Heterogeneity of neurogenic transcription factor expression in small cell lung cancer

Abstract

SCLC is an aggressive neuroendocrine (NE) cancer with a 7% 5-year survival rate. Molecular characterization of this disease has lagged behind other cancer types and standard of care treatment has remained largely unchanged for over three decades. SCLC is treated as a clinically homogenous disease, but evidence of its intra- and inter-tumor heterogeneity is increasing. Part of this heterogeneity is attributed to expression of neurogenic transcription factors (TFs) ASCL1 and NEUROD1, which correlate with SCLC pathogenesis and NE status, and are now included in a consensus for SCLC classification [1]. Our Circulating tumor cell patient Derived eXplant (CDX) models recapitulate disease heterogeneity in terms of neurogenic TF expression; furthermore, we recently identified a previously unrecognized subset within our CDX model panel expressing another neurogenic TF, ATOH1, mostly within NEUROD1 expressing models [2]. I am investigating the roles of the three NE TFs in SCLC CDX, to refine disease classification and explore rationale for new therapeutic approaches stratified to SCLC phenotypes. To do so, we have characterized ASCL1 and NEUROD1 expression in CDX models with multiplex immunofluorescence and flow cytometry. We have begun to interrogate the biology of ASCL1 and NEUROD1 in established SCLC cell lines via CRISPR- and shRNA-mediated genetic depletion of these TFs. Furthermore, we have recently developed antibodies to ATOH1 to support interrogation of its under-researched role in SCLC biology. We have found that CDX models express ASCL1 (30/38), NEUROD1 (3/38) and ATOH1 together with NEUROD1 (4/38) [2]. ASCL1 and NEUROD1 are co-expressed in several CDX but (with the exception of one CDX model) in different cell subpopulations. In contrast with published literature, genetic depletion of ASCL1 and NEUROD1 in established SCLC cell lines did not influence cell proliferation and response to chemotherapy. I have recently generated specific antibodies to ATOH1 and ongoing studies are focused on characterization of ATOH1 expression in CDX models and its impact on the pathogenesis and chemotherapy responses in SCLC. In conclusion, ASCL1 and NEUROD1 are expressed in a mutually exclusive way in CDX models, with the exception of one CDX; manipulation of their expression does not influence cell proliferation or response to chemotherapy in SCLC cell lines. Studies looking at the consequences of genetic depletion of these TFs, including ATOH1, in CDX models are underway.