Roadmap for the Emerging Field of Cancer Neuroscience.

Mounting evidence indicates that the nervous system plays a central role in cancer pathogenesis. In turn, cancers and cancer therapies can alter nervous system form and function. This Commentary seeks to describe the burgeoning field of "cancer neuroscience" and encourage multidisciplinary collaboration for the study of cancer-nervous system interactions.

Hypermutation of the inactive X chromosome is a frequent event in cancer.

Mutation is a fundamental process in tumorigenesis. However, the degree to which the rate of somatic mutation varies across the human genome and the mechanistic basis underlying this variation remain to be fully elucidated. Here, we performed a cross-cancer comparison of 402 whole genomes comprising a diverse set of childhood and adult tumors, including both solid and hematopoietic malignancies. Surprisingly, we found that the inactive X chromosome of many female cancer genomes accumulates on average twice and up to four times as many somatic mutations per megabase, as compared to the individual autosomes. Whole-genome sequencing of clonally expanded hematopoietic stem/progenitor cells (HSPCs) from healthy individuals and a premalignant myelodysplastic syndrome (MDS) sample revealed no X chromosome hypermutation. Our data suggest that hypermutation of the inactive X chromosome is an early and frequent feature of tumorigenesis resulting from DNA replication stress in aberrantly proliferating cells.

The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation.

Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:

Human fetal cerebellar cell atlas informs medulloblastoma origin and oncogenesis.

Medulloblastoma (MB) is the most common malignant childhood brain tumour1,2, yet the origin of the most aggressive subgroup-3 form remains elusive, impeding development of effective targeted treatments. Previous analyses of mouse cerebella3-5 have not fully defined the compositional heterogeneity of MBs. Here we undertook single-cell profiling of freshly isolated human fetal cerebella to establish a reference map delineating hierarchical cellular states in MBs. We identified a unique transitional cerebellar progenitor connecting neural stem cells to neuronal lineages in developing fetal cerebella. Intersectional analysis revealed that the transitional progenitors were enriched in aggressive MB subgroups, including group 3 and metastatic tumours. Single-cell multi-omics revealed underlying regulatory networks in the transitional progenitor populations, including transcriptional determinants HNRNPH1 and SOX11, which are correlated with clinical prognosis in group 3 MBs. Genomic and Hi-C profiling identified de novo long-range chromatin loops juxtaposing HNRNPH1/SOX11-targeted super-enhancers to cis-regulatory elements of MYC, an oncogenic driver for group 3 MBs. Targeting the transitional progenitor regulators inhibited MYC expression and MYC-driven group 3 MB growth. Our integrated single-cell atlases of human fetal cerebella and MBs show potential cell populations predisposed to transformation and regulatory circuitries underlying tumour cell states and oncogenesis, highlighting hitherto unrecognized transitional progenitor intermediates predictive of disease prognosis and potential therapeutic vulnerabilities.

Genome sequencing of pediatric medulloblastoma links catastrophic DNA rearrangements with TP53 mutations.

Genomic rearrangements are thought to occur progressively during tumor development. Recent findings, however, suggest an alternative mechanism, involving massive chromosome rearrangements in a one-step catastrophic event termed chromothripsis. We report the whole-genome sequencing-based analysis of a Sonic-Hedgehog medulloblastoma (SHH-MB) brain tumor from a patient with a germline TP53 mutation (Li-Fraumeni syndrome), uncovering massive, complex chromosome rearrangements. Integrating TP53 status with microarray and deep sequencing-based DNA rearrangement data in additional patients reveals a striking association between TP53 mutation and chromothripsis in SHH-MBs. Analysis of additional tumor entities substantiates a link between TP53 mutation and chromothripsis, and indicates a context-specific role for p53 in catastrophic DNA rearrangements. Among these, we observed a strong association between somatic TP53 mutations and chromothripsis in acute myeloid leukemia. These findings connect p53 status and chromothripsis in specific tumor types, providing a genetic basis for understanding particularly aggressive subtypes of cancer.

Quantum-enhanced nonlinear microscopy.

The performance of light microscopes is limited by the stochastic nature of light, which exists in discrete packets of energy known as photons. Randomness in the times that photons are detected introduces shot noise, which fundamentally constrains sensitivity, resolution and speed1. Although the long-established solution to this problem is to increase the intensity of the illumination light, this is not always possible when investigating living systems, because bright lasers can severely disturb biological processes2-4. Theory predicts that biological imaging may be improved without increasing light intensity by using quantum photon correlations1,5. Here we experimentally show that quantum correlations allow a signal-to-noise ratio beyond the photodamage limit of conventional microscopy. Our microscope is a coherent Raman microscope that offers subwavelength resolution and incorporates bright quantum correlated illumination. The correlations allow imaging of molecular bonds within a cell with a 35 per cent improved signal-to-noise ratio compared with conventional microscopy, corresponding to a 14 per cent improvement in concentration sensitivity. This enables the observation of biological structures that would not otherwise be resolved. Coherent Raman microscopes allow highly selective biomolecular fingerprinting in unlabelled specimens6,7, but photodamage is a major roadblock for many applications8,9. By showing that the photodamage limit can be overcome, our work will enable order-of-magnitude improvements in the signal-to-noise ratio and the imaging speed.

Design of the elusive proteinaceous oxygen donor copper site suggests a promising future for copper for MRI contrast agents.

We report the preparation and spectroscopic characterization of a highly elusive copper site bound exclusively to oxygen donor atoms within a protein scaffold. Despite copper generally being considered unsuitable for use in MRI contrast agents, which in the clinic are largely Gd(III) based, the designed copper coiled coil displays relaxivity values equal to, or superior than, those of the Gd(III) analog at clinical field strengths. The creation of this new-to-biology proteinaceous CuOx-binding site demonstrates the power of the de novo peptide design approach to access chemistry for abiological applications, such as for the development of MRI contrast agents.

Theoretical Advances in Polariton Chemistry and Molecular Cavity Quantum Electrodynamics.

When molecules are coupled to an optical cavity, new light-matter hybrid states, so-called polaritons, are formed due to quantum light-matter interactions. With the experimental demonstrations of modifying chemical reactivities by forming polaritons under strong light-matter interactions, theorists have been encouraged to develop new methods to simulate these systems and discover new strategies to tune and control reactions. This review summarizes some of these exciting theoretical advances in polariton chemistry, in methods ranging from the fundamental framework to computational techniques and applications spanning from photochemistry to vibrational strong coupling. Even though the theory of quantum light-matter interactions goes back to the midtwentieth century, the gaps in the knowledge of molecular quantum electrodynamics (QED) have only recently been filled. We review recent advances made in resolving gauge ambiguities, the correct form of different QED Hamiltonians under different gauges, and their connections to various quantum optics models. Then, we review recently developed ab initio QED approaches which can accurately describe polariton states in a realistic molecule-cavity hybrid system. We then discuss applications using these method advancements. We review advancements in polariton photochemistry where the cavity is made resonant to electronic transitions to control molecular nonadiabatic excited state dynamics and enable new photochemical reactivities. When the cavity resonance is tuned to the molecular vibrations instead, ground-state chemical reaction modifications have been demonstrated experimentally, though its mechanistic principle remains unclear. We present some recent theoretical progress in resolving this mystery. Finally, we review the recent advances in understanding the collective coupling regime between light and matter, where many molecules can collectively couple to a single cavity mode or many cavity modes. We also lay out the current challenges in theory to explain the observed experimental results. We hope that this review will serve as a useful document for anyone who wants to become familiar with the context of polariton chemistry and molecular cavity QED and thus significantly benefit the entire community.

The U1 spliceosomal RNA is recurrently mutated in multiple cancers.

Cancers are caused by genomic alterations known as drivers. Hundreds of drivers in coding genes are known but, to date, only a handful of noncoding drivers have been discovered-despite intensive searching1,2. Attention has recently shifted to the role of altered RNA splicing in cancer; driver mutations that lead to transcriptome-wide aberrant splicing have been identified in multiple types of cancer, although these mutations have only been found in protein-coding splicing factors such as splicing factor 3b subunit 1 (SF3B1)3-6. By contrast, cancer-related alterations in the noncoding component of the spliceosome-a series of small nuclear RNAs (snRNAs)-have barely been studied, owing to the combined challenges of characterizing noncoding cancer drivers and the repetitive nature of snRNA genes1,7,8. Here we report a highly recurrent A>C somatic mutation at the third base of U1 snRNA in several types of tumour. The primary function of U1 snRNA is to recognize the 5' splice site via base-pairing. This mutation changes the preferential A-U base-pairing between U1 snRNA and the 5' splice site to C-G base-pairing, and thus creates novel splice junctions and alters the splicing pattern of multiple genes-including known drivers of cancer. Clinically, the A>C mutation is associated with heavy alcohol use in patients with hepatocellular carcinoma, and with the aggressive subtype of chronic lymphocytic leukaemia with unmutated immunoglobulin heavy-chain variable regions. The mutation in U1 snRNA also independently confers an adverse prognosis to patients with chronic lymphocytic leukaemia. Our study demonstrates a noncoding driver in spliceosomal RNAs, reveals a mechanism of aberrant splicing in cancer and may represent a new target for treatment. Our findings also suggest that driver discovery should be extended to a wider range of genomic regions.

Retraction Note: A homing system targets therapeutic T cells to brain cancer.

This Article has been retracted; see accompanying Retraction.

Childhood cerebellar tumours mirror conserved fetal transcriptional programs.

Study of the origin and development of cerebellar tumours has been hampered by the complexity and heterogeneity of cerebellar cells that change over the course of development. Here we use single-cell transcriptomics to study more than 60,000 cells from the developing mouse cerebellum and show that different molecular subgroups of childhood cerebellar tumours mirror the transcription of cells from distinct, temporally restricted cerebellar lineages. The Sonic Hedgehog medulloblastoma subgroup transcriptionally mirrors the granule cell hierarchy as expected, while group 3 medulloblastoma resembles Nestin+ stem cells, group 4 medulloblastoma resembles unipolar brush cells, and PFA/PFB ependymoma and cerebellar pilocytic astrocytoma resemble the prenatal gliogenic progenitor cells. Furthermore, single-cell transcriptomics of human childhood cerebellar tumours demonstrates that many bulk tumours contain a mixed population of cells with divergent differentiation. Our data highlight cerebellar tumours as a disorder of early brain development and provide a proximate explanation for the peak incidence of cerebellar tumours in early childhood.

The molecular landscape of ETMR at diagnosis and relapse.

Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric embryonal brain tumours with a universally poor prognosis1. Here we collected 193 primary ETMRs and 23 matched relapse samples to investigate the genomic landscape of this distinct tumour type. We found that patients with tumours in which the proposed driver C19MC2-4 was not amplified frequently had germline mutations in DICER1 or other microRNA-related aberrations such as somatic amplification of miR-17-92 (also known as MIR17HG). Whole-genome sequencing revealed that tumours had an overall low recurrence of single-nucleotide variants (SNVs), but showed prevalent genomic instability caused by widespread occurrence of R-loop structures. We show that R-loop-associated chromosomal instability can be induced by the loss of DICER1 function. Comparison of primary tumours and matched relapse samples showed a strong conservation of structural variants, but low conservation of SNVs. Moreover, many newly acquired SNVs are associated with a mutational signature related to cisplatin treatment. Finally, we show that targeting R-loops with topoisomerase and PARP inhibitors might be an effective treatment strategy for this deadly disease.

Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu.

Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for the mechanisms of their biological functions as well as their potential as therapeutic targets.

Brain endothelial cells acquire blood-brain barrier properties in the absence of Vegf-dependent CNS angiogenesis.

During neurovascular development, brain endothelial cells (BECs) respond to secreted signals from the neuroectoderm that regulate CNS angiogenesis, the formation of new blood vessels in the brain, and barriergenesis, the acquisition of blood-brain barrier (BBB) properties. Wnt/ß-catenin signaling and Vegf signaling are both required for CNS angiogenesis; however, the relationship between these pathways is not understood. Furthermore, while Wnt/ß-catenin signaling is essential for barriergenesis, the role of Vegf signaling in this vital process remains unknown. Here, we provide the first direct evidence, to our knowledge, that Vegf signaling is not required for barriergenesis and that activation of Wnt/ß-catenin in BECs is independent of Vegf signaling during neurovascular development. Using double transgenic glut1b:mCherry and plvap:EGFP zebrafish (Danio rerio) to visualize the developing brain vasculature, we performed a forward genetic screen and identified a new mutant allele of kdrl, an ortholog of mammalian Vegfr2. The kdrl mutant lacks CNS angiogenesis but, unlike the Wnt/ß-catenin pathway mutant gpr124, acquires BBB properties in BECs. To examine Wnt/ß-catenin pathway activation in BECs, we chemically inhibited Vegf signaling and found robust expression of the Wnt/ß-catenin transcriptional reporter line 7xtcf-Xla.Siam:EGFP. Taken together, our results establish that Vegf signaling is essential for CNS angiogenesis but is not required for Wnt/ß-catenin-dependent barriergenesis. Given the clinical significance of either inhibiting pathological angiogenesis or stimulating neovascularization, our study provides valuable new insights that are critical for the development of effective therapies that target the vasculature in neurological disorders.

Longitudinal Follow-Up of Children With HLHS and Association Between Norwood Shunt Type and Long-Term Outcomes: The SVR III Study.

OBJECTIVE: In the SVR trial (Single Ventricle Reconstruction), newborns with hypoplastic left heart syndrome were randomly assigned to receive a modified Blalock-Taussig-Thomas shunt (mBTTS) or a right ventricle-to-pulmonary artery shunt (RVPAS) at Norwood operation. Transplant-free survival was superior in the RVPAS group at 1 year, but no longer differed by treatment group at 6 years; both treatment groups had accumulated important morbidities. In the third follow-up of this cohort (SVRIII [Long-Term Outcomes of Children With Hypoplastic Left Heart Syndrome and the Impact of Norwood Shunt Type]), we measured longitudinal outcomes and their risk factors through 12 years of age. METHODS: Annual medical history was collected through record review and telephone interviews. Cardiac magnetic resonance imaging (CMR), echocardiogram, and cycle ergometry cardiopulmonary exercise tests were performed at 10 through 14 years of age among participants with Fontan physiology. Differences in transplant-free survival and complication rates (eg, arrhythmias or protein-losing enteropathy) were identified through 12 years of age. The primary study outcome was right ventricular ejection fraction (RVEF) by CMR, and primary analyses were according to shunt type received. Multivariable linear and Cox regression models were created for RVEF by CMR and post-Fontan transplant-free survival. RESULTS: Among 549 participants enrolled in SVR, 237 of 313 (76%; 60.7% male) transplant-free survivors (mBTTS, 105 of 147; RVPAS, 129 of 161; both, 3 of 5) participated in SVRIII. RVEF by CMR was similar in the shunt groups (RVPAS, 51±9.6 [n=90], and mBTTS, 52±7.4 [n=75]; P=0.43). The RVPAS and mBTTS groups did not differ in transplant-free survival by 12 years of age (163 of 277 [59%] versus 144 of 267 [54%], respectively; P=0.11), percentage predicted peak Vo2 for age and sex (74±18% [n=91] versus 72±18% [n=84]; P=0.71), or percentage predicted work rate for size and sex (65±20% versus 64±19%; P=0.65). The RVPAS versus mBTTS group had a higher cumulative incidence of protein-losing enteropathy (5% versus 2%; P=0.04) and of catheter interventions (14 versus 10 per 100 patient-years; P=0.01), but had similar rates of other complications. CONCLUSIONS: By 12 years after the Norwood operation, shunt type has minimal association with RVEF, peak Vo2, complication rates, and transplant-free survival. RVEF is preserved among the subgroup of survivors who underwent CMR assessment. Low transplant-free survival, poor exercise performance, and accruing morbidities highlight the need for innovative strategies to improve long-term outcomes in patients with hypoplastic left heart syndrome. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT0245531.

A protein functionalization platform based on selective reactions at methionine residues.

Nature has a remarkable ability to carry out site-selective post-translational modification of proteins, therefore enabling a marked increase in their functional diversity1. Inspired by this, chemical tools have been developed for the synthetic manipulation of protein structure and function, and have become essential to the continued advancement of chemical biology, molecular biology and medicine. However, the number of chemical transformations that are suitable for effective protein functionalization is limited, because the stringent demands inherent to biological systems preclude the applicability of many potential processes2. These chemical transformations often need to be selective at a single site on a protein, proceed with very fast reaction rates, operate under biologically ambient conditions and should provide homogeneous products with near-perfect conversion2-7. Although many bioconjugation methods exist at cysteine, lysine and tyrosine, a method targeting a less-explored amino acid would considerably expand the protein functionalization toolbox. Here we report the development of a multifaceted approach to protein functionalization based on chemoselective labelling at methionine residues. By exploiting the electrophilic reactivity of a bespoke hypervalent iodine reagent, the S-Me group in the side chain of methionine can be targeted. The bioconjugation reaction is fast, selective, operates at low-micromolar concentrations and is complementary to existing bioconjugation strategies. Moreover, it produces a protein conjugate that is itself a high-energy intermediate with reactive properties and can serve as a platform for the development of secondary, visible-light-mediated bioorthogonal protein functionalization processes. The merger of these approaches provides a versatile platform for the development of distinct transformations that deliver information-rich protein conjugates directly from the native biomacromolecules.

A homing system targets therapeutic T cells to brain cancer.

Successful T cell immunotherapy for brain cancer requires that the T cells can access tumour tissues, but this has been difficult to achieve. Here we show that, in contrast to inflammatory brain diseases such as multiple sclerosis, where endothelial cells upregulate ICAM1 and VCAM1 to guide the extravasation of pro-inflammatory cells, cancer endothelium downregulates these molecules to evade immune recognition. By contrast, we found that cancer endothelium upregulates activated leukocyte cell adhesion molecule (ALCAM), which allowed us to overcome this immune-evasion mechanism by creating an ALCAM-restricted homing system (HS). We re-engineered the natural ligand of ALCAM, CD6, in a manner that triggers initial anchorage of T cells to ALCAM and conditionally mediates a secondary wave of adhesion by sensitizing T cells to low-level ICAM1 on the cancer endothelium, thereby creating the adhesion forces necessary to capture T cells from the bloodstream. Cytotoxic HS T cells robustly infiltrated brain cancers after intravenous injection and exhibited potent antitumour activity. We have therefore developed a molecule that targets the delivery of T cells to brain cancer.

Fetal dose assessment in a pregnant patient with brain tumor: A comparative study of proton PBS and 3DCRT/VMAT radiation therapy techniques.

PURPOSE: The treatment of brain tumors in pregnant patients poses challenges, as the out-of-field dose exposure to the fetus can potentially be harmful. A pregnant patient with prior radiation treatment was presented with a brain tumor at our clinic. This work reports on our pre-treatment study that compared fetal dose exposure between intensity-modulated proton therapy (IMPT) using pencil beam scanning (PBS) and conventional photon 3D conformal radiation therapy (3DCRT) and volumetric-modulated arc therapy (VMAT), and the subsequent pregnant patient's radiation treatment. MATERIALS AND METHODS: Pre-treatment measurements of clinical plans, 3DCRT, VMAT, and IMPT, were conducted on a phantom. Measurements were performed using a device capable of neutron detections, closely following AAPM guidelines, TG158. For photon measurements, fetus shielding was utilized. On patient treatment days, which was determined to be proton treatment, shielding was used only during daily imaging for patient setup. Additionally, an in vivo measurement was conducted on the patient. RESULTS: Measurements showed that IMPT delivered the lowest fetal dose, considering both photon and neutron out-of-field doses to the fetus, even when shielding was implemented for photon measurements. Additionally, the proton plans demonstrated superior treatment for the mother, a reirradiation case. CONCLUSION: The patient was treated with proton therapy, and the baby was subsequently delivered at full term with no complications. This case study supports previous clinical findings and advocates for the expanded use of proton therapy in this patient population.

Echocardiographic image collection and evaluation in infants with CHD: lessons learned from the imaging core lab for the Residual Lesion Score study.

Many factors affect patient outcome after congenital heart surgery, including the complexity of the heart disease, pre-operative status, patient specific factors (prematurity, nutritional status and/or presence of comorbid conditions or genetic syndromes), and post-operative residual lesions. The Residual Lesion Score is a novel tool for assessing whether specific residual cardiac lesions after surgery have a measurable impact on outcome. The goal is to understand which residual lesions can be tolerated and which should be addressed prior to leaving the operating room. The Residual Lesion Score study is a large multicentre prospective study designed to evaluate the association of Residual Lesion Score to outcomes in infants undergoing surgery for CHD. This Pediatric Heart Network and National Heart, Lung, and Blood Institute-funded study prospectively enrolled 1,149 infants undergoing 5 different congenital cardiac surgical repairs at 17 surgical centres. Given the contribution of echocardiographic measurements in assigning the Residual Lesion Score, the Residual Lesion Score study made use of a centralised core lab in addition to site review of all data. The data collection plan was designed with the added goal of collecting image quality information in a way that would permit us to improve our understanding of the reproducibility, variability, and feasibility of the echocardiographic measurements being made. There were significant challenges along the way, including the coordination, de-identification, storage, and interpretation of very large quantities of imaging data. This necessitated the development of new infrastructure and technology, as well as use of novel statistical methods. The study was successfully completed, but the size and complexity of the population being studied and the data being extracted required more technologic and human resources than expected which impacted the length and cost of conducting the study. This paper outlines the process of designing and executing this complex protocol, some of the barriers to implementation and lessons to be considered in the design of future studies.