ALK-induced lymphomagenesis: from CRISPR modeling to the identification of new regulators by OMICs analyses

Abstract : ALK+ anaplastic large cell lymphoma (ALCL) is a rare pediatric lymphoma harboring most frequently the t(2;5)(p23;q35) chromosomal translocation resulting in NPM1-ALK fusion protein. The first-line treatment is polychemotherapy which yields a high response rate but also presents severe side effects with 30% of relapses of poor prognosis. Second-line treatments now include targeted therapies, underscoring the need for a better comprehension of the pathology. In our team, we have developed a model of ALK+ ALCL transformation. Using CRISPR, we can induce the t(2;5)(p23;q35) translocation and reproduce ALK+ ALCL oncogenesis from healthy primary T lymphocytes to the tumors. Importantly, the edited tumors faithfully recapitulate the immune and histological phenotypes of patients. This original and robust model offers an extensive time frame to analyze every crucial stage of cell transformation, from the cell of origin to in vivo tumors, including pre-transformed cells. To obtain a comprehensive understanding of cell transformation, we are integrating OMIC data including transcriptomics (bulk and scRNAseq), epigenetics (ATAC-seq), and replication timing (Repli-seq). We have recently identified new candidate regulatory genes of ALK+ driven oncogenesis and are currently in the process of functionally validating them. Besides the t(2;5)(p23;q35) translocation, novel somatic mutations in patient tumors have recently been identified by a collaborator, however their exact role in oncogenesis remains unclear. To decipher their function, we are using a CRISPR KO approach in our model and already demonstrated a strong alteration of the transcriptomic oncogene signature. This includes the upregulation of several pathways, including the Hippo pathway involved in cell proliferation and apoptosis. Furthermore, these mutations strongly increased the migration and invasion potentials of cells. We are now conducting mouse experiments to assess the in vivo significance of these mutations.

Description of the novel role of kinesin-1 in dendritic cell migration : a bridge between microtubular dynamics and actin pools.

Abstract : Dendritic cell (DC) migration is a complex process crucial for the establishment, maintenance and development of immune responses. Immature DCs migrate using a biphasic migration pattern, alternating between a slow phase, necessary for the sampling of the antigen and the scanning of the immediate cell environment, and a fast phase, essential for the motility of the cell. While extensive research has been done on understanding DC migration and its necessity in the setting-up of immune responses in patients, the role of kinesin-1, a molecular motor using the microtubular network, in this process remains unclear. We previously showed that kinesin-1, is a key regulator in antitumoral responses and cross-presentation, a specific antigen presentation mechanism in DCs. Nevertheless nothing has yet been established about its implication regarding DC migration.
In this study, we investigated a novel role of kinesin-1 on DC migration, specifically during the establishment of the slow phase of their migratory behavior. Indeed, kinesin-1 deficient DCs display an erratic migratory behavior, lacking this slow phase, as well as an altered cell plasticity. Through live-cell imaging, microfluidics and genetic knockdown approaches, we discovered that kinesin-1 regulates the actin cytoskeleton dynamics, known for its essential role in the migratory behavior of DCs and in macropinocytosis, the engulfment of extracellular material. We hereby suggest that kinesin-1 acts as a cross-linker between microtubules and actin pools. Furthermore, our latest results, associated to drug testing, suggest that impairment of kinesin-1-related transport across the microtubular network could lead to the disruption of molecular pathways essential for the maintenance of the cytoskeletal dynamics. Thus, our work contributes to a better understanding of DC migration, and highlights the complex interplay between kinesin-1, actin dynamics, microtubular network, and its role on immune response modulation.

De novo mutations in the ATOH1 transcription factor induce a characteristic brainstem malformation

Abstract : Developmental disorders of the cerebellum (DDC) represent a heterogeneous group of diseases whose underlying mechanisms remain often unclear. Our recent works highlight early deregulations in the transcriptional control of neuronal fate specification as a pathological process that could contribute to a significant number of DDC cases. In our cohort of patients, we identified de novo variants in genes encoding key transcriptional regulators. Among those, we identified two de novo variants in ATOH1, associated with similar clinical presentations: cerebellar hypoplasia, a specific malformation of the pons, motor and cognitive impairments and deafness. In animal models, ATOH1 activity was shown to be necessary for neural fate specification in the cerebellum and brainstem and for mechanosensory hair cell differentiation, making ATOH1 a strong candidate causative gene for these patients. The two identified mutations lead to a frameshift, and thereby to a truncation of the C-terminal domain of ATOH1. Alterations of this domain have been associated with an increase in the stability of ATOH1 protein and we thus suspect unusual gain-of-function (GOF) effects of these de novo variants. Indeed, our in vitro results show an increased protein stability compared to the WT form, thus reinforcing our hypothesis. In parallel, we are investigating the variant’s transcriptional activity compared to the WT protein. We are also assessing in vivo the effect of truncating mutations in the C-terminal domain using a CRISPR/Cas9-based zebrafish model. Here, we observed a transient increase in the number of mechanosensory hair cells in the neuromasts of the lateral line at 48hpf, corroborating the hypothesis of a GOF and representing a possible link with the deafness of the patients. In the hindbrain, our preliminary results point to a premature differentiation of the neuronal population derived from atoh1a-expressing progenitors, which might be responsible for the brainstem malformation.

Interaction between intestinal bacterial and human amyloids in Parkinson’s disease

Abstract: Emerging evidence suggests Parkinson's disease (PD) may originate in the gut, with α-synuclein (A-SYN) aggregates found in the gastrointestinal tract prior to the onset of motor symptoms. Functional bacterial amyloids (FuBAs), notably Curli, accelerate PD progression by promoting A-SYN aggregation in experimental models. However, the precise mechanisms underlying PD pathology in the gut remain elusive. We hypothesize that enteroendocrine cells (EECs), expressing A-SYN and exposed to intestinal content, play a crucial role by internalizing FuBAs into lysosomes, fostering A-SYN aggregation and transmission to enteric neurons. We studied Curli/A-SYN interaction using the EEC line HuTu80 and human induced pluripotent stem cell (hiPSC)-derived enteric neurons and intestinal organoids (HIOs). CsgA-WT (monomers constituting curli), CsgA-R12343 (non-amyloidogenic control), and Curli were purified and characterized by thioflavin-T assay and electron microscopy. HuTu80 were treated with CsgA-WT, CsgA-R12343, or Curli. We found that CsgA monomers and Curli were localized into lysosomes in HuTu80. Furthermore, CsgA-WT, but not CsgA-R12343, is degraded in lysosomes as shown by bafilomycin A1 treatment. Preliminary data indicate that CsgA-WT and Curli induce A-SYN accumulation in HuTu80 co-treated with exogenous A-SYN monomers. The molecular mechanisms of Curli/A-SYN cross-seeding will be investigated by whole-cell and lysosome proteomics. Lysosomes are immunoprecipitated using HuTu80 stably expressing a tagged lysosomal membrane protein (TMEM192-3xHA). To study cross-seeded amyloid transfer between EECs and neurons, we established a microfluidic co-culture of hIPSC-derived enteric-like neurons and EECs. In summary, our results highlight the role of lysosomes in amyloid cross-seeding. Using HIOs co-cultured with enteric neurons, we now aim to validate our results in a disease-relevant model and identify pathways targeting early disease stage.