Eddy Pasquier

ALK-positive Anaplastic Large Cell Lymphoma (ALK+ ALCL) is a rare yet aggressive T-cell lymphoma, most often characterized by the NPM1::ALK fusion. Because few somatic variants have been identified in ALK+ ALCL, we conducted paired whole-exome sequencing of patient tumors with matched normal tissue. Compared with previous studies, we identified a higher frequency of mutations in cohesin complex genes, mostly STAG2 variants (6/16) predicted to impair protein function. Immunohistochemistry confirmed loss of STAG2 in additional patient tumor samples. Using an original model of NPM1::ALK-driven lymphomagenesis that recapitulates early steps of ALK-driven transformation initiated in primary T lymphocytes, we demonstrated that STAG2 defect accelerates oncogenesis, enhancing early cell proliferation of ALK-positive cells. Single-cell RNA-seq revealed that STAG2 depletion accelerates the early acquisition of the major ALK+ ALCL hallmarks and concomitantly reduces the oncogene-induced senescence signature. Despite higher ALK expression and robust cell transformation, STAG2 depletion resulted in the attenuation of the global ALK+ ALCL transcriptional program. Importantly, STAG2 expression was found decreased upon NPM1::ALK induction, indicating intrinsic downregulation during transformation even without genomic mutations. We also successfully identified STAG2-low/ALK-high cells in both patient-derived tumors and PDX models. Functionally, STAG2-depleted cells exhibit increased cell adhesion and migration, alongside constitutive YAP1 activation and upregulation of adhesion molecule such as ALCAM and CD44 but also increased PD-L1. Overall, our findings reveal that STAG2 loss plays a key role during the earliest stages of ALK+ ALCL lymphomagenesis by promoting cell tolerance to NPM1::ALK expression and increasing permissiveness to malignant transformation while also impacting cell adhesion, migration.

Three-dimensional cellular models such as organoids and spheroids hold major promise for developmental biology, disease modelling and precision medicine, yet their large-scale production and analysis remain constrained by handling-induced shear stress, sample fragility, positional instability and limited compatibility with advanced imaging workflows. Here, we introduce Microdomes, dense arrays of open-top, dome-shaped microcavities. Each cavity holds a single specimen in its own miniature aquarium, accessed through a narrow apical opening that admits cells, medium, matrix and staining reagents while shielding it from the shear generated during routine pipetting. Each Microdomes chip accommodates more than 100 spheroids or organoids, supporting diverse cell types, co-culture formats and both matrix-free and matrix-embedded culture. All specimens are retained through prolonged culture, repeated medium exchange, fixation and immunostaining. Because every specimen occupies a fixed, addressable position against a thin transparent film, the same spheroid can be relocated and re-imaged over weeks of culture and across microscopes, from array-wide overviews to subcellular details, without transfer, embedding or other perturbation. Microdomes also support AI-based automated segmentation, from whole-spheroid outlines to individual nuclei in 3D, using common image analysis software. Microdomes thereby turn each array into a self-contained quality control unit, providing specimen-level traceability that organoid production pipelines currently lack.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. The MYC‑amplified group 3 (G3) MB represents a high‑risk disease lacking effective therapies. A genetic screen identified the endoplasmic reticulum chaperone HSPA5 (BiP/GRP78) as a critical dependency in G3 MB. Its silencing induces apoptosis and suppresses tumor growth, with strong activation of the unfolded protein response (UPR). The UPR includes three branches, including the PERK–eIF2α axis, which transiently reduces global protein synthesis while enabling selective translation of stress‑responsive factors such as ATF4. We show that G3 MB requires sustained eIF2α activity for survival. Its inhibition triggers cell death through ATF4‑dependent and ‑independent transcriptional programs and translational reprogramming that deplete essential proteins and promote accumulation of detrimental factors. The eIF2α phosphatase PPP1R15B (CReP) maintains basal eIF2α activity and prevents apoptosis. These findings identify the eIF2α axis and its regulators as a crucial node and a targetable vulnerability in G3 MB.

Owing to chemoresistance, the prognosis of relapsed neuroblastoma is dismal with less than 10% of patients surviving after 5 years. We developed a reverse molecular pharmacology approach that is based on high-throughput drug screening coupled with chemo-informatic and transcriptomic analyses. This led to the identification of IRAK1 as a key chemoresistance factor in neuroblastoma. By performing functional and pharmacological drug combination screens targeting IRAK1, we revealed a synergy between IRAK1 inhibition/silencing and BET, EGFR and mTOR inhibitors as well as microtubule-targeting agents. The synergistic combination of microtubule-targeting agent, vincristine and IRAK inhibitors was then confirmed in tumor spheroids, patient-derived tumoroids and a syngeneic orthotopic mouse model. Mechanistically, IRAK inhibition potentiated the pro-apoptotic and cell cycle arrest properties of vincristine via a pathway involving the PIDDosome complex rather than its canonical MyDDosome axis. Altogether, this study represents a proof-of-concept of our reverse molecular pharmacology approach to quickly develop biology-guided drug combinations, that could be applied to any other human diseases.

All Supplementary Figures and their captions.

Microtubule Targeting Agents (MTAs) constitute a vital category of tubulin-binding compounds, deployed across anticancer therapies. Despite the array of MTA drugs developed by pharmaceutical entities, the quest for novel efficacious molecules continues unabated. We unveil an innovative in vitro MTA screening methodology employing nano differential scanning fluorimetry (nanoDSF), presenting distinct advantages over known assays. This novel approach not only assesses compound-tubulin binding but also quantitatively analyzes their impact on tubulin polymerization. Proposed nanoDSF assay was rigorously validated using the Prestwick Chemical Library, which encompasses 1,520 approved compounds, successfully identifying all previously known MTAs. Furthermore, this screening has unearthed potential anti-tubulin agents among drugs currently utilized for non-related medical conditions, offering insights into their mechanisms of action in inhibiting cancer cell proliferation and/or inducing cytotoxicity. These discoveries herald new opportunities for drug repositioning involving the newly identified MTAs and substantially streamline the process of screening extensive chemical libraries for MTAs featuring novel chemical structures.

Supplementary Data from ONC201 in combination with paxalisib for the treatment of H3K27-altered diffuse midline glioma

All Supplementary Figures and their captions.