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. Author manuscript; available in PMC: 2023 Jul 12.
Published in final edited form as: Pediatr Blood Cancer. 2021 Aug 28;68(11):e29304. doi: 10.1002/pbc.29304

In vivo evaluation of the Lysine-Specific Demethylase (KDM1A/LSD1) inhibitor SP-2577 (Seclidemstat) against Pediatric Sarcoma preclinical models - A report from the Pediatric Preclinical Testing Consortium (PPTC)

Raushan T Kurmasheva 1, Stephen W Erickson 2, Ruolan Han 3, Beverly A Teicher 4, Malcolm A Smith 4, Michael Roth 5, Richard Gorlick 5, Peter J Houghton 1
PMCID: PMC10338045  NIHMSID: NIHMS1907725  PMID: 34453478

Abstract

SP-2577 (seclidemstat), an inhibitor of lysine-specific demthylase KDM1A (LSD1) that is overexpressed in pediatric sarcomas, was evaluated against pediatric sarcoma xenografts. SP-2577 (100 mg/kg/day × 28 days) statistically significantly (P<0.05) inhibited growth of 3/8 EwS, 4/5 RMS, and 4/6 OS xenografts. The increase in EFS T/C was modest (< 1.5) for all models except RMS Rh10 (EFS T/C = 2.8). There were no tumor regressions or consistent changes in di-methyl Histone H3(K4), HOXM1, DAX1, c-MYC and N-MYC, or tumor histology/differentiation. SP-2577 has limited activity against these pediatric sarcoma models at the dose and schedule evaluated.

INTRODUCTION

Lysine-specific demethylase 1 (KDM1A also known as LSD1) is a histone demethylase that is overexpressed in numerous cancers,1,2,3,4 including pediatric sarcomas.5 Importantly, overexpression of KDM1A at the transcript level correlates with protein as determined by immunohistochemistry.6 KDM1A regulates many aspects of cell biology including self-renewal, differentiation and stem cell pluripotency.7–10 Of relevance to sarcoma, KDM1A regulates mesenchymal lineage differentiation for adipose and skeletal muscle fates.11, 12 In addition to its role as an epigenetic modifier, KDM1A is required for chromosome segregation in mitosis, and overexpression induces proliferation in non-transformed human cells, such as mesenchymal stem cells, supporting the idea that KDM1A can act as an oncogene in sarcoma.

In Ewing sarcoma, the EWS-FLI1 oncoprotein is reported to recruit KDM1A to the nucleosome remodeling and histone deacetylase (NuRD) complex,13 which leads to repression of EWS-FLI1 target genes, LOX, TGFBR2, c-MYC and DAX1. Pharmacologic inhibition of KDM1A by several small molecule inhibitors causes de-repression of these genes, and reduced cell viability of EwS cell lines14, suggesting enhanced sensitivity of EwS to KDM1A inhibition. Indeed, SP-2509 (also known as HCI-2509), a non-competitive, reversible inhibitor of KDM1A, suppressed growth of several EwS xenografts (A673, SK-N-MC, and SKES1) in mice13 suggesting that KDM1A may be a therapeutic target for both Ewing and other sarcomas. The clinical candidate, SP-2577 (Seclidemstat) is a potent non-competitive and reversible inhibitor of KDM1A that has similar in vitro potency compared to SP-2509, and in vivo activity in Ewing sarcoma models.1 Here we evaluated SP-2577 in panels of xenografts derived from pediatric sarcomas.

MATERIALS AND METHODS

In vivo testing:

C.B.17SC scid−/− (C.B-Igh-1b/IcrTac-Prkdcscid) female mice (Taconic, Germantown, NY) were used to propagate subcutaneous sarcoma xenografts. Tumor fragments (~ 9 mm3) were implanted in the left flank. All methods and analyses were as previously described.15 Details of the statistical analytic methods are provided in Appendix 1.

Tumor models:

All EwS lines used have a type 1 EWS-FLI1 translocation, and Rh10, Rh28, Rh30, Rh41 and Rh65 are alveolar RMS (t(2;13) fusion positive expressing Pax3-Foxo1). Rh36 is a Ras-mutated (HRASQ61L), fusion-negative RMS. Genomic characterization of most models used in this study have been recently reported.16

Western blotting:

Tissues were collected 4 hours after dose 7 (100 mg/kg daily) of SP-2577 for ES-4, EW-8 EwS models and Rh41 RMS. For the Rh10 RMS xenograft, tissue was collected 4 Hr after a single administration of SP2577. Western blotting was as described.17 In addition Rh10 control and SP-2577 treated tumor tissue was also taken for histologic evaluation 4 Hr following the last administration of drug (day 28).

Drugs and Formulation:

SP-2577 was provided to the PPTC by Salarius Pharmaceuticals. SP-2577 was diluted in the vehicle solution consisting of 1.6% DMA (dimethylacetamide), 5% ethanol (ETOH), 45% polyethylene glycol 400 (PEG400), 48.4% phosphate buffered saline (PBS) with a pH of 8 and administered intraperitoneal (IP) once daily for 28 days, as recommended by the supplier. A dose of 100 mg/kg was utilized based on recommendation from Salarius Pharmaceuticals.

RESULTS

In vivo evaluation:

Previously it was shown that SP-2509, a tool compound for SP-2577, could inhibit EwS xenograft growth at a dose of 30 mg/kg/day (footnote 1). SP-2577 is also active at the same dose in several EwS models. SP-2577 was tested at a daily dose of 100 mg/kg in 8 EwS, 5 RMS and 6 OS xenografts using procedures previously reported.18 Antitumor activity of SP-2577 against each sarcoma histotype is summarized in Table 1. Treatment was initiated when tumors were 200–400 mm3. SP-2577 statistically significantly (P<0.05) inhibited growth of 4/8 EwS, 4/5 RMS, and 6/6 OS xenografts. There were no tumor regressions, and with the exception of Rh10 (an alveolar RMS model), all responses were classified as Progressive Disease 1 (PD1) with less than 2-fold prolongation of median time to event (EFS T/C ratios 1.12–1.43), and 2.8 for Rh10. Data for RMS xenografts are shown in Figure 1, and EwS and OS in Supplemental Figures 1 and 2.

Table 1.

Summary of SP-2577 Activity Against Pediatric Sarcoma Xenografts

Tumor Group KM (Med) (days) EFS T-C (days) EFS T/C P-value (G/W)1 PD2 PD1 PD2 SD PR CR MCR ORR (%) Median Response
Ewing Sarcoma (EwS)
A673 A 6.3 10 0 0 0 0
B 8.7 2.4 1.37 0.037 9 8 1 0 0 0 0 0 PD1
ES-1 A 9.7 10 0 0 0 0
B 13.3 3.6 1.37 <0.001 8 5 3 0 0 0 0 0 PD1
ES-4 A 6.0 10 0 0 0 0
B 7.9 2.0 1.33 <0.001 10 8 2 0 0 0 0 0 PD1
ES-7 A 9.9 10 0 0 0 0
B 12.6 2.7 1.28 0.173 10 8 2 0 0 0 0 0 PD1
EW-5 A 9.5 10 0 0 0 0
B 9.6 0.1 1.02 0.327 10 10 0 0 0 0 0 0 PD1
EW-8 A 11.8 10 0 0 0 0
B 12.6 0.9 1.07 0.148 10 10 0 0 0 0 0 0 PD1
NCH-EWS-1 A 14.6 10 0 0 0 0
B 16.1 1.5 1.10 0.935 9 9 0 0 0 0 0 0 PD1
SK-NEP A 13.5 9 0 0 0 0
B 18.5 5.0 1.37 0.002 9 7 0 0 0 0 0 0 PD1
Rhabdomyosarcoma (RMS)
Rh10 A 7.8 10 0 0 0 0
B 21.8 14.0 2.79 <0.001 10 0 10 0 0 0 0 0 PD2
Rh28 A 12.3 10 0 0 0 0
B 17.6 5.2 1.43 0.007 6 4 0 0 0 0 0 0 PD1
Rh30 A 14.3 10 0 0 0 0
B 19.7 5.3 1.37 0.111 8 7 1 0 0 0 0 0 PD1
Rh36 A 20.2 10 0 0 0 0
B 23.8 3.6 1.18 0.040 9 8 0 0 0 0 0 0 PD1
Rh65 A 12.7 10 0 0 0 0 0
B 14.5 1.9 1.18 0.030 9 9 0 0 0 0 0 0 PD1
Osteosarcoma (OS)
OS-2 A 31.1 10 0 0 0 0
B 39.2 8.1 1.26 0.003 10 10 0 0 0 0 0 0 PD1
OS-9 A 15.3 10 0 0 0 0
B 19.0 3.6 1.24 0.003 10 10 0 0 0 0 0 0 PD1
OS-31 A 17.4 10 0 0 0 0
B 19.5 2.2 1.12 0.008 10 0 0 0 0 0 0 0 PD1
OS-33 A 19.8 10 0 0 0 0
B 26.4 6.7 1.34 0.004 10 10 0 0 0 0 0 0 PD1
OS-36 A 14.5 10 0 0 0
B 16.4 1.8 1.13 0.015 10 10 0 0 0 0 0 0 PD1
OS-60 A 30.1 10 0 0 0 0
B 35.8 5.6 1.19 0.036 10 0 0 0 0 0 0 0 PD1
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Figure 1.

Figure 1.

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Responses of rhabdomyosarcoma xenografts to SP-2577. Mice received SP-2577 (100 mg/kg daily × 28 days) when tumors were 200–400 mm3). Left panel, Lines show growth of individual tumors, Control (red); SP-2577 treated (blue). The solid (bold) lines show median response; Center panel, Relative tumor volume; Right panel, Kaplan-Meier probability plots for Event-Free Survival (EFS).

Pharmacodynamic study:

Expression of KDM1A in PPTC xenograft models and normal tissues is shown in Supplemental Figure 3. Median expression of KDM1A in PPTC xenografts is higher than in normal tissue, with the exception of testes. To assess target inhibition mice bearing ES-4, EW-8 or Rh41 xenografts were treated daily for 7 days with SP-2577 (100 mg/kg/day), and tumors excised 4 Hr after the final dose. Changes in EWS-FLI1 target gene products, DAX1, HOXM1 as well as Histone H3(K4 di-methyl), and Myc were determined after 7 days treatment ES-4, EW-8, Rh41), or 4Hr after a single drug administration (Rh10), Supplemental Figures 4 and 5. Histone H3(K4 di-methyl) marginally increased in ES-4 xenografts, but there were no drug related changes in DAX1, HOXM1 or N- or c-MYC.In Rh10 tumors there was a slight decrease of the c-MYC and DAX1 signals in treated tumors. There was no evidence of drug-induced differentiation at the end of 28 days dosing (Supplemental Figure 6).

DISCUSSION

Across the PPTC xenograft models, expression of KDM1A mRNA was quite variable (median 53 FPKM), but generally high in EwS (median 78 FPKM), RMS (median 70 FPKM) and OS (median 49 FPKM), relative to normal tissues, with the exception of testes.

In this study we evaluated SP-2577 against panels of molecularly characterized pediatric sarcoma xenografts. In contrast to the results reported with SP-2509 against three EwS xenografts (A673 SKES1 and SK-N-MC),13 the antitumor activity of SP-2577 against the EwS xenografts in our study, which included A673, was essentially negative as were results in OS models. For RMS models, SP-2577 extended EFS T/C by 2.8-fold in Rh10, but otherwise had little antitumor activity. A difference between the studies that could explain some of the discrepancies is the tumor volume at which time treatment was initiated (< 50 mm3 in the study by Sankar et. al13 compared to 200–300 mm3 in the current study). Another difference is that in the Sankar et al. study, assessment of drug efficacy was made at the end of treatment, whereas assessment in our study was made at day 42 (14 days after the last drug dose). Re-analysis of the current data set but comparing treated/control tumor volumes at the end of SP-2577 treatment (day 28), showed that tumors progressed through treatment, and that the difference in response (measured at day 42) was not the result of tumors accelerating their growth rate after treatment stopped.

Pharmacodynamic studies showed that there were higher levels of H3(K4) di-methylation in EW-8 and Rh41 tumors compared to ES-4 xenografts, and SP-2577 may have marginally increased demethylation in ES-4 tumors, but not the other models. Similarly, no drug-mediated changes in DAX1 were detected, but HOXM1 was slightly increased in SP-2577 treated EW-8 tumors compared to controls. Neither DAX1 nor HOXM1 were detected in Rh41 RMS xenografts, and drug treatment did not alter levels of N- or c-Myc in this model. Only very minor changes in c-MYC, H3K4 di-methylation and DAX1 were detected in Rh10 tumors. Similarly, there was no evidence for drug-induced differentiation after four weeks of treatment with SP-2577.

In summary, SP-2577 showed little preclinical activity and inconsistent pharmacodynamic effects. While SP-2577 had some activity against one alveolar RMS model, this activity may be through a mechanism independent of KMD1A demethylase inhibition.

Supplementary Material

Supplementary Figures

ACKNOWLEDGEMENTS

We thank Abhik Bandyophadhyay, Vanessa Del Pozo, Samson Ghilu, Edward Favours, Fuyang Li and Kathryn Bondra for technical assistance. Dr. Ruolan Han was an employee of Salarius Pharmaceuticals.

Abbreviation

EwS

Ewing sarcoma

RMS

Rhabdomyosarcoma

OS

Osteosarcoma

NuRD

nucleosome remodeling and histone deacetylase complex

Footnotes

1

SP-2577 (Seclidemstat) for the Treatment of Relapsed or Refractory Ewing Sarcoma, Oncologic Drugs Advisory Committee Pediatric Subcommittee Meeting, 17 June 2020. Advisory committee briefing materials: available for public release.

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Supplementary Materials

Supplementary Figures
NIHMS1907725-supplement-Supplementary_Figures.docx (34.4MB, docx)

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