Catalytic inhibition of KDM1A in Ewing sarcoma is insufficient as a therapeutic strategy

Ewing sarcoma and desmoplastic small round cell tumors (DSRCT) are rare and clinically aggressive sarcomas usually characterized by oncogenic fusion proteins involving EWS. Emerging studies of Ewing sarcoma have demonstrated EWS‐FLI1‐driven chromatin remodeling as a key aspect of tumorigenicity. In particular, the lysine‐specific demethylase KDM1A/LSD1 is linked to transcriptional regulation of target genes orchestrated by the EWS portion of the fusion protein interacting with repressive chromatin‐remodeling complexes. Consistent with this model, depletion of KDM1A supports it is a molecular therapeutic target in Ewing sarcoma cells, but effective drugs need to be identified.

Irreversible inhibitor covalently modifying the FAD cofactor (22) Irreversible inhibitor covalently modifying the FAD cofactor (23) Irreversible inhibitor covalently modifying the FAD cofactor (23) Reversible noncompetitive inhibitor (43) Clinical status Phase I/IIa study in AML (EudraCT Number: 2013-002447-29 ) and SCLC (NCT02913443) Phase I studies in SCLC (NCT02034123) and AML (NCT02177812) have been terminated because the risk benefit does not favor continuation. Phase I/II study in MDS (NCT02929498) NA NA of genomic regions and creating de novo enhancer elements that establish the Ewing sarcoma transcriptional signature. [8][9][10] In particular, the nucleosome remodeling deacetylase complex (NuRD; also known as Mi-2), a multi-subunit chromatin-remodeling complex commonly associated with repression of transcriptional activity, was identified to be relevant in the silencing of EWS-FLI1 target genes. 10,11 Essentially, the catalytic activity in the NuRD complex involves deacetylation by histone deacetylase 2 (HDAC2) and 3 (HDAC3), coupled with demethylation by lysine-specific demethylase 1 (KDM1A; also referred to as LSD1, AOF2, and BHC110). 9,10 The working model proposed by Sankar et al consists of EWS-FLI1 binding to promoters of repressed target genes, followed by the recruitment of the NuRD complex through interaction between its subunits chromodomain 4 (CHD4) and metastasis-associated protein 1 (MTA1) and the EWS portion of the fusion protein. Following treatment with SP2509, a tool compound targeting KDM1A, the EWS-FLI1driven transcriptional signature of both upregulated and downregulated genes was reversed. 10 Inhibition of KDM1A with SP2509 also resulted in apoptosis and disruption of the oncogenic phenotype. 10 KDM1A is a well-characterized histone lysine demethylase belonging to the family of flavin-dependent amine oxidases that has an important role in stem cell maintenance through transcriptional repression. [12][13][14] This demethylase is also overexpressed in Ewing sarcoma and other sarcomas including DSRCT and rhabdomyosarcoma. 15

3D spheroid culture
Three-dimensional spheroids were generated and cultured using the GravityTRAP ULA Plate 96-well (PerkinElmer, UK) unless otherwise indicated. Prior to seeding, wells were pre-wet with 40 µL of medium. One thousand cells in 70 µL were seeded into each well. Plates were spun for two minutes at 250 RCF to remove trapped air bubbles. Routine medium changes were performed every 48 hours by carefully aspirating the medium from the ledge at the inside wall of the well and replenishing it with fresh medium.

Statistical analysis
GraphPad PRISM 7 and R studio 3.3.2 were used to carry out the statistical analyses. Error bars represent means ± standard deviation from various independent experiments as indicated in figure legends. Statistical significance was measured by one-way ANOVA with post hoc Sidak test for multiple comparisons and two-way ANOVA with post hoc Dunnett test and Sidak test where applicable. P < 0.05 was considered significant and indicated by *, P < 0.01 is indicated by **, P < 0.001 is indicated by ***, and P < 0.0001 is indicated by ****.

RESULTS
Presently, there is no evidence demonstrating whether the clinical drug  Figure   S1F-S1H), 9,10 possibly due to the lower concentration used in our experiments.
To further demonstrate compound activity, we assessed the response of leukemia cells, previously shown to be sensitive to inhibition of KDM1A catalytic function by ORY-1001 in the nanomolar range. 22 In agreement with published findings, the clinical candidate ORY-1001, as well as chemical probe GSK-LSD1, showed a reduction in cell viability upon treatment with compound at 10 nM concentrations in two leukemia cell lines (Supporting Information Figure S2A and S2B). The clinical candidate GSK2879552 also decreased cell viability at 2 µM (Supporting Information Figure S2C).
To perform a more comprehensive evaluation of the effect of KDM1A inhibition, assessment of cell viability experiments was expanded to 3D models of Ewing sarcoma. Three-dimensional cell culture systems have become more prominent in preclinical studies due to their ability to more closely represent tissue compartments and enable longer experimental time frames. 27 Tumor spheroids, for example, can recapitulate aspects of the tumor microenvironment that ultimately influence drug response. 28 A673 spheroid cultures were treated with ORY-1001 and SP2509 for 10 days with a dose range between 0.3 and 10 µM (Figure 2 and 2B). Again, no effect on growth was observed with the irreversible inhibitor of KDM1A catalytic function ORY-1001, whereas SP2509 was active, albeit at higher concentrations compared with 2D cultures (Figure 2 and 2B). Drugs targeting epigenetic modifying enzymes often require prolonged inhibition for maximal drug potency and response to treatment. 22,23,29 We therefore additionally treated spheroids for up to 21 days with a maximum concentration of 100 µM of both clinical candidates (ORY-1001 and GSK2879552).
Both clinical candidates had no effect upon spheroid growth ( Figure 2 and 2E), even in the extended assay.
Finally, KDM1A has been reported to have a role in migration and invasion. 30 Figure S3). Again, we included the chemical probe GSK-LSD1 in the assay; it also had no effect on invasion (Figure 3 and 3B; Supporting Information Figure S3).

DISCUSSION
The Ewing sarcoma gene-expression profile heavily relies on EWS-  tor, also had no effect on the cell viability of Ewing sarcoma cells in 2D and 3D. Prolonged inhibition, as a means to achieve maximal efficacy, as in reports with inhibitors of Enhancer of Zeste Homolg 2 (EZH2) in lymphoma, did not alter the response to these inhibitors of KDM1A catalytic function. 22,23,29 KDM1A's reported role in modulating self-renewal and differentiation in embryonic stem cells also encompasses regulation of migration during normal mammalian development. 14

ACKNOWLEDGMENTS
We are grateful to our funders whose support made this work possible: the Elin Rose Appeal (AR), the Tom Bowdidge Foundation (EA), and the Hopkins family (SAG).

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.

AUTHORS' CONTRIBUTIONS
AR conducted the experiments and analyzed data. AR, EA, SAG, JS designed the project. SAG and JS supervised the project. AR drafted the manuscript and all authors participated in writing the manuscript and approved the final version.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.