Telomere maintenance by telomerase activation or alternative lengthening of telomeres (ALT) is a major determinant of poor outcome in neuroblastoma. Here, we screen for ALT in primary and relapsed neuroblastomas (n = 760) and characterize its features using multi-omics profiling. ALT-positive tumors are molecularly distinct from other neuroblastoma subtypes and enriched in a population-based clinical sequencing study cohort for relapsed cases. They display reduced ATRX/DAXX complex abundance, due to either ATRX mutations (55%) or low protein expression. The heterochromatic histone mark H3K9me3 recognized by ATRX is enriched at the telomeres of ALT-positive tumors. Notably, we find a high frequency of telomeric repeat loci with a neuroblastoma ALT-specific hotspot on chr1q42.2 and loss of the adjacent chromosomal segment forming a neo-telomere. ALT-positive neuroblastomas proliferate slowly, which is reflected by a protracted clinical course of disease. Nevertheless, children with an ALT-positive neuroblastoma have dismal outcome.

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In cross-linking mass spectrometry, the depth and sensitivity is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of low abundant cross-links, here we present a gas-phase separation strategy using high field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in gas phase using the FAIMS device, we increase the number of cross-link identification by 23% for a medium complex sample and 56% for strong cation exchange-fractionated HEK293 cell lysate. Furthermore, we show that for medium complex samples, FAIMS enables the collection of single-shot cross-linking data with comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.

Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors.

Reversible protein phosphorylation is a central signaling mechanism in eukaryotic cells. While the identification of canonical phosphorylation sites using mass-spectrometry (MS) based proteomics has become routine, annotation of non-canonical phosphorylation has remained a challenge. Here, we report a tailored pyrophosphoproteomics workflow to detect and reliably assign protein pyrophosphorylation in two human cell lines, providing the first direct evidence of endogenous protein pyrophosphorylation. Detection of protein pyrophosphorylation was reproducible, specific and consistent with previous biochemical evidence relating the installation of the modification to inositol pyrophosphates (PP-InsPs). We manually validated 148 pyrophosphosites across 71 human proteins, the most heavily pyrophosphorylated of which were the nucleolar proteins NOLC1 and TCOF1. A predictive workflow based on the MS data set was established to recognize putative pyrophosphorylation sequences, and UBF1, a nucleolar protein incompatible with the proteomics method, was biochemically shown to undergo pyrophosphorylation. When the biosynthesis of PP-InsPs was perturbed in a model cell line, proteins expressed in this background exhibited lower levels of pyrophosphorylation. Disruption of PP-InsP biosynthesis also significantly reduced rDNA transcription, potentially by lowering pyrophosphorylation on regulatory proteins NOLC1, TCOF1, and UBF1. Overall, protein pyrophosphorylation emerges as an archetype of non-canonical phosphorylation, and should be considered in future phosphoproteomic analyses.