Small cells - big issues: biological implications and preclinical advancements in small cell lung cancer

Abstract

Current treatment guidelines refer to small cell lung cancer (SCLC), one of the deadliest human malignancies, as a homogeneous disease. Accordingly, SCLC therapy comprises chemoradiation with or without immunotherapy. Meanwhile, recent studies have made significant advances in subclassifying SCLC based on the elevated expression of the transcription factors ASCL1, NEUROD1, and POU2F3, as well as on certain inflammatory characteristics. The role of the transcription regulator YAP1 in defining a unique SCLC subset remains to be established. Although preclinical analyses have described numerous subtype-specific characteristics and vulnerabilities, the so far non-existing clinical subtype distinction may be a contributor to negative clinical trial outcomes. This comprehensive review aims to provide a framework for the development of novel personalized therapeutic approaches by compiling the most recent discoveries achieved by preclinical SCLC research. We highlight the challenges faced due to limited access to patient material as well as the advances accomplished by implementing state-of-the-art models and methodologies.

Keywords: Molecular subtypes; Preclinical models; Small cell lung cancer; Translational progress.

Fig. 1

SCLC tumorigenesis from different cells of origin

Fig. 2

Molecular subclassification of SCLC.  SCLC subclassification discriminates between NE subtypes which are represented by ASCL1- or NEUROD1-driven tumors and non-NE subtypes characterized by POU2F3 or inflamed expression patterns. Distinct expression profiles show potential vulnerabilities of each SCLC subtype. Created with BioRender.com. TP53—Tumor protein 53; RB1 - Retinoblastoma 1; NE - neuroendocrine; PNEC - Pulmonary NE cells; ASCL1 - Achaete-scute homologue 1; NEUROD1 - Neurogenic differentiation factor 1; POU2F3 - POU class 2 homeobox 3; YAP1 - Yes-associated protein 1, QN - Quadruple negative, BCL2 - B-cell lymphoma 2, DLL3 - Delta-like protein 3, CHGA – Chromogranin A, EZH2 - Enhancer of zeste homologue 2, SOX2 - SRY-box transcription factor 2, CDH1 – E-cadherin, TTF-1 - Homeobox protein Nkx2.1, LSD1 - Lysine demethylase 1A, RET - Ret proto-oncogene, AURKA – Aurora kinase A, MYC – MYC proto-oncogene, NCAM1 - Neural cell adhesion molecule 1, NFIB - Nuclear factor 1 B, HES6 - Hairy and enhancer of split 6, ANTXR1 - Anthrax toxin receptor 1, INSM1 - Insulinoma-associated protein 1, ASCL2 - Achaete-scute homologue 2, IGF-1R - Insulin-growth factor receptor 1, SOX9 - SRY-box transcription factor 9, CHAT - Choline O-acetyltransferase, ATM - ATM serine/threonine kinase, TAZ - Homologue to YAP1, PLK - Polo-like kinase, PD-L1 - Programmed death-ligand 1, mTOR – Rapamycin, CDK4/6 - Cyclin-dependent kinases 4/6

Fig. 3

Intratumoral heterogeneity of SCLC based on endogenous or exogeneous influences. a Intratumoral subtype switching is facilitated by enriched MYC expressions and/or increased Notch activity. This endogenous transition is assumed to proceed from NE high tumors to non-NE phenotypes. Hence, ASCL1-driven SCLCs transform into NEUROD1 and, ultimately, into YAP1-high tumors. b Exogeneous subtype transition is caused by systemic therapy, leading to selection of subclones showing intrinsic or acquired resistance. Disease recurrence accompanied by chemoresistance represents a hallmark of SCLC. Created with BioRender.com. NE - Neuroendocrine; ASCL1 - Achaete-scute homologue 1; NEUROD1 - Neurogenic differentiation factor 1; MYC - MYC proto-oncogene

Fig. 4

Frequently altered signaling pathways in SCLC. a The Hippo signaling pathway discriminates between active (ON) and inactive (OFF) states. When Hippo signaling is on, the phosphorylation of SAV1 is mediated by MST1/2, both further activating MOB1A/B and LATS1/2 via phosphorylation. Ultimately, the YAP/TAZ complex is phosphorylated and degraded, resulting in transcriptional repression. Or in contrast, when Hippo signaling is off, the phosphorylation cascade does not take place. The YAP/TAZ complex translocates into the nucleus and interacts with TEAD, leading to transcriptional activation. b The Notch signaling pathway is mediated between signal-sending and signal-receiving cells via interaction of the Notch receptor and a Notch ligand (DLL or JAG). Consecutively, the Notch receptor is cleaved; NICD translocates into the nucleus and activates the transcription of target genes in signal-receiving cells (e.g. HES, HEY, and MYC). ASCL1 is categorized as a master regulator of NE differentiation via high expression of the non-functional DLL3 at the golgi apparatus. DLL3 acts as a dominant-negative inhibitor of Notch signaling and orchestrates the degradation of other Notch members. HES and HEY family members encode transcriptional repressors of ASCL1. Notch negatively regulates NE differentiation in SCLC. High ASCL1 expression levels significantly correlate with NE differentiation. c Hedgehog signaling is activated by the binding of SHH to PTCH1. This leads to the shift of inhibitory activity towards SUFU (green path). Subsequently, the GLI1 monomer translocates into the nucleus and promotes gene transcription. When PTCH1 exerts its inhibitory effects against SMO, SUFU and GLI1 build a complex which inactivates the expression of HH target genes (red path). d Epigenetic reprogramming plays an instrumental role in SCLC via methylation / acetylation of DNA/histones. Key chromatin modifiers are EZH2, the CREBBP/EP300 complex, and the KMT2 family proteins which each target the amino acids K27, K18, or K4 of histone 3, respectively. Created with BioRender.com. ASCL1 - Achaete-scute homologue 1; DLL3 - Delta-like protein 3; REST - RE1-silencing transcription factor; HES - HES family BHLH transcription factor 1; HEY - HES-related repressor protein 1; MYC - MYC proto-oncogene; CYCD1 - Cyclin D1; p21 - Cyclin dependent kinase inhibitor 1A; MOB1A/B - MOB kinase activator 1A/B; MST1/2 - Mammalian sterile 20-like kinases 1 and 2; LATS1/2 - Large tumor suppressor kinase 1/2; SAV1 - Salvador homolog 1; YAP1 - Yes-associated protein 1; TAZ - Tafazzin family protein; TEAD - TEA domain transcription factor; EZH2 - Enhancer of zeste homolog 2; Me - Methylation; Ac - Acetylation; CREBBP - CREB binding protein; EP300 - E1A binding protein P300; H2A/2B/3/4 - Histone 2A/2B/3/4; KMT2 - Lysine methyltransferase 2; SHH - Sonic hedgehog; GLI1 - GLI family zinc finger 1; SUFU - SUFU negative regulator of hedgehog signaling; PTCH1/2 - Patched 1; SMO - Smoothened, frizzled class receptor; BCL2 - B-cell lymphoma 2; SOX2 - SRY-box transcription factor 2

Fig. 5

Fundamental preclinical models for SCLC research. Preclinical research commonly relies on well-established assays based on two-dimensional (2D) or three-dimensional (3D) cell culture. Different cell compositions (cell lines, circulating tumor cells, tissues) can be furthermore examined in vivo via subcutaneous or orthotopic transplantation. Hence, CDX or PDX xenotransplantation mimic human disease regardless of immune microenvironment. The genetically engineered mouse model (GEMM) displays a platform to investigate tumor initiation and tumorigenesis of SCLC via genetic knock-out. In contrast, humanized mice present intact immunologic features that may be exploited to investigate the interaction between SCLC tumors and their microenvironment. Zebrafish larvae are frequently utilized to study the dynamic invasion of tumor cells in vivo or evaluate cost-effective chemical screenings. Sampling of representative tissue samples is rather rare in SCLC patients due to infrequently performed surgery. Rapid research autopsies should be used with greater frequency in SCLC research. Created with BioRender.com. CDX - Cell-derived xenograft; PDX – Patient-derived xenograft; GEMM – Genetically engineered mouse model