A DNA Hypomethylation Signature Predicts Antitumor Activity of LSD1 Inhibitors in SCLC

Epigenetic dysregulation has emerged as an important mechanism in cancer. Alterations in epigenetic machinery have become a major focus for new targeted therapies. The current report describes the discovery and biological activity of a cyclopropylamine containing inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552. This small molecule is a potent, selective, orally bioavailable, mechanism-based irreversible inhibitor of LSD1. A proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (SCLC) is sensitive to LSD1 inhibition. The subset of SCLC lines that undergo growth inhibition in response to GSK2879552 exhibit DNA hypomethylation of a signature set of probes suggesting this may be used as a predictive biomarker of activity. This targeted mechanism coupled with a novel predictive biomarker make LSD1 inhibition an exciting potential therapy for SCLC, a highly prevalent, rarely cured, tumor type representing approximately 15% of all lung cancers. Citation Format: Helai Mohammad, Ryan Kruger. A DNA hypomethylation signature predicts novel antitumor activity of LSD1 inhibition in SCLC. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR10.

ABSTRACTEpigenetic machinery have become a major focus for new targeted cancer therapies. Our previous report described the discovery and biological activity of a potent, selective, orally bioavailable, irreversible inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552. A proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (SCLC) was sensitive to LSD1 inhibition. The SCLC lines that undergo growth inhibition in response to GSK2879552 exhibit DNA hypomethylation of a signature set of probes suggesting this may be used as a predictive biomarker of activity. This targeted mechanism coupled with a novel predictive biomarker make LSD1 inhibition an exciting potential therapy for SCLC.

Survival and productivity of perennial plants in temperate zones are dependent on robust responses to prolonged and seasonal cycles of unfavorable conditions. Here we report whole-genome microarray, expression, physiological, and transgenic evidence in hybrid poplar (Populus tremula × Populus alba) showing that gibberellin (GA) catabolism and repressive signaling mediates shoot growth inhibition and physiological adaptation in response to drought and short-day (SD) induced bud dormancy. Both water deprivation and SDs elicited activation of a suite of poplar GA2ox and DELLA encoding genes. Poplar transgenics with up-regulated GA 2-oxidase (GA2ox) and DELLA domain proteins showed hypersensitive growth inhibition in response to both drought and SDs. In addition, the transgenic plants displayed greater drought resistance as evidenced by increased pigment concentrations (chlorophyll and carotenoid) and reductions in electrolyte leakage (EL). Comparative transcriptome analysis using whole-genome microarray showed that the GA-deficiency and GA-insensitivity, SD-induced dormancy, and drought response in poplar share a common regulon of 684 differentially-expressed genes, which suggest GA metabolism and signaling plays a role in plant physiological adaptations in response to alterations in environmental factors. Our results demonstrate that GA catabolism and repressive signaling represents a major route for control of growth and physiological adaptation in response to immediate or imminent adverse conditions.

The standard-of-care for limited stage and extensive stage SCLC has remained etoposide and a platinum complex for more than 30 years because 60-80% of patients respond; however, SCLC inevitably recurs. Recurrent SCLC has proven to be resistant to many therapeutics administered as second- or third-line treatments; therefore, combining therapies in the first instance may be a critically useful strategy. A high throughput screen was performed where 62 SCLC lines were exposed to etoposide (0.3uM)/carboplatin (3.7 uM) (E/C) with or without simultaneous exposure to a third agent (n = 220). Viability of the cells was measured using CellTiter-Glo after 96 hr exposure to 9 concentrations of each individual compound or combination with E/C. The test concentrations encompassed the clinical Cmax for each third agent, and the concentrations of E/C selected for the screen were systematically determined to produce SCLC kill that would allow observation of additivity/synergy upon addition of a third agent. IC50s were determined from the concentration response data and showed that the predominant effect of adding a third agent to E/C was additive. Less than additive effects occurred more frequently in SCLC lines that were sensitive to etoposide/carboplatin. Antagonism with E/C occurred in combination with taxanes and tubulin fragmenters such as vinorelbine. Effective single agents such as the nuclear kinase inhibitors (aurora kinase inhibitors, KSP/EG5 inhibitors and polo-like kinase inhibitors) were antagonistic in combination with E/C but were effective single agents. Greater than additive SCLC killing occurred with E/C in combination with several classes of agents. The combination of the Chk1 inhibitor rabusertib with E/C resulted in an IC50 that was >1 log lower than that of rabusertib alone in several SCLC lines. The GSK-3β inhibitor LY-2090314 produced greater than additive SCLC killing in combination with E/C. LY-2090314 had little effect on the SCLC lines alone but the simultaneous combination with E/C resulted in multi-log killing in selected SCLC lines. The BET bromodomain inhibitor MK-8628 was highly effective when combined with E/C as was the PARP1 inhibitor talazoparib in a small subset of the SCLC lines. While many agents have been tested in combination with E/C in SCLC and failed to improve patients’ survival, the findings of this study identified third agents that may represent new leads for the treatment of this recalcitrant disease. Distinct patterns of response in select subsets of the 62 SCLC lines tested may allow identification of biomarkers predictive of the responders to certain 3-drug regimens. This project has been funded in whole or in part with federal funds from the National Cancer Institute, NIH, under contract no. HHSN261200800001E. Note: This abstract was not presented at the meeting. Citation Format: Beverly A. Teicher, Michael Selby, Thomas Silvers, Julie Laudeman, Russell Reinhart, Rene Delosh, Chad Ogle, Ralph Parchment, Julia Krushkal, Dmitriy Sonkin, Joel Morris, Mark Kunkel, David Evans. Small cell lung carcinoma (SCLC) cell line screen of standard of care (etoposide/carboplatin) plus a third agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4831. doi:10.1158/1538-7445.AM2017-4831

Ewing sarcoma is an aggressive malignancy and the second most frequent bone tumor afflicting the adolescent and young adult population. Despite knowing the oncologic driving event of Ewing sarcoma for decades, outcomes and treatment options for this disease have not improved. A chromosomal translocation between chromosome 11 and 22, fusing the EWSR1 gene to the FLI1 gene (EWSR1::FLI1), is the cause of eighty five percent of Ewing sarcoma cases. Due to the inability to therapeutically target EWSR1::FLI1, the field has instead attempted to target critical co-regulators to disrupt the oncogenic program of EWSR1::FLI1. Histone specific lysine demethylase 1 (LSD1) is overexpressed in Ewing sarcoma and higher levels of LSD1 correlate with poor patient prognosis. Furthermore, LSD1 directly interacts with, colocalizes with, and targets a similar gene set as EWSR1::FLI1, making it a promising target for targeted therapy. Multiple LSD1 inhibitors have been studied with varying effects on the Ewing transcriptome. In particular, OG-L002, a tranylcypromine derivative with potent and specific LSD1 inhibition has little effect on Ewing sarcoma cells, while noncompetitive small molecule inhibitors targeting enzymatic and nonenzymatic functions of LSD1 kill Ewing sarcoma cells at sub micromolar concentrations. As such, we hypothesize that LSD1 has important nonenzymatic functions in Ewing sarcoma. We have curated a suite of molecular tools to deplete LSD1 DNA, mRNA, and protein to define the nonenzymatic effects of LSD1 in Ewing sarcoma. We have knocked out LSD1 via CRISPR genome editing at the DNA level, degraded LSD1 mature RNA via short hairpin RNA interference, degraded LSD1 protein with the UM171 protein degrader, and enzymatically inhibited LSD1 with OG-L002. In every condition we measured the effect on cellular proliferation with Incucyte growth assays, tested the impact on anchorage independent growth with soft agar transformation assays, and found the differentially expressed transcripts with RNA-sequencing. There are only 4 differentially expressed genes when cells are treated with the enzymatic inhibitor OG-L002, compared to hundreds of differentially expressed genes when nonenzymatic functions are depleted as well. In addition, 39 genes that are commonly targeted by depletion at the DNA, RNA, and protein level, corresponding to genes regulated by the nonenzymatic functions of LSD1. We rescued the LSD1 knock out cells with an enzymatically dead mutant (LSD1 K661A) to further specify genes regulated by LSD1 nonenzymatic functions. These genes are related to transcription factor coregulation, axon development, and synaptic organization and signaling. We believe LSD1 serves a scaffolding or recruitment role in various larger chromatin regulatory complexes that are essential for regulation of these genes in Ewing sarcoma oncogenesis that is distinct from LSD1 demethylase activity. Citation Format: Rachel D. Dreher, Ira Miller, Emily R. Theisen. The nonenzymatic effects of LSD1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1436.

Human lung cancer cells, including small cell lung carcinoma (SCLC), frequently lose expression of retinoic acid receptor beta (RAR-beta) and are resistant to the growth inhibitory activity of all-trans retinoic acid (RA). To elucidate the role of RAR-beta in the growth regulation of SCLC by retinoids, we restored RAR-beta expression in RAR-beta-negative H209 SCLC cells by retroviral transduction (H209-RAR-beta). We found that H209-RAR-beta, but not parental H209 cells, underwent growth inhibition upon RA treatment. RA-treated H209-RAR-beta cells arrested in G1 and displayed reduced L-myc expression and cyclin-dependent kinase 2 (cdk2) activity compared with untreated cells. RA treatment of H209-RAR-beta cells was also accompanied by increased expression of the cdk inhibitor p27Kip1, whereas no differences in the expression of L-myc or p27Kip1 were detected upon RA treatment of parental H209 cells. The RA-induced growth arrest of H82 SCLC cells, which express endogenous RAR-beta, was also associated with reduced c-myc and increased p27Kip1 expression. We found that ectopic expression of p27Kip1 induced growth inhibition in both H209 and H82 cells, and that sustained myc expression in H209-RAR-beta cells promoted the induction of apoptosis upon RA addition. Our observations indicate that RAR-beta gene transfer can restore RA sensitivity in SCLC cells and suggest that myc and p27Kip1 may represent critical mediators of the RA-induced cell cycle arrest in SCLC cells expressing RAR-beta.

The type II transforming growth factor-beta (TGF-beta) receptor Ser/Thr kinase (TbetaRII) is responsible for the initiation of multiple TGF-beta signaling pathways, and loss of its function is associated with many types of human cancer. Here we show that TbetaRII kinase is regulated intricately by autophosphorylation on at least three serine residues. Ser213, in the membrane-proximal segment outside the kinase domain, undergoes intra-molecular autophosphorylation which is essential for the activation of TbetaRII kinase activity, activation of TbetaRI and TGF-beta-induced growth inhibition. In contrast, phosphorylation of Ser409 and Ser416, located in a segment corresponding to the substrate recognition T-loop region in a three-dimensional structural model of protein kinases, is enhanced by receptor dimerization and can occur via an intermolecular mechanism. Phosphorylation of Ser409 is essential for TbetaRII kinase signaling, while phosphorylation of Ser416 inhibits receptor function. Mutation of Ser416 to alanine results in a hyperactive receptor that is better able than wild-type to induce TbetaRI activation and subsequent cell cycle arrest. Since on a single receptor either Ser409 or Ser416, but not both simultaneously, can become autophosphorylated, our results show that TbetaRII phosphorylation is regulated intricately and affects TGF-beta receptor signal transduction both positively and negatively.

Kdm1a promotes SCLC progression by transcriptionally silencing the tumor suppressor Rest

Epigenetic dysregulation is a fundamental cancer hallmark, and lysine demethylase 1 (LSD1) is a central target for cancer intervention. Developing novel LSD1 inhibitors with high selectivity, favorable bioavailability, and safety for acute myeloid leukemia (AML) remains challenging. We developed DC551040, a highly potent, selective irreversible LSD1 inhibitor with good tolerability in Phase I AML clinical trial (CTR20222026). DC551040-LSD1 complex crystal structure uncovered a new binding pocket, providing molecular insights for subsequent LSD1 inhibitor design. Given the significant role of LSD1 in epigenetic regulation, we performed comprehensive transcriptomic and proteomic analyses to investigate gene and protein expression dynamics following DC551040 treatment in an MV-4-11 xenograft model. These analyses revealed that multiple immune and inflammation related pathways are activated upon DC551040 treatment, including the key members STAT5, NF-κB, and AKT, suggesting the potential for adaptive resistance. Through a search of the Connectivity Map (CMAP) database, we identify homoharringtonine (HHT), an approved anti-leukemia drug, which mimics the anti-transcriptional activation of inflammatory pathways. Subsequent in vitro and in vivo experiments validated the efficacy of combining HHT with DC551040, demonstrating a synergistic antitumor effect and extended survival in MV-4-11 disseminated xenograft model mice. Together, this study not only introduces a novel LSD1 inhibitor but also delves into the molecular mechanisms underlying LSD1 inhibitors, while proposing a promising combination therapy for AML individuals in clinical trials.

Tumor suppressor Smad4/DPC4 is a central intracellular signal transducer for transforming growth factor-beta (TGF-beta) signaling. We recently reported that transcriptional potential of Smad4 was regulated by SUMOylation in transfected HeLa cells (1), but the precise mechanism and function of Smad4 SUMOylation in TGF-beta signaling remain to be elucidated. Here, we describe the regulation of TGF-beta signaling by SUMOylation through the control of Smad4 metabolic stability and subcellular localization. We found that SUMO-1 overexpression strongly increases Smad4 levels, while inhibition of SUMOylation by small interfering RNA (siRNA)-mediated knockdown of the E2 enzyme Ubc9 reduces endogenous Smad4 levels. Concomitantly, SUMO-1 overexpression enhances and Ubc9 knockdown reduces levels of intranuclear Smad4, growth inhibitory response, as well as transcriptional responses to TGF-beta. Comparison of wild type and mutant forms of Smad4 for SUMOylation, ubiquitination, and half-life allows the conclusion that SUMO-1 modification serves to protect Smad4 from ubiquitin-dependent degradation and consequently enhances the growth inhibitory and transcriptional responses of Smad4.

Neuroendocrine tumors (NETs) harbor neuroendocrine differentiation (ND) with specific markers including protein gene product 9.5 (PGP9.5) and Chromogranin A (CgA). In prostate cancers (PC), ND is induced by BRN2/SOX2 transcription factors. NET-like cells with low or absent androgen receptor (AR) signaling cause hormone therapy resistance and poor prognosis in PC. Small cell lung carcinoma (SCLC), a high-grade NET, presents with metastasis early and has poor survival. ONC201/TIC10 is a small molecule inducer of TRAIL signaling in clinical trials. ONC201 antagonizes dopamine D2 or D3 receptors (DRD2/DRD3) and is an agonist of mitochondrial caseinolytic protease P (ClpP) resulting in activation of DR5/TRAIL-dependent apoptosis involving the integrated stress response (ISR). ONC201 is active in various malignancies including H3K27M-mutated glioma and NETs expressing high levels of DRD2. We hypothesized that altered BRN2/SOX2 may impact NET apoptosis by ONC201 through the ISR and TRAIL/DR5. We analyzed the expression of neuroendocrine markers PGP9.5, CgA, SOX2, and BRN2, as well as markers of TRAIL signaling pathway markers ATF4, DR5, ClpP, ClpX, and DRD2/DRD3 in PC and SCLC cell lines (N=6) ± treatment with ONC201. Specifically, we compared pre-treatment protein expression levels with the IC50. Our results reveal that DU145 (IC50=3.11μM), PC3 (IC50=3.02μM), and LNCaP (IC50=1.33μM) are ONC201 sensitive. H1417 SCLC expresses CgA, unlike PC3 and DU145. PGP9.5 is expressed in these lines. PGP9.5 is expressed in PC3, DU145, H1417, and H1048 but not in LNCaP and 22RV1. BRN2 is expressed in PC3, H1417, and H1048 but not DU145, LNCaP, or 22RV1. ClpX is expressed in all 6 lines but at lower levels in SCLC. ClpP is expressed in the 6 lines. DR5 is expressed at higher levels in PC3, DU145, LNCaP, and 22RV1 PC versus H1417 and H1048 SCLC. SOX2 is expressed at high levels in H1417 cells. These results are establishing the landscape of ND in PC and SCLC lines for further experimentation and testing of our hypothesis. To characterize the association of BRN2 dysregulation with ONC201 sensitivity, we are performing BRN2/SOX2 knockdown experiments using siRNA and evaluating effects towards ONC201 sensitivity. Our results provide insights into molecular mechanisms of ND in PC and SCLC sensitivity to ONC201. We are also currently working to overexpress BRN2 and SOX2 in cell lines to analyze the impact on neuroendocrine differentiation and ONC201 sensitivity. Citation Format: Elizabeth C. Ding, Wafik S. El-Deiry. Neuroendocrine differentiation (ND) in sensitivity of neuroendocrine tumor (NET) cells to ONC201/TIC10 cancer therapeutic. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3734.

Abnormal levels of DNA methylation and/or histone modifications are observed in patients with a wide variety of chronic diseases. Methylation of lysines within histone tails is a key modification that contributes to increased gene expression or repression depending on the specific residue and degree of methylation, which is in turn controlled by the interplay of lysine methyl transferases and demethylases. Drugs that target these and other enzymes controlling chromatin modifications can modulate the expression of clusters of genes, potentially offering higher therapeutic efficacy than classical agents acting on downstream biochemical pathways that are susceptible to degeneracy. Lysine demethylases, first discovered in 2004, are the subject of increasing interest as therapeutic targets. This review provides an overview of recent findings implicating lysine demethylases in a range of therapeutic areas including oncology, immunoinflammation, metabolic disorders, neuroscience, virology and regenerative medicine, together with a summary of recent advances in structural biology and small molecule inhibitor discovery, supporting the tractability of the protein family for the development of selective druglike inhibitors.

To test the prognostic potential of tumor R2* with respect to radiotherapeutic outcome. Blood oxygenation level dependent (BOLD) MRI images are sensitive to changes in deoxyhemoglobin concentration through the transverse MRI relaxation rate R2* of tissue water, hence the quantitative measurement of tumor R2* may be related to tissue oxygenation. Tumor growth inhibition in response to radiation was established for both GH3 prolactinomas and RIF-1 fibrosarcomas with animals breathing either air or carbogen during radiation. In a separate cohort, the baseline R2* and carbogen (95% O2, 5% CO2)-induced DeltaR2* of rat GH3 prolactinomas and murine RIF-1 fibrosarcomas were quantified using multigradient echo (MGRE) MRI prior to radiotherapy, and correlated with subsequent tumor growth inhibition in response to ionizing radiation, while the animals breathed air. A radiation dose of 15 Gy caused pronounced growth delay in both tumor models and transient regression of the GH3 prolactinomas. When the animals breathed carbogen during radiation, the growth delay/regression was enhanced only in the GH3 prolactinomas. The GH3 prolactinomas, which exhibit a relatively fast baseline R2* and large DeltaR2* in response to carbogen breathing prior to radiotherapy, showed a substantial reduction in normalized tumor volume to 66 +/- 3% with air breathing and 36 +/- 5% with carbogen seven days after 15 Gy irradiation. In contrast, the effect of 15 Gy on the RIF-1 fibrosarcomas, which give a relatively slow baseline R2* and negligible DeltaR2* response to carbogen prior to treatment, showed a much smaller growth inhibition (143 +/- 3% with air, 133 +/- 12% with carbogen). Quantitation of tumor R2* and carbogen-induced DeltaR2* by MGRE MRI provides completely noninvasive prognostic indicators of a potential acute radiotherapeutic response.

In Arabidopsis thaliana, PROPEPs and their derived elicitor-active Pep epitopes provide damage-associated molecular patterns (DAMPs), which trigger defence responses through cell-surface receptors PEPR1 and PEPR2. In addition, Pep peptides induce root growth inhibition and root hair formation, however their relationships and coordinating mechanisms are poorly understood. Here, we reveal that Pep1-mediated root hair formation requires PEPR-associated kinases BAK1/BKK1 and BIK1/PBL1, ethylene, auxin and root hair differentiation regulators, in addition to PEPR2. Our analysis on 69 accessions unravels intraspecies variations in Pep1-induced root hair formation and growth inhibition. The absence of a positive correlation between the two traits suggests their separate regulation and diversification in natural populations of A. thaliana. Restricted PEPR2 expression to certain root tissues is sufficient to induce root hair formation and growth inhibition in response to Pep1, indicating the capacity of non-cell-autonomous receptor signalling in different root tissues. Of particular note, root hair cell-specific PEPR2 expression uncouples defence activation from root growth inhibition and root hair formation, suggesting a unique property of root hairs in root defence activation following Pep1 recognition.

Mammary tumor cells derived from vitamin D receptor (VDR) knock-out (KO) mice were engineered to stably express wild-type (WT) or mutated VDR for characterization of the mechanisms by which 1,25-dihydroxyvitamin D (1,25D), the VDR ligand, mediates growth regulation. Although KO cells were completely resistant to 1,25D, introduction of WT human VDR restored gene expression and growth inhibition in response to 1,25D and a variety of structural analogs. Pdgfb, Vegfa, and Nfkbi were identified as genomic targets of both human and murine VDR signaling in this cell model. KO cells expressing hVDRs containing point mutations (W286R, R274L) that reduce or abolish ligand binding did not exhibit changes in gene expression or growth in response to physiological doses of 1,25D but did respond to higher doses and more potent analogs. KO cells expressing hVDR with the G46D point mutation, which abrogates VDR binding to DR3 response elements, exhibited partial growth inhibition in response to 1,25D and synthetic vitamin D analogs, providing proof of principle that VDR signaling through alternative genomic or non-genomic mechanisms contributes to vitamin D mediated growth effects in transformed cells. We conclude that the 1,25D-VDR signaling axis that triggers anti-cancer effects is highly conserved between the murine and human systems despite differences in VDR protein, cofactors, and target genes and that these actions are not solely mediated via canonical VDRE signaling.