Single-cell profiling of prurigo nodularis demonstrates immune-stromal crosstalk driving profibrotic responses and reversal with nemolizumab.

BACKGROUND: Prurigo nodularis (PN) is a chronic neuroimmune skin disease characterized by bilaterally distributed pruritic hyperkeratotic nodules on extremities and trunk. Neuroimmune dysregulation and chronic scratching are believed to both induce and maintain the characteristic lesions. OBJECTIVES: This study sought to provide a comprehensive view of the molecular pathogenesis of PN at the single-cell level to identify and outline key pathologic processes and the cell types involved. Features that distinguish PN skin from the skin of patients with atopic dermatitis were of particular interest. We further aimed to determine the impact of the IL31RA antagonist, nemolizumab, and its specificity at the single-cell level. METHODS: Single-cell RNA-sequencing of skin from 15 healthy donors and nonlesional and lesional skin from 6 patients each with PN and atopic dermatitis, combined with spatial-sequencing using the 10x Visium platform. Integration with bulk RNA-sequencing data from patients treated with nemolizumab. RESULTS: This study demonstrates that PN is an inflammatory skin disease characterized by both keratinocyte proliferation and activation of profibrotic responses. This study also demonstrates that the COL11A1+ fibroblast subset is a major contributor to fibrosis and is predominantly found in the papillary dermis of PN skin. Activation of fibrotic responses is the main distinguishing feature between PN and atopic dermatitis skin. This study further shows the broad effect of nemolizumab on PN cell types, with a prominent effect driving COL11A1+ fibroblast and keratinocyte responses toward normal. CONCLUSIONS: This study provides a high-resolution characterization of the cell types and cellular processes activated in PN skin, establishing PN as a chronic fibrotic inflammatory skin disease. It further demonstrates the broad effect of nemolizumab on pathological processes in PN skin.

Modulation of Neuroimmune and Epithelial Dysregulation in Patients With Moderate to Severe Prurigo Nodularis Treated With Nemolizumab.

Importance: Prurigo nodularis (PN) is a debilitating skin disease characterized by intense pruritus and hyperkeratotic skin nodules. Nemolizumab, a monoclonal antibody targeting interleukin 31 receptor α, is a promising novel therapy for the treatment of moderate to severe PN. The biological mechanisms by which nemolizumab promotes improvement of itch and skin lesions in PN are unknown. Objective: To characterize changes in plasma protein biomarkers associated with clinical response to nemolizumab in patients with PN. Design, Setting, and Participants: This multicenter cohort study included patients recruited from Austria, France, Germany, Poland, and the US from a phase 2 clinical trial. Adults diagnosed with moderate to severe PN with severe pruritus for at least 6 months were included in the original trial. Patients in the nemolizumab group were included in the present study if they achieved at least a 4-point decrease in the Peak Pruritus Numerical Rating Scale (PP-NRS) from baseline to week 12 during nemolizumab treatment. Placebo controls did not experience a 4-point decrease in PP-NRS. Mass spectrometry with tandem mass tags to enrich skin-specific protein detection was used to characterize changes in plasma protein expression in nemolizumab and placebo groups. Data were collected from November 2, 2017, to September 26, 2018, and analyzed from December 6, 2019, to April 8, 2022. Intervention: As part of the clinical trial, patients were treated with 3 doses of nemolizumab or placebo at 0, 4, and 8 weeks. Main Outcomes and Measures: Changes in plasma and epidermal protein expression in nemolizumab-treated patients compared with the placebo group at 0, 4, and 12 weeks. Results: Among the 38 patients included in the analysis (22 women and 16 men; mean [SD] age, 55.8 [15.8] years), enrichment analysis of canonical pathways, biological functions, and upstream regulators showed downregulation of terms involving inflammation (IL-6, acute-phase response, signal transducer and activator of transcription 3, and interferon γ), neural processes (synaptogenesis signaling and neuritogenesis), tissue remodeling and fibrosis (transforming growth factor ß1 and endothelin-1), and epidermal differentiation (epithelial mesenchymal transition) in the plasma of nemolizumab group. Conclusions and Relevance: In this cohort study, differences between nemolizumab and placebo groups included modulation of inflammatory signaling, neural development, and epithelial differentiation, suggesting a promising potential approach for clinical management of PN.

Clonal expansion of intra-epithelial T cells in breast cancer revealed by spatial transcriptomics.

The spatial distribution of tumor-infiltrating lymphocytes (TIL) predicts breast cancer outcome and response to systemic therapy, highlighting the importance of an intact tissue structure for characterizing tumors. Here, we present ST-FFPE, a spatial transcriptomics method for the analysis of formalin-fixed paraffin-embedded samples, which opens the possibility of interrogating archival tissue. The method involves extraction, exome capture and sequencing of RNA from different tumor compartments microdissected by laser-capture, and can be used to study the cellular composition of tumor microenvironment. Focusing on triple-negative breast cancer (TNBC), we characterized T cells, B cells, dendritic cells, fibroblasts and endothelial cells in both stromal and intra-epithelial compartments. We found a highly variable spatial distribution of immune cell subsets among tumors. This analysis revealed that the immune repertoires of intra-epithelial T and B cells were consistently less diverse and more clonal than those of stromal T and B cells. T-cell receptor (TCR) sequencing confirmed a reduced diversity and higher clonality of intra-epithelial T cells relative to the corresponding stromal T cells. Analysis of the top 10 dominant clonotypes in the two compartments showed a majority of shared but also some unique clonotypes both in stromal and intra-epithelial T cells. Hyperexpanded clonotypes were more abundant among intra-epithelial than stromal T cells. These findings validate the ST-FFPE method and suggest an accumulation of antigen-specific T cells within tumor core. Because ST-FFPE is applicable for analysis of previously collected tissue samples, it could be useful for rapid assessment of intratumoral cellular heterogeneity in multiple disease and treatment settings.

The first comprehensive genomic characterization of rectal squamous cell carcinoma.

BACKGROUND: Rectal cancers represent 35% of colorectal cancers; 90% are adenocarcinomas, while squamous cell carcinoma accounts for 0.3% of them. Given its rarity, little is known concerning its pathogenesis, molecular profile and therapeutic management. The current treatment trend is to treat rectal squamous cell carcinoma by analogy to anal squamous cell carcinoma with definitive chemo-radiotherapy, setting aside surgery in case of local recurrence. METHODS: We performed an in-depth genomic analysis (next-generation sequencing, copy number variation, and human papilloma virus characterization) on 10 rectal squamous cell carcinoma samples and compared them in silico to those of anal squamous cell carcinoma and rectal adenocarcinoma. RESULTS: Rectal squamous cell carcinoma shows 100% HPV positivity. It has a mutational (PIK3CA, PTEN, TP53, ATM, BCL6, SOX2) and copy number variation profile (3p, 10p, 10q, 16q deletion and 1q, 3q, 5p, 8q, 20p gain) similar to anal squamous cell carcinoma. PI3K/Akt/mTOR is the most commonly affected signaling pathway similarly to anal squamous cell carcinoma. Most commonly gained or lost genes seen in rectal adenocarcinoma (FLT3, CDX2, GNAS, BCL2, SMAD4, MALT1) are not found in rectal squamous cell carcinoma. CONCLUSION: This study presents the first comprehensive genomic characterization of rectal squamous cell carcinoma. We confirm the existence of this rare histology and its molecular similarity with anal squamous cell carcinoma. This molecular proximity confirms the adequacy of therapeutic management based on histology and not localization, suggesting that rectal squamous cell carcinoma should be treated like anal squamous cell carcinoma and not as a rectal adenocarcinoma.

RAS activation induces synthetic lethality of MEK inhibition with mitochondrial oxidative metabolism in acute myeloid leukemia.

Despite recent advances in acute myeloid leukemia (AML) molecular characterization and targeted therapies, a majority of AML cases still lack therapeutically actionable targets. In 127 AML cases with unmet therapeutic needs, as defined by the exclusion of ELN favorable cases and of FLT3-ITD mutations, we identified 51 (40%) cases with alterations in RAS pathway genes (RAS+, mostly NF1, NRAS, KRAS, and PTPN11 genes). In 79 homogeneously treated AML patients from this cohort, RAS+ status were associated with higher white blood cell count, higher LDH, and reduced survival. In AML models of oncogenic addiction to RAS-MEK signaling, the MEK inhibitor trametinib demonstrated antileukemic activity in vitro and in vivo. However, the efficacy of trametinib was heterogeneous in ex vivo cultures of primary RAS+ AML patient specimens. From repurposing drug screens in RAS-activated AML cells, we identified pyrvinium pamoate, an anti-helminthic agent efficiently inhibiting the growth of RAS+ primary AML cells ex vivo, preferentially in trametinib-resistant PTPN11- or KRAS-mutated samples. Metabolic and genetic complementarity between trametinib and pyrvinium pamoate translated into anti-AML synergy in vitro. Moreover, this combination inhibited the propagation of RA+ AML cells in vivo in mice, indicating a potential for future clinical development of this strategy in AML.

Automated Detection of Arm-Level Alterations for Individual Cancer Patients in the Clinical Setting.

Copy number alterations are genetic events that promote tumor initiation and progression and are used in clinical care as diagnostic, prognostic, and predictive biomarkers. Based on the length of the alteration, they are roughly classified as focal and arm-level alterations. Although genome-wide techniques to detect arm-level alterations are gaining momentum in hospital laboratories, the high precision and novelty of these techniques pose new challenges: there is no consensus on the definition of an arm-level alteration and there is a lack of tools to compute them for individual patients. Based on 376 clinical samples analyzed with the OncoScan formalin-fixed, paraffin-embedded assay, a bimodal distribution of the percentage of bases with copy number alterations within a chromosomal arm was observed, with the second peak starting at 90% of arm length. Two approaches were tested for the definition of arm-level alterations: sum of altered segments (SoS) >90%, or the longest segment (LS) >90%. These approaches were validated against expert annotation of 25 clinical cases. The SoS method outperformed the LS method as indicated by a higher concordance (SoS, 95.2%; LS, 79.9%). Some of the discordances ultimately were attributed to human error, highlighting the advantages of automation. The increase in reliability led to the development of publicly available software and its inclusion into routine clinical practice at Geneva University Hospitals.

Insights into Islet Differentiation and Maturation through Proteomic Characterization of a Human iPSC-Derived Pancreatic Endocrine Model.

PURPOSE: Great progresses have been made for generating in vitro pluripotent stem cell pancreatic ß-like cells. However, the maturation stage of the cells still requires in vivo maturation to recreate the environmental niche. A deeper understanding of the factors promoting maturation of the cells is of great interest for clinical applications. EXPERIMENTAL DESIGN: Label-free mass spectrometry based proteomic analysis is performed on samples from a longitudinal study of differentiation of human induced pluripotent stem cells toward glucose responsive insulin producing cells. RESULTS: Proteome patterns correlate with specific transcription factor gene expression levels during in vitro differentiation, showing the relevance of the technology for identification of pancreatic-specific markers. The analysis of proteomes of the implanted cells in a longitudinal study shows that the neovascularization process linked to the extracellular matrix environment is time-dependent and conditions the proper maturation of the cells in ß-like cells secreting insulin in response to glucose. CONCLUSIONS AND CLINICAL RELEVANCE: Proteomic profiling is valuable to qualify and better understand in vivo maturation of progenitor cells toward ß-cells. This is critical for future clinical trials where in vivo maturation still needs to be improved for robustness and effectiveness of cell therapy. Novel biomarkers for predicting the efficiency of maturation represents noninvasive monitoring tools for following efficiency of the implant.

On-the-Fly QM/MM Docking with Attracting Cavities.

We developed a hybrid quantum mechanical/molecular mechanical (QM/MM) on-the-fly docking algorithm to address the challenges of treating polarization and selected metal interactions in docking. The algorithm is based on our classical docking algorithm Attracting Cavities and relies on the semiempirical self-consistent charge density functional tight-binding (SCC-DFTB) method and the CHARMM force field. We benchmarked the performance of this approach on three very diverse data sets: (1) the Astex Diverse set of 85 common noncovalent drug/target complexes formed both by hydrophobic and electrostatic interactions; (2) a zinc metalloprotein data set of 281 complexes, where polarization is strong and ligand/protein interactions are dominated by electrostatic interactions; and (3) a heme protein data set of 72 complexes, where ligand/protein interactions are dominated by covalent ligand/iron binding. Redocking performance of the on-the-fly QM/MM docking algorithm was compared to the performance of classical Attracting Cavities, AutoDock, AutoDock Vina, and GOLD. The results demonstrate that the QM/MM code preserves the high accuracy of most classical scores on the Astex Diverse set, while it yields significant improvements on both sets of metalloproteins at moderate computational cost.

Attracting cavities for docking. Replacing the rough energy landscape of the protein by a smooth attracting landscape.

Molecular docking is a computational approach for predicting the most probable position of ligands in the binding sites of macromolecules and constitutes the cornerstone of structure-based computer-aided drug design. Here, we present a new algorithm called Attracting Cavities that allows molecular docking to be performed by simple energy minimizations only. The approach consists in transiently replacing the rough potential energy hypersurface of the protein by a smooth attracting potential driving the ligands into protein cavities. The actual protein energy landscape is reintroduced in a second step to refine the ligand position. The scoring function of Attracting Cavities is based on the CHARMM force field and the FACTS solvation model. The approach was tested on the 85 experimental ligand-protein structures included in the Astex diverse set and achieved a success rate of 80% in reproducing the experimental binding mode starting from a completely randomized ligand conformer. The algorithm thus compares favorably with current state-of-the-art docking programs.

Toward on-the-fly quantum mechanical/molecular mechanical (QM/MM) docking: development and benchmark of a scoring function.

We address the challenges of treating polarization and covalent interactions in docking by developing a hybrid quantum mechanical/molecular mechanical (QM/MM) scoring function based on the semiempirical self-consistent charge density functional tight-binding (SCC-DFTB) method and the CHARMM force field. To benchmark this scoring function within the EADock DSS docking algorithm, we created a publicly available dataset of high-quality X-ray structures of zinc metalloproteins ( http://www.molecular-modelling.ch/resources.php ). For zinc-bound ligands (226 complexes), the QM/MM scoring yielded a substantially improved success rate compared to the classical scoring function (77.0% vs 61.5%), while, for allosteric ligands (55 complexes), the success rate remained constant (49.1%). The QM/MM scoring significantly improved the detection of correct zinc-binding geometries and improved the docking success rate by more than 20% for several important drug targets. The performance of both the classical and the QM/MM scoring functions compare favorably to the performance of AutoDock4, AutoDock4Zn, and AutoDock Vina.

Snail and slug mediate radioresistance and chemoresistance by antagonizing p53-mediated apoptosis and acquiring a stem-like phenotype in ovarian cancer cells.

The transcriptional repressors Snail and Slug contribute to cancer progression by mediating epithelial-mesenchymal transition (EMT), which results in tumor cell invasion and metastases. We extend this current understanding to demonstrate their involvement in the development of resistance to radiation and paclitaxel. The process is orchestrated through the acquisition of a novel subset of gene targets that is repressed under conditions of stress, effectively inactivating p53-mediated apoptosis, while another subset of targets continues to mediate EMT. Repressive activities are complemented by a concurrent derepression of specific genes resulting in the acquisition of stem cell-like characteristics. Such cells are bestowed with three critical capabilities, namely EMT, resistance to p53-mediated apoptosis, and a self-renewal program, that together define the functionality and survival of metastatic cancer stem cells. EMT provides a mechanism of escape to a new, less adverse niche; resistance to apoptosis ensures cell survival in conditions of stress in the primary tumor; whereas acquisition of "stemness" ensures generation of the critical tumor mass required for progression of micrometastases to macrometastases. Our findings, besides achieving considerable expansion of the inventory of direct genes targets, more importantly demonstrate that such elegant cooperative modulation of gene regulation mediated by Snail and Slug is critical for a cancer cell to acquire stem cell characteristics toward resisting radiotherapy- or chemotherapy-mediated cellular stress, and this may be a determinative aspect of aggressive cancer metastases.