Achaete-Scute complex homolog 1 (ASCL1) is a multi-faceted pro-neural transcription factor, playing a role in several processes during embryonic development and into adulthood, including neural progenitor proliferation and neuronal differentiation. This versatility is achieved through tightly controlled expression of ASCL1, either via integrating intracellular signalling cues or stabilisation at the protein level. The role of kinases in ASCL1-mediated neurogenesis is emerging, but to date few kinases have been attributed to act directly or indirectly on ASCL1.
To address this, we designed a cell-based high-throughput screen to identify kinase inhibitors that enhance ASCL1 protein levels. From this screen, two kinase inhibitors were identified to increase ASCL1 stability and transcriptional activity, and subsequent validation indicated that the effect was driven indirectly through Janus kinase family members.
These compounds may serve as useful tools for further investigating the role played by kinases in regulating neurogenesis and ultimately enable better understanding of how ASCL1 integrates different signalling cues to orchestrate with high precision the differentiation of progenitor cells into neurons.
© 2025. Merck & Co., Inc., Rahway, NJ, USA and its affiliates.

Complex RNA-seq signatures involving the transcription factors ASCL1, NEUROD1, and POU2F3 classify Small Cell Lung Cancer (SCLC) into four subtypes: SCLC-A, SCLC-N, SCLC-P, and SCLC-I (triple negative or inflamed). Preliminary studies suggest that identifying these subtypes can guide targeted therapies and potentially improve outcomes. This study aims to evaluate whether the expression levels of these three key transcription factors can effectively classify SCLC subtypes, comparable to the use of individual antibodies in immunohistochemical (IHC) analysis of formalin-fixed, paraffin-embedded (FFPE) tumor samples. We analyzed preclinical models of increasing complexity, including eleven human and five mouse SCLC cell lines, six patient-derived xenografts (PDXs), and two circulating tumor cell (CTC)-derived xenografts (CDXs) generated in our laboratory. RT-qPCR conditions were established to detect the expression levels of ASCL1, NEUROD1, and POU2F3. Additionally, protein-level analysis was performed using Western blot for cell lines and IHC for FFPE samples of PDX and CDX tumors, following our experience with patient tumor samples from the CANTABRICO trial (NCT04712903). We found that the analyzed SCLC cell line models predominantly expressed ASCL1, NEUROD1, and POU2F3, or showed no expression, as identified by RT-qPCR, consistently matching the previously assigned subtypes for each cell line. The classification of PDX and CDX models demonstrated consistency between RT-qPCR and IHC analyses of the transcription factors. Our results show that single-gene analysis by RT-qPCR from FFPE-extracted RNA simplifies SCLC subtype classification. This approach provides a cost-effective alternative to IHC staining or expensive multi-gene RNA sequencing panels, making SCLC subtyping more accessible for both preclinical research and clinical applications.

Geometrically controlled stem cell differentiation promotes reproducible pattern formation. Here, we present a protocol to fabricate elastomeric stencils for patterned stem cell differentiation. We describe procedures for using photolithography to produce molds, followed by molding polydimethylsiloxane (PDMS) to obtain stencils with through holes. We then provide instructions for culturing cells on stencils and, finally, removing stencils to allow colony growth and cell migration. This approach yields reproducible two-dimensional organoids tailored for quantitative studies of growth and pattern formation. For complete details on the use and execution of this protocol, please refer to Lehr et al.1.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Lineage plasticity mediates resistance to androgen receptor pathway inhibitors (ARPIs) and progression from adenocarcinoma to neuroendocrine prostate cancer (NEPC), a highly aggressive and poorly understood subtype. Neuronal transcription factor ASCL1 has emerged as a central regulator of the lineage plasticity driving neuroendocrine differentiation. Here, we showed that ASCL1 was reprogrammed in ARPI-induced transition to terminal NEPC and identified that the ASCL1 binding pattern tailored the expression of lineage-determinant transcription factor combinations that underlie discrete terminal NEPC identity. Notably, we identified FOXA2 as a major cofactor of ASCL1 in terminal NEPC, which is highly expressed in ASCL1-driven NEPC. Mechanistically, FOXA2 and ASCL1 interacted and worked in concert to orchestrate terminal neuronal differentiation. We identified that prospero homeobox 1 was a target of ASCL1 and FOXA2. Targeting prospero homeobox 1 abrogated neuroendocrine characteristics and led to a decrease in cell proliferation in vitro and tumor growth in vivo. Our findings provide insights into the molecular conduit underlying the interplay between different lineage-determinant transcription factors to support the neuroendocrine identity and nominate prospero homeobox 1 as a potential target in ASCL1-high NEPC.

ABSTRACT The subependymal zone (SEZ) of the mammalian brain is the most active germinal area that continues to generate newborn neurons throughout life. This area harbors a population of neural stem cells (NSCs) that can be found in different states of activation, each differing in proliferative capacity and molecular signature: quiescent NSCs (qNSCs), primed NSCs (pNSCs), and activated NSCs (aNSCs). There is currently a void in terms of the specific markers available to effectively discern between these transient states. Likewise, the molecular signaling mechanisms controlling the transition from quiescence to activation remain largely unexplored, as do the factors influencing the decision between differentiation and self-renewal during NSC division. Here, we present evidence that the metabotropic P2Y 13 purinergic receptor plays a critical role in regulating adult neurogenesis. We found that P2Y 13 is specifically expressed in NSCs within the adult SEZ and that its levels can be used to distinguish qNSCs from aNSCs. Functionally, P2Y 13 signaling promotes NSC activation, enhancing lineage progression, while dampening their self-renewal capacity. Conversely, pharmacological blockade or genetic silencing of the P2Y 13 receptor favors NSC quiescence. Thus, we identified the metabotropic P2Y 13 purinergic receptor as a pivotal modulator of NSC dynamics, influencing both the balance between NSC quiescence and activation and the mode of NSC division at the subependymal zone.