LSD1 is highly expressed in small cell lung cancer relative to other lung cancer subtypes
Over-expression of chromatin modifiers has been shown to influence their function in cancer . In lung cancer, LSD1 mRNA expression is highly expressed in SCLC relative to other lung cancer cell lines (Fig. 1A). Expression datasets from Clinical Lung Cancer Genome Project (CLCGP)  confirms LSD1 is also expressed highly relative to other lung cancer subtypes in primary patient samples (Fig. 1B). To further confirm LSD1 expression levels at the protein level, we conducted immunohistochemistry (IHC) on a lung cancer tissue microarray. IHC staining confirmed a statistically significantly higher staining intensity in SCLC specimens relative to that of squamous cell carcinoma, adenocarcinoma, and large cell carcinoma (1-way ANOVA Fig. 1C, 1D).
SCLC cell lines show heterogeneous responses to LSD1 inhibitors
Resulting from the observation of high expression of LSD1 in SCLC, we assessed whether SCLC cell lines were dependent on LSD1 activity for their growth. Using two shRNAs targeting LSD1 we observed growth inhibition in the NCI-H526 cell line that became strongest after 14 days of target knockdown (Figure S1A). Interestingly, DMS-114 cells did not appear sensitive to LSD1 knockdown over 14 days even though there was a significant loss of LSD1 protein level by western blot (Figure S1B).
Several reports have recently described sensitivity in SCLC cell lines to LSD1 inhibitors. To study this further, we profiled drug sensitivity to the FAD reversible inhibitor GSK690 (Ki = 4nM)  as well as the FAD irreversible compound OG-86 (IC50 = 47nM)  in a panel of SCLC cell lines (Figure S2). Due to the delayed onset of proliferation changes observed in shRNA experiments we tested the effects of GSK690 and OG-86 on SCLC cell line growth in long-term proliferation assays of 17–21 days. Consistent with shRNA results, we observed differential responses in many SCLC cell lines. In NCI-H1417, NCI-H187, NCI-H889 cells, dose-dependent growth inhibition was initially observed at 7-to-10 days of both GSK690 and OG-86 treatment with continued effects observed until day 17–21 (Figure S3A-C). DMS-114 cells, which were insensitive to LSD1 shRNA knockdown, were also insensitive to both GSK690 and OG-86 up to 17 days of treatment at concentrations up to 1.0µM OG-86 and 1.0µM GSK690 (Figure S3D). Interestingly, the anti-proliferative effects of LSD1 inhibitors plateau in their percent inhibition even after 17–21 days of continuous drug treatment due to the emergence of a subpopulation of slowly proliferative persister cells in NCI-H1417 and NCI-H187 cells (Figure S3). These data suggest that SCLC cell lines possess both intrinsic and acquired resistance mechanisms to LSD1 inhibitors.
Neuroendocrine and mesenchymal expression signatures stratify sensitivity to LSD1 inhibition
Across the SCLC cell line panel, 12 out of 29 tested SCLC cell lines showed greater than 50% growth inhibition in response to 0.3µM GSK690 treatment at day 17 (Fig. 2A). A non-enzymatic mechanism of action has recently been proposed to account for the anti-tumor activity of LSD1 inhibitors in SCLC . In our cell panel we identified that all SCLC cell lines sensitive to GSK690 express SNAG domain proteins INSM1 or GFI1B, however we also observed examples of INSM1 or GFI1B-expressing cell lines that were also resistant to the drug (Fig. 2C). Thus, expression of SNAG-domain proteins alone is insufficient to define cell lines that will respond to GSK690.
To identify additional biomarkers that predict sensitivity to GSK690 we explored both mutational and gene expression features of SCLC cell lines from available CCLE data  LSD1 expression level alone did not correlate with drug sensitivity (Fig. 2B, 2C). Additionally, using both univariate and multivariate association methods, no mutation significantly correlated with cell line sensitivity (data not shown). Analysis of the cell line gene expression by principal component analysis suggests that cell lines sensitive to GSK690 stratify in a group with distinct transcriptional state compared to insensitive cell lines with exception of NCI-H526 and NCI-H1963 cells that appear to be outliers (Figure S5A). SCLC cell lines can be stratified by neuroendocrine/epithelial (NE) and mesenchymal-like (ML) features. Using established gene expression markers of these SCLC cell states [16, 17] we found enrichment in GSK690 sensitive cell lines in expression of epithelial genes, MYCL and EPCAM, and neuroendocrine genes, GRP, DDC, ASCL1 (Fig. 2B-C, S4). SCLC cell lines resistant to GSK690 featured higher expression of mesenchymal genes MYC, ZEB1, and VIM (Fig. 2B, 2C, S4). These data suggest that neuroendocrine-like SCLC cell lines maybe more sensitive to LSD1 inhibition while mesenchymal-like SCLC states may confer resistance to LSD1 inhibitors.
To experimentally address the intrinsic drug resistance of mesenchymal-shifted SCLC cells to LSD1 inhibitors, we treated a mesenchymal variant form of NCI-H69 cells, NCI-H69V  with GSK690. NCI-H69 cells grow in suspension and show enrichment in NE transcription factors FOXA2, SOX2, LEF1, NCAM, E-cadherin (Fig. 2D). In contrast, NCI-H69V cells grow as adherent cells and express a ML signature enriched transcription factors SMAD3, MYC, ZEB1, VIM  (Fig. 2D). In line with our hypothesis, mesenchymal-shifted NCI-H69V appeared to be resistant to GSK690 treatment, showing only 22% growth inhibition compared to 85% growth inhibition observed in parental NCI-H69 cells (Fig. 2D).
To further refine the gene expression signature associated with LSD1 inhibitor resistance, we performed differential expression analysis between LSD1 inhibition sensitive and resistant SCLC cells from CCLE cell line data. Using this approach, we identified a differential gene expression signature (FDR < 5% and FC > 2) stratifying cell lines sensitive and resistant to LSD1 inhibitors (Fig. 3A). By performing Gene Set Enrichment Analysis (GSEA)  on these differential genes, we found that gene sets of ASCL1 targets, epithelial differentiation, and ZEB1 repressive sites are up-regulated in the sensitive cells, whereas EMT, TGF beta and MYC pathway gene sets are significantly up-regulated in the resistant cells (Fig. 3B).
Since sensitivity to LSD1 inhibition was associated with presence or absence of expression of canonical neuroendocrine and mesenchymal markers in our previous analysis, we compared the GSK690 differentially expressed gene signature to an established gene co-expression network that delineates tumor heterogeneity observed in SCLC tumor samples as Neuroendocrine (NE) and Mesenchymal-Like (ML) [18, 19]. Aligning the LSD1 inhibition differential expression signature to genes defining the NE (n = 1102) or ML (n = 2663) co-expression networks , we found that sensitive cell lines significantly up-regulated NE network genes, while resistant lines significantly up-regulated ML network genes (Fig. 3C). In addition, genes up-regulated in LSD1 inhibitor sensitive cell lines significantly overlapped with NE network genes while genes up-regulated in LSD1 inhibitor resistant cell lines significantly overlapped with the ML network genes. (Figure S5B). Overall, these data highlight the context specificity of GSK690 activity in neuroendocrine SCLC subtypes and explain the heterogenous drug responses observed in SCLC cell lines.
We next assessed the potential of the LSD1 sensitivity signature in identification of SCLC patients that might be sensitive or resistant to LSD1 inhibition. Using the differential gene expression signatures that stratify cell lines sensitive and resistant to LSD1 inhibitors (Fig. 3A), TCGA 2015 UCologne SCLC patient dataset was analyzed. Patients with high expression of NE genes correlated with genes increased in expression in the LSD1 sensitivity signature score. Conversely, patients enriched in ML signature overlapped with LSD1 inhibitor resistance gene expression (Fig. 3D). Thus the LSD1 inhibitor gene expression signature defined distinct subsets of SCLC patients that are predicted to be either sensitive or resistant to LSD1 inhibitors.
Drug tolerant cells with mesenchymal-like states emerge after LSD1 inhibitor treatment
SCLC cell lines grow in floating cell aggregates however a subset of cell lines will grow attached to tissue culture plastic [44, 45]. Cell lines which grow in suspension tend to express neuroendocrine markers while adherently growing cell lines are enriched in mesenchymal biomarkers . SCLC cells that persist after day 10 of 0.3µM GSK690 treatment show profound morphologic changes. NCI-H526 cells changed from loose floating aggregates to tightly adherent spheres (Fig. 4A). COR-L88 cells changed morphologically and gained adherence to tissue culture treated plates at day 12 of GSK690 treatment (Fig. 4A).
Since morphological changes are known to occur in SCLC cells that transition to a mesenchymal state , we assessed if LSD1 inhibitor persister populations shift to a mesenchymal differentiation state. We performed bulk RNA-seq on 4 sensitive cell lines (NCI-H889, NCI-H1417, NCI-H69 and COR-L88) and 2 insensitive cell lines (NCI-H82 and NCI-H1694). At day 10 of GSK690 treatment, a larger number of genes show changes in all four sensitive cell lines, with the majority of genes showing up-regulation (FDR < 0.05, fold change > = 2) (Fig. 4B). Interestingly, only 5 genes and 43 genes were significantly altered after 10 days of GSK690 treatment in 2 GSK690 insensitive models, NCI-H82 and NCI-H1694, respectively (Fig. 4B). Evaluation of common transcriptional changes occurring in sensitive models revealed only seven genes as consistently altered (FDR < 0.05, fold change > = 2) in expression at day 10 in all four cells lines (Fig. 4C). However, the data showed more convergence at a pathway level with several common pathways identified showing consistent changes due to GSK690 treatment in the four sensitive cell lines. Pathways associated with genes up-regulated by GSK690 treatment are enrichment in epithelial-mesenchymal transition pathway, TGF beta signaling, NOTCH signaling as well as focal adhesion related signaling consistent with the observed changes in adhesion (Fig. 4D, S5C). Conversely, pathways enriched in genes down-regulated by GSK690 treatment did not show overlap between different cell lines and include various signaling pathways (Fig. 4D, S5C).
Changes in neuroendocrine and mesenchymal markers were also observed at the protein level in GSK690 sensitive cell models (Figure S6). COR-L88 cells showed the strongest shift into a mesenchymal-like state showing down-regulation of neuroendocrine proteins NSE, GRP, NCAM, and CHGA and neuroendocrine transcription factors FOXA2, ASCL1, and SOX2. COR-L88 cells also showed strong up-regulation of the mesenchymal protein VIM. Other GSK690 sensitive cell models showed only small changes in protein levels across our panel with GRP reduction and ZEB1 and CDH2 up-regulation being the most consistent markers of response (Figure S6). We observed no consistent changes in the protein levels of any neuroendocrine and mesenchymal markers in GSK690 insensitive cell lines. Importantly, the extent of mesenchymal gene induction in GSK690 treated SCLC cells does appear comparatively lower relative to what we observed in mesenchymal-shifted NCI-H69V cells and thus may represent a ‘partial EMT’ .
LSD1 inhibitor persister cells evolve from epigenetically distinct subpopulations
To define the origin of mesenchymal-like persister cells, we conducted single cell RNA-seq analysis on NCI-H69 cells treated with DMSO or 0.3 µM GSK690 for 21 days. Harmony  was applied to integrate DMSO and treatment groups so that gene expression within each single cell subpopulation can be directly compared to obtain subpopulation specific differentially expressed (DE) genes after treatment (Fig. 5A). To identify distinct cell subpopulations, unsupervised cell clustering was performed and five clusters were selected manually after visualization using uniform manifold approximation and projection (UMAP) (Fig. 5B and S7A). Cell subpopulations in Cluster 0 and 3 were increased in 0.3 µM GSK690 treated group relative to the DMSO group (Figure S7A). In contrast, cell subpopulations in Cluster 1 and 2 in DMSO group were reduced after 21 days of LSD1 inhibitor treatment (Figure S7A). There was no significant difference in the Cluster 4 cell subpopulation between DMSO and treated groups. Cluster 2 was identified as a neuroendocrine-like subpopulation, as neuroendocrine transcriptional factors GRP and ASCL1 were specifically up-regulated in this subpopulation (Fig. 5B, S8A). Both GRP and ASCL1 expression were strongly reduced in Cluster 2 after treatment (Figure S7B). In addition, the mean expression of neuroendocrine markers (NE score) including ASCL1, CHGA, GRP, INSM1, NCAM1, FOXA2 and SOX2 were significantly reduced in Cluster 2 after treatment (Fig. 5C, left), suggesting cells in this cluster are losing NE features. On the other hand, the mean expression of 76 EMT marker genes from the Broad MSigDB Hallmark Signatures (EMT score) was up-regulated in multiple cell clusters after treatment, but most significantly increased in Cluster 0 (Fig. 5C, right). This observation is supported by the significant up-regulation of EMT and lung cancer survival pathways in Cluster 0 after treatment (Figure S8B). Altogether, these results highlight the loss of NE cell subpopulation, and the emergence of several transcriptionally distinct, mesenchymal-like populations in NCI-H69 cells resistant to LSD1 inhibitors.
In an attempt to assess the reversibility of the mesenchymal shift observed in LSD1 inhibitor persister cells we performed drug washout experiments. After 14 days of GSK690 treatment in NCI-H526 and NCI-H69 cells, anti-proliferative effects occurred concomitant with the up-regulation of mesenchymal markers CDH2, SNA1, MYC, VIM, and ZEB1 and the down-regulation of neuroendocrine markers GRP, ASCL1, and CHGA (Figure S9). After 7 days of drug washout, gene expression signatures recovered in both cell lines to levels similar to that observed in DMSO controls. Moreover, after 7 days of drug wash-out, cells regained sensitivity to LSD1 inhibition to a similar extent as untreated cell lines (Figure S9). These data suggest that the mesenchymal shift in SCLC cells treated with LSD1 inhibitors is reversible and caused by an “epi-stable” mesenchymal-like differentiation state.
LSD1 inhibitor resistance occurs through chromatin accessibility changes for genes associated with neuroendocrine and mesenchymal like programs
We next sought to define the extent of epigenetic reprogramming in LSD1 inhibitor drug tolerant cells by employing an assay for transposase-accessible chromatin using sequencing (ATAC-seq). ATAC-seq data in NCI-H69 cells treated with 0.3 µM GSK690 for 21 days was highly reproducible between biological replicates and showed clear enrichment at specific genomic regions (Figure S10A). Consistent with observed changes in gene expression, differentially open or closed chromatin accessibility peaks were primarily identified at distal regulatory regions of genes after 21 days of GSK690 treatment compared to controls (Fig. 6A, S10B). GREAT gene ontology analysis  for genes acquiring open chromatin accessibility peaks near regulatory regions suggests enrichment in pathways involved in mesenchyme morphogenesis and regulation of epithelial cell differentiation (Fig. 6B). In contrast, genes that acquire closed chromatin do not show any significant pathway enrichment.
To study the transcription factor programs regulating drug resistant SCLC cells, we identified the transcription factor motifs represented in differentially accessible, closed or open, chromatin regions after GSK690 treatment. We used ChromVar , a package designed for inferring transcription factor activity from ATAC-seq based on chromatin accessibility levels at TF binding motifs genome-wide (Fig. 6C). Our analysis demonstrated that NEUROD1/NEUROD2 and ASCL1 DNA binding motifs were significantly closed following GSK690 treatment. The DNA binding motif for TEAD4 is the only significantly opened region with the treatment. TEAD4 is a downstream mediator of YAP1 activity, which has been previously identified as a possible phenotypic modulator in a subset of non-neuroendocrine SCLC cell lines . These data indicate that mesenchymal-like drug tolerance in SCLC cell lines occurs through epigenetic reprogramming mediated through changes in transcription factor programs and chromatin accessibility changes that alter gene expression.