LSD1 inhibitor, TAS1440, disrupts INSM1-LSD1 complex activating tumor-suppressive pathways via transcriptional reprogramming in neuroendocrine SCLC

Domain: biology
Published: 2026-03-25
Editorial outcome: minor_revision

Abstract
Small cell lung cancer (SCLC) is aggressive with limited treatment options, requiring new therapies. Lysine-specific histone demethylase 1A (LSD1) maintains neuroendocrine state by repressing NOTCH/TGF-β signaling; their reactivation suppresses proliferation and induces differentiation. However, mechanisms of LSD1 inhibition and chemoresistance remain unclear. Here we developed TAS1440, a histone H3-competitive LSD1 inhibitor, using structure-based engineering to improve specificity and reduce off-target effects. Unlike irreversible inhibitors targeting the flavin adenine dinucleotide site, TAS1440 non-covalently targets the H3-binding pocket to enhance safety and efficacy. TAS1440 suppressed proliferation in INSM1/ASCL1-high SCLC-A cells and induced tumor regression in xenografts. TAS1440 acts through dual mechanisms: inhibiting LSD1 activity and disrupting LSD1-repressive complexes, remodeling histone marks and activating transcription factors INSM1 and SMAD2. These actions reprogram tumor-suppressive TGF-β/NOTCH signaling, supporting TAS1440 as epigenetic therapy for SCLC. Loss of LSD1 enzymatic activity or INSM1 knockout abrogated TAS1440 effects, defining its mode of action and chemoresistance. These findings support TAS1440 as a next-generation epigenetic therapy candidate for INSM1-high SCLC-A.

Introduction
Small-cell lung cancer (SCLC) is an aggressive, high-grade neuroendocrine carcinoma with a poor prognosis. It characteristically expresses neuroendocrine markers, including achaete-scute homolog 1 (ASCL1), neurogenic differentiation 1 (NEUROD1), and insulinoma-associated protein 1 (INSM1)1. First-line platinum-based chemotherapy is initially effective; however, relapse is common, and the 5‑year survival rate remains <7%2, underscoring the need for new therapeutic strategies. Contemporary transcriptomic frameworks delineate SCLC into ASCL1‑ (SCLC‑A), NEUROD1‑ (SCLC‑N), and POU2F3‑driven (SCLC‑P) states, with an inflamed SCLC‑I state being increasingly recognized3,4. Among tumor-suppressive circuits silenced in SCLC, NOTCH signaling—central to cell-fate decisions—functions as a tumor suppressor: its loss sustains the neuroendocrine phenotype and promotes lineage plasticity and progression5, whereas NOTCH reactivation in preclinical models suppresses proliferation, induces cell death, and drives transdifferentiation toward non-neuroendocrine states6. Therefore, NOTCH repression constitutes a fundamental vulnerability, particularly in neuroendocrine-high SCLC-A.

As a strategy to restore NOTCH signaling, lysine-specific demethylase 1A (LSD1/KDM1A)—a flavin-dependent amine oxidase and epigenetic regulator—has emerged as a promising therapeutic target in SCLC. LSD1 represses NOTCH‑associated genes predominantly by demethylating histone H3 lysine 4 (H3K4me1/2)7, thereby shaping chromatin states and transcriptional output. LSD1 requires flavin adenine dinucleotide (FAD) for its enzymatic activity and supports transcriptional repression via H3K4 demethylation8. Notably, LSD1 functions within multi‑protein corepressor assemblies, including the REST corepressor (CoREST)9 and the nucleosome remodeling and deacetylase (NuRD) complex10.

Elevated LSD1 expression is observed across multiple cancers, including SCLC, where its activity is associated with proliferation, differentiation, and epithelial–mesenchymal transition11. Pharmacological inhibition of LSD1 activates NOTCH signaling and downregulates neuroendocrine-associated genes such as ASCL1, suppressing SCLC cell proliferation in vitro and demonstrating antitumor efficacy in mouse xenograft models12. Nonetheless, the detailed mechanisms by which LSD1 inhibitors act in SCLC, and the determinants of therapeutic resistance, remain incompletely defined—motivating further development of next‑generation inhibitors and mechanistic dissection.

LSD1 inhibitors are broadly classified as covalent FAD‑directed agents and non‑covalent chemotypes that compete at the histone H3‑binding pocket13. Covalent inhibitors such as ORY-1001 and GSK2879552 irreversibly modify the FAD cofactor to block catalysis14; however, irreversible binding raises concerns about off‑target effects, limited adaptability for rapidly turning‑over enzymes, and potential immunogenicity4,13,15. In contrast, non-covalent inhibitors such as CC-9001116 compete with histone H3 at its binding site, enabling improved selectivity, tunable pharmacokinetics, and potentially wider therapeutic windows; importantly, they can also perturb protein–protein interactions within LSD1 complexes, suggesting mechanistic distinctions from covalent agents that extend beyond enzyme inhibition13,17.

Here, we report TAS1440, a potent histone H3–competitive non-covalent LSD1 inhibitor, which showed a favorable selectivity profile in our preclinical assays. TAS1440 exhibited preferential activity in SCLC‑A models. Mechanistically, our data support a dual mode of action: (i) direct inhibition of LSD1 catalytic activity, remodeling the chromatin landscape via altered H3 methylation, and (ii) disruption of LSD1‑containing repressive complexes (e.g., CoREST and NuRD), relieving INSM1‑tethered repression and enhancing SMAD2‑dependent signaling. Together, these effects reprogram transcriptional networks in tumor‑suppressive pathways, including TGF‑β and NOTCH, supporting TAS1440 as a potential therapeutic candidate for SCLC—particularly the SCLC‑A subtype characterized by high INSM1 expression.

Results
Molecular Subtype-Driven Therapeutic Vulnerabilities in SCLC: TAS1440 as a Selective LSD1 Inhibitor for SCLC-A

SCLC is a rapidly growing tumor with poor prognosis and limited treatment options. SCLC comprises transcriptionally defined states with distinct vulnerabilities. We profiled 22 SCLC cell lines by bulk RNA sequencing (RNA‑seq) to stratify models by subtype signatures and lineage‑defining transcription factors (Fig. 1a). Consistent with previous reports1, our cluster analysis classified the cell lines into four molecular states annotated by ASCL1 (SCLC‑A), NEUROD1 (SCLC‑N), POU2F3 (SCLC‑P), and a YAP1‑high group (Fig. 1b–d). As expected, SCLC‑A lines showed higher expression of neuroendocrine markers (e.g., INSM1, DLL3), as well as genes enriched in SCLC‑A (e.g., SYK, ETS2, CRACDL); moreover, ASCL1 expression was the highest in SCLC‑A (Fig. 1e). These findings highlight the molecular heterogeneity of SCLC and the distinct transcriptional characteristics that may underlie differential responsiveness to LSD1‑regulated programs.

Given reports that YAP1‑high lines are frequently misclassified (e.g., SMARCA4‑deficient undifferentiated tumors or sarcomatoid neoplasms3,4) and are not considered a consensus SCLC subtype, we examined our dataset and confirmed markedly reduced SMARCA4 expression in these lines (Fig. 1e). To avoid confounding in downstream analyses, we excluded these models and re‑analyzed the remaining 19 SCLC lines, which retained the SCLC‑A/N/P assignments (Supplementary Fig. 1a–d). This 19‑line framework provided a robust platform for evaluating LSD1‑regulated transcriptional programs and therapeutic vulnerabilities.

To evaluate the effects of LSD1 inhibitors across SCLC subtypes, we profiled TAS1440 and two covalent inhibitors (ORY-1001 and GSK2879552) in 19 SCLC cell lines using single‑concentration challenge at 7,500 nM to assess drug resistance, with cell growth expressed as % of DMSO control (Fig. 2a, b). Across subtypes, TAS1440 showed the strongest growth suppression in SCLC‑A (Fig. 2a, b), highlighting it as a promising agent for neuroendocrine‑high disease.

Within SCLC‑A, NCI‑H1417, NCI‑H510A, NCI‑H146, and COR‑L51 were particularly sensitive to TAS1440 and displayed steep dose–response curves relative to other SCLC-A lines or subtypes (Fig. 2c and Supplementary Fig. 2a). Consistent with these responses, these lines yielded the lowest TAS1440 IC₅₀ values in the panel, whereas the covalent comparators frequently showed IC₅₀ ≥ 1,000 nM and were often right‑censored at the upper testing limit (7,500 nM) (Fig. 2d and Supplementary Fig. 2b). To benchmark TAS1440 against reversible, non‑covalent inhibitors, we compared it with SP250918 and CC‑9001116 across the same panel (Supplementary Fig. 3). TAS1440 and CC‑90011 (both H3‑competitive) showed broadly similar inhibition patterns, with TAS1440 exhibiting greater potency in the most sensitive lines; in contrast, SP2509 produced broad cytotoxicity at higher concentrations, consistent with reported off‑target liabilities (Supplementary Fig. 3).

TAS1440 is a small-molecule LSD1 inhibitor with a [1,1':2',1''-terphenyl]-4'-carboxamide scaffold (Fig. 2e) and a defined synthetic route (Supplementary Fig. 4a). Unlike tranylcypromine (TCP) analogs such as ORY‑1001 and GSK2879552, which covalently modify the FAD cofactor, TAS1440 functions as a histone H3‑competitive inhibitor. In biochemical assays, TAS1440 showed marked selectivity for LSD1 (IC₅₀ = 4.8 nM) over related amine oxidases (LSD2, MAO‑A, MAO‑B; all IC₅₀ > 10,000 nM; Fig. 2f) and a broader panel of epigenetic enzymes (e.g., JARID1A, DNMT1, EZH1/EED/SUZ12, HDAC1, JMJD2B/3, PRMT4; all IC₅₀ > 10,000 nM; Supplementary Fig. 4b), indicating a favorable selectivity profile.

Consistent with classical competitive inhibition at the histone‑substrate site, the apparent IC₅₀ of TAS1440 for LSD1 increased as the H3K4me1 peptide concentration increased (e.g., 100 nM vs. 2,000 nM substrate), whereas ORY‑1001 remained essentially substrate‑insensitive (Fig. 2g). These biochemical findings are consistent with structural data presented later, where TAS1440 engages the H3‑binding pocket of LSD1.

TAS1440 Modulates Global Gene Expression and Activates Tumor-Suppressive Pathways in SCLC

The potent inhibitory effects of TAS1440 in SCLC‑A are likely dependent on LSD1‑regulated neuroendocrine programs driven by lineage‑defining factors such as ASCL1 and INSM1. To characterize TAS1440-induced transcriptional changes, we performed bulk RNA-seq across 19 SCLC cell lines, comparing TAS1440-sensitive and -insensitive groups (Fig. 3a). In the TAS1440-sensitive NCI-H1417 cell line, 1017 genes were upregulated, whereas ≤500 genes were upregulated in insensitive lines (Fig. 3a, b). At baseline, KDM1A/LSD1 expression was considerably higher in the insensitive group than in the sensitive group across all SCLC lines and within SCLC‑A (Fig. 3c), suggesting that elevated LSD1 abundance may diminish effective target engagement given that TAS1440 competes at the histone H3‑binding pocket.

To link these transcriptional changes with growth-inhibitory effects, we correlated gene‑wise log (fold‑change) (TAS1440 vs. DMSO) with cell growth (% of DMSO) across the 19 lines (Pearson correlation coefficient |R| ≥ 0.5, P < 0.05). This analysis identified 207 genes with strong correlations (|R| ≥ 0.5; P < 0.05), with the top 100 genes averaging a correlation coefficient of more than 0.5 (Fig. 3e and Supplementary Fig. 5a–c). TGF‑β‑related genes—including SMAD6, BMP5, FBN1, and TGFB2—were positively correlated with TAS1440 efficacy (i.e., more up‑regulated with stronger growth inhibition), whereas neuroendocrine/ASCL1‑axis genes such as DLL3, INSM1, and ASCL1 showed negative correlations (i.e., more down‑regulated with stronger growth inhibition) (Fig. 3f). Consistently, pathway‑level gene‑set enrichment analysis (GSEA) demonstrated higher normalized enrichment scores for NOTCH signaling in TAS1440‑sensitive lines—both across all SCLC models and within the SCLC‑A subset—further linking NOTCH activation to drug response (Fig. 3d). Collectively, these findings demonstrate that TAS1440 induces a coordinated reprogramming of gene expression, characterized by activation of NOTCH/TGF‑β pathways and suppression of the neuroendocrine program, which collectively underlie its antiproliferative activity.

TGF-β and NOTCH Pathway Activation by TAS1440 Mediates Antitumor Effects in SCLC-A

To define the temporal dynamics of TAS1440‑induced transcriptional rewiring, we performed RNA‑seq in three TAS1440-sensitive SCLC‑A lines, using full time courses in NCI‑H1417 and NCI‑H146 (days 0, 1, 2, 3, 5, and 7) and a baseline/endpoint design in NCI‑H510A (days 0 and 7). Global expression changes were modest on days 1–3 and became most pronounced by days 5–7 (Fig. 4a and Supplementary Fig. 6a). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and GSEA consistently showed activation of tumor‑suppressive programs—most notably TGF‑β and NOTCH—with peak enrichment at later time points (Fig. 4b, f; Supplementary Fig. 6b, f; and Supplementary Fig. 7b). Concordantly, TAS1440 upregulated key pathway components and targets (e.g., NOTCH1/3, JAG1, TGFB2, SMAD3/6, HES1) (Fig. 4a and Supplementary Fig. 6a and Supplementary Fig. 7a). Pathways associated with neuroendocrine phenotypes1, such as those observed in pancreatic and prostate cancer, were enriched among genes upregulated by TAS1440 treatment (Fig. 4b and Supplementary Fig. 6b).

At the protein level, TAS1440 rapidly induced SMAD2 phosphorylation, peaking around 30–45 min in NCI‑H1417 and NCI‑H146 (Fig. 4c; Supplementary Fig. 6c). Additionally, NOTCH1 expression increased more slowly, peaking at 3 days post-treatment in both models (Fig. 4c and Supplementary Fig. 6c).

Immunocytochemistry confirmed increased nuclear p‑SMAD2 across all three models (Fig. 4d, Supplementary Fig. 6d, and Supplementary Fig. 7c) and enhanced nuclear accumulation of the NOTCH intracellular domain (NICD) (Fig. 4e, Supplementary Fig. 6e, and Supplementary Fig. 7d). These findings indicate TAS1440’s ability to modulate global gene expression and activate tumor-suppressive pathways, particularly through engagement of TGF-β and NOTCH signaling19,20, underscoring its therapeutic potential in SCLC-A.

To test pathway dependence, we perturbed each axis pharmacologically and genetically. The NOTCH inhibitor LY411575, the TGF-β inhibitor SB431542, and SMAD2 siRNA (siSMAD2) partially reversed TAS1440‑mediated growth inhibition, whereas dual inhibition nearly abrogated the antiproliferative effect (Fig. 4g, Supplementary Fig. 6g, and Supplementary Fig. 7e). LY411575 attenuated TAS1440‑induced NOTCH1/HES1 upregulation, and SMAD2 knockdown blunted HES1, TGFB2, and TGFBR2 induction across three cell lines (Fig. 4h, Supplementary Fig. 6h, and Supplementary Fig. 7f). To corroborate these pharmacologic findings at the transcriptome level, we performed RNA-seq analysis on SMAD2-knockdown NCI-H1417 and NCI-H146 cells following TAS1440 treatment (Supplementary Fig. 8). SMAD2 depletion was verified by reduced SMAD2 FPKM in both models (Supplementary Fig. 8a). Relative to siNC, siSMAD2 cells displayed substantially fewer TAS1440‑responsive differentially expressed genes (DEGs), and a blunted global signature by heatmap (Supplementary Fig. 8b–d). Importantly, KEGG TGF‑β signaling was no longer strongly enriched under siSMAD2, with dampened induction of canonical components (e.g., TGFB2, TGFBR2, SMAD6/7, ID1/3) (Supplementary Fig. 8e, f), indicating that SMAD2 is required for the full TGF‑β program elicited by TAS1440.

Finally, comparison with four LSD1 inhibitors (ORY‑1001, GSK2879552, CC‑90011, and SP2509) showed that TAS1440 induced more pronounced genome‑wide expression changes (Fig. 4i and Supplementary Fig. 6i) and stronger activation of p‑SMAD2 and NOTCH1/NICD than comparators at the tested concentrations and time points (Fig. 4j, k; Supplementary Fig. 6j; and Supplementary Fig. 7g). While such cross‑compound comparisons in cell‑based systems are limited by differences in antiproliferative potency (IC₅₀) and potential variations in mechanism of action, these findings highlight TAS1440’s distinctive capacity—as an H3‑competitive LSD1 inhibitor—to activate TGF‑β/NOTCH programs and suppress growth in SCLC‑A.

TAS1440 Attenuates Neuroendocrine Identity and Modulates Immune-Related and Surface Antigen Pathways

Given that LSD1 blockade can restore MHC‑I antigen presentation and enhance intrinsic immunogenicity, with NOTCH‑linked attenuation of neuroendocrine features in SCLC21, we evaluated whether TAS1440 modulates lineage identity and immune/surface‑antigen programs across SCLC models. TAS1440 consistently suppressed ASCL1 and the neuroendocrine (NE) marker INSM1 in SCLC‑A lines (ASCL1: Supplementary Fig. 9a; INSM1: Fig. 3f), whereas NEUROD1, YAP1, and POU2F3 remained largely unchanged (Supplementary Fig. 9a). Sample–sample correlation matrices and lineage‑marker profiles did not support transitions to SCLC‑N, ‑P, or ‑Y (Supplementary Fig. 9b, c), indicating a loss of NE identity without subtype switching.

We next assessed the immunomodulatory effects of TAS1440. Across 19 lines, TAS1440 induced selective immunogenic rewiring: transcripts encoding MHC‑I/antigen‑processing components (B2M, TAP1/2, HLA‑A/B/C) and IFN‑γ–response genes (IRF1, STAT1, CXCL9/10) were upregulated, while CD274/PD‑L1 decreased (Supplementary Fig. 9d). GSEA revealed enrichment of NOTCH, JAK–STAT, and innate inflammatory pathways in a subset of lines (Supplementary Fig. 9f); however, these pathway changes did not consistently correlate with growth inhibition (the cell‑growth vs. DMSO (%) indicator in Supplementary Fig. 9d, f), consistent with context-dependent pharmacodynamics in cell line systems.

Regarding clinically actionable antigens, TAS1440 downregulated DLL3—the target of the T‑cell engager tarlatamab—while leaving SEZ6, CD276 (B7‑H3), and TACSTD2 (TROP2) largely unchanged (Supplementary Fig. 9e). In ASCL1‑expressing SCLC‑A lines, TAS1440‑induced NOTCH pathway enrichment negatively correlated with ASCL1 and DLL3 expression changes, indicating NOTCH‑linked repression of the ASCL1–DLL3 axis (Supplementary Fig. 9g). These transcript‑level findings suggest caution with concurrent DLL3‑directed approaches (e.g., tarlatamab) but support antibody-drug conjugate (ADC) strategies targeting SEZ6, B7‑H3, or TROP2 during TAS1440 exposure. Future confirmation will require cell‑surface protein quantification (e.g., flow cytometry/proteomics) and validation in orthotopic/PDX models and, where feasible, clinical cohorts.

Dual Mechanisms of TAS1440 in SCLC‑A: Inhibition of LSD1 Catalysis and Disruption of INSM1–LSD1 and SMAD2–LSD1 Complexes

To elucidate the multifaceted roles of the LSD1 complex in SCLC and the basis for differential responses to LSD1 inhibitors, we profiled LSD1-interacting proteins with or without inhibitors. NCI‑H1417 cells were transfected with a 3×FLAG–Myc‑tagged human LSD1 vector or an empty vector; after 46 h, cells were treated for 2 h with 0.1% DMSO or 300 nM LSD1 inhibitors (TAS1440, ORY‑1001, GSK2879552) and subjected to FLAG immunoprecipitation followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) (Fig. 5a). In untreated cells, we identified 422 nuclear and 67 epigenetic factors bound to LSD1 (Fig. 5b). Enrichment analysis highlighted HDAC-containing corepressor complexes (e.g., CoREST-HDAC, NuRD, HCF-1, ALL-1 supercomplex) among LSD1 interactors (Supplementary Fig. 10a). Following TAS1440 treatment, 498 nuclear and 77 epigenetic factors were recovered (Fig. 5b, right). Similarly, 455/70 and 442/66 interactors were identified following ORY-1001 and GSK2879552 treatment, respectively (Supplementary Fig. 10b).

Quantitative interactome analysis revealed that TAS1440 caused dissociation of 78 nuclear factors (13 epigenetic) from LSD1 (Fig. 5c, red), while 344 (54) remained stable (Fig. 5c). Dissociated proteins included transcription factors (e.g., INSM1, SOX2, SMAD2) and chromatin modifiers (e.g., CHD4, SMARCA2) (Fig. 5c, d). RCOR1 binding was largely unchanged, whereas HDAC1 (and to a lesser extent, HDAC2) binding decreased; this result is consistent with immunoblot validation (Fig. 5e, f). ORY-1001 and GSK2879552 induced dissociation of 90 (12) and 87 (18) nuclear factors, respectively (Fig. 5d, Supplementary Fig. 10c–e). Across inhibitors, 35 proteins were commonly dissociated, whereas 22, including INSM1 and SMAD2, were TAS1440-specific (Supplementary Fig. 10f).

Co-immunoprecipitation and immunoblotting confirmed robust TAS1440-mediated INSM1 dissociation (mean ± SD; P < 0.01), exceeding other inhibitors (Fig. 5e, f). Dose–response analyses showed progressive loss of the INSM1–LSD1 interaction with increasing TAS1440 concentrations (Supplementary Fig. 10g, h), and at ≥300 nM, TAS1440 caused stronger INSM1 dissociation than other compounds (Supplementary Fig. 10i, j). TAS1440 also more strongly dissociated SMAD2 than comparators, consistent with downstream SMAD2 activation (Fig. 5d–f, Fig. 4j, Supplementary Fig. 6j, and Supplementary Fig. 7g).

To explore structural underpinnings, we performed molecular docking simulation using the LSD1–FAD complex (PDB: 2HKO) (Fig. 5g). Across replicate runs and scoring functions, TAS1440 consistently achieved more favorable docking scores (−10.1 kcal/mol) than comparators and adopted a pose along the histone H3–binding channel proximal to FAD, positioning its core within the substrate tunnel. To relate the small‑molecule pocket to repressor engagement, we applied AlphaFold3 modeling to map partner‑binding interfaces on LSD1 (Fig. 5h). In these models, the TAS1440‑defined pocket lies adjacent to regions predicted to engage INSM1 and SMAD2, providing a structural rationale for the observed dissociation of these factors from LSD1 upon TAS1440 treatment. While these predictions are computational, they converge with our biochemical and proteomic readouts.

Finally, X-ray co-crystal analysis shows occupancy of the H3-binding site in vitro (Fig. 5i), consistent with the substrate‑competition observed in Fig. 2g. Furthermore, a comparison with the previously reported LSD1–CC-90011 structure (PDB: 6W4K)16, another non-covalent H3-competitive inhibitor, demonstrates that TAS1440 and CC-90011 occupy nearly identical regions within this substrate-binding pocket, supporting a common mechanism. Superposition with LSD1–GSK2879552 (PDB: 6NQU) and an LSD1–INSM1 peptide complex (PDB: 3ZMS) shows that TAS1440 overlaps the H3 pocket and sterically intersects the INSM1 footprint, providing a direct steric basis for destabilizing the LSD1–INSM1 interaction. By contrast, GSK2879552 forms a covalent adduct with FAD at a shifted position that minimally overlaps the INSM1 site, consistent with its distinct FAD‑directed covalent mode. Collectively, these structural data support a dual-mechanism for TAS1440—inhibition of LSD1 demethylase activity and disruption of the INSM1–LSD1 complex—and, together with our modeling (Fig. 5h), are consistent with potential interference at SMAD2‑interacting surfaces.

Coordinated Mechanisms of TAS1440 via INSM1-LSD1 Complex-Mediated Transcription and Epigenetic Networks

Focusing on the histone demethylase function and epigenetic regulation of LSD1, we performed ChIP‑seq analysis of histone modifications in NCI‑H1417 and NCI‑H146 cells to identify transcription factors (TFs) involved in TAS1440‑mediated epigenetic reprogramming. TAS1440 markedly increased genome‑wide metaprofiles of H3K4me1, H3K4me2, and H3K27ac compared to the DMSO control (Fig. 6a and Supplementary Fig. 11h). In contrast, H3K4me3 levels remained unchanged or were slightly reduced, likely reflecting LSD1’s substrate specificity and preference for H3K4me1 and H3K4me2 over H3K4me3, as previously reported22.

Similar effects on histone modifications were observed with ORY‑1001 and GSK2879552. However, TAS1440 produced markedly larger increases in H3K4me1 and H3K4me2 meta‑signals (Fig. 6a and Supplementary Fig. 11h). By contrast, ORY‑1001 and GSK2879552 caused a more substantial reduction in H3K4me3 levels than TAS1440; the basis for this difference remains to be clarified. Overall, these data indicate global epigenetic reprogramming consistent with LSD1 catalytic inhibition and emphasize TAS1440’s capacity to modulate histone methylation, acetylation, and downstream transcription in SCLC‑A.

Motif analysis of regions with increased H3K4me2 identified 247 enriched motifs in NCI‑H1417 and 226 enriched motifs in NCI‑H146 (Fig. 6b and Supplementary Fig. 11i). Analogous analyses for H3K4me1 and H3K27ac yielded overlapping motif sets in both cell lines. Notably, four motifs consistently enriched across all three sets corresponded to the transcription factors INSM1, SMAD2, SP4, and YY1 in NCI‑H1417 and three motifs—INSM1, SMAD2, and SP4—in NCI‑H146 (Fig. 6b, c and Supplementary Fig. 11i, j). These transcription factors dissociated from the LSD1 complex upon TAS1440 treatment, indicating their pivotal role at the intersection of LSD1 demethylase‑dependent epigenetic regulation and transcription factor activation following LSD1 repressor-complex disruption. Accordingly, we conducted further analyses to elucidate the roles of these transcription factors and their interactions with downstream signaling pathways in the context of antitumor responses.

Network mapping revealed candidate LSD1–TFs complex binding sites near the transcription start sites (TSSs) of genes involved in the NOTCH and TGF‑β pathways (Fig. 6d and Supplementary Fig. 11k). Specifically, potential LSD1–TFs complex binding sites were detected for these target genes in NCI‑H1417 cells: 33 sites for INSM1, 26 for SMAD2, 35 for SP4, and 9 for YY1. For example, we identified several potential INSM1 binding sites near the TSSs of NOTCH1 and NOTCH3, and these sites became more accessible, as indicated by increased H3K4me2 and, at selected loci, H3K4me1 and H3K27ac after LSD1 inhibitor treatment (Fig. 6e, f and Supplementary Fig. 11a, b). Similarly, potential INSM1 binding sites were identified near the TSS of the TGFB2 gene, which also transitioned to an epigenetically active state after treatment with LSD1 inhibitors (Supplementary Fig. 11d, e). Similar results were observed in NCI‑H146 cells, confirming that these effects are not cell line‑specific (Supplementary Fig. 11l–o).

These results were further supported by INSM1‑ and SMAD2‑ChIP assays. INSM1‑ChIP showed that TAS1440 increased INSM1 binding near the TSSs of NOTCH1 (TSS1, −458 bp; TSS2, +482 bp), NOTCH3 (TSS2, +588 bp), and TGFB2 (+470 bp) and, compared with other LSD1 inhibitors, produced stronger enrichment at these promoter‑proximal sites except at NOTCH3 TSS1 (−533 bp) (Fig. 6g and Supplementary Fig. 11c, f). SMAD2‑ChIP likewise confirmed increased SMAD2 occupancy near the NOTCH1 (TSS1, −458 bp; TSS2, +482 bp) and TGFB2 (+470 bp) promoters, again exceeding the enrichment observed with other LSD1 inhibitors (Fig. 6h and Supplementary Fig. 11g). These findings support a model in which TAS1440 coordinates activation of NOTCH and TGF‑β signaling by combining catalytic inhibition of LSD1 with disruption of INSM1–LSD1 and SMAD2–LSD1 corepressor complexes.

Collectively, these findings suggest the potential of TAS1440 as a therapeutic strategy for SCLC‑A by targeting INSM1–LSD1 complex‑mediated transcriptional and epigenetic networks; by inducing global epigenetic reprogramming together with de‑repression of key TFs, TAS1440 preferentially activates tumor‑suppressive pathways, including NOTCH and TGF‑β.

LSD1 Catalytic Activity is Required for TAS1440-Mediated Effects

LSD1 promotes oncogenesis through its demethylation activity and non‑enzymatic scaffolding functions. To dissect the contribution of enzymatic activity to TAS1440’s mechanism, we generated a catalytically impaired LSD1 mutant (LSD1-K661A) that hinders demethylase function9. Using CRISPR–Cas9, we first knocked out endogenous LSD1 (LSD1‑KO) in NCI‑H1417 and NCI‑H146 cells. These LSD1‑KO cells were then reconstituted with 3×FLAG–Myc–LSD1‑WT, LSD1-K661A, or empty vector (Fig. 7a, b, d and Supplementary Fig. 12a, b, d). Immunoblotting confirmed loss of H3K4me1/2 demethylase activity in LSD1-K661A (Fig. 7d and Supplementary Fig. 12d; compare lane 4 vs. lane 5).

Comparison of the dose–response curves showed that TAS1440 suppresses growth within the tested concentration range (100–3,000 nM) in parental cells (WT) with endogenous LSD1 (red), whereas LSD1‑KO cells (black) remained largely refractory, indicating that TAS1440’s antiproliferative activity is LSD1‑dependent (Fig. 7c and Supplementary Fig. 12c). To further assess target selectivity, we tested SP2509, a known LSD1 inhibitor with reported off‑target activity23. Unlike TAS1440, SP2509 retained partial growth inhibition in LSD1‑KO cells (green), indicating both LSD1‑dependent and ‑independent actions (Fig. 7c and Supplementary Fig. 12c).

Functionally, both LSD1‑WT and LSD1-K661A considerably rescued proliferation in LSD1‑KO cells under vehicle (DMSO) (Fig. 7e, f and Supplementary Fig. 12e, f), although LSD1-K661A was less effective. TAS1440 inhibited cell growth in both reconstituted lines, suggesting that enzymatic inhibition contributes to, but does not fully account for, growth effects. At the pathway level, disruption of the LSD1 corepressor complex by KO produced a mild basal induction of NOTCH1 and pSMAD2 (Fig. 7g, lane 2). This increase was completely reversed by LSD1‑WT (lane 3) and only partially reversed by LSD1‑K661A (lane 4). Upon comparing TAS1440 responses across genotypes, TAS1440 induced NOTCH1 and pSMAD2 in both reconstituted lines (Fig. 7g); however, relative to the parental cells (gray), responsiveness (fold change; TAS1440 vs DMSO) was abolished in LSD1‑KO (Empty; yellow), fully restored by LSD1‑WT (blue), and not restored by LSD1‑K661A (magenta) (Fig. 7h). Consistently, RT-qPCR analysis confirmed reduced activation of NOTCH and TGF‑β programs in K661A (Fig. 7i).

RNA‑seq showed that TAS1440 had little to no impact on DEGs in LSD1‑KO (Empty) cells, consistent with on‑target specificity (Fig. 7k, l). By contrast, DEG counts were markedly lower in LSD1‑K661A than in LSD1‑WT (Fig. 7j, k), and LSD1‑WT exhibited the broadest transcriptional response (Fig. 7k, l). KEGG/GSEA showed NOTCH and TGF‑β pathway enrichment predominantly in LSD1‑WT, attenuated in LSD1-K661A (Fig. 7m). Finally, co‑immunoprecipitation showed that TAS1440 disrupts INSM1/SMAD2–LSD1 complexes regardless of catalytic status (Fig. 7n), supporting a dual mechanism of TAS1440—direct inhibition of LSD1 demethylase activity plus co‑repressor complex disassembly.

INSM1 Depletion Abrogates TAS1440-Mediated Transcriptional Regulation in SCLC-A and Highlights Its Role in Drug Resistance

To evaluate the requirement of INSM1 for the antitumor effects of TAS1440, we generated INSM1-knockout SCLC-A cell lines using the CRISPR/Cas9 system (Fig. 8a). NCI-H1417 and NCI-H146 cells were electroporated with Cas9/sgRNA complexes targeting INSM1, followed by FACS sorting and validation of INSM1 depletion at days 0 and 10. Immunoblotting confirmed loss of INSM1 protein expression (Fig. 8b and Supplementary Fig. 13a), and RT-qPCR corroborated mRNA depletion (Fig. 8c).

We then assessed the impact of INSM1 loss on TAS1440 sensitivity using real-time live-cell imaging (IncuCyte® SX5). TAS1440 robustly inhibited growth in wild‑type (INSM1‑proficient) cells, whereas this effect was strongly attenuated in INSM1‑knockout cells (Fig. 8d and Supplementary Fig. 13b), indicating that INSM1 contributes to the growth‑suppressive effects of TAS1440 in SCLC‑A.

To elucidate the molecular basis of this drug resistance, we profiled TAS1440‑induced gene expression. In wild-type cells, TAS1440 upregulated tumor-suppressive genes in the NOTCH/TGF-β targets (NOTCH1, HES1, TGFBR2, TGFB2, and ID3), whereas these responses were markedly blunted in INSM1-deficient cells (Fig. 8e and Supplementary Fig. 13c). Furthermore, TAS1440-dependent suppression of ASCL1 expression, observed in wild-type cells, was also impaired in INSM1-knockout NCI-H1417 cells, suggesting that INSM1 is required for TAS1440-induced repression of neuroendocrine identity. Overall, TAS1440-induced gene expression changes in these pathways were markedly attenuated in INSM1-knockout cells compared to wild-type cells (Fig. 8e and Supplementary Fig. 13c).

RNA-seq identified 1,588 DEGs in TAS1440‑treated wild‑type versus 234 DEGs in INSM1‑knockout cells (Fig. 8f, g, Supplementary Fig. 14a), with NOTCH/TGF‑β enrichment primarily in wild‑type (Fig. 8h); NCI‑H146 showed similar patterns (799 vs. 161 DEGs; Supplementary Fig. 13d–f, Supplementary Fig. 15a). Nevertheless, pathway analysis revealed enrichment of TGF-β and NOTCH signaling upon TAS1440 treatment in both WT and INSM1-knockout cells across NCI-H1417 and NCI-H146 cell lines (Supplementary Fig. 14b and Supplementary Fig. 15b). Interestingly, even in the absence of TAS1440, INSM1-knockout cells displayed upregulation of several TGF-β and NOTCH target genes, such as NOTCH1 (Supplementary Fig. 14c, e; Supplementary Fig. 15c), consistent with derepression of INSM1-repressed targets. However, TAS1440 failed to further enhance the expression of these genes, suggesting that INSM1 is necessary for dynamic transcriptional reprogramming in response to LSD1 inhibition, rather than maintaining static expression levels.

To determine whether transcription factor activation was also disrupted, we assessed nuclear accumulation of phospho-SMAD2 and NICD. Immunocytochemistry revealed that TAS1440-induced nuclear accumulation of both factors was evident in wild-type cells but absent in INSM1-knockout cells (Fig. 8i, j and Supplementary Fig. 16a, b). These results were supported by immunoblotting (Supplementary Fig. 14d, e), reinforcing the idea that INSM1 loss impairs signal transduction downstream of LSD1 inhibition by TAS1440.

To confirm the functional role of INSM1, we reintroduced INSM1 into the knockout cells (Fig. 8k and Supplementary Fig. 13g). Restoration of INSM1 expression rescued TAS1440 sensitivity in both NCI-H1417 and NCI-H146 cells, as measured by cell viability assays (Fig. 8l; Supplementary Fig. 13h), and reinstated the expression of NOTCH and TGF-β downstream targets, including NOTCH1, HES1, and TGFB2 (Fig. 8m and Supplementary Fig. 13i). Conversely, combination of INSM1-knockout with SMAD2 knockdown markedly abrogated TAS1440‑induced growth suppression and transcriptional changes (Fig. 8l, m and Supplementary Fig. 13g–i), highlighting their cooperative role in mediating TAS1440 sensitivity.

To further dissect the apparent dual role of INSM1 in regulating NOTCH1 expression, we investigated chromatin-level changes associated with INSM1 loss using ChIP-seq for histone modifications. Given that INSM1 knockdown reduces TAS1440 sensitivity in SCLC-A cells (Supplementary Fig. 13j, k), we next investigated its impact on histone modifications. Consistent with its lack of intrinsic demethylase activity, INSM1 knockdown did not alter H3K4me1/2/3 levels at TSSs (Fig. 8n), indicating that it does not directly regulate methylation. In contrast, we observed a pronounced increase in H3K27ac levels at TSSs in INSM1-knockout cells, suggesting derepression of transcriptional targets. At the gene level, representative tumor-suppressive targets such as NOTCH1 and TGFB2 exhibited increased H3K27ac enrichment and transcriptional upregulation in the absence of INSM1 (Fig. 8o–r). These findings support a model in which INSM1 functions as an LSD1‑coupled repressor under basal conditions, and its loss both dampens TAS1440‑driven dynamic induction and rewires epigenetic states associated with resistance.

INSM1 Overexpression Increases TAS1440 Sensitivity in Non-Neuroendocrine and TAS1440-Resistant Neuroendocrine Cells

To complement our loss‑of‑function studies, we tested whether gain of INSM1 enhances responsiveness to TAS1440. We ectopically expressed INSM1 in two resistant non‑neuroendocrine models—NCI‑H211 (SCLC‑P) and NCI‑H196 (YAP1‑high, non‑NE)—and confirmed robust protein induction by immunoblotting (Supplementary Fig. 17a). We also evaluated DMS79 (SCLC‑A, neuroendocrine), an intrinsically TAS1440‑refractory background, to determine whether enforced INSM1 further increases sensitivity. INSM1 overexpression shifted TAS1440 dose–response curves leftward and increased growth suppression in the two non‑NE models relative to empty‑vector controls; in DMS79, the enhancement was present but more modest (Supplementary Fig. 17b).

In parallel RNA‑seq analyses, INSM1 overexpression did not alter baseline expression of the NE marker ASCL1 or of YAP1 and POU2F3 (Supplementary Fig. 17c). Regarding drug responsiveness, INSM1 overexpression increased the number of TAS1440‑induced DEGs in the non‑neuroendocrine (non‑NE) lines NCI‑H211 and NCI‑H196, as well as in DMS79—which expresses moderate endogenous INSM1—relative to empty‑vector controls (Supplementary Fig. 17d and e). KEGG and GSEA further indicated that INSM1 overexpression augmented TAS1440‑mediated enrichment of NOTCH and TGF‑β signaling in the non‑NE models (Supplementary Fig. 17f). Consistent with these transcriptomic findings, immunoblotting showed that ectopic INSM1 enhanced TAS1440‑induced increases in pSMAD2 and the induction of NOTCH1, albeit with cell line–dependent variation, in both previously less‑responsive cell lines (Supplementary Fig. 17g, h). Examination of downstream pathway activation by heatmap profiling further revealed that, in the insensitive state (empty vector), few genes changed between DMSO and TAS1440, whereas with INSM1 overexpression, both pathways were partially activated following treatment (Supplementary Fig. 17i, j). Although the dominant downstream effectors of NOTCH/TGF‑β may vary by cell line, these findings support a link between the INSM1–LSD1 axis and pharmacologic sensitivity.

Collectively, these data support a model in which INSM1 is both a determinant and an amplifier of TAS1440 response—such that INSM1 gain converts resistant non‑NE states toward a drug‑responsive, NE‑like program and modestly enhances TAS1440 effects even in an NE‑positive context.

TAS1440 Inhibits SCLC-A Tumor Growth in Vivo and Demonstrates Prognostic and Therapeutic Relevance of INSM1–LSD1 Axis in SCLC

To evaluate the in vivo efficacy of TAS1440, we tested four SCLC‑A xenograft models (NCI‑H1417, NCI‑H146, NCI‑H510A, and COR‑L51). TAS1440 significantly reduced tumor growth in all models (dose group: 16.7 or 50 mg/kg/day in NCI‑H1417; 50 mg/kg/day in the others) compared with vehicle (Fig. 9a–e, g; Supplementary Fig. 18e, i). In the NCI-1417 model, ORY‑1001 also reduced tumor growth at the tested dose (0.01–0.03 mg/kg/day) (Fig. 9a). Consistent with in vitro findings, immunoblotting of xenograft tumors showed NOTCH1 upregulation and increased pSMAD2 after TAS1440 (Fig. 9h,i; Supplementary Fig. 18f, g, j, k). RT-qPCR confirmed induction of NOTCH/TGF‑β‑pathway genes (NOTCH1, HES1, TGFB2, TGFBR2, ID3) and reduction of ASCL1 and DLL3, with some markers showing borderline significance (e.g., TGFBR2 P = 0.06) (Fig. 9j; Supplementary Fig. 18h, l).

To evaluate INSM1 dependence in vivo, INSM1-wild-type (WT) NCI-H146 xenografts were responsive to TAS1440, whereas INSM1-knockout (KO) NCI-H146 xenografts were non-responsive (Fig. 9f), indicating that INSM1 expression confers TAS1440 sensitivity in vivo. In an orthotopic NCI‑H146 lung model, TAS1440 reduced pulmonary tumor burden, as shown by micro‑computed tomography (CT) images and volumetric quantification (Fig. 9k).

Body‑weight monitoring showed modest decreases in some TAS1440 groups, with no overt loss relative to vehicle throughout the treatment period (Supplementary Fig. 18a–d).

Finally, survival analysis in a public SCLC RNA‑seq cohort 24 showed that high KDM1A expression associates with worse overall survival (HR 2.17, P = 0.0082), whereas INSM1 exhibited a non‑significant trend; similar non‑significant trends were observed within SCLC‑A (Fig. 9l, m). These data support the prognostic and therapeutic relevance of the INSM1–LSD1 axis in SCLC.

Methods
Materials Availability
This study did not generate new unique reagents.

Cell Lines and Cell Culture
NCI-H1417 (Cat. No. CRL-5869), NCI-H209 (HTB-172), SHP-77 (CRL-2195), NCI-H211 (CRL-5824), NCI-H196 (CRL-5823), NCI-H146 (HTB-173), NCI-H187 (CRL-5804), NCI-H69 (HTB-119), NCI-H446 [H446] (HTB-171), DMS 79 (CRL-2049), NCI-H510A (HTB-184), DMS 114 (CRL-2066), DMS 53 (CRL-2062), NCI-H2081 (CRL-5920), NCI-H524 [H524] (CRL-5831), NCI-H128 (HTB-120), NCI-H526 (CRL-5811), and NCI-H1694 (CRL-5888) cell lines were purchased from the American Type Culture Collection. Lu-135 (JCRB0170), COR-L51 (92031916), CPC-N (ACC 306), and SBC-5 (JCRB0819) cell lines were obtained from the Japanese Collection of Research Bioresources, the European Collection of Authenticated Cell Cultures, the German Collection of Microorganisms and Cell Cultures GmbH, and the Health Science Research Resources Bank, respectively. All of the above are small-cell lung cancer (SCLC) cell lines and were cultured in medium supplemented with fetal bovine serum (FBS) (Cat#: 10270106, Gibco, Thermo Fisher Scientific). The medium was conditioned in high-glucose RPMI 1640 (Wako, Cat#: 187-02705) with 10% FBS for NCI-H146, NCI-H187, NCI-H69, NCI-H446, NCI-H526, NCI-H524, and DMS 79; in McCoy’s 5A (Gibco, Cat#: 16600-082) with 10% FBS for CPC-N; minimum essential medium (Cat#: 21442-25, Nacalai Tesque, Kyoto, Japan) with 10% FBS for SBC-5; F-12K medium (Gibco, Cat#: 11765-054) with 10% FBS for NCI-H510A; Waymouth’s medium (Gibco, Cat#: 11220-035) with 10% FBS for DMS 114 and DMS 53; and IMDM medium (Gibco, Cat#: 12440-053) with 20% FBS for NCI-H128 or HITES medium (Gibco, 11330-032) with 10% FBS, insulin (FUJIFILM, Cat#: 093-06351), transferrin (Cat#: 10652202001, Roche Diagnostics, Rotkreuz, Switzerland), hydrocortisone (Cat#: H6909-10ML, Sigma-Aldrich, St. Louis, MO, USA), GlutaMAX (Gibco, Cat#: 35050-061), sodium selenite (FUJIFILM, Cat#: 190-10844), and β-estradiol (Sigma-Aldrich, Cat#: E2758-250MG) for NCI-H2081 and NCI-H1694. Criteria and Rationale for Inclusion or Exclusion of SCLC Cell Lines Used in This Study are provided in Supplementary Table S1.

In Vitro Cytotoxicity Assay
SCLC cell lines were seeded in 96‑well plates. The following day, LSD1 inhibitors were serially diluted with DMSO and mixed with culture medium. The mixtures were then added to each well. The final DMSO concentration was 0.075%. Cells were maintained for 8 days with the inhibitors without medium change. Cell viability was assessed using CellTiter‑Glo® 2.0 (Promega, Madison, WI, USA).

Cell Proliferation Assay
To analyze cell proliferation after LSD1 inhibition, 5,000 SCLC cells were seeded in 96‑well plates and maintained at 37 °C. Live cells were monitored using an IncuCyte® SX5 Live‑Cell Imaging and Analysis Instrument (Sartorius, Göttingen, Germany). Cells were labeled with NucLight Rapid NIR Reagent (Cat. No. 4804, Sartorius). IncuCyte® enabled automated counting of monolayer cell cultures.

Chemical Compounds and Antibodies
TAS1440 was designed and synthesized at Taiho Pharmaceutical Co., Ltd. or at Aptuit (Oxford, UK) Ltd. ORY‑1001 and GSK2879552 were synthesized by Taiho Pharmaceutical Co., Ltd., and Sanyu Chemical Co., Ltd., respectively. Pulrodemstat (CC‑90011) and SP2509 were purchased from Selleckchem (Houston, TX, USA). LY411575 was purchased from Sigma‑Aldrich. SB431542 was purchased from Cayman Chemical Co., Ltd. (Ann Arbor, MI, USA). The anti‑GAPDH antibody (Cat. No. GTX100118) was purchased from GeneTex, Inc. (Irvine, CA, USA). Antibodies including anti‑FLAG (Cat. No. 14793), anti‑LSD1 (Cat. No. 2184), anti‑CHD4 (Cat. No. 4245), anti‑HDAC1 (Cat. No. 5356), anti‑HDAC2 (Cat. No. 57156), anti‑SMAD2 (Cat. No. 5339), anti‑NOTCH1 (Cat. No. 3608), anti‑CoREST (Cat. No. 14567), rabbit anti‑mouse IgG (HRP‑conjugated, Cat. No. 58802), and mouse anti‑rabbit IgG (HRP‑conjugated, Cat. No. 93702) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‑INSM1 (Cat. No. sc‑271408) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti‑SMAD2 (phospho‑Ser467; ab280888) was purchased from Abcam, Ltd. (Waltham, MA, USA). Rabbit anti‑mouse immunoglobulins (HRP‑conjugated, Cat. No. P0260) were purchased from Agilent (Santa Clara, CA, USA). Donkey anti‑rabbit IgG (HRP‑conjugated, Cat. No. NA934) was purchased from Cytiva (Marlborough, MA, USA). siRNA oligonucleotides targeting SMAD2 and INSM1 (Cat. No. AM16708) were purchased from Ambion, Thermo Fisher Scientific. NCI‑H1417, NCI‑H146, or NCI‑H510A cells were transfected with siRNAs using Lipofectamine™ RNAiMAX (Thermo Fisher Scientific, Cat. No. 13778150) following the manufacturer’s protocol. At 24 h after transfection, reagents were added, and subsequent experiments were performed.

Enzyme Assays
Recombinant human LSD1 (Cat. No. ab80379) and LSD2 (Cat. No. 31479) were purchased from Abcam and Active Motif (Carlsbad, CA, USA), respectively, and used in enzymatic assays. Inhibition of enzymatic activity by TAS1440 was tested using LANCE® time‑resolved FRET (TR‑FRET). Biotin‑H3K4me1 peptide (Cat. No. AS‑64355, AnaSpec, Fremont, CA, USA) was used as a substrate, and its demethylation by LSD1 or LSD2 was detected by the TR‑FRET signal generated when a europium‑labeled demethylation‑site‑specific antibody (Eu‑labeled anti‑H3K4 as energy donor, Cat. No. TRF040‑M, PerkinElmer, Shelton, CT, USA) and streptavidin‑conjugated Alexa Fluor™ 647 (energy acceptor; Thermo Fisher Scientific, Cat. No. S21374) were in molecular proximity. The substrate concentrations used for substrate‑competition assays were 100 nM or 2,000 nM (Fig. 2g) and 200 nM for IC50 determination (Fig. 2f). Assays for histone lysine demethylases (JARID1A, JARID1B, UTX, JMJD2B, and JMJD3), histone lysine methyltransferases (SMYD2 and EZH1/EED/SUZ12), histone deacetylase (HDAC1), DNA methyltransferase (DNMT1), and protein arginine N‑methyltransferase (PRMT4) were performed by Eurofins Cerep SA (Celle‑Lévescault, France). MAO‑A and MAO‑B assays were performed by Eurofins Panlabs Discovery Services Taiwan, Ltd. (Taipei, Taiwan).

RNA-Seq and Data Analysis
SCLC cell lines were treated for the indicated durations, with different LSD1 inhibitors (TAS1440, 300 nM; ORY‑1001, 100 nM; GSK2879552, 3000 nM; CC‑90011, 300 nM; SP2509, 300 nM). Total RNA was extracted using TRIzol™ (Invitrogen, Thermo Fisher Scientific). RNA integrity and concentration were measured using a 4200 TapeStation system (Agilent, Santa Clara, CA, USA). RNA‑seq libraries were prepared using the NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) with SPRIselect (Cat. No. B23318, Beckman Coulter, Indianapolis, IN, USA) following the manufacturers’ protocols. Deep sequencing was performed on an Illumina NovaSeq 6000 (paired‑end). Sequenced reads were aligned using Bowtie 2 and TopHat (version 1.3.2), and Cufflinks (version 2.0.2) was used for transcriptome assembly. Gene expression levels were expressed as fragments per kilobase of exon per million mapped reads (FPKM). Differentially expressed genes (DEGs) were identified using R (version 4.2.0). Heatmaps and dendrograms were generated using R, GraphPad Prism 9.0, or ChiPlot (https://www.chiplot.online/). DEG‑related Kyoto Encyclopedia of Genes and Genomes (KEGG) terms were analyzed using gene set enrichment analyses (GSEA; version 4.2.3).

RT–qPCR
Total RNA was extracted using TRIzol™ reagent (Thermo Fisher Scientific). Quantity and purity were evaluated with a NanoDrop spectrophotometer (Thermo Fisher Scientific), and the A260/A280 ratio was used to assess purity. cDNA was synthesized using ReverTra Ace™ qPCR RT Master Mix (TOYOBO, Osaka, Japan). RT–qPCR was performed using Fast SYBR™ Green Master Mix (Thermo Fisher Scientific, Cat. No. 4385614) on a StepOnePlus™ Real‑Time PCR System (Thermo Fisher Scientific) using the default fast‑mode setting (stage 1: 95.0 °C for 20 s; stage 2: 40 cycles at 95.0 °C for 1 s and 60.0 °C for 20 s). Relative gene expression was quantified using the 2 −ΔΔCt method and normalized to ACTB expression. Primers are shown in Supplementary Table S2.

Plasmid Construction
To obtain 3×FLAG‑Myc, the myc sequence was seamlessly inserted at the end of the 3×FLAG sequence of p3×FLAG‑CMV10 (Cat. No. E7658, Sigma‑Aldrich). To generate 3×FLAG‑Myc‑LSD1, the LSD1 coding sequence from pIDS‑LSD1fl (#109157, Addgene, Watertown, MA, USA) was subcloned downstream of the Myc sequence in p3×FLAG‑Myc‑CMV10 using NEBuilder HiFi DNA Assembly (Cat. No. E5520S, New England Biolabs). Seamless joining was verified by Sanger sequencing. The backbone was then changed to pcDNA3.1+ (V79020, Invitrogen, Thermo Fisher Scientific). pcDNA3‑3×FLAG‑Myc‑LSD1 K661A was generated using NEBuilder HiFi after amplifying fragments harboring the missense mutation by PCR from the pcDNA3‑LSD1 plasmid. pcDNA3‑EGFP was a gift from Doug Golenbock (Addgene plasmid #13031). To subclone pcDNA3‑GFP‑hINSM1 and pLX304‑GFP‑hINSM1, the coding sequence of human INSM1 was obtained from cDNA and cloned into pcDNA3 with a C‑terminal HA tag (pcDNA3‑hINSM1‑HA). Based on this construct, two GFP‑tagged variants were derived: pLX304‑hINSM1‑GFP, in which the HA tag was replaced with GFP and the sequence cloned into the pLX304 lentiviral vector, and pcDNA3‑hINSM1‑GFP, in which the GFP‑tagged INSM1 sequence was introduced into the pcDNA3 backbone by Gibson Assembly (New England Biolabs). pLenti‑PGK‑Venus‑Fluc (pSuro) was a gift from Roland Friedel (Addgene plasmid #140328; RRID:Addgene_140328)42.

LSD1 Immunoprecipitation and LC‒MS/MS Analysis
NCI‑H1417 cells were electrotransfected with either pcDNA3‑3×FLAG‑Myc‑empty or pcDNA3‑3×FLAG‑Myc‑LSD1. 46 hours later, cells were treated with LSD1 inhibitors for 2 h, harvested, and solubilized in immunoprecipitation buffer: 50 mM Tris‑HCl (pH 7.5), 150 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) Tween‑20, 1 mM EDTA, 10 mM NaF, 1 mM DTT, and protease inhibitor cocktail (Cat#: 25955‑24, Nacalai Tesque). Cells were lysed on ice for 30 min with gentle shaking and sonicated on a SFX150 Sonifier (Branson, Brookfield, CT, USA) for cycles of 15 s sonication at 50% amplitude followed by 45 s rest. Precipitates were removed by centrifugation at 20,000 × g for 10 min at 4 °C, and immunoprecipitation was performed with 50 µL ANTI‑FLAG® M2 Affinity Agarose Gel (Cat. No. A2220, Sigma‑Aldrich) overnight at 4 °C. After washing with PBS, proteins were eluted with 150 µg/mL 3×FLAG® peptide (Cat. No. F4799, Sigma‑Aldrich). Eluates were acetone‑precipitated, resuspended in 1× SDS sample buffer, and separated by SDS–PAGE. Gel bands were excised, washed twice with 100 mM ammonium bicarbonate in acetonitrile, and digested with trypsin for 8 h at 37 °C. Peptides (in 0.1% formic acid) were analyzed by LC–MS/MS using an Advance UHPLC (Bruker, Billerica, MA, USA) and an Orbitrap Velos Pro (Thermo Fisher Scientific). Data were analyzed with Mascot (version 2.5.1, Matrix Science, Boston, MA, USA). A Mascot score >100 indicates that the absolute probability of a random match is <1 × 10−10.

ChIP
NCI‑H1417 cells were treated with DMSO or 300 nM TAS1440 for 5 days and cross‑linked at 37 °C for 10 min with 1% formaldehyde. Cross‑linking was quenched to a final concentration of 125 mM glycine. Cells were lysed in swelling buffer (25 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, and 0.1% (v/v) NP‑40) containing protease inhibitors and centrifuged at 5,000 × g for 5 min at 4 °C. Nuclei were resuspended in sonication buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X‑100, 0.1% (w/v) SDS, and 0.1% (w/v) sodium deoxycholate; ~5 × 106 cells/mL) and sonicated on a Q700 (Qsonica, Newtown, CT, USA) for 20–25 cycles of 15 s on/45 s off at amplitude 60 (total 6,000 J). Insoluble material was removed by two rounds of centrifugation at 16,000 × g for 10 min at 4 °C. Supernatants were immunoprecipitated with either IgG (Thermo Fisher Scientific, Cat. No. 10003D), 5 µg SMAD2 antibody (Cell Signaling Technology, Cat. No. 5339) or 5 µg INSM1 antibody (Santa Cruz, Cat. No. sc‑271408) prebound to Dynabeads Protein G (Invitrogen) for 24 h at 4 °C, washed, and eluted for 6 h at 65 °C in ChIP elution buffer (50 mM Tris‑HCl pH 7.5, 1% (w/v) SDS, 1 mM EDTA). Cross‑links were reversed by adding NaCl and RNase A to 160 mM and 20 µg/mL, respectively, and incubating at 65 °C overnight. EDTA was increased to 5 mM, Proteinase K (200 µg/mL) was added, and samples incubated at 45 °C for 2 h. DNA was purified using a Gel/PCR DNA Isolation System (Cat. No. GP1001, VioGen, Taipei, Taiwan). Samples were analyzed by qPCR at the NOTCH1 promoter (primer sequences in Table S2).

ChIP‑Seq and Data Analysis
NCI‑H1417 cells were treated with DMSO or 300 nM LSD1 inhibitor (TAS1440, ORY‑1001, GSK2879552) for 5 days and processed following the protocol mentioned prior. Libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs) with SPRIselect (Cat. No. B23318, Beckman Coulter). Sequencing was performed on an Illumina NovaSeq 6000 (paired‑end). Reads were mapped to GRCh37.p13 (hg19) using Bowtie 2. Peaks were called using MACS2 with the broad option; peaks identified in three replicates were used for further analyses. Quantification used the “Annotate Peaks” command in Partek Flow (https://www.partek.com/). Differentially enriched regions (DERs) were detected using the “Gene‑Specific Analysis” command in Partek Flow. IGV (Integrative Genomics Viewer) was used for visualization.

Western Blotting
Cells were washed with chilled PBS and lysed in RIPA buffer (25 mM Tris‑HCl, pH 7.4; 150 mM NaCl; 1% (v/v) NP‑40; 1 mM EDTA; 5% (v/v) glycerol) supplemented with protease and phosphatase inhibitors. Proteins were separated by SDS–PAGE and electrotransferred onto PVDF membranes (Mini‑PROTEAN® Tetra system, Bio‑Rad, Hercules, CA, USA). Membranes were blocked with 10% (w/v) skim milk followed by 1% (w/v) BSA at 23 °C for 1 h, probed with primary antibodies at 4 °C overnight, washed three times with TBS‑T, and incubated with secondary antibodies (Cat. No. NA934, Cytiva; Cat. No. P0260, Agilent) for 1 h at 23 °C. Signals were visualized using enhanced chemiluminescence (Cytiva, Cat. No. RPN2232) and captured with Fusion FX (Vilber Lourmat, Marne‑la‑Vallée, France).

For Fig. 4c, anti‑GAPDH (Cat. No. GTX100118; 1:5000, GeneTex), anti‑SMAD2 (phospho‑Ser467, ab280888; 1:1000, Abcam), and anti‑NOTCH1 (Cat. No. 3608; 1:1000, Cell Signaling Technology) were used. For Fig. 5e, anti‑FLAG (Cat. No. 14793; 1:1000), anti‑LSD1 (Cat. No. 2184; 1:1000), anti‑HDAC1 (Cat. No. 5356; 1:1000), anti‑HDAC2 (Cat. No. 57156; 1:1000), anti‑SMAD2 (Cat. No. 5339; 1:1000), and anti‑CoREST (Cat. No. 14567; 1:1000) were obtained from Cell Signaling Technology, whereas anti‑INSM1 (Cat. No. sc‑271408; 1:1000) was obtained from Santa Cruz Biotechnology.

Expression and Purification of LSD1–CoREST
An N‑terminal His6 tag, SUMOstar tag, and TEV protease cleavage site fused to LSD1 (residues S172–M833), and its cofactor CoREST (residues R311–E443), were subcloned into MCS1 and MCS2 of pETDuet‑1. E. coli BL21 (DE3) CodonPlus cells were transformed and cultured in TB medium containing 50 µg/mL ampicillin at 37 °C to OD600 = 0.6. Expression was induced with 0.1 mM IPTG and continued at 16 °C overnight. Cells were lysed in Buffer A (50 mM Tris‑HCl pH 7.5, 300 mM NaCl, 10% (v/v) glycerol, 0.1 mM TCEP), sonicated, and clarified by centrifugation at 16,000 × g for 30 min. The supernatant was applied to a HiTrap TALON column equilibrated with Buffer A and eluted with ~50 mM imidazole. Excess FAD was added to the fraction. The N‑terminal tag was removed using His6‑tagged TEV protease during dialysis in Buffer A for 40 h. The dialysate was applied to a second TALON column to remove the cleaved tag and TEV protease; the flow‑through was collected and further purified on a HiLoad 26/600 Superdex 200 pg column equilibrated with 25 mM potassium phosphate (pH 7.2) containing 5% (v/v) glycerol. The LSD1–CoREST complex was concentrated to 18 mg/mL for crystallization (Bradford method).

Structure Analysis of TAS1440-bound LSD1–CoREST
LSD1–CoREST was initially crystallized by hanging‑drop vapor diffusion; small seed crystals (50–200 µm) were obtained from a precipitant containing 50–100 mM N‑(2‑acetamido)iminodiacetic acid (ADA), pH 6.5, and 1.18–1.28 M sodium potassium tartrate. Larger soakable crystals (200–400 µm) were obtained by macroseeding from a precipitant containing 100 mM ADA (pH 6.5) and 1.22 M sodium potassium tartrate. Crystals were soaked in 100 mM ADA (pH 6.5), 1.22 M sodium potassium tartrate, 10% (v/v) glycerol, and 2 mM TAS1440 at 20 °C for 2 h. Diffraction data were collected at KEK Photon Factory BL‑1A and processed using iMOSFLM in the CCP4 suite. The space group was I222. The structure of TAS1440‑bound LSD1–CoREST was solved by molecular replacement with MOLREP (CCP4) using PDB 5YJB as a search model and refined with REFMAC5. Manual rebuilding and map interpretation were performed in Coot. Final R‑values were R_work = 19.3% and R_free = 23.0%. Coordinates were deposited in the PDB under 8JF5 (statistics in Supplementary Table S3).

Immunofluorescence Analysis
NCI‑H1417 or NCI‑H146 cells were cultured in 4‑well chamber slides (2 × 104 cells per well) and treated with 0.1% (v/v) DMSO or 300 nM TAS1440 for the indicated times. Cells were fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% (v/v) Triton X‑100 in PBS for 5 min at 23 °C and blocked with 3% (v/v) FBS in PBS for 60 min. Primary antibodies encompassed cleaved NOTCH1 (Val1744; 1:100, Cell Signaling, Cat. No. 4147S), SMAD2 (1:200, Cell Signaling, Cat. No. 5339S), and INSM1 (1:100, Santa Cruz, Cat. No. sc‑271408). Secondary antibodies were donkey anti‑mouse IgG Alexa Fluor™ 488 (1:500, Invitrogen, Cat. No. A32766), donkey anti‑mouse IgG Alexa Fluor™ 647 (1:500, Invitrogen, Cat. No. A32787), and donkey anti‑rabbit IgG Alexa Fluor™ 594 (1:500, Invitrogen, Cat. No. A32754). Primary incubations were performed at 4 °C overnight, whereas secondary incubations at room temperature for 2 h. Nuclei were stained with DAPI (1 µg/mL; Cat. No. H‑1200, Vector Laboratories, Newark, CA, USA). Images were acquired on a Zeiss LSM 980 confocal microscope (Axio Observer 7; 20× Plan‑Apochromat or 63× Plan‑Apochromat Oil) and analyzed using ZEN Blue v3.2. Nuclear phospho‑SMAD2‑ and NICD‑positive cells were quantified on a BZ‑X800 fluorescence microscope (KEYENCE, Osaka, Japan) equipped with the BZ‑H4C image cytometer module.

INSM1-KO or LSD1-KO NCI-H1417 and NCI-H146 Cells
The CRISPR/Cas9 system was employed as previously described 35,42. The backbone vector pSpCas9(BB)‑2A‑GFP (PX458) was obtained from Addgene. The gRNA sequence targeting INSM1 was 5′‑CTCGCCGCCGCGGACCCGGT‑3′, and that targeting LSD1 was 5′‑CCGGCCCTACTGTCGTGCCT‑3′. Oligos were annealed and phosphorylated using T4 DNA Ligase Reaction Buffer and T4 Polynucleotide Kinase (New England Biolabs) at 37 °C for 30 min and 95 °C for 5 min, respectively. PX458 was digested with BbsI (Thermo Fisher Scientific) at 37 °C for 30 min and purified using a QIAquick Gel Extraction Kit (QIAGEN). PX458 and the annealed oligos were ligated at room temperature for 10 min using a Quick Ligation Kit (New England Biolabs) and treated with PlasmidSafe™ exonuclease (Cambio, Cambridge, UK) at 37 °C for 30 min. Plasmids were transformed into One Shot™ Stbl3™ chemically competent E. coli, and amplified plasmids purified with NucleoBond Xtra Midi (Takara Bio, Shiga, Japan). The INSM1 or LSD1 CRISPR/Cas9 KO plasmid (10 µg) was electroporated into NCI‑H1417 or NCI‑H146 cells (1 × 10^6) using a NEPA21 (Nepa Gene Co., Ltd., Chiba, Japan) in a 2-mm gap cuvette. The electroporation parameters were as follows: Poring pulse (Voltage: 125 V, Pulse length: 5 ms, Pulse interval: 50 ms, Number of pulses: 2, Decay rate: 10%, Polarity: +) and Transfer pulse (Voltage: 20 V, Pulse length: 50 ms, Pulse interval: 50 ms, Number of pulses: 5, Polarity: ±). Two days later, GFP‑positive cells were sorted by BD FACSMelody™ (BD Biosciences, San Jose, CA, USA) and cultured. TAS1440 (300 nM) or DMSO was added the day after sorting. INSM1‑KO was confirmed by western blotting (anti‑INSM1; Fig. 8b and Supplementary Fig. 13a). LSD1‑KO was confirmed by western blotting (anti‑LSD1; Fig. 7b and Supplementary Fig. 12b).

Generation of Stable Fluc- and INSM1-expressing SCLC cell lines
For lentivirus preparation, the vector of interest was co‑transfected with packaging components VSV‑G, gag/pol, and rev into Lenti‑X 293T (Clontech) cells at ~70% confluency using Lipofectamine 2000 (Thermo Fisher Scientific). The medium was changed the next day. After 48 h, the viral supernatant was harvested and filtered (0.45 µm). NCI‑H146 cells were infected at ~50% confluency and selected with 0.2 µg/mL puromycin for 7 days before analysis. NCI‑H196 and NCI‑H211 cells were infected similarly and selected with 2 µg/mL and 4 µg/mL blasticidin, respectively, for 10 days before analysis.

In Vivo Efficacy Studies
NCI‑H1417, NCI‑H146, NCI‑H510A, COR-L51 and NCI-H146 INSM1-KO cells were suspended in PBS and mixed 1:1 with Matrigel (Corning, NY, USA). Suspensions were injected subcutaneously into six‑week‑old male C.B-17 SCID mice (Charles River Laboratories Japan, Inc. and CLEA Japan, Inc.). Upon tumor establishment, mice were randomized into control and experimental groups (n = 4–6 per group) using MiSTAT Grouping to balance mean baseline tumor volumes. TAS1440 was administered at 16.7 or 50 mg/kg/day. The ORY-1001 dose (0.01–0.03 mg/kg/day) was chosen based on previously reported efficacy in the SCLC PDX models12. Tumor volume was calculated as length × width2 / 2 (length: longest diameter; width: shortest diameter). The maximum tumor diameter permitted was 1.5 cm; all tumors remained ≤1.5 cm. To examine the pulmonary environment, an intrapulmonary implantation model was established: a small skin incision was made on the left chest wall (seventh intercostal space); after exposing the left lung via the pleura, NCI‑H146 (1 × 107) cells in 40 µL PBS/Matrigel (3:1) were injected into the lung. Two months later, mice were treated orally with TAS1440 (50 mg/kg/day) or vehicle for 4 weeks. Tumor growth was monitored by micro‑CT (SkyScan 1276, Bruker, USA) under anesthesia (50 kV, 200 µA, 2048 × 2048). Mice were euthanized when tumor burden met criteria or at end of treatment; tumors were excised and weighed. Mice were maintained under SPF conditions (20–26 °C; 30–70% humidity; 12‑h light cycle) with ad libitum CE‑2 chow and water. TAS1440 was administered orally once daily for 3 weeks; tumor volume and body weight were measured every 3 days. All animal experiments were approved by the IACUCs of Taiho Pharmaceutical Co., Ltd. (Permit No. AE21‑425-01 and 18TB14) and Chiba University (No. A7‑284) and conducted in accordance with institutional guidelines.

Docking Simulations
The crystal structure of LSD1 complexed with FAD (PDB 2HKO) was used. Molecular docking between LSD1 and inhibitors was performed using AutoDock Vina (AutoDockTools v1.5.6). Docking scores are reported as binding energies (kcal/mol), with lower values indicating more stable interactions; the lowest‑energy pose was selected. Protein–protein docking between LSD1 and INSM1 or SMAD2 was performed using AlphaFold3, and molecular graphics were generated in UCSF ChimeraX.

Survival Analysis
Survival analysis used RNA‑seq expression and clinical data from a previously published cohort of patients with SCLC reported by Liu et al.12. Overall survival was analyzed using the Kaplan–Meier method and compared by log‑rank test. For each gene, patients were stratified into High and Low based on the median TPM. Hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated using the Cox proportional hazards model and expressed as the relative risk of death in the High‑expression group versus Low‑expression. The log‑rank p‑value was reported for each gene.

Statistical Analyses
Quantitative data are presented as mean ± SD or mean ± SEM. Comparisons between groups were performed using a two‑tailed Student’s t‑test or one‑way analysis of variance unless otherwise specified. Correlations were determined using Pearson’s correlation. GraphPad Prism 9 (https://www.graphpad.com) was used for statistical analyses. Statistical significance was set at P < 0.05.

Synthesis of TAS1440 Benzoate Salt
Abbreviations: TEA, triethylamine; MeOH, methanol; EtOH, ethanol; HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; DMA, N,N-dimethylacetamide; Ac, acetyl; dppf, 1,1'-Bis(diphenylphosphino)ferrocene; PPh3, triphenylphosphine; NMR, nuclear magnetic resonance spectroscopy; MS, mass spectrometry; RT, room temperature; aq., aqueous.
General experiments and information: NMR spectra were recorded using a Varian Mercury 400 or Bruker AV Neo 400 NMR spectrometer. Mass spectra were obtained using an ACQUITY QDa Mass Detector (Waters, Framingham, MA, USA).
Reagents and conditions: (a) TEA, HATU, DMA, RT, 1 h, 74%; (b) Pd(PPh3)4, 2 M Na2CO3 aq., 1,4-Dioxane, 90 °C, overnight, 90%; (c) Pd(OAc)2, Silica-SMAP, KOAc, 1,4-Dioxane, 150 °C, 18 h, 61%; (d) PdCl2(dppf), 2 M Na2CO3 aq., 120 °C, 0.5 h, 78%; (e) 12 M HCl aq., MeOH, RT, 0.5 h, 63%; (f) benzoic acid, ethanol, water, 1 °C, 13 h, 91%.
Step 1: tert-Butyl (S)-(1-(3-bromo-4-chlorobenzoyl)pyrrolidin-3-yl)carbamate (S2)
3-Bromo-4-chloro-benzoic acid (SC1) (10 g) was dissolved in DMA (85 mL). HATU (24 g), TEA (12 mL), and tert-Butyl N-[(3S)-pyrrolidin-3-yl]carbamate (S1.1) (8.7 g) were added to the solution, which was stirred at room temperature for 1 h. Ethyl acetate was added to the mixture, and the resulting mixture was washed with water and brine. The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated. The residue was purified by chromatography using a silica-gel column (mobile phase: hexane/ethyl acetate) to obtain the title compound (12.7 g) as a solid. ESI MS m/z 403, 405, 407 [M+H]+.
Step 2: tert-Butyl N-[(3S)-1-[4-chloro-3-(4-cyano-3-fluoro-phenyl)benzoyl]pyrrolidin-3-yl]carbamate (S3).
tert-Butyl (S)-(1-(3-bromo-4-chlorobenzoyl)pyrrolidin-3-yl)carbamate (S2) (14 g) was dissolved in 1,4-dioxane (87 mL). (4-Cyano-3-fluoro-phenyl)boronic acid (S2.2) (6.3 g), Pd(PPh3)4 (1.2 g), and 2 M Na2CO3 aqueous solution (43 mL) were added to the solution and stirred at 90 °C overnight. Ethyl acetate was added to the mixture, and the resulting mixture was washed with water and brine. The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated. The residue was purified by chromatography using a silica-gel column (mobile phase: hexane/ethyl acetate) to obtain the title compound (13.9 g) as a solid. ESI MS m/z 444, 446 [M+H]+.
Step 3: tert-Butyl N-[(3S)-1-[3-(4-cyano-3-fluoro-phenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl]pyrrolidin-3-yl]carbamate (S4).
tert-Butyl N-[(3S)-1-[4-chloro-3-(4-cyano-3-fluoro-phenyl)benzoyl]pyrrolidin-3-yl]carbamate (S3) (4 g) was dissolved in 1,4-dioxane (45 mL). At room temperature, Pd(OAc)2 (0.40 g), KOAc (2.7 g), bis(pinacolato)diboron (4.6 g), and Silica-SMAP (0.72 g) were added to the mixture and stirred at 150 °C for 18 h. The mixture was cooled to room temperature. Subsequently, the mixture was filtered, and filtrate evaporated. The resulting residue was purified by chromatography using a silica-gel column (mobile phase: hexane/ethyl acetate) to obtain the title compound (2.95 g) as a solid. ESI MS m/z 536 [M+H]+.
Step 4: tert-Butyl N-[(3S)-1-[3-(4-cyano-3-fluoro-phenyl)-4-[2-fluoro-4-(2-hydroxy-2-methyl-propyl)phenyl]benzoyl]pyrrolidin-3-yl] carbamate (S5).
tert-Butyl N-[(3S)-1-[3-(4-cyano-3-fluoro-phenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl]pyrrolidin-3-yl]carbamate (S4) (8.00 g) and 1-(4-bromo-3-fluoro-phenyl)-2-methyl-propan-2-ol (S4.1) (4.43 g) were dissolved in 1,4-dioxane (75 mL). PdCl2(dppf) (273 mg) and 2 M Na2CO3 aq. (37 mL) were added to the mixture, which was stirred at 120 °C for 0.5 h. The mixture was cooled to RT. The reaction mixture was filtered, and filtrate evaporated. Ethyl acetate was added to the resulting mixture. The organic layer was washed with water and brine, dried over anhydrous sodium sulfate, and the filtrate was evaporated. The residue was purified by chromatography using a silica-gel column (mobile phase: chloroform/methanol) to obtain the title compound (6.70 g) as a solid. ESI MS m/z 576 [M+H]+.
Step 5: 4-[5-[(3S)-3-Aminopyrrolidine-1-carbonyl]-2-[2-fluoro-4-(2-hydroxy-2-methyl-propyl)phenyl]phenyl]-2-fluoro-benzonitrile (TAS1440).
tert-Butyl N-[(3S)-1-[3-(4-cyano-3-fluoro-phenyl)-4-[2-fluoro-4-(2-hydroxy-2-methyl-propyl)phenyl]benzoyl]pyrrolidin-3-yl] carbamate (S5) (6.70 g) was dissolved in methanol (67 mL). A 12 M aqueous HCl solution (67 mL) was added to the solution and stirred at RT for 30 min. The reaction mixture was neutralized by adding water (67 mL) and 2 M aqueous sodium hydroxide (500 mL) (the solution was confirmed to be basic using a pH indicator). Chloroform was added to the mixture, and the organic layer was washed with water and brine and dried over anhydrous sodium sulfate. The filtrate was evaporated to obtain the title compound (4.50 g) as a solid.
1H-NMR (500 MHz, DMSO-d6) δ: 7.79 (1H, t, J = 7.5 Hz), 7.70–7.67 (1H, m), 7.63 (1H, dd, J = 6.6, 1.5 Hz), 7.52 (1H, d, J = 8.1 Hz), 7.27 (1H, d, J = 10.3 Hz), 7.21 (1H, t, J = 7.9 Hz), 7.14 (1H, dd, J = 8.1, 1.5 Hz), 7.06 (1H, d, J = 7.7 Hz), 6.95 (1H, d, J = 11.4 Hz), 4.39 (1H, s), 3.68–3.55 (2H, m), 3.54–3.41 (2H, m), 3.21–3.12 (1H, m), 2.65 (2H, s), 2.03–1.88 (1H, m), 1.75 (2H, s), 1.70–1.59 (1H, m), 1.04 (6H, s); ESI MS m/z 476 [M+H]+.
Step 6: 4-[5-[(3S)-3-Aminopyrrolidine-1-carbonyl]-2-[2-fluoro-4-(2-hydroxy-2-methyl-propyl)phenyl]phenyl]-2-fluoro-benzonitrile benzoate salt (TAS1440 benzoate salt).
Ethanol (42 mL) and water (18 mL) were added to 4-[5-[(3S)-3-aminopyrrolidine-1-carbonyl]-2-[2-fluoro-4-(2-hydroxy-2-methyl-propyl)phenyl]phenyl]-2-fluoro-benzonitrile (TAS1440) (10 g) and benzoic acid (2.57 g), and stirred at 60 °C for 20 min to dissolve the mixture. Subsequently, water (45 mL) was added dropwise to the solution for 3 min at 60 °C and cooled to 1 °C. The suspension was then stirred at 1 °C for 13 h. The resulting precipitate was collected by filtration, washed with a mixture of ethanol (6 mL) and water (14 mL), and dried to obtain the title compound (11.5 g) as an off-white solid.
1H NMR (400 MHz, DMSO-d6) δ 7.90 (d, J = 7.29 Hz, 2H), 7.78 (t, J = 7.15 Hz, 1H), 7.61–7.72 (m, 2H), 7.45–7.55 (m, 2H), 7.36–7.42 (m, 2H), 7.17–7.29 (m, 2H), 7.13 (d, J = 8.43 Hz, 1H), 7.06 (dd, J = 1.28, 7.88 Hz, 1H), 6.95 (dd, J = 1.10, 11.36 Hz, 1H), 3.26–3.80 (m, 5H), 2.65 (s, 2H), 1.99–2.14 (m, 1H), 1.75–1.92 (m, 1H) , 1.04 (s, 6H); ESI MS m/z 476 [M+H]+.

Data Availability
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. The RNA-seq data generated in this study have been deposited in the Gene Expression Omnibus (GEO) database under accession code GSE272002 and the ChIP-seq data generated in this study have been deposited in the GEO database under accession code GSE272003. The mass spectrometry proteomics data were deposited in the Japan Proteome Standard repository/database JPST003190. Proteomics data supporting the findings of this study are available within the article and in Supplementary Data 1. Source data are provided with this paper.

Code Availability
No custom code was generated in this study.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "There is no effective molecular targeted therapies for SCLC yet. Here Machida et al. developed TAS1440, a novel histone H3-competitive LSD1 inhibitor to test its efficacy in treatment of SCLC, especially the A subtype. The authors found that TAS1440 non-covalently targeted the histone H3-binding pocket of LSD1 and exhibited enhanced anti-proliferative activity in SCLC cells with high INSM1 and ASCL1 expression. Mechanistically, they revealed that TAS1440 exerted its anti-tumor effects through dual mechanisms: direct inhibition of LSD1 enzymatic activity and disruption of LSD1-INSM1 complexes, thereby activating tumor suppressive pathways including TGF-β and NOTCH signaling. Collectively, these findings highlight TAS1440 as a potential targeted therapy for SCLC, especially in the SCLC-A subtype with high INSM1 expression. This work is novel and interesting. I list some of my concerns below.\n1. In Fig. 2a-b, the method used to calculate cell growth (%) is weird (Lines 539-546). The authors tend to exaggerate the impacts of inhibitors using two different formula dependent on the cell number of treatment groups when compared to the control.\n2. The authors proposed that TAS1440 could serve as a therapeutic option for SCLC, particularly for the SCLC-A subtype with high INSM1 expression. While their data showed that several SCLC-A cell lines were sensitive to TAS1440, some other SCLC-A lines, including NCI-H69, DMS79, SHP-77, and NCI-H2081, appear resistant to TAS1440 (Fig. 2b and Extended Data Fig. 2a). What are the potential mechanisms underlying the differential responses among different subtype-A cell lines to TAS1440 treatment? Could this variability be related to the differences of baseline INSM1 levels, or attributed to the variations of NOTCH or TGF-β signaling activity?\n3. In Fig. 5g, what is the difference between the left and right panels? They seem identical.\n4. Only one cell line is used to evaluate the efficacy of TAS1440 in vivo (Fig. 7j). At least two should be studied.\n5. In Fig. 7l, the authors assessed the prognostic value of KDM1A, NOTCH1 and TGFB2 in NSCLC but not SCLC patients. To strengthen their conclusions, the authors should perform survival analyses using SCLC datasets. Although clinical data for SCLC are limited, studies with larger sample sizes of SCLC with RNA-seq data are available (PMID: 38181741; PMID: 27093186). Moreover, since the authors concluded that “This demonstrates the prognostic and therapeutic implications of targeting the INSM1-LSD1 complex in small cell lung cancer, particularly in SCLC-A subtype” (Lines 420-422), it would be ideal to evaluate these survival analyses specifically in the SCLC-A subtype. The evaluation of prognostic implications of INSM1 in SCLC would also be informative.\n6. Fig. s8e showed that TAS1440-induced NOTCH1 upregulation was significantly impaired in INSM1-deficient cells (lane 3), suggesting that INSM1 is required for TAS1440-induced NOTCH1 upregulation. However, the same data also showed that NOTCH1 expression was markedly upregulated in INSM1-deficient cells without TAS1440 treatment (lane 2), indicative a suppressive role of INSM1 in NOTCH1 induction. How do the authors reconcile these seemingly contradictory effects of INSM1 on NOTCH1 expression?\n7. The authors stated that “TAS1440 treatment resulted in the dissociation of 78 nuclear factors, of which 13 were epigenetic factors, from LSD1 (Fig. 5c, red)” (Lines 264-266). However, only 6 genes are labeled in red in Fig. 5c.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The work by Machida et al. highlights the development of a novel LSD1 inhibitor (TAS1440) and demonstrate its potential efficacy in small cell lung cancer. The work on the validation of the drug, including its mechanism of action, the exertion of its effect through INSM1 and the connection to NOTCH signaling is very convincing. The authors have done an excellent job of characterizing a novel epigenetic modifier.\nWhere the manuscript struggles, however, is the focus on small cell lung cancer. That is not to say that small cell lung cancer is not a worthy target of this focus – quite the contrary – but the authors familiarity with small cell lung cancer appears outdated and the manuscript’s impact suffers mightily from this. There has been significant progress in small cell lung cancer – both preclinical and clinically – in the past 3-4 years and the authors seem completely unaware of this, as evidenced by the fact that their most recent citation for small cell lung cancer is the Ireland et al. Cancer Cell manuscript from 2020. This includes even recent publications on LSD1 inhibition in small cell lung cancer (e.g. Nguyen et al. J Thorac Oncol 2022).\nDespite the limitations in the authors command of the SCLC literature, there is a convincing case that the activity of TAS1440 is most robust in the neuroendocrine-high subtypes, especially SCLC-A. Their in vitro and in vivo data support this well.\nSignificant small cell lung cancer-focused revisions to text and experiments would be necessary, in my opinion, to make this a valid addition to the small cell lung cancer body of literature, including:\nMajor Issues:\n(1) The authors focus on the SCLC subtyping proposed by Rudin et al. It should be noted that this was a review and no longer reflects the consensus subtyping approach. In particular, while Rudin et al. proposed YAP1 as a subtype-defining transcription factor, this has been largely dismissed, including from work by Dr. Rudin’s group (see Baine et al. J Thorac Oncol 2020), as well as Gay et al. Cancer Cell 2021 and Ng et al., Clin Cancer Res, 2024. In particular, the three cell lines chosen by the authors here to represent the supposed SCLC-Y subtype were shown in the Ng et al. manuscript to be SMARCA4-deficient undifferentiated tumor in two cases and a sarcomatoid spindle cell neoplasm in the other case (see Table 1). At minimum, the figures should be revised to exclude these cell lines and the SCLC-Y subtype completely.\n(2) Once the authors appropriately reconcile their subtypes in Figure 1 and beyond with the current state of the SCLC literature, an key, overlooked direction is how LSD1 inhibition impacts the subtype landscape. This has, to a degree, already been addressed for earlier LSD1 inhibitors – see Nguyen et al. manuscript referenced above. It’s not clear why the authors seem to ignore this manuscript, rather than follow its format and experiments to highlight the ways in which their LSD1 inhibitor may be superior.\na. The authors make no mention of SCLC-I, the inflamed fourth subtype of SCLC. The earlier work by Nguyen et al. suggests that LSD1 inhibition can shift uninflamed, neuroendocrine high SCLC (e.g. SCLC-A) to a more inflamed state reminiscent of SCLC-I. This would be critical to explore here.\nb. Do the tumors that are SCLC-A and effectively treated with TAS1440 shift to SCLC-N? SCLC-P? SCLC-I? ASCL1 goes down, but what arises as a result? Even YAP1 is a possibility here, as demonstrated previously by Ireland et al. (which the authors do cite).\nc. As LSD1 inhibitors are unlikely to serve as successful single-agent therapies in SCLC, the possibility of combination therapies and the rationale for them is underexplored. If the tumors become more inflamed, could this agent be combined with immune checkpoint blockade? That experiment may be beyond the scope of the publication due to requirement for immunocompetent models, but at least some data supporting more inflammatory features (MHC expression, IFN-y, etc. a la Nguyen et al.) would be appropriate.\nd. Combinations with chemotherapies? Impact on expression of high-profile targets (e.g. DLL3 – the target of tarlatamab – or several ADC targets including B7H3, SEZ6, TROP2). Maybe it’s not a good thing to downregulate ASCL1 (and, presumably, DLL3 and SEZ6) since the standard of care for SCLC is now tarlatamab.\n(3) In the final paragraph of the results, the authors shift to NSCLC for survival analysis. This is clearly not appropriate to support the implications of targeting LSD1 in SCLC-A. Despite the authors comments, there are data available with transcriptional and clinical data for SCLC, including George et al. Nature 2015, Nabet et al. Cancer Cell 2024, and several others. If the authors wish to include clinical data, they need to gain access to one of several publicly available SCLC datasets.\nMinor Issues:\n(1) Page 6, line 102. Should this read “potently” rather than “potentially”?\n(2) Figure 2B would be easier to interpret if the cell line subtypes were labeled.\n(3) The in-text reference for Figure 2D arrives after the in-text reference for Figure 2E.\n(4) Page 15, line 317 seems to contain a typographical error “and GSK2879552”.", "reviewer_4": "In this manuscript, Machida et al. studied the therapeutic effect and understand the mechanism of the new LSD1 inhibitor TAS1440 in neuroendocrine SCLC. Earlier studies showed that LSD1 inhibition derepresses notch and TGFb signaling resulting in suppression of SCLC growth. This study presents the first development of a new LSD1 inhibitor, TAS1440 which is a histone H3 competitive inhibitor of LSD1. TAS1440 demonstrated superior antiproliferative activity in SCLC-A subtype cells and exhibited in vivo activity in subcutaneous xenograft models. Mechanistically, TAS effects are attributed to both direct inhibition of LSD1 enzymatic activity as well as disruption of LSD1-repressive complexes leading to coordinated regulation of histone modifications and activation of transcription factors INSM1 and SMAD2 which reprograms the NOTCH and TGFb tumor suppressor pathways. Further depletion of INSM1 resulted in the abrogation of TAS1440 effects, suggesting the dependency of INSM1 for TAS1440 functions.\nThis study is interesting as TAS has the dual function of inhibition of LSD1 enzymatic activity and disrupting LSD1 complexes and holds significant promise as a new targeted therapy for the SCLC-A subtype with high INSM1 expression. Data was generated rigorously using multiple cell lines and demonstrated the superior activity of TAS1440 than existing LSD1 inhibitors, and mechanistic studies were thoroughly conducted using RNA-seq of 22 cell lines, as well as ChIP-seq and mass spec studies and rigorous validation in multiple cell lines. The figures are good quality and easy to follow and the manuscript is well written.\nHowever, there are several issues noted in the manuscript and several claims are overstated without concrete evidence. The novelty of the study is moderate as LSD1's role in SCLC, and the utility of other LSD1 inhibitors was thoroughly investigated, although these inhibitors are associated with significant toxicity. The authors claim that TAS1440 has high specificity, less off-target effects, and is a safer drug, which could be a breakthrough for this study. Unfortunately, there was not enough data presented to support this statement. Further the objective states the identification of chemoresistance drivers, however no such studies using chemoresistance cells were conducted. In vivo studies are preliminary in nature, and only one subcutaneous xenograft model was used.\nThe on-target specificity of the compound should be established using LSD1-KO cells. Further, it is not clear if TAS1440 directly interacts with LSD1. This can be done using biotin-labeled TAS-1440 and LSD1 protein using avidin pulldown.\nIn vivo studies are conducted using subcutaneous models which do not mimic the actual microenvironment. Only one xenograft model was used. Studies using orthotopic models of SCLC and syngenic models with intact immune systems should be conducted to establish the superior activity of TAS1440. Further, there are no PDX models were utilized to account for the heterogeneity of SCLC.\nThere were no details provided pertaining to how the in vivo dose of TAS1440 was determined, what its MTD, MED. Concerning toxicity, it appears that this compound also reduces body weight, suspecting the potential toxicity of this compound. It is important to study whether this compound has any hematologic toxicity as commonly observed with other LSD1 inhibitors.\nTAS has dual activity of LSD1 enzymatic inhibition and complex disruptor. However, it is essential to determine if the enzymatic activity is important for TAS effects. Efficacy and mechanistic studies need to be conducted using enzymatic dead mutant LSD1 (K661A) to ascertain whether enzymatic activity is essential for TAS1440 effects.\nIt is not clear how it is different from existing reversible noncovalent inhibitors such as SP2509.\nSome of the data was generated using only one cell line, particularly Figure 4g,h,j,k. and 6d,e. This should be done using at least one more cell line.\nAuthors claim that TAS1440 dissociates TFs such as INSM1 and SMAD2 from the LSD1 complex and synergistically promotes the activation of key signaling pathways such as NOTCH and TGF-b via epigenetic reprogramming. However, no data was provided to support this conclusion except cell viability data.\nData shows that TAS dissociates LSD1 interaction with INSM1 and SMAD2. However, much of the studies are conducted with only INSM1 manipulation. It is not clear what is the role of SMAD2 in TAS actions.\nIP data show that in addition to TAS, ORY-1001, and GSK2879552 also reduce the LSD1 interaction with INSM1 despite the latter being catalytic inhibitors. How do the authors explain this? How is TAS1440's superior activity justified here?\nStudies that address the molecular basis of resistance using INSM1-KO need to be verified using reexpression of INSM1 in INSM1-KO background and show that reinstating INSM1 expression restores TAS1440 efficacy.\nFigure 7I has no significance as this data was generated using NSCLC. I think this data can be removed from the manuscript as earlier studies have already demonstrated the LSD1 survival correlation in NSCLC.", "reviewer_5": "This manuscript describes a new LSD1 (histone demethylase) inhibitor and its potential pharmacological activity against a sub-type of small-cell lung cancer. The bottom line is that the LSD1 inhibitor disrupts the interaction between the demethylase and INSM1. In this way, this transcription factor looses its ability to function as a repressor of NOTCH and TGF-β signaling. These pathways are thereby activated and induce an epigenetic reprogramming that halts cell proliferation. The authors must be congratulated for the extensive amount of work that they describe in this submission. The experiments are extremely well illustrated and described by the narrative. My enthusiasm is, however, considerably decreased by the consideration that the manuscript almost ignores previous literature. TAS1440, the hereby reported inhibitor, is chemically very similar to CC-90011, a non covalent inhibitor described 15 years ago (10.1021/acs.jmedchem.0c00978) that was moved forwards to clinical trials (doi.org/10.1021/acs.jmedchem.3c02133). Moreover, there is already quite some literature describing the interplay between INSM1 and LSD1 in SCLC. For instance, doi.org/10.1158/0008-5472.CAN-16-3502 shows that inhibition of the LSD1-INSM1 interaction \"inhibits expression of neuroendocrine-associated genes, such as ASCL1\", anticipating the conclusion of this work. Similar conclusions were given in reference 10 whereas reference 24 demonstrates that \"T-3775440 (an irreversible LSD1 inhibitor) blocks proliferation of SCLC cells in vitro and retards SCLC tumor growth in vivo. T-3775440 disrupts the interaction between LSD1 and the transcriptional repressor INSM1, thereby inhibiting expression of neuroendocrine-associated genes, such as ASCL1. INSM1 silencing phenocopies the effects of T-3775440 on gene expression and cell proliferation, consistent with the likelihood T-3775440 mediated its effects in SCLC by inhibiting INSM1\". These are virtually the same take-home message offered by this manuscript. The relevance is this work is the demonstration that stratification of the cancer sub-type is critical and that the levels of ASCL1 can be prognostic. The manuscript rationalizes this phenomenon describing a plausible molecular mechanism.\nWhether this manuscript meets the degree of novelty expected by the journal is an editorial decision. I recommend that the Authors extensively revise their manuscript highlighting their new findings while properly referring to and comparing with previous work whenever this is relevant. I think that it would be important to compare TAS1440 with CC-90011 with regard to their antiproliferative activity and cell-type selectivity. Given their structural similarity, it may well be possible that they behave the same or with only marginal differences."}, "author_response": "We would like to thank Reviewer #1 for their careful and expert appraisal from the perspectives of targeted therapy, drug resistance, and ChIP‑seq. We have undertaken a substantial revision to address the points raised, including (i) standardizing the cell‑growth calculation and updating all affected figures, (ii) adding in vivo efficacy across multiple SCLC‑A models, (iii) strengthening on‑target specificity with LSD1 knockout and catalytic‑dead rescue, (iv) mechanistic clarifications with ChIP/ChIP‑seq and co‑IP, and (v) survival analyses in SCLC (including SCLC‑A). Please refer to our point‑by‑point responses below.\n#1-1\n1.In Fig. 2a-b, the method used to calculate cell growth (%) is weird (Lines 539-546). The authors tend to exaggerate the impacts of inhibitors using two different formula dependent on the cell number of treatment groups when compared to the control.\nResponse: We appreciate the reviewer’s insightful comment. We agree that our original calculation of “cell growth (%)”—which switched between two formulas when post‑treatment cell numbers fell below the initial seeding density—could inflate apparent inhibition, particularly at high doses with substantial cytotoxicity. To eliminate this ambiguity and ensure consistent comparability across conditions, we have adopted a single definition throughout the manuscript:\nCell growth (%) = (cell number in each drug treatment group / cell number in DMSO control group) × 100.\nWe have recalculated and updated all affected figures and legends (e.g., revised Fig. 2a–d; Fig. S2a, b; Fig. S3a; and other panels quantifying growth inhibition) and revised the Methods accordingly. This uniform approach aligns with standard practice, improves clarity and reproducibility, and does not change the study’s main conclusions. For transparency, we now also clarify in the Methods how values are interpreted under strongly cytotoxic conditions and provide the exact time‑points and seeding parameters used for normalization.\n#1-2\n2.The authors proposed that TAS1440 could serve as a therapeutic option for SCLC, particularly for the SCLC-A subtype with high INSM1 expression. While their data showed that several SCLC-A cell lines were sensitive to TAS1440, some other SCLC-A lines, including NCI-H69, DMS79, SHP-77, and NCI-H2081, appear resistant to TAS1440 (Fig. 2b and Extended Data Fig. 2a). What are the potential mechanisms underlying the differential responses among different subtype-A cell lines to TAS1440 treatment? Could this variability be related to the differences of baseline INSM1 levels, or attributed to the variations of NOTCH or TGF-β signaling activity?\nResponse: We appreciate the reviewer’s thoughtful question. To understand why a subset of SCLC-A lines remains refractory to TAS1440, we stratified our panel into TAS1440-sensitive vs. -insensitive groups by dose–response behavior (revised Fig. 3a) and examined baseline and on-treatment features that track with drug response.\n(i) Target abundance (baseline).\nAcross the full panel and within SCLC-A, KDM1A/LSD1 expression was significantly higher in the insensitive group (revised Fig. 3c), consistent with reduced effective target engagement for an H3-substrate-competitive inhibitor at standard doses. In contrast, baseline INSM1 (and ASCL1) did not differ between sensitive and insensitive SCLC-A lines—likely reflecting uniformly high expression within this lineage (Fig. R1 for Reviewer).\n(ii) On-treatment derepression capacity.\nDrug response correlated strongly with the magnitude of INSM1 downregulation on treatment (ΔINSM1) rather than with baseline INSM1 (revised Fig. 3f; correlation statistics are reported in the legend). Consistently, GSEA of RNA-seq after TAS1440 showed NOTCH pathway enrichment in sensitive lines (both across the panel and within SCLC-A), whereas TGF-β enrichment was not consistently different between groups (revised Fig. 3d; Fig. R1 for reviewer). These data support a model in which TAS1440 efficacy appears to require the ability to derepress NOTCH signaling downstream of LSD1 inhibition in an INSM1-high neuroendocrine context.\n(iii) Functional perturbations.\nINSM1 knockdown in two sensitive SCLC-A lines (NCI-H1417, NCI-H510A) reduced TAS1440 sensitivity (revised Fig. S13j, k). Conversely, INSM1 overexpression in two resistant non-neuroendocrine lines (NCI-H211/SCLC-P and NCI-H196/SMARCA4-deficient) increased TAS1440 sensitivity and induced NE features (revised Fig. S17a–j). In the resistant SCLC-A line DMS79, ectopic INSM1 increased TAS1440-responsive DEGs and yielded partial post-treatment activation of both pathways (revised Fig. S17e–j).\nConclusion.\nHeterogeneous TAS1440 sensitivity within SCLC-A appears to reflect a combination of (1) target abundance (higher LSD1 dampens a substrate-competitive inhibitor) and (2) on-treatment derepression competence—specifically, the ability to downregulate INSM1 and reactivate NOTCH upon LSD1 inhibition. This framework explains why several SCLC-A lines (e.g., NCI-H69, DMS79, SHP-77, NCI-H2081) are less responsive despite shared lineage identity. Full statistics (effect sizes, n, tests, and P-val) are provided in the revised figure legends; definitions of sensitive/insensitive and dose–response modeling are detailed in the Methods.\n#1-3.\n3. In Fig. 5g, what is the difference between the left and right panels? They seem identical.\nResponse: Thank you for pointing this out. Originally, this figure was designed to appear three-dimensional, but it has been changed to a standard two-dimensional structure to avoid confusion. Please refer to the revised Fig. 5i.\n#1-4\n4. Only one cell line is used to evaluate the efficacy of TAS1440 in vivo (Fig. 7j). At least two should be studied.\nResponse: We thank the reviewer for this important suggestion. In response, we have broadened our in‑vivo evaluation beyond the original NCI‑H1417 model to include three additional SCLC‑A xenograft models—NCI‑H146, NCI‑H510A, COR‑L51 and a INSM1 knockout (KO) NCI-H146 xenograft model. TAS1440 significantly suppressed tumor growth across all SCLC-A additional models (revised Fig. 9a-e; Fig. 9g and Fig. S18e, i), consistent with our initial H1417 findings (revised Fig. 9a), whereas INSM1 knockout (KO) NCI-H146 xenografts were non responsive (revised Fig. 9f). To address microenvironmental relevance, we have conducted an orthotopic study by implanting SCLC‑A cells into the lung parenchyma. TAS1440 treatment markedly lowered intrathoracic tumor burden. CT imaging revealed a 66.7% reduction in tumor area at the maximal radius (revised Fig.9k). These concordant results strengthen the generalizability of TAS1440’s in‑vivo efficacy within SCLC‑A and support its subtype‑selective therapeutic potential. Details of dose, schedule, sample size, randomization/blinding, effect sizes with 95% CIs, and statistical tests are reported in the revised figure legends and Methods sections.\nThe following text has been added to the manuscript (Results):\n“To evaluate the in‑vivo efficacy of TAS1440, we assessed tumor growth inhibition in xenograft mouse models derived from four SCLC‑A cell lines (NCI‑H1417, NCI‑H146, NCI‑H510A, COR‑L51). TAS1440 treatment significantly reduced tumor volume vs. vehicle across all models (Fig. 9a-e; Fig. 9g and Fig. S18e, i).” Study design parameters (dose/schedule), randomization and blinding procedures, n, and statistical analyses are provided in the figure legends and Methods.\n#1-5\n5. In Fig. 7l, the authors assessed the prognostic value of KDM1A, NOTCH1 and TGFB2 in NSCLC but not SCLC patients. To strengthen their conclusions, the authors should perform survival analyses using SCLC datasets. Although clinical data for SCLC are limited, studies with larger sample sizes of SCLC with RNA-seq data are available (PMID: 38181741; PMID: 27093186). Moreover, since the authors concluded that “This demonstrates the prognostic and therapeutic implications of targeting the INSM1-LSD1 complex in small cell lung cancer, particularly in SCLC-A subtype” (Lines 420-422), it would be ideal to evaluate these survival analyses specifically in the SCLC-A subtype. The evaluation of prognostic implications of INSM1 in SCLC would also be informative.\nResponse: We appreciate the reviewer’s insightful comment. In response, we have removed the prior NSCLC‑based analysis and performed overall‑survival analyses in a publicly available SCLC RNA‑seq cohort (Liu et al. 2024; PMID: 38181741).\nIn the entire SCLC cohort, higher KDM1A (LSD1) expression was significantly associated with shorter overall survival, supporting its potential prognostic relevance (revised Fig. 9l). By contrast, lower INSM1 showed a trend toward longer survival that did not reach statistical significance in the overall cohort (revised Fig. 9l).\nTo address the request for subtype‑restricted evaluation, we analyzed the SCLC‑A subgroup defined by an ASCL1‑high transcriptional program, consistent with current consensus. Directionally similar trends were observed for both KDM1A and INSM1, but associations did not reach statistical significance, likely reflecting limited power within subtype‑restricted strata (revised Fig. 9m). These findings are directionally concordant with prior pathology‑based reports linking lower INSM1/LSD1 expression to better outcomes in SCLC (Sakakibara R, et al. Am J Surg Pathol 44:757-764,2020., Xu et al. Pathol Res Pract;229:153693,2022.).\nFor transparency, we now restrict clinical inferences to SCLC datasets. Analytic details were pre‑specified: median expression cut‑offs, Kaplan–Meier log‑rank tests, and Cox models reporting hazard ratios with 95% confidence intervals; multiple‑testing control is indicated in the figure legends and Methods sections. We note the inherent limitations of available resources (lack of uniform pretreatment/response annotation, bulk RNA‑seq averaging of intra‑tumoral heterogeneity, and modest sample sizes for subtype‑specific analyses). While clinical prognostication is not the primary aim of this mechanistic work, these results support—but do not overstate—the clinical relevance of the INSM1–LSD1 axis in SCLC, particularly within SCLC‑A.\n#1-6\n6. Fig. s8e showed that TAS1440-induced NOTCH1 upregulation was significantly impaired in INSM1-deficient cells (lane 3), suggesting that INSM1 is required for TAS1440-induced NOTCH1 upregulation. However, the same data also showed that NOTCH1 expression was markedly upregulated in INSM1-deficient cells without TAS1440 treatment (lane 2), indicative a suppressive role of INSM1 in NOTCH1 induction. How do the authors reconcile these seemingly contradictory effects of INSM1 on NOTCH1 expression?\nResponse: We appreciate this incisive question and agree that the observations can be reconciled within a single, context‑dependent derepression model. Under basal conditions, INSM1 tethers an LSD1‑containing corepressor complex, establishing a poised repressive state over tumor‑suppressive programs such as NOTCH and TGF‑β. Accordingly, loss of INSM1 derepresses these loci, elevating baseline NOTCH1 (revised Fig. S14e, lane 2) by removing the repressive scaffold. Upon TAS1440 exposure, inhibition of LSD1 catalytic activity together with disruption of the INSM1–LSD1 complex lifts repression and induces pathway activation. However, in INSM1‑deficient cells, the TAS‑sensitive repressive complex is already absent; baseline NOTCH1 is partially elevated, and the dynamic range for further TAS‑mediated induction is reduced, yielding the attenuated upregulation observed (revised Fig. S14e, lane 3).\nConsistent with this model, INSM1 knockdown did not alter H3K4me1/2/3 at TSSs, whereas H3K27ac was significantly increased, indicating release of repression; NOTCH1 and TGFB2 showed corresponding increases in H3K27ac and transcript levels (revised Fig. 8n–r). Baseline repression is further supported by INSM1‑overexpression experiments in H196 and DMS79, where ectopic INSM1 enhanced TAS1440‑induced increases in pSMAD2 and the induction of NOTCH1, albeit with cell line-dependent variation. (revised Fig. S17g-h, lane3 vs. lane1).\nTogether, these data support INSM1 acting primarily as a chromatin‑tethered repressor at these loci, with TAS1440 functioning by derepressing INSM1/LSD1‑programmed sites. This framework parsimoniously explains both (i) the baseline NOTCH1 increase upon INSM1 loss and (ii) the blunted incremental induction by TAS1440 in the absence of INSM1.\nTo improve clarity, we have (i) added baseline and on‑treatment NOTCH1 quantification (fold‑change, effect sizes, and P values) to illustrate the dynamic‑range effect and (ii) revised the text to explicitly state this derepression model (revised Fig. S14d–e; Results, pp. 23–24).\n#1-7\n7. The authors stated that “TAS1440 treatment resulted in the dissociation of 78 nuclear factors, of which 13 were epigenetic factors, from LSD1 (Fig. 5c, red)” (Lines 264-266). However, only 6 genes are labeled in red in Fig. 5c.\nResponse: Thank you for drawing this to our attention. We agree that the labeling in the original (former) Fig. 5c was not sufficiently clear. In the revised Fig. 5c, all 13 epigenetic factors that dissociate from LSD1 upon TAS1440 exposure are now consistently highlighted in red, matching the statement in the text. To aid interpretation, we have boxed the five interactors prioritized for experimental validation (INSM1, SMAD2, HDAC1, HDAC2, RCOR1). As shown by the accompanying co‑immunoprecipitation/immunoblot analyses (revised Fig. 5e, f), these proteins indeed dissociate from LSD1 following TAS1440 treatment.\nFor transparency, the figure legend now defines the color/box annotations and specifies the criteria for “dissociation” (significance threshold and fold‑change), and the Source Data file lists the full set of 78 nuclear factors and the 13 epigenetic factors.\nReviewer #2 (Remarks to the Author): An early career researcher co-reviewer\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We appreciate Nature Communications’ Early Career Researcher co‑review initiative and thank the co‑reviewer for their contribution.\nReviewer #3 (Remarks to the Author): neuroendocrine SCLC and targeted therapy\nThe work by Machida et al. highlights the development of a novel LSD1 inhibitor (TAS1440) and demonstrate its potential efficacy in small cell lung cancer. The work on the validation of the drug, including its mechanism of action, the exertion of its effect through INSM1 and the connection to NOTCH signaling is very convincing. The authors have done an excellent job of characterizing a novel epigenetic modifier.\nWhere the manuscript struggles, however, is the focus on small cell lung cancer. That is not to say that small cell lung cancer is not a worthy target of this focus – quite the contrary – but the authors familiarity with small cell lung cancer appears outdated and the manuscript’s impact suffers mightily from this. There has been significant progress in small cell lung cancer – both preclinical and clinically – in the past 3-4 years and the authors seem completely unaware of this, as evidenced by the fact that their most recent citation for small cell lung cancer is the Ireland et al. Cancer Cell manuscript from 2020. This includes even recent publications on LSD1 inhibition in small cell lung cancer (e.g. Nguyen et al. J Thorac Oncol 2022). Despite the limitations in the authors command of the SCLC literature, there is a convincing case that the activity of TAS1440 is most robust in the neuroendocrine-high subtypes, especially SCLC-A. Their in vitro and in vivo data support this well.\nSignificant small cell lung cancer-focused revisions to text and experiments would be necessary, in my opinion, to make this a valid addition to the small cell lung cancer body of literature, including:\nResponse: We sincerely appreciate the constructive critique and agree that our initial submission did not sufficiently reflect recent advances in SCLC biology and therapy. We have therefore comprehensively realigned the manuscript to current consensus by:\n Updating SCLC subtyping (restricting to SCLC-A/N/P, removing the legacy SCLC-Y/SMARCA4-deficientlines from canonical analyses per recent reports) and revising the figures accordingly (from revised Fig. 2 onward).\n Expanding in-vivo validation beyond H1417 to three additional SCLC-A xenografts (revised Fig. 9). Consistent antitumor activity was also confirmed in an orthotopic lung model.\n Replacing the prior NSCLC survival panel with SCLC-only overall-survival analyses, including SCLC-A–restricted evaluation (Liu et al., 2024; PMID: 38181741; revised Fig. 9l, m).\n Strengthening on-target and mechanistic evidence (LSD1 KO dependency; WT/K661A rescue; complex disruption; epigenomic readouts).\n Reframing the work within the modern SCLC literature, explicitly integrating Nguyen et al. (JTO 2022) and other key studies (Baine 2020; Gay 2021; Ng 2024; George 2015; Nabet 2024) into the Introduction/Discussion.\n Where appropriate, we now discuss SCLC-I–related inflammatory programs and the rationale for ICB combinations in the context of LSD1 inhibition.\nPlease refer to our detailed, point-by-point responses below.\nMajor Issues:\n#3-1\n(1) The authors focus on the SCLC subtyping proposed by Rudin et al. It should be noted that this was a review and no longer reflects the consensus subtyping approach. In particular, while Rudin et al. proposed YAP1 as a subtype-defining transcription factor, this has been largely dismissed, including from work by Dr. Rudin’s group (see Baine et al. J Thorac Oncol 2020), as well as Gay et al. Cancer Cell 2021 and Ng et al., Clin Cancer Res, 2024. In particular, the three cell lines chosen by the authors here to represent the supposed SCLC-Y subtype were shown in the Ng et al. manuscript to be SMARCA4-deficient undifferentiated tumor in two cases and a sarcomatoid spindle cell neoplasm in the other case (see Table 1). At minimum, the figures should be revised to exclude these cell lines and the SCLC-Y subtype completely.\nResponse: We thank the reviewer for raising this important point. We agree that the YAP1‑defined “SCLC‑Y” category proposed in the 2019 review by Rudin et al. no longer reflects current consensus. Subsequent studies—including Baine et al. (J Thorac Oncol, 2020), Gay et al. (Cancer Cell, 2021), and Ng et al. (Clin Cancer Res, 2024; Table 1)—indicate that cell lines historically labeled “SCLC‑Y” were misclassified, corresponding instead to SMARCA4‑deficient undifferentiated tumors or sarcomatoid spindle‑cell neoplasms. In line with this literature, we re‑examined our transcriptomic data and confirmed markedly reduced SMARCA4 expression in the previously designated SCLC‑Y lines (revised Fig. 1e), supporting their exclusion from canonical SCLC analyses.\nAccordingly, we have removed the SCLC‑Y subtype and all associated cell lines from downstream analyses. From revised Fig. 2 onward, we focus exclusively on the currently recognized subtypes—SCLC‑A, SCLC‑N, and SCLC‑P—and have updated all figures, legends, and text to align with this framework. For transparency, we provide a supplementary table listing included/excluded cell lines with the rationale for each decision (Supplementary Table S3). We believe these revisions improve rigor, interpretability, and alignment with contemporary SCLC biology.\n#3-1-a\n(2) Once the authors appropriately reconcile their subtypes in Figure 1 and beyond with the current state of the SCLC literature, an key, overlooked direction is how LSD1 inhibition impacts the subtype landscape. This has, to a degree, already been addressed for earlier LSD1 inhibitors – see Nguyen et al. manuscript referenced above. It’s not clear why the authors seem to ignore this manuscript, rather than follow its format and experiments to highlight the ways in which their LSD1 inhibitor may be superior.\na. The authors make no mention of SCLC-I, the inflamed fourth subtype of SCLC. The earlier work by Nguyen et al. suggests that LSD1 inhibition can shift uninflamed, neuroendocrine high SCLC (e.g. SCLC-A) to a more inflamed state reminiscent of SCLC-I. This would be critical to explore here.\nResponse: Thank you for this insightful suggestion. We fully acknowledge the clinical relevance of the inflamed SCLC‑I subtype and prior evidence that LSD1 inhibition can shift neuroendocrine‑high states (e.g., SCLC‑A) toward a more inflamed phenotype (Nguyen et al., J Thorac Oncol 2022). Because SCLC‑I designation relies on tumor–immune context (immune‑cell infiltration, IFN‑γ pathway activity, antigen presentation)—features not faithfully recapitulated in 2D cell‑line systems—we did not assign an SCLC‑I label in vitro to avoid over‑interpretation.\nNevertheless, we have adopted an analytic framework consistent with Nguyen et al. and systematically queried immunologic rewiring under TAS1440. We have also profiled canonical markers (IFNG, CXCL9/CXCL10, HLA‑A/B/C, B2M, TAP1/TAP2, STAT1, IRF1, CD274/PD‑L1) and performed GSEA on RNA‑seq data. TAS1440 decreased CD274 (PD‑L1) while increasing MHC‑I components (e.g., B2M) and IFN‑γ–responsive genes (e.g., IRF1); inflammatory signatures were activated in a subset of lines, although unsupervised clustering did not yield a robust SCLC‑I‑like group in vitro (revised Fig. S9d, f). Where applicable, NES and P values are reported in the figure legend to aid interpretation. Importantly, a decrease in PD‑L1 transcripts does not preclude enhanced immunogenicity when antigen‑presentation and IFN‑γ programs increase concurrently, which can raise tumor visibility to cytotoxic T cells.\nTaken together, while TAS1440 induces MHC‑I/IFN‑γ–responsive genes in a subset of lines (for example, B2M, TAP1/2, IRF1; Fig. S9d, f), strict assignment to the SCLC‑I subtype is not possible in vitro using bulk RNA‑seq. We therefore avoided SCLC‑I labeling and instead reported pathway‑level changes with appropriate caveats. Future work will test orthotopic/PDX and immune co‑culture models to evaluate ICB combinations with predefined pharmacodynamic readouts. We have added these analyses and a tempered discussion to the revised manuscript (Results/Methods/Discussion; revised Fig. S9d, f).\n#3-1-b\nb. Do the tumors that are SCLC-A and effectively treated with TAS1440 shift to SCLC-N? SCLC-P? SCLC-I? ASCL1 goes down, but what arises as a result? Even YAP1 is a possibility here, as demonstrated previously by Ireland et al. (which the authors do cite).\nResponse: Thank you for this thoughtful question on subtype plasticity after TAS1440. Across multiple SCLC‑A cell lines, TAS1440 consistently downregulated ASCL1 and INSM1, consistent with disruption of the LSD1–INSM1–ASCL1 axis. To determine whether this suppression precipitates a lineage switch, we have profiled subtype‑defining programs—NEUROD1 (SCLC‑N), POU2F3 (SCLC‑P), and YAP1 activity (assessed as a pathway readout rather than as a distinct “SCLC‑Y” subtype)—before and after TAS1440 (revised Fig. S9a). We did not observe reproducible induction of NEUROD1, POU2F3, or YAP1‑associated signatures. Unsupervised clustering based on canonical marker sets likewise indicated that overall transcriptional identity remained largely stable, with no coordinated transition from SCLC‑A to other subtypes (revised Fig. S9b, c; enrichment statistics are reported in the legend).\nAs these experiments reflect short‑term (≤7‑day) in‑vitro exposure, delayed plasticity under chronic dosing and/or microenvironmental cues cannot be excluded. Within the tested window, TAS1440‑mediated ASCL1 suppression attenuates the neuroendocrine program but does not, by itself, drive a switch to SCLC‑N, SCLC‑P, or an SCLC‑I‑like state. These results and their limitations have been incorporated into the revised Results/Discussion (revised Fig. S9a–c).\n#3-2-c\nc. As LSD1 inhibitors are unlikely to serve as successful single-agent therapies in SCLC, the possibility of combination therapies and the rationale for them is underexplored. If the tumors become more inflamed, could this agent be combined with immune checkpoint blockade? That experiment may be beyond the scope of the publication due to requirement for immunocompetent models, but at least some data supporting more inflammatory features (MHC expression, IFN-y, etc. a la Nguyen et al.) would be appropriate.\nResponse: We thank the reviewer for this valuable comment. We agree that LSD1 inhibitors are unlikely to deliver durable benefit as monotherapies in SCLC and that rational combinations—particularly those leveraging immunologic reprogramming—are essential for translation. Prior reports (e.g., Nguyen et al., J Thorac Oncol 2022; Lin et al., Cell Reports 2024) have shown that LSD1 inhibition can enhance antigen‑presentation programs and IFN‑γ–responsive transcription.\nTo assess whether TAS1440 elicits similar effects, we analyzed RNA‑seq from 19 SCLC cell lines and profiled MHC‑I components, IFN‑γ signaling, and immune‑checkpoint genes after treatment. TAS1440 downregulated CD274 (PD‑L1) while upregulating MHC‑I pathway genes (e.g., B2M) and IFN‑γ–responsive genes (e.g., IRF1) (revised Fig. S9d). Gene‑set enrichment analysis further showed activation of inflammatory/immune‑related signatures in a subset of models (revised Fig. S9f); NES and FDR values are reported in the legend. As expected in cell‑line systems lacking an intact tumor–immune microenvironment, these changes were not uniformly correlated with growth sensitivity; however, they are consistent with a shift toward greater antigen‑presentation capacity, even with decreased PD‑L1 transcripts.\nConsistent with this concept, studies of other reversible LSD1 inhibitors (e.g., Bomedemstat, SP‑2577, CC‑90011) reported increased CD8⁺ T‑cell infiltration, induction of IFN‑γ–related genes, and improved responses to anti–PD‑1 in preclinical settings. Although direct combination experiments are beyond the scope of our current in‑vitro study, these transcriptomic data (revised Fig. S9d, f) provide a mechanistic rationale to test TAS1440 + immune checkpoint blockade (ICB) in microenvironment‑preserving systems (orthotopic, syngeneic, or humanized/PDX), with prespecified pharmacodynamic endpoints (MHC‑I upregulation, IFN‑γ‑response gene induction, intratumoral CD8⁺ T‑cell infiltration) and objective tumor‑control readouts. We have added these data and an expanded discussion to the revised manuscript.\n#3-2-d\nd. Combinations with chemotherapies? Impact on expression of high-profile targets (e.g. DLL3 – the target of b – or several ADC targets including B7H3, SEZ6, TROP2). Maybe it’s not a good thing to downregulate ASCL1 (and, presumably, DLL3 and SEZ6) since the standard of care for SCLC is now tarlatamab.\nResponse: Thank you for raising this important point regarding how TAS1440 may influence clinically actionable surface targets and, by extension, combination strategies.\nUsing RNA‑seq, we compared expression of relevant antigens before and after TAS1440 (revised Fig. S9e): DLL3 (tarlatamab, a DLL3‑CD3 bispecific T‑cell engager), SEZ6 (investigational ADCs), B7‑H3/CD276 (enoblituzumab, MGC018), and TROP2/TACSTD2 (sacituzumab govitecan). Across multiple SCLC‑A lines, TAS1440 significantly downregulated DLL3, whereas SEZ6, CD276 (B7‑H3), and TACSTD2 (TROP2) remained largely unchanged across SCLC‑A lines (revised Fig. S9e). Consistent with DLL3 being downstream of ASCL1, which itself is suppressed by LSD1 inhibition, NOTCH pathway enrichment (NES) negatively correlated with ASCL1 and DLL3 fold changes (R = −0.66 and R = −0.63, respectively; Fig. S9g). This aligns with our mechanistic model whereby TAS1440 disrupts the LSD1–INSM1–ASCL1 axis and indirectly activates NOTCH signaling. In contrast, SEZ6, B7‑H3, and TROP2 were largely unchanged after TAS1440, suggesting these ADC targets may remain exploitable in combination regimens (summary statistics are reported in the legend).\nPractical implications: Because DLL3 declines under TAS1440, concurrent TAS1440–tarlatamab administration is unlikely to be optimal. A more rational approach would be sequencing—deploying tarlatamab when DLL3 expression is highest and reserving TAS1440 for windows aimed at transcriptional reprogramming—rather than simultaneous dosing. More broadly, the relative stability of SEZ6, B7‑H3, and TROP2 supports the feasibility of ADC‑based combinations that do not rely on DLL3 while TAS1440 is on board. We note that these are transcript‑level observations; surface protein levels were not directly quantified here.\nRegarding chemotherapy combinations, although outside the scope of the present work, future studies could evaluate TAS1440 with standard cytotoxics (e.g., platinum–etoposide or topotecan), using prespecified synergy metrics and schedule optimization, while monitoring pharmacodynamic markers (ASCL1/DLL3 modulation, DNA‑damage/repair signatures) and non‑overlapping toxicity. These considerations provide a clear framework for biomarker‑guided sequencing and combination strategies in SCLC.\n#3-3\n(3) In the final paragraph of the results, the authors shift to NSCLC for survival analysis. This is clearly not appropriate to support the implications of targeting LSD1 in SCLC-A. Despite the authors comments, there are data available with transcriptional and clinical data for SCLC, including George et al. Nature 2015, Nabet et al. Cancer Cell 2024, and several others. If the authors wish to include clinical data, they need to gain access to one of several publicly available SCLC datasets.\nResponse: We appreciate this thoughtful comment. In response, we removed the prior NSCLC‑based analysis and performed overall survival analyses in a publicly available SCLC RNA‑seq cohort (PMID: 38181741). In the full SCLC cohort, higher KDM1A (LSD1) expression was significantly associated with shorter overall survival, supporting the prognostic relevance of LSD1 (revised Fig. 9l). By contrast, lower INSM1 expression showed a trend toward improved survival that did not reach statistical significance in the overall cohort (revised Fig. 9l).\nTo meet the request for subtype‑restricted evaluation, we further analyzed the SCLC‑A subgroup (defined by an ASCL1‑high transcriptional program aligned with current consensus). Directionally similar trends were observed for KDM1A and INSM1, but associations did not reach statistical significance, likely reflecting limited power within subtype‑restricted strata (revised Fig. 9m).\nThese findings are directionally concordant with prior pathology‑based reports linking lower INSM1/LSD1 expression to better outcomes in SCLC, while differences in cohort composition and therapy may contribute to variability across studies. For transparency, we now restrict all clinical inferences to SCLC datasets. Analytic details were prespecified (median expression cut‑offs; Kaplan–Meier log‑rank tests; Cox proportional‑hazards models reporting hazard ratios with 95% CIs; multiple‑testing control as appropriate), and cohort size/event counts are reported in the figure legends and Methods sections. Although clinical prognostication is not the primary aim of this mechanistic study, future work—where feasible—could extend survival analyses to additional public SCLC resources (e.g., George et al., 2015; Nabet et al., 2024) to further refine the clinical implications of the INSM1–LSD1 axis.\nMinor Issues:\n(1) Page 6, line 102. Should this read “potently” rather than “potentially”?\n(2) Figure 2B would be easier to interpret if the cell line subtypes were labeled.\n(3) The in-text reference for Figure 2D arrives after the in-text reference for Figure 2E.\n(4) Page 15, line 317 seems to contain a typographical error “and GSK2879552”.\nResponse: We thank the reviewer for the careful reading and helpful suggestions. All items have been corrected in the revised manuscript, as follows:\n(1) “potentially” has been corrected to “potently” (p. 6, line 102).\n(2) Cell‑line subtypes are now explicitly labeled in Fig. 2b (panel and legend) to improve interpretability.\n(3) The in‑text figure citations have been reordered so that Fig. 2d is cited before Fig. 2e at first mention.\n(4) The typographical error on p. 15, line 317 has been corrected (removal of the extraneous “and” before “GSK2879552”).\nReviewer #4 (Remarks to the Author): LSD1 inhibitor, epigenetic targeting and mass spectrometry\nIn this manuscript, Machida et al. studied the therapeutic effect and understand the mechanism of the new LSD1 inhibitor TAS1440 in neuroendocrine SCLC. Earlier studies showed that LSD1 inhibition derepresses notch and TGFb signaling resulting in suppression of SCLC growth. This study presents the first development of a new LSD1 inhibitor, TAS1440 which is a histone H3 competitive inhibitor of LSD1. TAS1440 demonstrated superior antiproliferative activity in SCLC-A subtype cells and exhibited in vivo activity in subcutaneous xenograft models. Mechanistically, TAS effects are attributed to both direct inhibition of LSD1 enzymatic activity as well as disruption of LSD1-repressive complexes leading to coordinated regulation of histone modifications and activation of transcription factors INSM1 and SMAD2 which reprograms the NOTCH and TGFb tumor suppressor pathways. Further depletion of INSM1 resulted in the abrogation of TAS1440 effects, suggesting the dependency of INSM1 for TAS1440 functions.\nThis study is interesting as TAS has the dual function of inhibition of LSD1 enzymatic activity and disrupting LSD1 complexes and holds significant promise as a new targeted therapy for the SCLC-A subtype with high INSM1 expression. Data was generated rigorously using multiple cell lines and demonstrated the superior activity of TAS1440 than existing LSD1 inhibitors, and mechanistic studies were thoroughly conducted using RNA-seq of 22 cell lines, as well as ChIP-seq and mass spec studies and rigorous validation in multiple cell lines. The figures are good quality and easy to follow and the manuscript is well written.\nHowever, there are several issues noted in the manuscript and several claims are overstated without concrete evidence. The novelty of the study is moderate as LSD1's role in SCLC, and the utility of other LSD1 inhibitors was thoroughly investigated, although these inhibitors are associated with significant toxicity. The authors claim that TAS1440 has high specificity, less off-target effects, and is a safer drug, which could be a breakthrough for this study. Unfortunately, there was not enough data presented to support this statement. Further the objective states the identification of chemoresistance drivers, however no such studies using chemoresistance cells were conducted. In vivo studies are preliminary in nature, and only one subcutaneous xenograft model was used.\nResponse: We are grateful to Reviewer #4 for their insightful comments emphasizing (i) firm evidence of on‑target engagement, (ii) the relative novelty of TAS1440 in the context of prior LSD1 inhibitors, and (iii) the need for broader in vivo support. Guided by these suggestions, we have added genetic and biochemical validation of TAS1440–LSD1 engagement, expanded comparative profiling vs. other inhibitors, and strengthened our in vivo dataset. We have also revised the text to avoid overstatement and to align claims with the new evidence. Please refer to our detailed responses below.\n#4-1\nThe on-target specificity of the compound should be established using LSD1-KO cells. Further, it is not clear if TAS1440 directly interacts with LSD1. This can be done using biotin-labeled TAS-1440 and LSD1 protein using avidin pulldown.\nResponse: We appreciate the reviewer’s important comment regarding the on‑target specificity of TAS1440.\nGenetic dependency: To directly assess target dependency, we generated CRISPR–Cas9 LSD1‑knockout (KO) derivatives of NCI‑H1417 and NCI‑H146 (revised Fig. 7a, b; Fig. S12a, b). Upon TAS1440 exposure, LSD1‑KO cells were completely refractory to growth inhibition, in contrast to parental controls (revised Fig. 7c; Fig. S12c), indicating that TAS1440’s antiproliferative activity is LSD1‑dependent.\nAs a selectivity benchmark, we compared SP2509, a reported LSD1 inhibitor with off‑target liabilities: SP2509 retained partial activity in LSD1‑KO cells, consistent with mixed LSD1‑dependent and ‑independent effects, whereas TAS1440 lost activity entirely in the absence of LSD1 (revised Fig. 7c; Fig. S12c), reinforcing high target specificity.\nTranscriptomic concordance: RNA‑seq further supported on‑target engagement. In LSD1‑KO NCI‑H1417 cells reconstituted with empty vector, TAS1440 induced minimal differential expression (MA plot, revised Fig. 7k), whereas a broad transcriptional response was observed in LSD1‑WT cells (revised Fig. 7i).\nGSEA/KEGG showed robust enrichment of TAS1440‑associated pathways—most notably TGF‑β and NOTCH signaling—in WT but substantially attenuated responses in KO‑empty cells (revised Fig. 7m).\nBiochemical selectivity and mechanism: Using recombinant enzymes, TAS1440 inhibited LSD1 with an IC₅₀ of 4.8 nM, while showing minimal inhibition of LSD2, MAO‑A, MAO‑B, and a panel of chromatin regulators (HDAC1, DNMT1, PRMT4, SMYD2, JmjC demethylases), each with IC₅₀ > 10,000 nM (revised Fig. 2f, g; Fig. S4b). Substrate‑competition assays with biotinylated H3K4me1 peptide showed that increasing substrate concentration shifted the TAS1440 IC₅₀ to higher values (rightward shift), consistent with an H3‑competitive mechanism. By contrast, the covalent inhibitor ORY‑1001 displayed substrate‑insensitive IC₅₀ values, as expected for an irreversible FAD‑directed agent.\nStructural evidence for direct binding: X‑ray co‑crystal analysis shows TAS1440 occupying the histone H3‑binding pocket of LSD1 (revised Fig. 5i), providing a structural rationale for both enzymatic inhibition and disruption of the LSD1–INSM1 complex. Molecular docking further supports stable interactions within the H3 groove and proximity to INSM1/SMAD2 interaction surfaces (revised Fig. 5g, h).\nWe appreciate the suggestion to use a biotin‑labeled TAS1440 for avidin pulldown. Given the narrow H3‑binding groove observed in the co‑crystal, appending a bulky biotin is likely to perturb binding geometry and underestimate affinity/engagement. In lieu of this, we provide orthogonal lines of evidence—genetic KO abrogation, biochemical selectivity and substrate competition, and direct co‑crystal structural data—collectively establishing on‑target engagement of TAS1440 with LSD1.\n#4-2\nIn vivo studies are conducted using subcutaneous models which do not mimic the actual microenvironment. Only one xenograft model was used. Studies using orthotopic models of SCLC and syngenic models with intact immune systems should be conducted to establish the superior activity of TAS1440. Further, there are no PDX models were utilized to account for the heterogeneity of SCLC.\nResponse: We appreciate this important suggestion. In the revision, we have expanded the in vivo efficacy assessment beyond NCI‑H1417 to three additional SCLC‑A xenograft models (NCI‑H146, NCI‑H510A, and COR‑L51) and a INSM1 knockout (KO) NCI-H146 xenograft model. INSM1 wild type (WT) NCI-H146 xenografts were responsive to TAS1440, whereas INSM1 knockout (KO) NCI-H146 xenografts were non responsive (Fig. 9a-f); dose, schedule, sample size, randomization/blinding, and statistics are reported in the figure legends (revised Fig. 9), reinforcing subtype‑selective activity within SCLC‑A across multiple lines.\nTo address microenvironmental relevance, we have conducted an orthotopic study by implanting SCLC‑A cells into the lung parenchyma. TAS1440 treatment markedly lowered intrathoracic tumor burden. CT imaging revealed a 66.7% reduction in tumor area at the maximal radius (revised Fig.9k). These results are concordant with our subcutaneous data and support on‑target activity within the lung microenvironment. Methods for the orthotopic model (surgical procedure, imaging schedules, predefined endpoints, randomization/blinding) have been added to the revised manuscript.\nWith respect to immunocompetent (syngeneic) models, we have performed pilot implantations to explore drug evaluation in an intact immune context. Human‑derived SCLC cell lines failed to engraft in immunocompetent mouse strains, precluding assessment of TAS1440 activity—an outcome consistent with xenogeneic rejection. Accordingly, true syngeneic evaluation would require murine SCLC allografts or genetically engineered mouse model (GEMM)–derived lines (e.g., Rb1/Trp53 ± Rbl2 backgrounds) or de novo GEMM cohorts. Securing these resources, establishing colonies, and completing the necessary protocol amendments would exceed the current major‑revision timeline.\nRegarding patient‑derived xenograft (PDX) models, we agree they would better capture inter‑patient heterogeneity; however, acquisition, engraftment, and stabilization of early‑passage SCLC PDXs likewise extend beyond the present revision. Accordingly, we have (i) added three additional subcutaneous models and the orthotopic pilot (above) and (ii) revised the Discussion to acknowledge these limitations and to outline next‑step studies in immunocompetent and PDX settings with prespecified pharmacodynamic endpoints (e.g., pathway engagement, antigen‑presentation markers) and objective tumor‑control readouts.\n#4-3\nThere were no details provided pertaining to how the in vivo dose of TAS1440 was determined, what its MTD, MED. Concerning toxicity, it appears that this compound also reduces body weight, suspecting the potential toxicity of this compound. It is important to study whether this compound has any hematologic toxicity as commonly observed with other LSD1 inhibitors.\nResponse: We thank the reviewer for raising this important point. In the current study, TAS1440 was administered orally (p.o.) at 16.7 mg/kg/day and 50 mg/kg/day, guided by preliminary tolerability/exposure data and commonly used preclinical dosing ranges for non‑covalent LSD1 inhibitors. These regimens were generally tolerated in CB17/Icr‑Prkdc^scid/CrlCrlj mice and produced consistent antitumor effects across multiple SCLC‑A xenograft models.\nWe did not conduct formal maximum tolerated dose (MTD) or minimum effective dose (MED) studies in this work. No overt clinical signs (e.g., skin lesions, lethargy, sudden death) were observed; however, we observed body‑weight loss in TAS1440‑treated cohorts (revised Fig. S18a-d), indicating the need for careful dose optimization to balance efficacy and tolerability in future studies. To address the reviewer’s question about hematologic toxicity, we added a peripheral blood analysis. Compared with vehicle, TAS1440 produced significant reductions in red cell parameters—RBC, hemoglobin, and hematocrit—while WBC and platelets showed no significant change at the tested doses/time point; MCV, MCH, and MCHC were likewise unchanged (Fig. R2 for reviewer). This pattern is consistent with a normocytic, normochromic anemia without leukopenia or thrombocytopenia under the present conditions.\nConsistent with prior experience for reversible LSD1 inhibitors, hematologic toxicity has been documented clinically (e.g., CC‑90011: anemia 51.1%, thrombocytopenia 46.6%; Grade ≥ 3 in 13.6% and 19.3%, respectively; Rudin et al., J Thorac Oncol. 2023). These adverse events have generally been manageable with dose modification.\nWe have added these clarifications to the revised Discussion section and acknowledge the need for follow‑up toxicity profiling, including hematologic assessments and formal dose‑ranging studies, in future work.\n#4-4\nTAS has dual activity of LSD1 enzymatic inhibition and complex disruptor. However, it is essential to determine if the enzymatic activity is important for TAS effects. Efficacy and mechanistic studies need to be conducted using enzymatic dead mutant LSD1 (K661A) to ascertain whether enzymatic activity is essential for TAS1440 effects.\nResponse: We thank the reviewer for this important, mechanistically focused suggestion. LSD1 contributes to SCLC biology via both canonical (enzymatic demethylation) and non‑canonical (scaffolding/complex‑forming) functions. To isolate the role of catalytic activity in TAS1440 responses, we engineered a catalytically inactive LSD1 mutant (K661A).\nGenetic system and validation: Using CRISPR–Cas9, we ablated endogenous LSD1 and reconstituted NCI‑H1417 and NCI‑H146 with 3×FLAG–MYC–LSD1‑WT, 3×FLAG–MYC–LSD1‑K661A, or empty vector (EV). Immunoblotting confirmed increased global H3K4me1/2 in K661A‑expressing cells, consistent with loss of demethylase activity, and WT/K661A expression levels were matched by densitometry (revised Fig. 7a, b; Fig. S12a, b).\nProliferation rescue: Both WT and K661A partially restored proliferation in LSD1‑KO cells, with a smaller rescue by K661A, indicating that catalytic activity contributes to LSD1‑driven growth (revised Fig. 7e, f; Fig. S12e, f).\nTAS1440 activity in WT vs. K661A: TAS1440 inhibited growth in both WT‑ and K661A‑expressing cells, showing that activity is not solely attributable to catalytic blockade. As expected, TAS1440 increased NOTCH1 and pSMAD2 was attenuated in K661A relative to WT (revised Fig. 7g, h). qPCR for canonical NOTCH/TGF‑β targets recapitulated these trends (revised Fig. 7i).\nTranscriptomic profiling: TAS1440 elicited broader differential‑expression changes and NOTCH/TGF‑β enrichment in WT cells, whereas responses were blunted in K661A and minimal in EV (RNA‑seq; KEGG/GSEA; revised Fig. 7j–m).\nComplex disruption: Co‑immunoprecipitation (co‑IP) showed that LSD1‑K661A retains binding to INSM1 and SMAD2, and TAS1440 disrupts these interactions in both WT and K661A contexts (revised Fig. 7n), supporting a catalysis‑independent complex‑disruptor component.\nConclusion: Collectively, these data indicate that LSD1 catalytic activity is required for the full extent of TAS1440‑induced transcriptional reprogramming and pathway activation, while disruption of LSD1‑containing repressive complexes provides a second, catalysis‑independent contribution–supporting a dual‑function mechanism.\n#4-5\nIt is not clear how it is different from existing reversible noncovalent inhibitors such as SP2509.\nResponse: Thank you for raising this important question. To delineate how TAS1440 differs from existing reversible, non‑covalent LSD1 inhibitors, we benchmarked TAS1440 against SP2509 and CC‑90011 across orthogonal assays (cell growth, genetic dependency, transcriptomics, complex disruption, and structural modeling).\n1) On‑target dependency and growth phenotype: Across a panel of SCLC cell lines (revised Fig. S3a), TAS1440 and CC‑90011 produced broadly similar antiproliferative profiles, with TAS1440 showing greater potency in several sensitive lines. By contrast, SP2509 induced broad cytotoxicity at higher concentrations. Consistent with reported off‑target liabilities, SP2509 retained partial activity in LSD1‑knockout cells, whereas TAS1440 activity was abolished in the absence of LSD1 (revised Fig. 7c, Fig. S12c), supporting tighter on‑target dependence for TAS1440.\n2) Transcriptional programs and pathway activation: RNA‑seq of NCI‑H1417 and NCI‑H146 treated with five LSD1 inhibitors revealed distinct expression signatures among TAS1440, CC‑90011, and SP2509 (revised Fig. 4i, Fig. S6i). GSEA indicated that TAS1440 uniquely and robustly enriched NOTCH and TGF‑β pathways (Fig. R3 for reviewer), which was concordant with immunoblot evidence of stronger NOTCH1 and pSMAD2 induction by TAS1440 (revised Fig. 4j, k).\n3) Disruption of LSD1 repressive complexes: Co‑immunoprecipitation showed that TAS1440 efficiently disrupted LSD1–INSM1, LSD1–SMAD2, LSD1–HDAC1, and LSD1–HDAC2, whereas SP2509 did not affect LSD1–INSM1 or LSD1–SMAD2 and instead selectively perturbed LSD1–RCOR1 (revised Fig. 5e, f), indicating a distinct complex‑disruption fingerprint.\n4) Structural rationale: Docking placed TAS1440 within the histone H3‑binding pocket of LSD1, adjacent to surfaces implicated in INSM1/SMAD2 interactions, whereas SP2509 docked at a topologically distinct site (revised Fig. 5g, h), providing a plausible basis for the divergent functional effects.\nConclusion: Together, these data differentiate TAS1440 from SP2509 (and from CC‑90011) by (i) tighter on‑target dependence, (ii) dual NOTCH/TGF‑β pathway activation, and (iii) broader disruption of LSD1‑corepressor complexes, establishing a distinct pharmacologic profile among reversible, non‑covalent LSD1 inhibitors.\n#4-6\nSome of the data was generated using only one cell line, particularly Figure 4g,h,j,k. and 6d,e. This should be done using at least one more cell line.\nResponse: We thank the reviewer for this constructive suggestion. In response, we have replicated each of the single–cell-line findings in at least one additional SCLC-A model to confirm reproducibility and generalizability, mapping directly to the figures noted in the comment (former Fig. 4g, h, j, k and 6d, e):\n In vitro signaling and rescue (former Fig. 4g, h, j, k–related assays): We have repeated the rescue experiments and the comparative analyses of NOTCH1 and pSMAD2 activation under different LSD1 inhibitors in NCI-H146 (revised Fig. S6g–j) and performed parallel rescue/drug-comparison assays in NCI-H510A, yielding results concordant with NCI-H1417 (revised Fig. S8a–g). Experimental conditions (doses/timepoints) were matched to the original assays.\n Epigenomics (mechanistic support): We have conducted ChIP-seq for H3K4me1/2/3 and H3K27ac in NCI-H146, which recapitulated the TAS1440-induced chromatin changes observed in NCI-H1417 (revised Fig. S11h–o).\n Assays corresponding to Fig. 6d, e (complex disruption/pathway readouts): We have reproduced the experiments underlying Fig. 6d, e in NCI-H146 (added in revised Fig. S11h–o, as noted in the legends) and observed the same direction and magnitude of effects as in NCI-H1417, including the expected complex-disruption and pathway-activation patterns with TAS1440.\n In vivo generalization (context): Beyond NCI-H1417, we have expanded xenograft efficacy to NCI-H146, NCI-H510A, COR-L51 and INSM1 knockout (KO) NCI-H146 xenografts; INSM1 wild type (WT) xenografts were responsive to TAS1440 (revised Fig. 9a-e; Fig. 9g and Fig. S18e, i), consistent with our initial H1417 findings (revised Fig. 9a) whereas INSM1 knockout (KO) NCI-H146 xenografts were non responsive (revised Fig. 9f); TAS1440 reduced tumor growth across all models (dose, schedule, sample size, randomization/blinding, and statistics in revised Fig. 9), supporting reproducibility within SCLC-A across independent lines.\nFor all new experiments, we now report biological replicates (n), error metrics, effect sizes with 95% CIs, and statistical tests in the figure legends and Methods sections.\nTaken together, these additions directly address the request to verify key results in ≥1 additional cell line per assay and support the robustness of TAS1440’s effects across independent SCLC-A models.\n#4-7\nAuthors claim that TAS1440 dissociates TFs such as INSM1 and SMAD2 from the LSD1 complex and synergistically promotes the activation of key signaling pathways such as NOTCH and TGF-b via epigenetic reprogramming. However, no data was provided to support this conclusion except cell viability data.\nResponse: We thank the reviewer for this important comment. Beyond cell-viability assays, we now provide orthogonal evidence that TAS1440 disrupts the LSD1–INSM1/SMAD2 repressive complex and reactivates NOTCH and TGF-β via epigenetic reprogramming.\n1) Direct complex dissociation (biochemistry/proteomics).\nExogenous co-immunoprecipitation in NCI-H1417 showed reduced co-precipitation of INSM1 and SMAD2 with LSD1 after TAS1440 (empty-vector–normalized densitometry; fold-decrease: INSM1 = 5.81, SMAD2 = 2.40; n=3; two-tailed paired t-test; INSM1: P=0.0015, SMAD2: P=0.046; revised Fig. 5e). Affinity-purification mass spectrometry of the LSD1 interactome further demonstrated loss of multiple nuclear cofactors, including epigenetic regulators; INSM1 and SMAD2 were among the dissociated interactors (log fold-change indicating decrease: INSM1 = –6.77, 2 SMAD2 = –5.13; revised Fig. 5c).\n2) Genetic dependency (necessity/sufficiency).\n• INSM1: siRNA knockdown in NCI-H1417/NCI-H510A significantly attenuated TAS1440 response (ΔGI%: H510A = 17.63%, n=4, two-tailed unpaired t-test, P=0.003; H1417 = 10.99%, n=4, P=0.025; revised Fig. S13j, k). Conversely, INSM1 overexpression in resistant non-NE lines (NCI-H211 [SCLC-P] and NCI-H196 [SMARCA4-deficient]) restored NE features and increased TAS1440 sensitivity (EC50 shift: H211 = 49.63%, n=4, P<0.0001; H196 = 22.66%, n=4, P<0.0001; revised Fig. S17a, b). RNA-seq in these OE models showed baseline repression of NOTCH/TGF-β programs by INSM1 (e.g., TGF-β GSEA NES: H196 = –1.36, P<0.001) and on-treatment reactivation with TAS1440 (NOTCH GSEA NES: H211 = 1.30, P<0.001; H196 = 1.40, P<0.001; ΔDEGs: H211 = 958; H196 = 2230; revised Fig. S17c–f).\n• SMAD2.:siRNA knockdown in NCI-H1417/H146/H510A conferred resistance to TAS1440 (ΔGI%: H1417 = 36.57%, n=8, P<0.0001; H146 = 8.92%, n=4, P=0.005; H510A = 27.02%, n=4, P<0.0001; revised Fig. 4g; Fig. S6g; Fig. S8c), and blunted TAS1440-induced transcriptional changes (ΔDEGs: H1417 = 619; H146 = 350; revised Fig. S7b), with selective attenuation of TGF-β-responsive genes (e.g., GSEA NES in H1417 from 1.34 (P<0.001) to 1.11 (P=0.22); H146 from 1.65 (P<0.001) to 1.22 (P=0.10); revised Fig. S7c–f). Double knockdown (INSM1+SMAD2) produced near-complete resistance to TAS1440 (n=4; revised Fig. 8l; S13h) and loss of pathway induction by qPCR (n=3; revised Fig. 8m; S13i).\n3) Epigenomic reprogramming (ChIP-seq/ChIP-qPCR).\nIn NCI-H1417, INSM1 depletion did not change H3K4me1/2/3 at TSSs, but significantly increased H3K27ac genome-wide (revised Fig. 8n), with increased H3K27ac and transcripts at NOTCH1 and TGFB2 (revised Fig. S8o–r). SMAD2 ChIP-qPCR showed enhanced occupancy at NOTCH1/TGFB2 specifically with TAS1440, exceeding other LSD1 inhibitors (n=3; revised Fig. 6h; Fig. S11g).\nConclusion: Taken together, these orthogonal datasets demonstrate that TAS1440 dissociates INSM1/SMAD2 from LSD1 and reactivates NOTCH and TGF-β through coordinated epigenetic reprogramming. We have added statistical details (n, tests, effect sizes, P values) and clarified figure references in the revised manuscript accordingly.\n#4-8\nData shows that TAS dissociates LSD1 interaction with INSM1 and SMAD2. However, much of the studies are conducted with only INSM1 manipulation. It is not clear what is the role of SMAD2 in TAS actions.\nResponse: Thank you for this important comment. To define the role of SMAD2—beyond our INSM1 data—in mediating TAS1440 activity, we have performed complementary genetic, genomic, and epigenomic analyses across multiple SCLC-A models (NCI-H1417, NCI-H146, and NCI-H510A).\n1) Genetic dependency (siRNA).\nSMAD2 knockdown significantly reduced TAS1440 sensitivity in NCI-H146 and NCI-H510A, consistent with NCI-H1417 (revised Fig. S6g–h, S8e-f). We report biological replicates (n), effect sizes with 95% CIs, and two-sided tests in the figure legends.\n2) Genomic occupancy (ChIP-qPCR).\nUpon TAS1440 treatment, SMAD2 occupancy at the NOTCH1 and TGFB2 loci increased, and did so more prominently than with other reversible LSD1 inhibitors tested (revised Fig. 6h; Fig. S11g), indicating enhanced recruitment under TAS1440 exposure.\n3) Transcriptomic response (RNA-seq).\nIn NCI-H1417 and NCI-H146, siSMAD2 blunted TAS1440‑induced differential expression (total DEGs, up + down): NCI‑H1417 1,789 → 1,170 and NCI‑H146 675 → 325 in siNC vs. siSMAD2 (revised Fig. S7b).\n4) Genetic epistasis (double knockdown).\nDual INSM1+SMAD2 knockdown in NCI-H1417 and NCI-H146 conferred near-complete resistance to TAS1440 and abrogated induction of NOTCH/TGF-β signatures by qPCR (revised Fig. 8k–m; S13g–i).\nConclusion.\nAcross orthogonal assays, SMAD2 is required for the full transcriptional and phenotypic response to TAS1440—non-redundant yet cooperative with INSM1 following LSD1-complex disruption. We have added statistical details (n, tests, effect sizes, and FDR where applicable) and clarified figure references in the revised manuscript accordingly.\n#4-9\nIP data show that in addition to TAS, ORY-1001, and GSK2879552 also reduce the LSD1 interaction with INSM1 despite the latter being catalytic inhibitors. How do the authors explain this? How is TAS1440's superior activity justified here?\nResponse: Thank you for raising this important point. While our co-immunoprecipitation (IP) experiments show that several LSD1 inhibitors—including TAS1440, ORY-1001, and GSK2879552—can reduce the LSD1–INSM1 interaction, the extent of dissociation is dose-dependent and compound-specific. To create a fair comparison, we benchmarked all three agents at matched concentrations (100, 300, 500 nM) under identical lysis/wash conditions with input-normalized densitometry. TAS1440 produced greater complex disruption at lower concentrations (revised Fig. S10g–j), indicating superior potency for breaking the LSD1–INSM1 complex at equimolar exposure.\nMechanistic rationale: Our structural data place TAS1440 within the histone H3-binding pocket of LSD1 (revised Fig. 5i), in proximity to residues engaged by H3-like peptides and reported INSM1-contacting regions. This geometry provides a direct steric basis for antagonizing INSM1 binding. By contrast, ORY-1001 and GSK2879552 are FAD-directed covalent (catalytic-site) inhibitors that occupy a topologically shifted region with limited spatial overlap with the INSM1 interface; the observed reduction in co-precipitation with these covalent inhibitors is therefore plausibly indirect/allosteric and, in our hands, less efficient at equimolar doses.\nFunctional consequence: Consistent with stronger complex disruption, TAS1440 drives more robust downstream reprogramming—including NOTCH1 and pSMAD2 induction and NOTCH/TGF-β pathway enrichment—relative to comparator agents (revised Fig. 4j, k). Together, these data support a dual-function mechanism for TAS1440 (potent catalytic inhibition plus direct competition at/near the H3/INSM1 interface) that explains both its stronger complex dissociation and greater transcriptional effects.\nWe have clarified these points in the revised manuscript, including the matched-dose IP methodology, normalized quantification, and figure cross-references (revised Fig. S10g–j; Fig. 5i).\n#4-10\nStudies that address the molecular basis of resistance using INSM1-KO need to be verified using reexpression of INSM1 in INSM1-KO background and show that reinstating INSM1 expression restores TAS1440 efficacy.\nResponse: Thank you for this insightful suggestion. To directly test whether loss-of-function phenotypes in INSM1-KO cells are rescued by INSM1 re-expression, we performed add-back experiments in NCI-H1417 and NCI-H146.\n Re-expression system and controls: INSM1-KO cells were transduced with INSM1 or empty vector (EV) (revised Fig. 8k, l; Fig. S13g, h, with replicate details in the legends).\n Functional rescue of drug response: Upon ectopic INSM1 re-expression, TAS1440 sensitivity was restored toward parental levels in dose–response viability assays (revised Fig. 8l; Fig. S13h), whereas EV controls remained resistant. Effect sizes (e.g., EC50 shift/AUC change), n (biological replicates), and statistical tests are provided in the figure legends.\n Pharmacodynamic rescue: Consistently, qPCR (and immunoblot where applicable) demonstrated re-activation of NOTCH/TGF-β pathway targets in the reconstituted lines, concordant with recovery of on-target pharmacodynamic signaling (revised Fig. 8m; Fig. S13i).\nThese data indicate that INSM1 is required for the full TAS1440 response and that re-expression restores TAS1440 efficacy in the INSM1-KO background within the tested context, thereby validating the conclusions drawn from our INSM1-KO models.\n#4-11\nFigure 7I has no significance as this data was generated using NSCLC. I think this data can be removed from the manuscript as earlier studies have already demonstrated the LSD1 survival correlation in NSCLC.\nResponse: We agree that NSCLC survival data are not appropriate to support SCLC‑specific claims. Accordingly, we removed the NSCLC survival panel (former Fig. 7I) and now restrict clinical inference to SCLC datasets. As a replacement, we present overall survival (OS) analyses in a publicly available SCLC RNA‑seq cohort (PMID: 38181741). In the overall SCLC cohort, higher KDM1A expression was associated with shorter OS, supporting the prognostic relevance of LSD1, whereas lower INSM1 showed a trend toward improved OS that did not reach statistical significance. In SCLC‑A, the direction of associations was concordant but non‑significant, likely reflecting limited power in subtype‑restricted strata. These directions are in line with prior pathology‑based reports, while inter‑cohort differences in clinical background and therapy may contribute to variability across studies. We report hazard ratios with 95% CIs, log‑rank P values, the prespecified cut‑point, cohort size, and event counts in the revised figure legends and Methods sections (revised Fig. 9l-m).\n#5-1\nReviewer #5 (Remarks to the Author): structural biology, may know medicinal chemistry\nThis manuscript describes a new LSD1 (histone demethylase) inhibitor and its potential pharmacological activity against a sub-type of small-cell lung cancer. The bottom line is that the LSD1 inhibitor disrupts the interaction between the demethylase and INSM1. In this way, this transcription factor looses its ability to function as a repressor of NOTCH and TGF-β signaling. These pathways are thereby activated and induce an epigenetic reprogramming that halts cell proliferation. The authors must be congratulated for the extensive amount of work that they describe in this submission. The experiments are extremely well illustrated and described by the narrative. My enthusiasm is, however, considerably decreased by the consideration that the manuscript almost ignores previous literature. TAS1440, the hereby reported inhibitor, is chemically very similar to CC-90011, a non covalent inhibitor described 15 years ago (10.1021/acs.jmedchem.0c00978) that was moved forwards to clinical trials (doi.org/10.1021/acs.jmedchem.3c02133). Moreover, there is already quite some literature describing the interplay between INSM1 and LSD1 in SCLC. For instance, doi.org/10.1158/0008-5472.CAN-16-3502 shows that inhibition of the LSD1-INSM1 interaction \"inhibits expression of neuroendocrine-associated genes, such as ASCL1\", anticipating the conclusion of this work. Similar conclusions were given in reference 10 whereas reference 24 demonstrates that \"T-3775440 (an irreversible LSD1 inhibitor) blocks proliferation of SCLC cells in vitro and retards SCLC tumor growth in vivo. T-3775440 disrupts the interaction between LSD1 and the transcriptional repressor INSM1, thereby inhibiting expression of neuroendocrine-associated genes, such as ASCL1. INSM1 silencing phenocopies the effects of T-3775440 on gene expression and cell proliferation, consistent with the likelihood T-3775440 mediated its effects in SCLC by inhibiting INSM1\". These are virtually the same take-home message offered by this manuscript. The relevance is this work is the demonstration that stratification of the cancer sub-type is critical and that the levels of ASCL1 can be prognostic. The manuscript rationalizes this phenomenon describing a plausible molecular mechanism.\nWhether this manuscript meets the degree of novelty expected by the journal is an editorial decision. I recommend that the Authors extensively revise their manuscript highlighting their new findings while properly referring to and comparing with previous work whenever this is relevant. I think that it would be important to compare TAS1440 with CC-90011 with regard to their antiproliferative activity and cell-type selectivity. Given their structural similarity, it may well be possible that they behave the same or with only marginal differences.\nResponse: We thank the reviewer for underscoring two key points—(i) the extensive prior literature on LSD1–INSM1 biology and LSD1 inhibition in SCLC, and (ii) the need for an explicit, head-to-head comparison with CC-90011 given their chemical relatedness. We have revised the Introduction/Discussion to more fully cite and contextualize prior work (including studies demonstrating INSM1–LSD1 interplay and neuroendocrine gene repression), and we now provide the following side-by-side analyses.\nAntiproliferative activity and subtype selectivity.\nAcross a panel of SCLC cell lines (revised Fig. S3a), TAS1440 and CC-90011 showed broadly similar inhibition profiles, with TAS1440 exhibiting greater potency in several SCLC-A–sensitive lines. Stratified analyses by molecular subtype indicate stronger activity in SCLC-A relative to non-A subtypes for both agents (summary statistics reported in the legend). In contrast, SP2509 displayed broad cytotoxicity at higher concentrations and retained residual activity in LSD1-KO cells (revised Fig. 7c; Fig. S12c), consistent with reported off-target liabilities.\nTranscriptional programs and pathway activation.\nRNA-seq in NCI-H1417 and NCI-H146 treated with five LSD1 inhibitors revealed distinct expression signatures among TAS1440, CC-90011, and SP2509 (revised Fig. 4i; Fig. S6i). GSEA showed that TAS1440 more robustly enriched NOTCH and TGF-β pathways (Fig. R3 for reviewer), concordant with stronger NOTCH1 and pSMAD2 induction by immunoblot (revised Fig. 4j, k).\nDisruption of LSD1 repressive complexes.\nCo-immunoprecipitation/immunoblotting (revised Fig. 5e, f) demonstrated that TAS1440 efficiently disrupts LSD1–INSM1, LSD1–SMAD2, LSD1–HDAC1, and LSD1–HDAC2 interactions. SP2509 left LSD1–INSM1/SMAD2 largely unaffected but perturbed LSD1–RCOR1, indicating a distinct complex-disruption fingerprint (matched-dose conditions and input-normalized densitometry are detailed in the legends).\nStructural rationale.\nDocking places TAS1440 within the histone H3-binding pocket of LSD1 (revised Fig. 5g, h), proximal to surfaces implicated in INSM1/SMAD2 engagement, providing a plausible steric basis for complex disruption. By contrast, SP2509 adopts a topologically distinct pose, consistent with its divergent functional effects and off-target profile.\nConclusion.\nTogether, these data position TAS1440—relative to CC-90011 and SP2509—as having tighter on-target dependence, more robust NOTCH/TGF-β pathway activation, and broader disruption of LSD1 corepressor complexes, while explicitly situating our findings within the prior SCLC LSD1–INSM1 literature highlighted by the reviewer.\nWe have updated the manuscript accordingly and report effect sizes, replicates, and statistics in the figure legends and Methods sections.\nUltimately, we would like to express our sincere gratitude to the editors and reviewers for their positive and constructive criticism. The manuscript has vastly benefited from your valuable and insightful comments and suggestions. We look forward to hearing from you and would be happy to address any further concerns, if required. We hope this further pushes the manuscript closer to publication in your esteemed journal.\nFigure R1 for reviewer\nBox plots showing INSM1 and ASCL1 gene expression levels between the sensitive and insensitive groups among the SCLC-A cell lines and the normalized enrichment scores (NES) of TGF-Beta Signaling Pathway between the sensitive and insensitive groups (n = 3 for each cell line; min–max, Student’s t-test).\nFigure R2 for reviewer\nPeripheral blood hematology after TAS1440 treatment.\nBar graphs compare vehicle (gray) and TAS1440 (red) for the following parameters: white blood cell count (WBC, /μL), red blood cell count (RBC, ×104/μL), hemoglobin (Hb, g/dL), hematocrit (HCT, %), mean corpuscular volume (MCV, fL), mean corpuscular hemoglobin (MCH, pg), mean corpuscular hemoglobin concentration (MCHC, %), and platelet count (PLT, ×104/μL). Each dot denotes one animal; bars represent mean ± SD. Group differences were evaluated by two‑sided Student’s t‑test with significance indicated as ***P < 0.001, **P < 0.01 and N.S. not significant.\nFigure R3 for reviewer\n(upper) Gene set enrichment analysis by KEGG pathway analysis showing NOTCH or TGF-β signaling pathways that were differently activated by five LSD1 inhibitors in NCI-H1417 and NCI-H146 cells.\n(lower) RNA-seq heatmap depicting NOTCH or TGF-β signaling changes in mRNA expression between by five LSD1 inhibitors in NCI-H1417 and NCI-H146 cells.\nResponse to reviewers’ comments\nWe thank the reviewers for their thoughtful comments and suggestions. We have addressed all points and revised the manuscript accordingly.\nReviewer #4\nFigure 9F: The authors claim that TAS1440 outperforms ORY-1001 in terms of efficacy. However, the dose of ORY-1001 used (≤0.03 mg) is substantially lower compared to the 50 mg dose of TAS1440. Therefore, it may not be appropriate to directly compare their efficacy under such disparate dosing conditions. It is unclear why such low doses of ORY-1001 were chosen, and no explanation is provided in the Methods section.\nResponse:\nWe thank the reviewer for the constructive comments and insights. As the reviewer noted, the substantial difference in dose levels between TAS1440 and ORY-1001 meant that our original text did not adequately justify a direct efficacy comparison. To avoid implying a like-for-like efficacy comparison under disparate dosing, we removed the phrase “outperformed ORY-1001” from the main text and now report only tumor-growth inhibition at the tested doses for each compound. We also added the rationale to the Methods section for the ORY-1001 dose range (0.01–0.03 mg/kg/day), citing prior studies demonstrating efficacy in SCLC PDX models (Reference 12).\nRevised text (Results; Page 25, Lines 505–509):\n“To evaluate the in vivo efficacy of TAS1440, we tested four SCLC‑A xenograft models (NCI‑H1417, NCI‑H146, NCI‑H510A, and COR‑L51). TAS1440 significantly reduced tumor growth in all models (dose group: 16.7 or 50 mg/kg/day in NCI‑H1417; 50 mg/kg/day in the others) compared with vehicle and outperformed ORY-1001 (Fig. 9a–e, g; Supplementary 18e, i). In the NCI-1417 model, ORY‑1001 also reduced tumor growth at the tested dose (0.01–0.03 mg/kg/day) (Fig. 9a).”\nRevised text (Methods; Page 57, Lines 1239–1242):\n“Upon tumor establishment, mice were randomized into control and experimental groups (n = 3–5 per group) using MiSTAT Grouping to balance mean baseline tumor volumes. TAS1440 was administered at 16.7 or 50 mg/kg/day. The ORY-1001 dose (0.01-0.03 mg/kg/day) was chosen based on previously reported efficacy in the SCLC PDX models12.”\nReviewer #5\n1. The structure of LSD1 bound to CC-90011 was reported a few years ago (reference 16 in the manuscript). The description of the crystal structure of LSD1-TAS1440 on page 16 should mention this and compare the binding of the two inhibitors, which, I guess is very similar.\nResponse:\nWe appreciate the reviewer's constructive suggestions. As proposed, both TAS1440 and CC-90011 are non-covalent, H3-competitive inhibitors, and an explicit side-by-side comparison would be informative. We therefore compared LSD1–TAS1440 co-crystal structure (PDB: 8JF5; Fig. 5i) with the previously reported LSD1–CC-90011 structure (PDB: 6W4K; Reference 16). Consistent with the reviewer’s expectation, the two inhibitors occupy nearly identical regions within the histone H3 substrate-binding pocket, supporting a common non-covalent, H3-competitive mechanism (Figure for Reviewer). We have added a description of this comparison to the Results (Page 16, Line 309-321).\nRevised text (Results; Page 16, Lines 309-321):\n“Finally, X-ray co-crystal analysis shows occupancy of the H3-binding site in vitro (Fig. 5i), consistent with the substrate‑competition observed in Fig. 2g. Furthermore, a comparison with the previously reported LSD1–CC-90011 structure (PDB: 6W4K)16, another non-covalent H3-competitive inhibitor, demonstrates that TAS1440 and CC-90011 occupy nearly identical regions within this substrate-binding pocket, supporting a common mechanism. Superposition with LSD1–GSK2879552 (PDB: 6NQU) and an LSD1–INSM1 peptide complex (PDB: 3ZMS) shows that TAS1440 overlaps the H3 pocket and sterically intersects the INSM1 footprint, providing a direct steric basis for destabilizing the LSD1–INSM1 interaction. By contrast, GSK2879552 forms a covalent adduct with FAD at a shifted position that minimally overlaps the INSM1 site, consistent with its distinct FAD‑directed covalent mode. Collectively, these structural data support a dual-mode mechanism of action for TAS1440—inhibition of LSD1 demethylase activity and disruption of the INSM1–LSD1 complex—and, together with our modeling (Fig. 5h), are consistent with potential interference at SMAD2‑interacting surfaces.”\nFigure for the reviewers.\nStructural comparison of TAS1440 and CC-90011 binding modes in the LSD1 active site. Superposition of the LSD1–TAS1440 co-crystal structure (PDB: 8JF5) and the LSD1–CC-90011 co-crystal structure (PDB: 6W4K). Structures were aligned on the LSD1 backbone (gray cartoon). TAS1440 (8JF5) is shown as red sticks, and CC-90011 (6W4K) as yellow sticks. The superposition indicates that both non-covalent, H3-competitive inhibitors occupy nearly identical regions within the H3 substrate-binding pocket.\n2. Line 287 on page 19 states that the mutant K166A is inactive.\n10.1016/j.molcel.2020.04.019 has shown that \"LSD1(K661A) frequently used as a catalytically inactive mutant in vivo (based on in vitro peptide studies) actually retains substantial H3K4 demethylase activity on nucleosome substrates.\" Therefore, the mutant should be described as \"less active\", a subtle but critical difference that explains the data presented in figure 7 showing that LSD1(K661A) partly rescues LSD1 activity.\nResponse:\nWe appreciate the reviewer’s precise and important comment. As correctly noted by the reviewer, reference 9 (Kim et al., 2020) shows that although LSD1(K661A) is inactive on peptide substrates, it retains substantial H3K4 demethylase activity on nucleosomal substrates. Accordingly, describing the mutant as “catalytically inactive” in our manuscript was imprecise in this context. We have therefore replaced this wording with “catalytically impaired” throughout the Results and the Figure 7 legend, which clarifies the partial rescue observed with LSD1(K661A) in Figure 7. We also note a typographical confusion (K166A vs. K661A); our study uses K661A throughout, and any instances of K166A have been corrected.\nRevised text (Results; Page 19, Lines 378–379):\n“we generated a catalytically impaired LSD1 mutant (LSD1-K661A) that impairs demethylase function.”\nRevised text (Figure legends; Page 40, Line 858):\n“LSD1‑K661A (catalytically impaired)”\nRevised text (Figure legends Page 41, Line 874):\n“catalytically impaired LSD1‑K661A”\n3. The introduction indicates that LSD1 can also be a H3K9 demethylase. It has been shown by multiple studies by many groups that LSD1 is devoid of any direct K9 activity (e.g. 10.1016/j.molcel.2020.04.019).\nResponse:\nWe appreciate the reviewer's comments regarding the accuracy of the description of LSD1's function in the Introduction. Statements implying direct H3K9 demethylase activity by LSD1 may conflict with recent studies (Reference. 9). Current evidence suggests that any apparent activity toward H3K9 is likely indirect and, if observed, occurs within specific complexes. Following the reviewer’s feedback, we removed the potentially confusing H3K9me1/2 language from the Introduction. This change clarifies LSD1’s primary role in SCLC in our study—H3K4 demethylation and repression of NOTCH signaling, which is the focus of the paper.\nRevised text (Introduction Page 5, Line 79-86):\n“As a strategy to restore NOTCH signaling, lysine-specific demethylase 1A (LSD1/KDM1A)—a flavin-dependent amine oxidase and epigenetic regulator—has emerged as a promising therapeutic target in SCLC. LSD1 represses NOTCH‑associated genes predominantly by demethylating histone H3 lysine 4 (H3K4me1/2) and, in other contexts, can demethylate H3K9me1/27, thereby shaping chromatin states and transcriptional output. LSD1’s enzymatic activity requires flavin adenine dinucleotide (FAD) and can support transcriptional repression via H3K4 demethylation or activation via H3K9 demethylation8. Notably, LSD1 also functions within multi‑protein corepressor assemblies, including the REST corepressor (CoREST) 9 and the nucleosome remodeling and deacetylase (NuRD) complex10.”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed my concerns and the manuscript is appropriate for the publication.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "I commend the authors on their thorough revision of the manuscript. In particular, the authors have vastly improved the contextualization of this work within the prior body of literature on LSD1 inhibition in SCLC and on SCLC transcriptional diversity. Specifically, the exclusion of the YAP1 subtype, inclusion of SCLC-specific survival data, inclusion of relevant SCLC-specific therapeutic implications (e.g. impact on DLL3 but not SEZ6 expression) all render this work more appropriate for publication.\nI am satisfied with their responses to my queries and to those queries of my fellow reviewers.", "reviewer_4": "Authors did an excellent job in addressing most of the previously raised concerns.\nMinor comment:\nFigure 9F: The authors claim that TAS1440 outperforms ORY-1001 in terms of efficacy. However, the dose of ORY-1001 used (≤0.03 mg) is substantially lower compared to the 50 mg dose of TAS1440. Therefore, it may not be appropriate to directly compare their efficacy under such disparate dosing conditions. It is unclear why such low doses of ORY-1001 were chosen, and no explanation is provided in the Methods section.", "reviewer_5": "The authors have addressed all points raised by the reviewers including the comments made in previous review.\nThe manuscript presents a massive amount of data supporting the notion that LSD1 inhibition may represent a valuable avenue for CLC-A treatment and that TAS1440 may be a powerful starting molecule towards this aim.\nThe authors may like to consider a couple of minor additional points:\n-the structure of LSD1 bound to CC-90011 was reported a few years ago (reference 16 in the manuscript). The description of the crystal stucture of LSD1-TAS1440 on page 16 should mention this and compare the binding of the two inhibitors, which, I guess is very similar.\n-Line 287 on page 19 states that the mutant K166A is inactive. 10.1016/j.molcel.2020.04.019 has shown that \"LSD1(K661A) frequently used as a catalytically inactive mutant in vivo (based on in vitro peptide studies) actually retains substantial H3K4 demethylase activity on nucleosome substrates.\" Therefore, the mutant should be described as \"less active\", a subtle but critical difference that explains the data presented in figure 7 showing that LSD1(K661A) partly rescues LSD1 activity.\n-The introduction indicates that LSD1 can also be a H3K9 demethylase. It has been shown by multiple studies by many groups that LSD1 is devoid of any direct K9 activity (e.g. 10.1016/j.molcel.2020.04.019)."}, "author_response": "Response to reviewers’ comments\nWe thank the reviewers for their thoughtful comments and suggestions. We have addressed all points and revised the manuscript accordingly.\nReviewer #4\nFigure 9F: The authors claim that TAS1440 outperforms ORY-1001 in terms of efficacy. However, the dose of ORY-1001 used (≤0.03 mg) is substantially lower compared to the 50 mg dose of TAS1440. Therefore, it may not be appropriate to directly compare their efficacy under such disparate dosing conditions. It is unclear why such low doses of ORY-1001 were chosen, and no explanation is provided in the Methods section.\nResponse:\nWe thank the reviewer for the constructive comments and insights. As the reviewer noted, the substantial difference in dose levels between TAS1440 and ORY-1001 meant that our original text did not adequately justify a direct efficacy comparison. To avoid implying a like-for-like efficacy comparison under disparate dosing, we removed the phrase “outperformed ORY-1001” from the main text and now report only tumor-growth inhibition at the tested doses for each compound. We also added the rationale to the Methods section for the ORY-1001 dose range (0.01–0.03 mg/kg/day), citing prior studies demonstrating efficacy in SCLC PDX models (Reference 12).\nRevised text (Results; Page 25, Lines 505–509):\n“To evaluate the in vivo efficacy of TAS1440, we tested four SCLC‑A xenograft models (NCI‑H1417, NCI‑H146, NCI‑H510A, and COR‑L51). TAS1440 significantly reduced tumor growth in all models (dose group: 16.7 or 50 mg/kg/day in NCI‑H1417; 50 mg/kg/day in the others) compared with vehicle and outperformed ORY-1001 (Fig. 9a–e, g; Supplementary 18e, i). In the NCI-1417 model, ORY‑1001 also reduced tumor growth at the tested dose (0.01–0.03 mg/kg/day) (Fig. 9a).”\nRevised text (Methods; Page 57, Lines 1239–1242):\n“Upon tumor establishment, mice were randomized into control and experimental groups (n = 3–5 per group) using MiSTAT Grouping to balance mean baseline tumor volumes. TAS1440 was administered at 16.7 or 50 mg/kg/day. The ORY-1001 dose (0.01-0.03 mg/kg/day) was chosen based on previously reported efficacy in the SCLC PDX models12.”\nReviewer #5\n1. The structure of LSD1 bound to CC-90011 was reported a few years ago (reference 16 in the manuscript). The description of the crystal structure of LSD1-TAS1440 on page 16 should mention this and compare the binding of the two inhibitors, which, I guess is very similar.\nResponse:\nWe appreciate the reviewer's constructive suggestions. As proposed, both TAS1440 and CC-90011 are non-covalent, H3-competitive inhibitors, and an explicit side-by-side comparison would be informative. We therefore compared LSD1–TAS1440 co-crystal structure (PDB: 8JF5; Fig. 5i) with the previously reported LSD1–CC-90011 structure (PDB: 6W4K; Reference 16). Consistent with the reviewer’s expectation, the two inhibitors occupy nearly identical regions within the histone H3 substrate-binding pocket, supporting a common non-covalent, H3-competitive mechanism (Figure for Reviewer). We have added a description of this comparison to the Results (Page 16, Line 309-321).\nRevised text (Results; Page 16, Lines 309-321):\n“Finally, X-ray co-crystal analysis shows occupancy of the H3-binding site in vitro (Fig. 5i), consistent with the substrate‑competition observed in Fig. 2g. Furthermore, a comparison with the previously reported LSD1–CC-90011 structure (PDB: 6W4K)16, another non-covalent H3-competitive inhibitor, demonstrates that TAS1440 and CC-90011 occupy nearly identical regions within this substrate-binding pocket, supporting a common mechanism. Superposition with LSD1–GSK2879552 (PDB: 6NQU) and an LSD1–INSM1 peptide complex (PDB: 3ZMS) shows that TAS1440 overlaps the H3 pocket and sterically intersects the INSM1 footprint, providing a direct steric basis for destabilizing the LSD1–INSM1 interaction. By contrast, GSK2879552 forms a covalent adduct with FAD at a shifted position that minimally overlaps the INSM1 site, consistent with its distinct FAD‑directed covalent mode. Collectively, these structural data support a dual-mode mechanism of action for TAS1440—inhibition of LSD1 demethylase activity and disruption of the INSM1–LSD1 complex—and, together with our modeling (Fig. 5h), are consistent with potential interference at SMAD2‑interacting surfaces.”\nFigure for the reviewers.\nStructural comparison of TAS1440 and CC-90011 binding modes in the LSD1 active site. Superposition of the LSD1–TAS1440 co-crystal structure (PDB: 8JF5) and the LSD1–CC-90011 co-crystal structure (PDB: 6W4K). Structures were aligned on the LSD1 backbone (gray cartoon). TAS1440 (8JF5) is shown as red sticks, and CC-90011 (6W4K) as yellow sticks. The superposition indicates that both non-covalent, H3-competitive inhibitors occupy nearly identical regions within the H3 substrate-binding pocket.\n2. Line 287 on page 19 states that the mutant K166A is inactive.\n10.1016/j.molcel.2020.04.019 has shown that \"LSD1(K661A) frequently used as a catalytically inactive mutant in vivo (based on in vitro peptide studies) actually retains substantial H3K4 demethylase activity on nucleosome substrates.\" Therefore, the mutant should be described as \"less active\", a subtle but critical difference that explains the data presented in figure 7 showing that LSD1(K661A) partly rescues LSD1 activity.\nResponse:\nWe appreciate the reviewer’s precise and important comment. As correctly noted by the reviewer, reference 9 (Kim et al., 2020) shows that although LSD1(K661A) is inactive on peptide substrates, it retains substantial H3K4 demethylase activity on nucleosomal substrates. Accordingly, describing the mutant as “catalytically inactive” in our manuscript was imprecise in this context. We have therefore replaced this wording with “catalytically impaired” throughout the Results and the Figure 7 legend, which clarifies the partial rescue observed with LSD1(K661A) in Figure 7. We also note a typographical confusion (K166A vs. K661A); our study uses K661A throughout, and any instances of K166A have been corrected.\nRevised text (Results; Page 19, Lines 378–379):\n“we generated a catalytically impaired LSD1 mutant (LSD1-K661A) that impairs demethylase function.”\nRevised text (Figure legends; Page 40, Line 858):\n“LSD1‑K661A (catalytically impaired)”\nRevised text (Figure legends Page 41, Line 874):\n“catalytically impaired LSD1‑K661A”\n3. The introduction indicates that LSD1 can also be a H3K9 demethylase. It has been shown by multiple studies by many groups that LSD1 is devoid of any direct K9 activity (e.g. 10.1016/j.molcel.2020.04.019).\nResponse:\nWe appreciate the reviewer's comments regarding the accuracy of the description of LSD1's function in the Introduction. Statements implying direct H3K9 demethylase activity by LSD1 may conflict with recent studies (Reference. 9). Current evidence suggests that any apparent activity toward H3K9 is likely indirect and, if observed, occurs within specific complexes. Following the reviewer’s feedback, we removed the potentially confusing H3K9me1/2 language from the Introduction. This change clarifies LSD1’s primary role in SCLC in our study—H3K4 demethylation and repression of NOTCH signaling, which is the focus of the paper.\nRevised text (Introduction Page 5, Line 79-86):\n“As a strategy to restore NOTCH signaling, lysine-specific demethylase 1A (LSD1/KDM1A)—a flavin-dependent amine oxidase and epigenetic regulator—has emerged as a promising therapeutic target in SCLC. LSD1 represses NOTCH‑associated genes predominantly by demethylating histone H3 lysine 4 (H3K4me1/2) and, in other contexts, can demethylate H3K9me1/27, thereby shaping chromatin states and transcriptional output. LSD1’s enzymatic activity requires flavin adenine dinucleotide (FAD) and can support transcriptional repression via H3K4 demethylation or activation via H3K9 demethylation8. Notably, LSD1 also functions within multi‑protein corepressor assemblies, including the REST corepressor (CoREST) 9 and the nucleosome remodeling and deacetylase (NuRD) complex10.”"}]

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