Nfib Promotes Metastasis through a Widespread Increase in Chromatin Accessibility.

Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer (SCLC) to investigate the mechanisms that drive the metastatic spread of this lethal cancer. Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions. Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression.

Genetic requirement for Mycl and efficacy of RNA Pol I inhibition in mouse models of small cell lung cancer.

Small cell lung cancer (SCLC) is a devastating neuroendocrine carcinoma. MYCL (L-Myc) is frequently amplified in human SCLC, but its roles in SCLC progression are poorly understood. We isolated preneoplastic neuroendocrine cells from a mouse model of SCLC and found that ectopic expression of L-Myc, c-Myc, or N-Myc conferred tumor-forming capacity. We focused on L-Myc, which promoted pre-rRNA synthesis and transcriptional programs associated with ribosomal biogenesis. Deletion of Mycl in two genetically engineered models of SCLC resulted in strong suppression of SCLC. The high degree of suppression suggested that L-Myc may constitute a therapeutic target for a broad subset of SCLC. We then used an RNA polymerase I inhibitor to target rRNA synthesis in an autochthonous Rb/p53-deleted mouse SCLC model and found significant tumor inhibition. These data reveal that activation of RNA polymerase I by L-Myc and other MYC family proteins provides an axis of vulnerability for this recalcitrant cancer.

Phase I Study of Entinostat, Atezolizumab, Carboplatin, and Etoposide in Previously Untreated Extensive-Stage Small Cell Lung Cancer, ETCTN 10399.

BACKGROUND: CREBBP and EP300 mutations occur at a frequency of 15% and 13%, respectively, in small cell lung cancer (SCLC), and preclinical models demonstrated susceptibility to targeting with HDAC inhibitors. METHODS: Patients with treatment-naïve extensive-stage SCLC, ECOG ≤2 were enrolled and treated with entinostat orally weekly (4 dose levels, DL) in combination with standard dose carboplatin, etoposide, and atezolizumab. Cohort allocation was determined by Bayesian optimal interval (BOIN) design targeting an MTD with a DLT rate of 20%. RESULTS: Three patients were enrolled and treated at DL1 with entinostat 2 mg. Patients were aged 69-83; 2 male, 1 female; 2 were ECOG 1, and 1 was ECOG 0. The most common adverse events (AEs) were anemia (3), neutropenia (3), thrombocytopenia (2), leukopenia (2), and hypocalcemia (2). Two experienced DLTs during cycle 1: (1) grade (Gr) 4 febrile neutropenia, and (1) Gr 5 sepsis. BOIN design required stopping accrual to DL1, and the trial was closed to further accrual. Entinostat and atezolizumab pharmacokinetics were both comparable to historical controls. CONCLUSION: Addition of entinostat to atezolizumab, carboplatin, and etoposide is unsafe and resulted in early onset and severe neutropenia, thrombocytopenia. Further exploration of entinostat with carboplatin, etoposide, and atezolizumab should not be explored. (ClinicalTrials.gov Identifier: NCT04631029).

BCAT1 affects mitochondrial metabolism independently of leucine transamination in activated human macrophages.

In response to environmental stimuli, macrophages change their nutrient consumption and undergo an early metabolic adaptation that progressively shapes their polarization state. During the transient, early phase of pro-inflammatory macrophage activation, an increase in tricarboxylic acid (TCA) cycle activity has been reported, but the relative contribution of branched-chain amino acid (BCAA) leucine remains to be determined. Here, we show that glucose but not glutamine is a major contributor of the increase in TCA cycle metabolites during early macrophage activation in humans. We then show that, although uptake of BCAAs is not altered, their transamination by BCAT1 is increased following 8 h lipopolysaccharide (LPS) stimulation. Of note, leucine is not metabolized to integrate into the TCA cycle in basal or stimulated human macrophages. Surprisingly, the pharmacological inhibition of BCAT1 reduced glucose-derived itaconate, α-ketoglutarate and 2-hydroxyglutarate levels without affecting succinate and citrate levels, indicating a partial inhibition of the TCA cycle. This indirect effect is associated with NRF2 (also known as NFE2L2) activation and anti-oxidant responses. These results suggest a moonlighting role of BCAT1 through redox-mediated control of mitochondrial function during early macrophage activation.

Reprogramming fatty acyl specificity of lipid kinases via C1 domain engineering.

C1 domains are lipid-binding modules that regulate membrane activation of kinases, nucleotide exchange factors and other C1-containing proteins to trigger signal transduction. Despite annotation of typical C1 domains as diacylglycerol (DAG) and phorbol ester sensors, the function of atypical counterparts remains ill-defined. Here, we assign a key role for atypical C1 domains in mediating DAG fatty acyl specificity of diacylglycerol kinases (DGKs) in live cells. Activity-based proteomics mapped C1 probe binding as a principal differentiator of type 1 DGK active sites that combined with global metabolomics revealed a role for C1s in lipid substrate recognition. Protein engineering by C1 domain swapping demonstrated that exchange of typical and atypical C1s is functionally tolerated and can directly program DAG fatty acyl specificity of type 1 DGKs. Collectively, we describe a protein engineering strategy for studying metabolic specificity of lipid kinases to assign a role for atypical C1 domains in cell metabolism.

The H3K4 methyltransferase SETD1A is required for proliferation of non-small cell lung cancer cells by promoting S-phase progression.

Epigenetic dysregulation has been strongly implicated in carcinogenesis and is one of the mechanisms that contribute to the development of lung cancer. Using genome-wide CRISPR/Cas9 library screening, we showed SET domain-containing protein 1A (SETD1A) is an essential epigenetic modifier of the proliferation of NSCLC H1299 cells. Depletion of SETD1A strikingly inhibited the proliferation of NSCLC cells. IHC staining and bioinformatics showed that SETD1A is upregulated in lung cancer. Kaplan-Meier survival analysis indicated that high expression of SETD1A is associated with poor prognosis of patients with NSCLC. We revealed that loss of SETD1A inhibits DNA replication and induces replication stress accompanied by impaired fork progression. In addition, transcription of CDC7 and TOP1, which are involved in replication origin activation and fork progression, respectively, was significantly reduced by knockdown of SETD1A. Taken together, these findings demonstrated SETD1A is a critical epigenetic modifier of NSCLC cell proliferation by promoting the transcription of a subset of DNA replication-associated genes.

Comprehensive genomic profiles of small cell lung cancer.

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

Genetically-engineered mouse models of small cell lung cancer: the next generation.

Small cell lung cancer (SCLC) remains the most fatal form of lung cancer, with patients in dire need of new and effective therapeutic approaches. Modeling SCLC in an immunocompetent host is essential for understanding SCLC pathogenesis and ultimately discovering and testing new experimental therapeutic strategies. Human SCLC is characterized by near universal genetic loss of the RB1 and TP53 tumor suppressor genes. Twenty years ago, the first genetically-engineered mouse model (GEMM) of SCLC was generated using conditional deletion of both Rb1 and Trp53 in the lungs of adult mice. Since then, several other GEMMs of SCLC have been developed coupling genomic alterations found in human SCLC with Rb1 and Trp53 deletion. Here we summarize how GEMMs of SCLC have contributed significantly to our understanding of the disease in the past two decades. We also review recent advances in modeling SCLC in mice that allow investigators to bypass limitations of the previous generation of GEMMs while studying new genes of interest in SCLC. In particular, CRISPR/Cas9-mediated somatic gene editing can accelerate how new genes of interest are functionally interrogated in SCLC tumorigenesis. Notably, the development of allograft models and precancerous precursor models from SCLC GEMMs provides complementary approaches to GEMMs to study tumor cell-immune microenvironment interactions and test new therapeutic strategies to enhance response to immunotherapy. Ultimately, the new generation of SCLC models can accelerate research and help develop new therapeutic strategies for SCLC.

Genetic disruption of ATAT1 causes RhoA downregulation through abnormal truncation of C/EBPß.

Microtubule acetylation has been shown to regulate actin filament dynamics by modulating signaling pathways that control actin organization, although the precise mechanisms remain unknown. In this study, we found that the downregulation of microtubule acetylation via the disruption ATAT1 (which encodes α-tubulin N-acetyltransferase 1) inhibited the expression of RhoA, a small GTPase involved in regulating the organization of actin filaments and the formation of stress fibers. Analysis of RHOA promoter and chromatin immunoprecipitation assays revealed that C/EBPß is a major regulator of RHOA expression. Interestingly, the majority of C/EBPß in ATAT1 knockout (KO) cells was found in the nucleus as a 27-kDa fragment (referred to as C/EBPßp27) lacking the N-terminus of C/EBPß. Overexpression of a gene encoding a C/EBPßp27-mimicking protein via an N-terminal deletion in C/EBPß led to competitive binding with wild-type C/EBPß at the C/EBPß binding site in the RHOA promoter, resulting in a significant decrease of RHOA expression. We also found that cathepsin L (CTSL), which is overexpressed in ATAT1 KO cells, is responsible for C/EBPßp27 formation in the nucleus. Treatment with a CTSL inhibitor led to the restoration of RHOA expression by downregulation of C/EBPßp27 and the invasive ability of ATAT1 KO MDA-MB-231 breast cancer cells. Collectively, our findings suggest that the downregulation of microtubule acetylation associated with ATAT1 deficiency suppresses RHOA expression by forming C/EBPßp27 in the nucleus through CTSL. We propose that CTSL and C/EBPßp27 may represent a novel therapeutic target for breast cancer treatment. [BMB Reports 2024; 57(6): 293-298].

CRACD loss induces neuroendocrine cell plasticity of lung adenocarcinoma.

Tumor cell plasticity contributes to intratumoral heterogeneity and therapy resistance. Through cell plasticity, some lung adenocarcinoma (LUAD) cells transform into neuroendocrine (NE) tumor cells. However, the mechanisms of NE cell plasticity remain unclear. CRACD (capping protein inhibiting regulator of actin dynamics), a capping protein inhibitor, is frequently inactivated in cancers. CRACD knockout (KO) is sufficient to de-repress NE-related gene expression in the pulmonary epithelium and LUAD cells. In LUAD mouse models, Cracd KO increases intratumoral heterogeneity with NE gene expression. Single-cell transcriptomic analysis showed that Cracd KO-induced NE cell plasticity is associated with cell de-differentiation and stemness-related pathway activation. The single-cell transcriptomic analysis of LUAD patient tumors recapitulates that the distinct LUAD NE cell cluster expressing NE genes is co-enriched with impaired actin remodeling. This study reveals the crucial role of CRACD in restricting NE cell plasticity that induces cell de-differentiation of LUAD.

CRACD suppresses neuroendocrinal plasticity of lung adenocarcinoma.

Tumor cell plasticity contributes to intratumoral heterogeneity and therapy resistance. Through cell plasticity, lung adenocarcinoma (LUAD) cells transform into neuroendocrinal (NE) tumor cells. However, the mechanisms of NE cell plasticity remain unclear. CRACD, a capping protein inhibitor, is frequently inactivated in cancers. CRACD knock-out (KO) de-represses NE-related gene expression in the pulmonary epithelium and LUAD cells. In LUAD mouse models, Cracd KO increases intratumoral heterogeneity with NE gene expression. Single-cell transcriptomic analysis showed that Cracd KO-induced NE plasticity is associated with cell de-differentiation and activated stemness-related pathways. The single-cell transcriptomes of LUAD patient tumors recapitulate that the distinct LUAD NE cell cluster expressing NE genes is co-enriched with SOX2, OCT4, and NANOG pathway activation, and impaired actin remodeling. This study reveals an unexpected role of CRACD in restricting NE cell plasticity that induces cell de-differentiation, providing new insights into cell plasticity of LUAD.

CRACD loss promotes small cell lung cancer tumorigenesis via EZH2-mediated immune evasion.

The mechanisms underlying immune evasion and immunotherapy resistance in small cell lung cancer (SCLC) remain unclear. Herein, we investigate the role of CRACD tumor suppressor in SCLC. We found that CRACD is frequently inactivated in SCLC, and Cracd knockout (KO) significantly accelerates SCLC development driven by loss of Rb1, Trp53, and Rbl2. Notably, the Cracd-deficient SCLC tumors display CD8+ T cell depletion and suppression of antigen presentation pathway. Mechanistically, CRACD loss silences the MHC-I pathway through EZH2. EZH2 blockade is sufficient to restore the MHC-I pathway and inhibit CRACD loss-associated SCLC tumorigenesis. Unsupervised single-cell transcriptomic analysis identifies SCLC patient tumors with concomitant inactivation of CRACD, impairment of tumor antigen presentation, and downregulation of EZH2 target genes. Our findings define CRACD loss as a new molecular signature associated with immune evasion of SCLC cells and proposed EZH2 blockade as a viable option for CRACD-negative SCLC treatment.

Effect of chromatin modifiers on the plasticity and immunogenicity of small-cell lung cancer.

Tumor suppressor genes (TSGs) are often involved in maintaining homeostasis. Loss of tumor suppressor functions causes cellular plasticity that drives numerous types of cancer, including small-cell lung cancer (SCLC), an aggressive type of lung cancer. SCLC is largely driven by numerous loss-of-function mutations in TSGs, often in those encoding chromatin modifiers. These mutations present a therapeutic challenge because they are not directly actionable. Alternatively, understanding the resulting molecular changes may provide insight into tumor intervention strategies. We hypothesize that despite the heterogeneous genomic landscape in SCLC, the impacts of mutations in patient tumors are related to a few important pathways causing malignancy. Specifically, alterations in chromatin modifiers result in transcriptional dysregulation, driving mutant cells toward a highly plastic state that renders them immune evasive and highly metastatic. This review will highlight studies in which imbalance of chromatin modifiers with opposing functions led to loss of immune recognition markers, effectively masking tumor cells from the immune system. This review also discusses the role of chromatin modifiers in maintaining neuroendocrine characteristics and the role of aberrant transcriptional control in promoting epithelial-to-mesenchymal transition during tumor development and progression. While these pathways are thought to be disparate, we highlight that the pathways often share molecular drivers and mediators. Understanding the relationships among frequently altered chromatin modifiers will provide valuable insights into the molecular mechanisms of SCLC development and progression and therefore may reveal preventive and therapeutic vulnerabilities of SCLC and other cancers with similar mutations.

Opa1 and Drp1 reciprocally regulate cristae morphology, ETC function, and NAD+ regeneration in KRas-mutant lung adenocarcinoma.

Oncogenic KRas activates mitochondrial fission through Erk-mediated phosphorylation of the mitochondrial fission GTPase Drp1. Drp1 deletion inhibits tumorigenesis of KRas-driven pancreatic cancer, but the role of mitochondrial dynamics in other Ras-driven malignancies is poorly defined. Here we show that in vitro and in vivo growth of KRas-driven lung adenocarcinoma is unaffected by deletion of Drp1 but is inhibited by deletion of Opa1, the GTPase that regulates inner membrane fusion and proper cristae morphology. Mechanistically, Opa1 knockout disrupts cristae morphology and inhibits electron transport chain (ETC) assembly and activity, which inhibits tumor cell proliferation through loss of NAD+ regeneration. Simultaneous inactivation of Drp1 and Opa1 restores cristae morphology, ETC activity, and cell proliferation indicating that mitochondrial fission activity drives ETC dysfunction induced by Opa1 knockout. Our results support a model in which mitochondrial fission events disrupt cristae structure, and tumor cells with hyperactive fission activity require Opa1 activity to maintain ETC function.

Regulation of UHRF1 acetylation by TIP60 is important for colon cancer cell proliferation.

BACKGROUND: Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is upregulated in colon cancer cells and associated with silencing tumor suppressor genes (TSGs) to promote colon cancer cell proliferation. OBJECTIVE: To investigate epigenetic modification of UHRF1 by TIP60. Whether UHRF1 acetylation by TIP60 can induce cell proliferation in colon cancer cells. METHODS: Acetylation sites of UHRF1 by TIP60 was predicted by ASEB (Acetylation Set Enrichment Based) method and identified by immunoprecipitation assay using anti-pan-acetyl lysine antibody and in vitro acetylation assay. Based on this method, UHRF1 acetylation-deficient mimic 4KR (K644R, K646R, K648R, K650R) mutant was generated to investigate effects of UHRF1 acetylation by TIP60. shRNA system was used to generate stable knockdown cell line of UHRF1. With transient transfection of UHRF1 WT and 4KR, the effects of UHRF1 4KR mutant on Jun dimerization protein 2 (JDP2) gene expression, cell proliferation and cell cycle were investigated by RT-qPCR and FACS analysis in shUHRF1 colon cancer cell line. RESULTS: Downregulation of TIP60-mediated UHRF1 acetylation is correlated with suppressed cell cycle progression. Acetylation-deficient mimic of UHRF1 showed poor cell growth through increased expression of JDP2 gene. CONCLUSIONS: Acetylation of UHRF1 4K residues by TIP60 is important for colon cancer cell growth. Furthermore, upregulated JDP2 expression by acetylation-deficient mutant of UHRF1 might be an important epigenetic target for colon cancer cell proliferation.

KIX domain determines a selective tumor-promoting role for EP300 and its vulnerability in small cell lung cancer.

EP300, a transcription coactivator important in proliferation and differentiation, is frequently mutated in diverse cancer types, including small cell lung cancer (SCLC). While these mutations are thought to result in loss of EP300 function, the impact on tumorigenesis remains largely unknown. Here, we demonstrate that EP300 mutants lacking acetyltransferase domain accelerate tumor development in mouse models of SCLC. However, unexpectedly, complete Ep300 knockout suppresses SCLC development and proliferation. Dissection of EP300 domains identified kinase inducible domain-interacting (KIX) domain, specifically its interaction with transcription factors including MYB, as the determinant of protumorigenic activity. Ala627 in EP300 KIX results in a higher protein-binding affinity than Asp647 at the equivalent position in CREBBP KIX, underlying the selectivity of KIX-binding partners for EP300. Blockade of KIX-mediated interactions inhibits SCLC development in mice and cell growth. This study unravels domain-specific roles for EP300 in SCLC and unique vulnerability of the EP300 KIX domain for therapeutic intervention.

WNT5A-RHOA Signaling Is a Driver of Tumorigenesis and Represents a Therapeutically Actionable Vulnerability in Small Cell Lung Cancer.

WNT signaling represents an attractive target for cancer therapy due to its widespread oncogenic role. However, the molecular players involved in WNT signaling and the impact of their perturbation remain unknown for numerous recalcitrant cancers. Here, we characterize WNT pathway activity in small cell lung cancer (SCLC) and determine the functional role of WNT signaling using genetically engineered mouse models. ß-Catenin, a master mediator of canonical WNT signaling, was dispensable for SCLC development, and its transcriptional program was largely silenced during tumor development. Conversely, WNT5A, a ligand for ß-catenin-independent noncanonical WNT pathways, promoted neoplastic transformation and SCLC cell proliferation, whereas WNT5A deficiency inhibited SCLC development. Loss of p130 in SCLC cells induced expression of WNT5A, which selectively increased Rhoa transcription and activated RHOA protein to drive SCLC. Rhoa knockout suppressed SCLC development in vivo, and chemical perturbation of RHOA selectively inhibited SCLC cell proliferation. These findings suggest a novel requirement for the WNT5A-RHOA axis in SCLC, providing critical insights for the development of novel therapeutic strategies for this recalcitrant cancer. This study also sheds light on the heterogeneity of WNT signaling in cancer and the molecular determinants of its cell-type specificity. SIGNIFICANCE: The p130-WNT5A-RHOA pathway drives SCLC progression and is a potential target for the development of therapeutic interventions and biomarkers to improve patient treatment.

Fragmentation of Small-Cell Lung Cancer Regulatory States in Heterotypic Microenvironments.

Small-cell lung cancers derive from pulmonary neuroendocrine cells, which have stem-like properties to reprogram into other cell types upon lung injury. It is difficult to uncouple transcriptional plasticity of these transformed cells from genetic changes that evolve in primary tumors or secondary metastases. Profiling of single cells is also problematic if the required sample dissociation activates injury-like signaling and reprogramming. Here we defined cell-state heterogeneities in situ through laser capture microdissection-based 10-cell transcriptomics coupled with stochastic-profiling fluctuation analysis. In labeled cells from a small-cell lung cancer mouse model initiated by neuroendocrine deletion of Rb1-Trp53, variations in transcript abundance revealed cell-to-cell differences in regulatory state in vitro and in vivo. Fluctuating transcripts in spheroid culture were partly shared among Rb1-Trp53-null models, and heterogeneities increased considerably when cells were delivered intravenously to colonize the liver. Colonization of immunocompromised animals drove a fractional appearance of alveolar type II-like markers and poised cells for paracrine stimulation from immune cells and hepatocytes. Immunocompetency further exaggerated the fragmentation of tumor states in the liver, yielding mixed stromal signatures evident in bulk sequencing from autochthonous tumors and metastases. Dozens of transcript heterogeneities recurred irrespective of biological context; their mapped orthologs brought together observations of murine and human small-cell lung cancer. Candidate heterogeneities recurrent in the liver also stratified primary human tumors into discrete groups not readily explained by molecular subtype but with prognostic relevance. These data suggest that heterotypic interactions in the liver and lung are an accelerant for intratumor heterogeneity in small-cell lung cancer. SIGNIFICANCE: These findings demonstrate that the single-cell regulatory heterogeneity of small-cell lung cancer becomes increasingly elaborate in the liver, a common metastatic site for the disease.See related articles by Singh and colleagues, p. 1840 and Sutcliffe and colleagues, p. 1868.

SPDEF regulates goblet cell hyperplasia in the airway epithelium.

Goblet cell hyperplasia and mucous hypersecretion contribute to the pathogenesis of chronic pulmonary diseases including cystic fibrosis, asthma, and chronic obstructive pulmonary disease. In the present work, mouse SAM pointed domain-containing ETS transcription factor (SPDEF) mRNA and protein were detected in subsets of epithelial cells lining the trachea, bronchi, and tracheal glands. SPDEF interacted with the C-terminal domain of thyroid transcription factor 1, activating transcription of genes expressed selectively in airway epithelial cells, including Sftpa, Scgb1a1, Foxj1, and Sox17. Expression of Spdef in the respiratory epithelium of adult transgenic mice caused goblet cell hyperplasia, inducing both acidic and neutral mucins in vivo, and stainined for both acidic and neutral mucins in vivo. SPDEF expression was increased at sites of goblet cell hyperplasia caused by IL-13 and dust mite allergen in a process that was dependent upon STAT-6. SPDEF was induced following intratracheal allergen exposure and after Th2 cytokine stimulation and was sufficient to cause goblet cell differentiation of Clara cells in vivo.

FGFR1 Is Critical for RBL2 Loss-Driven Tumor Development and Requires PLCG1 Activation for Continued Growth of Small Cell Lung Cancer.

Small cell lung cancer (SCLC) remains a recalcitrant disease where limited therapeutic options have not improved overall survival, and approved targeted therapies are lacking. Amplification of the tyrosine kinase receptor FGFR1 (fibroblast growth factor receptor 1) is one of the few actionable alterations found in the SCLC genome. However, efforts to develop targeted therapies for FGFR1-amplified SCLC are hindered by critical gaps in knowledge around the molecular origins and mediators of FGFR1-driven signaling as well as the physiologic impact of targeting FGFR1. Here we show that increased FGFR1 promotes tumorigenic progression in precancerous neuroendocrine cells and is required for SCLC development in vivo. Notably, Fgfr1 knockout suppressed tumor development in a mouse model lacking the retinoblastoma-like protein 2 (Rbl2) tumor suppressor gene but did not affect a model with wild-type Rbl2. In support of a functional interaction between these two genes, loss of RBL2 induced FGFR1 expression and restoration of RBL2 repressed it, suggesting a novel role for RBL2 as a regulator of FGFR1 in SCLC. Additionally, FGFR1 activated phospholipase C gamma 1 (PLCG1), whereas chemical inhibition of PLCG1 suppressed SCLC growth, implicating PLCG1 as an effector of FGFR1 signaling in SCLC. Collectively, this study uncovers mechanisms underlying FGFR1-driven SCLC that involve RBL2 upstream and PLCG1 downstream, thus providing potential biomarkers for anti-FGFR1 therapy. SIGNIFICANCE: This study identifies RBL2 and PLCG1 as critical components of amplified FGFR1 signaling in SCLC, thus representing potential targets for biomarker analysis and therapeutic development in this disease.