to ALK inhibitor treatment lead to collateral vulnerabilities in neuroblastoma cells

Background: Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. Methods: Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. Results: Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. Conclusions: Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells

Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4–8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions

Click to enter:activation of oligo-arginine cell-penetrating peptides by bioorthogonal tetrazine ligations

\u3cp\u3eCell-penetrating peptides are able to transport a wide variety of cargo across cell membranes. Although promising, they are not often considered for therapeutic purposes as they lack controllable activity and cell selectivity. We have developed an activation strategy based on a split octa-arginine cell-penetrating peptide (CPP) that can be activated by means of bioorthogonal ligation. To this end we prepared two non-penetrating tetra-arginine halves, functionalized either with a tetrazine or with a complementary bicyclo[6.1.0]nonyne (BCN) group. We demonstrate that an active octa-arginine can be reconstituted in situ upon mixing the complementary split peptides. The resulting activated peptide is taken up as efficiently as the well-established cell-penetrating peptide octa-arginine. The activation of the oligo-arginines can also be achieved using trans-cyclooctene (TCO) as a ligation partner, while norbornene appears too kinetically slow for use in situ. We further show that this strategy can be applied successfully to transport a large protein into living cells. Our results validate a promising first step in achieving control over cell penetration and to use CPPs for therapeutic approaches.\u3c/p\u3

Subcellular Protein Labeling by a Spatially Restricted Arylamine <i>N</i>‑Acetyltransferase

Mapping proteins at a specific subcellular location is essential to gaining detailed insight on local protein dynamics. We have developed an enzymatic strategy to label proteins on a subcellular level using arylamine <i>N</i>-acetyltransferase (NAT). The NAT enzyme activates an arylhydroxamic acid functionality into a nitrenium ion that reacts fast, covalently, and under neutral conditions with nucleophilic residues of neighboring proteins. The electron density on the aromatic ring proved important for probe activation as strong labeling was only observed with an arylhydroxamic acid bearing an electron donating substituent. We further demonstrate that, using this electron rich arylhydroxamic acid, clear labeling was achieved on a subcellular level in living cells that were transfected with a genetically targeted NAT to the nucleus or the cytosol

Modular, bioorthogonal strategy for the controlled loading of cargo into a protein nanocage

Virus capsids, i.e., viruses devoid of their genetic material, are suitable nanocarriers for biomedical applications such as drug delivery and diagnostic imaging. For this purpose, the reliable encapsulation of cargo in such a protein nanocage is crucial, which can be accomplished by the covalent attachment of the compounds of interest to the protein domains positioned at the interior of the cage. This approach is particularly valid for the capsid proteins of the cowpea chlorotic mottle virus (CCMV), which have their N-termini located at the inside of the capsid structure. Here, we examined several site-selective modification methods for covalent attachment and encapsulation of cargo at the N-terminus of the CCMV protein. Initially, we explored approaches to introduce an N-terminal azide functionality, which would allow the subsequent bioorthogonal modification with a strained alkyne to attach the desired cargo. As these methods showed compatibility issues with the CCMV capsid proteins, a strategy based on 2-pyridinecarboxaldehydes for site-specific N-terminal protein modification was employed. This method allowed the successful modification of the proteins, and was applied for the introduction of a bioorthogonal vinylboronic acid moiety. In a subsequent reaction, the proteins could be modified further with a fluorophore using the tetrazine ligation. The application of capsid assembly conditions on the functionalized proteins led to successful particle formation, showing the potential of this covalent encapsulation strategy