Identifying the Origins of Heterogeneity and Plasticity in Small Cell Lung Cancer and Other Neuroendocrine Tumors

Abstract

Small cell lung cancer (SCLC) and olfactory neuroblastoma (ONB) are aggressive malignancies with neuroendocrine (NE) features, poor outcomes, and limited therapeutic options. Despite being clinically treated as a uniform entity, SCLC exhibits remarkable molecular and phenotypic heterogeneity, including distinct subtypes defined by the lineage-related transcription factors ASCL1, NEUROD1, POU2F3, and controversially— YAP1 (SCLC-A, -N, -P, and -Y). Similarly, ONB—a rare tumor arising in the olfactory epithelium—displays histologic diversity and variable NE differentiation, yet its origins and classification remain poorly defined. In both SCLC and ONB, there are major gaps in knowledge surrounding the cellular and molecular determinants of phenotypic heterogeneity. Clarifying whether SCLC and ONB phenotypic diversity stems from genetic drivers, distinct cells of origin, dynamic plasticity, or a combination of these factors is essential for advancing classification frameworks and identifying more effective, targeted treatments for these diseases.To address these knowledge gaps, we leveraged insights from normal development and tissue repair alongside time-series single-cell transcriptomics and lineage tracing, genetically-engineered mouse models (GEMMs) and organoids, and extensive single-cell and bulk analyses of primary human tumors. We began investigating SCLC subtype diversity from a NE cell of origin—the widely accepted origin for SCLC. We developed a powerful ex vivo platform to isolate early NE-high lesions from MYC- high and -low SCLC GEMMs that lack Rb1 and Trp53, enabling study of SCLC tumor progression in multiple genetic contexts. This, paired with studies in human cell lines, led us to discover that MYC is sufficient to drive SCLC subtype evolution from SCLC-A to - N to -Y states from NE cells of origin. However, regardless of genetic context, we find that NE cells fail to generate the full spectrum of phenotypic diversity observed in human tumors, including SCLC-P, motivating us to explore alternative cells of origin. We next investigated the lung basal cell as an origin for SCLC, given the ability of basal cells to generate all lung cell types, including rare ASCL1/NEUROD1+ NE cells and POU2F3+ tuft cells that mirror SCLC-A, -N, and -P phenotypes, respectively. We create highly-tractable basal-origin models and perform in vivo lineage-tracing under multiple relevant, genetic contexts to find that basal cells can produce the full spectrum of known SCLC phenotypes—including SCLC-A, -N, -P, -Y, and a recently identified ATOH1+ state. Moreover, we discover mixed NE/neuronal, hybrid tuft-ionocyte-progenitor (“TIP”-like), and inflammatory basal-like states in our models that are conserved in human SCLC tumors, but previously unrecognized. Single-cell clonal analyses reveal an immense capacity of SCLC for plasticity and uncover preferred plasticity trajectories in SCLC and putative plasticity drivers. Altogether, our findings establish basal cells as the most likely cell of origin for SCLC—fundamentally reframing our understanding of SCLC heterogeneity and plasticity and offering new avenues for modeling and treating the disease. In parallel, we developed a novel GEMM of ONB using Rb1/Trp53/Myc (RPM) alterations that generates high-grade, NEUROD1+, metastatic ONB. We identify the globose basal cell (GBC)—a progenitor population capable of regenerating neuronal and non-neuronal cell types in the olfactory epithelium—as a permissive and likely cell of origin for ONB. We demonstrate that ASCL1 loss in autochthonous RPM GEMMs and GBC organoids permits emergence of non-neuronal states, including POU2F3+ tuft-like lineages, alongside NEUROD1+ neuronal states. Consistently, analysis of human ONB tumors using scRNA-seq, spatial transcriptomics, and immunofluorescence reveals tumor heterogeneity, including NEUROD1+ neuronal and POU2F3+ microvillar states, reminiscent of normal GBC differentiation trajectories. Thus, highly similar to SCLC, mouse and human ONB tumors arise from a basal-like cell of origin, harbor mutually exclusive NEUROD1 and POU2F3-like states, and exhibit extensive plasticity between neuronal and non-neuronal lineages. Altogether, this body of work, informed by principles of normal development and injury repair, resolves the origins and lineage relationships of SCLC subtypes and reveals molecular parallels between SCLC and ONB that have important therapeutic implications. These results challenge the traditional view of fixed lineage identity in NE tumors and reveal that tumor heterogeneity and plasticity are collectively influenced by cell of origin, genetic alterations, and normal developmental or tissue repair pathways. Insights gleaned from this work establish a new conceptual framework for studying and treating SCLC, ONB, and other similar NE tumors.