ADVISORY COMMITTEE BRIEFING MATERIALS:
AVAILABLE FOR PUBLIC RELEASE
Oncologic Drugs Advisory Committee Pediatric Subcommittee Meeting
17 June 2020
SP-2577 (Seclidemstat) for the Treatment of Relapsed or Refractory Ewing Sarcoma
Salarius Pharmaceuticals 2450 Holcombe Blvd, Suite J-608 Houston, TX 77021 Phone: 346-772-0346 Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 2 of 58
Table of Contents List of Abbreviations ...... 5 1 SP-2577 (Seclidemstat): Introduction ...... 7 2 Relapsed / Refractory Ewing Sarcoma ...... 7 2.1 SP-2577: A novel LSD1 inhibitor in relapsed / refractory Ewing sarcoma patients ...... 13 3 Drug ...... 16 3.1 SP-2577: Description of the drug ...... 16 4 Nonclinical Studies in Ewing Sarcoma Models...... 19 4.1 Background ...... 19 4.2 SP-2577 and SP-2509 inhibit LSD1 activity ...... 20 4.3 SP-2577 and SP-2509 reduce tumor cell viability in vitro ...... 22 4.4 SP-2577 and SP-2509 reduce tumor burden in vivo ...... 23 4.5 SP-2577 and SP-2509 are effective in combination with epigenetic and chemotherapeutic agents ...... 30 4.6 Summary of nonclinical studies ...... 39 5 SP-2577: IND Enabling Toxicology and Pharmacology Studies ...... 39 5.1 SP-2577 GLP toxicology studies in rats and dogs ...... 39 5.2 Nonclinical pharmacokinetics and rationale for starting dose ...... 39 6 Seclidemstat: Clinical Human Experience to Date ...... 40 6.1 Compassionate use study ...... 40 6.2 Seclidemstat: Phase 1 studies...... 41 6.2.1 Patients with relapsed or refractory Ewing sarcoma ...... 41 6.2.2 Patients with advanced solid tumors ...... 45 6.2.3 Combined pharmacokinetic data in SALA-002-EW16 and SALA-003-AC19 ...... 47 6.3 Seclidemstat: Summary of preliminary Phase 1 data ...... 50 7 Future Clinical Development of Seclidemstat ...... 51 8 Summary ...... 53 9 References ...... 54
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List of Figures Figure 1: LSD1 expression in Ewing sarcoma vs survival ...... 14 Figure 2: LSD1 inhibition reverses oncogenic gene expression pattern in Ewing sarcoma models ...... 15 Figure 3: Chemical structure of SP-2577 ...... 16
Figure 4: IC50 of SP-2577 against LSD1 enzymatic activity ...... 17 Figure 5: Diagram of LSD1 catalytic activity ...... 18 Figure 6: SP-2509 and SP-2577 demonstrate similar efficacy in vitro ...... 19 Figure 7: SP-2577 destabilizes LSD1 and cMyc protein ...... 20 Figure 8: SP-2509 reverses LSD1/EWS/FLI1-driven gene expression more robustly than irreversible LSD1 inhibitors ...... 21 Figure 9: SP-2509 LSD1 inhibition reduces cell viability more robustly than irreversible inhibitors and impedes anchorage-independent growth ...... 22 Figure 10: SP-2577 in vivo pharmacokinetics ...... 23 Figure 11: In vivo efficacy of 30 mg/kg treatment of SP-2577 in SK-N-MC xenograft model of Ewing sarcoma ...... 25 Figure 12: In vivo efficacy of 30 mg/kg treatment of SP-2577 in A673 xenograft model of Ewing sarcoma ...... 26 Figure 13: In vivo efficacy of i.p. and oral-administered SP-2577 in SK-N-MC xenograft mouse model ...... 27 Figure 14: Responses of Ewing sarcoma xenografts to SP-2577...... 29 Figure 15: Combination of SP-2509 and SP-2577 with chemotherapy ...... 31 Figure 16: LSD1 is over-expressed in various sarcomas ...... 32 Figure 17: Event-free survival, study PPTC-1705-01 ...... 35 Figure 18: SP-2577 in hematological malignancies ...... 37 Figure 19: SP-2577 in combination with checkpoint blockade inhibition ...... 38 Figure 20: Cycle 1, single-dose concentration versus time profiles by dose level in SALA-002-EW16 in fasted conditions ...... 45 Figure 21: Cycle 1, single-dose concentration versus time profiles by dose level in SALA-003-AC19 in fasted conditions ...... 47 Figure 22: Cycle 1, single-dose concentration versus time profiles by dose level in SALA-002-EW16 and SALA-003-AC19 combined in fasted conditions ...... 48 Figure 23: Steady-state pharmacokinetic modeling based on Cycle 1, Day 1 fasting pharmacokinetics in SALA-002-EW16 and SALA-003-AC19 ...... 50
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List of Tables Table 1: Conventional chemotherapeutics being studied for recurrent / relapsed Ewing sarcoma ...... 10 Table 2: IGF-1R antibody responses in Ewing sarcoma ...... 11 Table 3: Summary of in vivo efficacy studies ...... 24 Table 4: Study results PPTC study PPTC-1705-01; SALA Pharm 015 ...... 30 Table 5: Study results PPTC-1705-01 ...... 34 Table 6: SALA-002-EW16 patient demographics ...... 43 Table 7: SALA-002-EW16 disease characteristics ...... 43 Table 8: Summary Cycle 1, Day 1 fasted pharmacokinetics in SALA-002-EW16 ...... 45 Table 9: Summary Cycle 1, Day 1 fasted pharmacokinetics in SALA-003-AC19 ...... 47 Table 10: Summary Cycle 1, Day 1 fasted pharmacokinetics in SALA-002-EW16 and SALA-003-AC19 combined ...... 48
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List of Abbreviations AML acute myeloid leukemia AOD amine oxidase domain AUC area under the plasma concentration over time curve BID twice daily cfDNA cell-free deoxyribonucleic acid CI confidence interval
Cmax peak plasma concentrations CR complete response CT computed tomography CTC circulating tumor cells CTCAE Common Terminology Criteria for Adverse Events CTOS Connective Tissue Oncology Society COG Children’s Oncology Group DFI disease-free interval DLT dose-limiting toxicity DoR duration of response ECOG Eastern Cooperative Oncology Group EFS event-free survival EFS T/C event-free survival treated/control EVR endogenous retroviruses FAD flavin adenine dinucleotide FDA Food and Drug Administration
GI50 50% growth inhibitory concentration HDAC histone deacetylase HSPC hematopoietic stem and progenitor cell
IC50 half maximal inhibitory concentration ICH International Council for Harmonisation IGF-1R insulin-like growth factor 1 IND investigational new drug application i.p. intraperitoneal, intraperitoneally IQR interquartile range KM Kaplan-Meier LSD1 lysine-specific histone demethylase 1 MDS myelodysplastic syndrome MRI magnetic resonance imaging MTD maximum tolerated dose NuRD nucleosome remodeling and deacetylase Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 6 of 58
ORR overall response rate PARP poly ADP ribose polymerase PD progressive disease PFS progression-free survival PK pharmacokinetics PO oral PPTC NCP Pediatric Preclinical Testing Consortium PR partial response RECIST response evaluation criteria in solid tumors rEECur randomized study by the Euro Ewing Consortium RP2D recommended Phase 2 dose SRC Safety Review Committee SWIRM α-helical domain of about 85 residues in chromosomal proteins such as Swi3p, Rsc8p SAHA suberanilohydroxamic acid TCP tranylcypromine VDC/IE vincristine, doxorubicin, cyclophosphamide / ifosfamide and etoposide Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 7 of 58
1 SP-2577 (Seclidemstat): Introduction Salarius Pharmaceuticals holds an investigational new drug application (IND) for SP-2577 (seclidemstat), which is currently being studied in a Phase 1 clinical study as a single agent administered orally in patients at least 12 years of age with relapsed or refractory Ewing sarcoma (SALA-002-EW16). Seclidemstat is also being studied in an ongoing Phase 1 study in patients at least 12 years of age with advanced solid tumors (SALA-003-AC19). The Food and Drug Administration (FDA) granted seclidemstat orphan drug designation in January 2017 and fast track designation in December 2019 for relapsed or refractory Ewing sarcoma. Prior to the active IND (March 2018) for Phase 1, Salarius completed an IND compassionate use protocol with seclidemstat for a single patient with metastatic Ewing sarcoma (SALA-CP01-16- 001-CC) who was treated over a 3-month period (April-July 2016). The Phase 1 studies for Ewing sarcoma and advanced solid tumors are both currently recruiting patients. The Ewing sarcoma study started in 3Q 2018 and is further detailed below (Section 6). The advanced solid tumor study started in 2Q 2019. Salarius believes that seclidemstat’s mechanism of action provides strong scientific rationale for studying the drug’s therapeutic activity in the relapsed/refractory Ewing sarcoma population. Salarius’ ongoing clinical studies are focused on studying seclidemstat’s safety/pharmacokinetics as well as collecting exploratory pharmacodynamic biomarker data. To successfully develop seclidemstat, Salarius is seeking guidance on selecting the appropriate patient population, defining the necessary clinical endpoints and designing innovative trials for assessment of seclidemstat’s activity in the relapsed/refractory Ewing sarcoma population. Additionally, Salarius seeks input on the best approach for future submission of a Proposed Pediatric Study Request.
2 Relapsed / Refractory Ewing Sarcoma Ewing sarcoma is an uncommon illness with a poor prognosis that is poorly served by available therapies. Advances in the understanding of the disease and its pathogenesis has led to newer therapies. These therapies must be brought to the patients and this will need a close coordination and collaboration between the drug developers, investigators, and regulators. Ewing sarcoma is a rare, aggressive, small round blue cell tumor typically presenting as a primary bone tumor in children and young adults. It has an incidence of ~3.0 cases per million (Gorlick et al., 2013; Esiashvili et al., 2008). Ewing sarcoma is the second most common malignant bone tumor in children and adolescents and is considered a pediatric cancer. Ewing sarcoma has a peak incidence rate during the second decade of life (10-20 years of age) with a median age of diagnosis being 15 years (Esiashvilli et al., 2008; Ginsberg et al., 2010; Gurney et al., 1995; Liddy et al., 2003). Advances in multimodality treatment consisting of surgery, radiation and chemotherapy have improved the 5-year survival rate of patients presenting with Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 8 of 58 localized disease to 70%-80% (Xu et al., 2019). However, despite the intensity of current treatment regimens, in the ~30% of patients that initially present with metastatic disease the rate of treatment failure ranges from 50% to 80%. Furthermore, about 25% of patients with initially localized Ewing sarcoma will relapse, with the majority (70%) relapsing within two years (Van Mater et al., 2019). Collectively, for these refractory/relapsed patients, there have not been improvements in treatment options and long-term survival remains low at around 20% (Van Mater et al., 2019). The prognosis for relapsed/refractory Ewing sarcoma patients is distressingly poor, a fact further accentuated by the young population that is affected by the disease. There is no standardized second-line treatment, highlighting the lack of clarity for optimal treatment options (Xu et al., 2019). The most indicative prognostic factor within the recurrent population is the time between diagnosis and the first disease relapse, referred to as the disease-free interval (DFI). About 70% of relapse patients have a DFI of less than two years (Van Mater et al., 2019). For relapsed patients with a DFI of less than two years, the 2-year overall survival is only about 7%, with a median survival duration as low as three months (Shanker et al., 2003). The current low survival rate and short median survival times for these patients are unacceptably low and define this patient population as a group with a high unmet medical need. There is a need for the development of new therapeutic options to improve patient outcomes in the relapsed/refractory Ewing sarcoma population. Current practices to improve outcomes for patients with relapsed/refractory Ewing sarcoma consist of conventional cytotoxic agents or enrollment into studies studying novel approaches. The Phase 2/3 rEECur study, an international randomized trial for the treatment of recurrent and refractory Ewing sarcoma, which is conducted by the EURO Ewing Consortium, is evaluating four routinely used chemotherapy regimens to determine which will produce the best response. rEECur is evaluating 1) gemcitabine/docetaxel, 2) cyclophosphamide/topotecan, 3) high dose ifosfamide and 4) temozolomide/irinotecan. Patients will be randomized to receive one of up to four treatment regimens, their response assessed, and the worst of the treatment regimens will be eliminated via two interim assessments until two regimens remain. The remaining two regimens will be studied in a Phase 3 study. The first interim reported that gemcitabine/docetaxel was less effective than the other three. All four treatment regimens produced substantial side effects with ~60% of patients reporting ≥ Grade 3 adverse events. The second interim report is expected in the next few months and will reveal which of the two treatment regimens will be studied in the Phase 3 portion of the study (McCabe et al., ASCO 2019). The Phase 2/3 rEECur study will randomize a total of 525 patients across European sites. It started recruitment in January 2015 and is expected to complete by 2025 (https://www.cancerresearchuk.org/about-cancer/find-a- clinical-trial/a-trial-looking-at-chemotherapy-for-ewings-sarcoma-reecur). At the first interim analysis, 71% (157/220) of patients had either primary refractory or first recurrence in less than two years, while an additional 14% (30/220) had first recurrence more or equal to two years and Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 9 of 58 the remaining 15% had second or subsequent relapse (McCabe et al., ASCO 2019). Safety and efficacy data from the stopped gemcitabine/docetaxel arm, combined three remaining arms and the combined four arms, demonstrate an overall response rate (ORR) (partial response [PR] and complete response [CR]) of 11% (7/62), 24% (29/123) and 20% (36/185), respectively, for patients with evaluable measurable disease. The overall CR rate for the combined four arms is low (4/195, 2.1%). Of the 220 patients treated, 71% had progressed. There was considerable toxicity with approximately 60% of all randomized patients experiencing Grade 3 or higher adverse events. The therapies chosen for the rEECur study were chosen because they represented the best-known chemotherapies at the time, mainly doublet chemotherapy, to treat a group of recurrent/refractory Ewing sarcoma patients who meet an unmet medical need. The chemotherapy regimens chosen for the rEECur study have been previously studied, along with other chemotherapeutic regimens, and the cumulative response rates are shown in Table 1. These cumulative response rates are the summation of multiple, small-in-number, usually single- institution retrospective analyses or single-arm Phase 2 studies. As shown, the temozolomide/irinotecan combination results in the best cumulative response rate. Unfortunately, the median time to progression is only approximately five months across seven studies to date (Van Mater et al., 2019). Additionally, there are severe short and long-term cytotoxic side effects associated with chemotherapeutic regimens including cardiomyopathy, renal insufficiency, reduced fertility and increased chance of developing a secondary malignancy (Grunewald et al., 2018). The ongoing rEECur study will provide the safety and efficacy data for the proposed chemotherapy regimens in the setting of a multi-national, randomized, controlled study of chemotherapy, which will prospectively define the appropriate chemotherapy regimen to use at the time of treatment for patients with recurrent or primary refractory Ewing sarcoma. While determining the optimal second-line chemotherapy regimen will help standardize treatment for this patient population, the proposed regimens each have low response rates with rare complete responses, responses are of limited duration, and the majority of patients will progress within one year. Additionally, there are severe short and long-term cytotoxic side effects associated with chemotherapeutic regimens including cardiomyopathy, renal insufficiency, reduced fertility and increased chance of developing a secondary malignancy (Grunewald et al., 2018). Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 10 of 58
Table 1: Conventional chemotherapeutics being studied for recurrent / relapsed Ewing sarcoma
Agent(s) Number of Cumulative Cumulative Published Data Patients response rate
Cyclophosphamide / topotecan 3 79 32%
Gemcitabine/docetaxel 3 24 29%
Ifosfamide 1 35 34%
Temozolomide/irinotecan 7 166 47%
Etoposide with carbo- or cisplatin 1 107 29%
Oral etoposide 1 58 19%
Adapted from Van Mater et al., 2019. Investigators have turned toward targeted agents to help improve relapsed/refractory Ewing sarcoma patient outcomes. Collectively, single agent targeted therapies have shown acceptable safety profiles in Phase 1 studies but have failed to show any meaningful responses in Phase 1/2 or Phase 2 studies (Xu et al., 2019). As a result, only a few of these agents have moved forward in clinical development as monotherapy beyond a Phase 1 and none beyond a Phase 2. However, targeted agents are now being combined with chemotherapeutic backbones, such as insulin-like growth factor 1 (IGF-1R) targeted therapy. Originally, IFG-1R was seen as a promising target due to the high expression in Ewing sarcoma cells (Van Mater et al., 2019). However, Phase 2 studies studying IGF-1R antibodies have produced modest responses (Table 2). A Phase 3 study of combination chemotherapy (vincristine, doxorubicin, cyclophosphamide, ifosfamide and etoposide [VDC/IE]) with or without ganitumab (monoclonal antibody directed against IGF-1R) in patients with newly diagnosed, metastatic Ewing sarcoma as a first-line therapy has been reported in preliminary manner, with similar outcomes between randomized arms (Connective Tissue Oncology Society [CTOS] annual meeting, 2019). Other studies of targeted agents including poly ADP ribose polymerase (PARP) inhibitors, kinase inhibitors, and agents targeting the transcriptional machinery are ongoing, many not due to finish recruitment until after 2021 and beyond (https://clinicaltrials.gov/). Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 11 of 58
Table 2: IGF-1R antibody responses in Ewing sarcoma
Intervention No. of pts evaluated ORR (%) Reference
Figitumab 106 14 Juergens et al., 2011
Ganitumab 18 5 Tap et al., 2012
R1507 115 10 Pappo et al., 2011
The Children’s Oncology Group (COG) recently reported on the treatment outcomes of 128 patients with relapsed or progressive Ewing sarcoma enrolled from January 1997 until October 2007 on seven Phase 2 COG studies utilizing single-agent treatments (median number of patients per study 18 pts, range 9-26) (Collier et al., 2019). These seven treatments were predominantly cytotoxic agents (6) including docetaxel, topotecan, irinotecan, a rebeccamycin analogue, oxaliplatin and ixabepilone and targeted therapy (1) including imatinib. Treatment response outcomes included event-free survival (EFS) (defined as time from study enrollment until first date of disease progression, date of detection of disease at a previously uninvolved site, date of death or date of late follow-up) using the method of Kaplan and Meier. For all studies combined, for a total of 124 events including 106 relapses and 18 deaths, the 6-month EFS was 12.7% (95% confidence interval [CI] 7.6-19%). There was no difference in EFS based on age at relapse, age at diagnosis, number of prior chemotherapy regimens or patient sex. Only one study which treated patients with docetaxel, met the protocol-specified goal for activity defined by radiographic response rate with 11.5% (3/26 patients) PRs. However, the 6-month progression- free survival (PFS) in the docetaxel study was 15% (95% CI 4.8-31%). Comparing the EFS of this study to the other six studies combined, there was no difference in EFS (12%, 95% CI: 6.6- 19.2%; p=0.253). The higher response rate for docetaxel did not translate into superior disease control as assessed by EFS. Partial responses were demonstrated in two other studies, but were minimal, 2/22 (9.1%) for topotecan and 1/26 (3.8%) for imatinib. These results demonstrate that currently available chemotherapy/targeted therapies have poor overall treatment outcomes in this patient population and further support that these patients meet the criteria for an unmet medical need. The authors suggest that the pooled 6-month EFS of 12.7% from the seven studies could be utilized as the EFS benchmark for assessing activity of single agents studied in future trials conducted in recurrent Ewing sarcoma. The potential to modulate the immune compartment from a tumor permissive environment to an antitumor environment is an area of significant clinical interest and promise, However, the success of immunotherapy has only been observed in a small number of patients, whose tumor microenvironments are such that checkpoint blockade inhibition can be effective. Ewing sarcoma cells have low mutagenicity and tumors have minimal T cell infiltration resulting in “cold” tumors that do not respond to checkpoint inhibitors (Van Mater et al., 2019). This is also Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 12 of 58 true for other sarcomas which are characterized as having quite low immunogenicity (Heymann et al., 2020). A review of checkpoint blockade inhibitor studies in pediatric populations revealed that of 84 patients with sarcoma, only one patient demonstrated a partial response to therapy (Pearson et al., 2020) These dismal results indicate that there is a dire need for understanding how to unlock the immunogenic potential of these patients. During the third multi-stakeholder Pediatric Strategy Forum, jointly organized by ACCELERATE and the EMA, it was recommended that pediatric patients no longer be subjected to failed mono-therapy studies, but that hypothesis-driven combination therapies be explored. Though epigenetic modulating therapies are known to promote antitumor activity through affecting expression of tumor suppressor genes and oncogenes, recent investigation of these therapeutic modalities has uncovered an additional mechanism of antitumor activity through their ability to activate the expression of human endogenous retroviruses (ERVs) (Yau et al., 2019). The Shi lab published that lysine-specific histone demethylase 1 (LSD1) normally demethylates and stabilizes members of the RNA-induced silencing complex (RISC) and that depletion of LSD1 in cancer cells leads to accumulation of double stranded RNA (dsRNA) and activation of an antiviral innate immune response, a state that is termed viral mimicry (Sheng et al., 2018). Depletion of LSD1 in the “cold” B16-F10 syngeneic murine melanoma tumor model, promoted response of these cells to anti-PD-1 therapy. In vitro studies demonstrated that SP-2577 treated cancer cells exhibited increased expression of ERVs and cytokines shown to promote recruitment of anti-tumor immune cells. Organoid studies further confirmed SP-2577’s immunomodulatory effects by demonstrating an increase of immune cell migration across transwell plates into the organoids when cultured with SP-2577 conditioned media (Soldi et al., 2020). One immuno-oncology approach that has progressed to the clinic is Vigil. Vigil is an autologous cell vaccine platform designed to stimulate the patient’s immune system. In a Phase 1 study, Vigil demonstrated improved response rates compared to historical controls of chemotherapy and is now being investigated in an ongoing Phase 3 study in combination with temozolomide and irinotecan (Ghisoli et al., 2016). The Phase 3 study is a randomized study of intradermal autologous Vigil immunotherapy in combination with irinotecan and temozolomide vs combination chemotherapy alone in patients with metastatic Ewing Sarcoma Family of Tumors (ESFT) refractory/intolerant or recurrent to one prior line of chemotherapy as a second-line regimen (N=114 pts). This study started August 2018 with an estimated study completion date of July 2022 (https://clinicaltrials.gov/). For the relapsed/refractory Ewing sarcoma population, the lack of substantial improvements in patient outcomes and the concerning side effects of conventional regimens demonstrates a high unmet need for this population. There is an urgent need to: 1) identify novel targeted agents hat show activity and clinical benefit in the refractory or relapsed patient population, 2) identify agents with an improved therapeutic benefit over traditional chemotherapy, and 3) identify agents that are well tolerated. Given the urgency, the rarity of the illness and poor outcomes with Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 13 of 58 the current available therapies, it is important for a close collaboration between the sponsors and the FDA as they develop these new agents. These interactions will allow for clarity on optimal study designs, including endpoints, patient selection, as well as regulatory pathways for accelerated registration in patients that optimize the number of patients and time to endpoints for relapsed or refractory Ewing sarcoma.
2.1 SP-2577: A novel LSD1 inhibitor in relapsed / refractory Ewing sarcoma patients Ewing sarcoma is characterized by a chromosomal translocation (11;22) involving the EWSR1 gene and an ETS family transcription factor gene. The resulting fusion protein, most commonly EWSR1/FLI1 (85% of cases), acts as the molecular driver for tumor development by regulating genes involved in cell-cycle regulation, cell migration, cell differentiation, signal transduction and other cancer promoting functions (Thiesen et al., 2016; Sorensen et al., 1994; Cidre- Aranaz 2015). EWS/FLI functions as both a transcriptional activator and as a transcriptional repressor in Ewing sarcoma (Sankar et al., 2013). Prevention of EWS-FLI transcriptional activity is believed to be a viable therapeutic target for the treatment of Ewing sarcoma. However, the onco-fusion protein is intrinsically disordered with no enzymatic site, making it difficult to target directly. As such, targeting transcriptional co-regulators, proteins the EWS/FLI fusion associates with to modulate gene expression, is more readily achievable. Lysine-specific histone demethylase 1, a flavin-dependent monoamine oxidase, is a histone demethylase, which can demethylate mono- and di-methylated lysines 4 and 9 on the tail of histone H3 (H3K4 and H3K9). LSD1 is required for EWS/FLI mediated oncogenesis and inhibition of LSD1 affords a unique opportunity to target the immediate molecular machinery the EWS/FLI transcription factor relies on. In conjunction with EWS/FLI, LSD1 utilizes its enzymatic and scaffolding (i.e. structural role in protein complexes) functions to carry out both gene activation and repression, leading to oncogenic transformation (Sankar et al., 2014). Ewing sarcoma, like other sarcomas, exhibit some of the highest LSD1 protein expression levels out of any cancers. A clinical study looking at Ewing sarcoma patient samples (n=293) showed that about 90% of samples have LSD1 expression with 60% exhibiting moderate to high levels. (Bennani-Baiti et al., 2012). Additionally, LSD1 expression level is correlated to Ewing sarcoma patient prognosis with high LSD1 expression associated with decreased overall survival (p=0.03) (Figure 1) (Pishas et al., 2018). LSD1’s extensive involvement in EWS/FLI mediated transcription and its high expression make LSD1 inhibition an attractive approach for treatment of Ewing sarcoma. Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 14 of 58
Figure 1: LSD1 expression in Ewing sarcoma vs survival
Effective targeting of Ewing sarcoma cells via LSD1 inhibition requires comprehensive inhibition of LSD1’s two functions, its enzymatic (i.e., catalytic) activity and scaffolding function (i.e., structural role in protein complexes). Recent preclinical work demonstrates that catalytic inhibitors of LSD1 are insufficient as a therapeutic strategy for Ewing sarcoma (Romo- Morales et al., 2019). However, in several Ewing sarcoma cell lines depletion of LSD1 results in significant cell perturbation with anti-proliferative effects, demonstrating the therapeutic potential of complete LSD1 inhibition (Pishas et al., 2018). It is essential for pharmacological LSD1 inhibitors to inhibit LSD1’s catalytic and scaffolding functions to have a therapeutic impact. Salarius developed SP-2577, as a selective and reversible LSD1 inhibitor. SP-2577 is distinct from other LSD1 inhibitors currently in the clinic in that SP-2577 robustly inhibits LSD1’s scaffolding properties in addition to its catalytic activity. An SP-2577 analog, demonstrated reversal of genes that are up-regulated and down-regulated by the EWS/FLI oncoprotein in Ewing sarcoma through LSD1-mediated interactions with transcriptional co-activating proteins or transcriptional repressive protein complexes such as the Nucleosome Remodeling Deacetylase (NuRD) complex, respectively (Figure 2) (Sankar et al., 2014). The NuRD complex also Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 15 of 58 contains other epigenetic enzymes, i.e., Histone deacetylase (HDAC)1/2. In a study by Garcia- Dominguez and colleagues, the SP-2577 analog demonstrated synergy when combined with the HDAC inhibitor, suberanilohydroxamic acid (SAHA), in in vitro models and in in vivo patient- derived xenograft models of Ewing sarcoma. The synergistic result further validates the importance of disrupting LSD1’s scaffolding functions as increased disruption of remodeling complexes by targeting several of the core components may be therapeutically beneficial. SP-2577’s ability to work in both directions (enhancing and repressing expression) translates into potent therapeutic activity across a range of Ewing sarcoma cell lines both in vitro and in vivo (data detailed in subsequent sections). In addition, the reversible binding mechanism of SP-2577 should not cause severe hematological toxicity unlike other LSD1 inhibitors that are irreversible binders of LSD1. Irreversible LSD1 inhibitors may cause hematological toxicity due to LSD1’s normal role in hematopoietic stem cell homeostasis and its requirement in terminal differentiation of erythroid, megakaryocytic and granulocytic lineages (Mould et al., 2015). Thus SP-2577’s reversible binding mechanism and differential inhibition of LSD1 favors SP-2577’s potential for an improved therapeutic index. Salarius’ Phase 1 trial is enriching for Ewing sarcoma patients with gene rearrangements in EWSR1. At dose expansion, tumor biopsies will be taken pre-treatment and during treatment to retrospectively analyze for correlation between LSD1 expression levels and patient response.
Figure 2: LSD1 inhibition reverses oncogenic gene expression pattern in Ewing sarcoma models
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3 Drug
3.1 SP-2577: Description of the drug Salarius has developed SP-2577, seclidemstat, a selective and reversible LSD1 inhibitor, with low nanomolar activity against LSD1 and no monoamine oxidase activity. SP-2577 is an off- white to yellow color powder with the chemical name (E)-N'-(1-(5- chloro-2-hydroxy phenyl) ethylidene)-3-((4-methyl piperazin-1-yl) sulfonyl) benzohydrazide mesylate. The chemical structure of the mesylate salt form of SP-2577 is provided in the Figure 3. SP-2577 is provided for the clinical trials as a 75 mg enteric-coated tablet.
Figure 3: Chemical structure of SP-2577
SP-2577 and related compounds were developed utilizing in silico design and fragment-based screening approaches and validated using enzymatic and cellular assays. In cell free demethylase
LSD1 enzyme coupling fluorescence assays, the half maximal inhibitory concentration (IC50) of SP-2577 is between 20-49 nM, while the irreversible LSD1 inhibitor, tranylcypromine (TCP) has an IC50 of 386 nM (Figure 4). When tested against other epigenetic targets, SP-2577 has no activity, demonstrating its specificity for LSD1. Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 17 of 58
Figure 4: IC50 of SP-2577 against LSD1 enzymatic activity
Compounds were tested in 10-dose IC50 mode with a 3-fold serial dilution starting at 10 μM. Assay was performed using SP-2577 four times and TCP (tranylcypromine) once. All compounds were pre-incubated for 30 minutes with LSD1 enzyme before adding substrate to start the reaction. The production of flavin adenine dinucleotide (FAD)- dependent H2O2 as a result of demethylase activity of LSD1 was measured by coupling with horseradish peroxidase (HRP) and Amplex Red. The results shown here are a compilation of two separate experiments.
Salarius’ compounds are quite distinct from competitive inhibitors, which target LSD1’s enzymatic activity. LSD1’s enzymatic activity is mediated by FAD-dependent oxidation, requiring FAD as a cofactor for the catalytic activity of the amine oxidation domain (AOD) (Figure 5). Traditional LSD1 inhibitors were designed as derivatives of tranylcypromine, acting at LSD1’s catalytic pocket where they covalently bind to the FAD cofactor and inhibit LSD1’s enzymatic activity. These inhibitors are irreversible, binding throughout the enzyme’s normal life cycle and have been shown to subsequently lead to on-target adverse events, namely hematological toxicities, such as thrombocytopenia and neutropenia. As LSD1-mediated repression of hematopoietic stem and progenitor cell (HSPC) genes is essential for normal hematopoiesis, irreversible inhibition of LSD1’s activity, logically leads to hematological toxicities. In fact, studies in which LSD1 was biochemically or genetically depleted result in de- repression of HSPC genes, which led to compromised differentiation and maturation of blood cells (Sprussel et al., 2012; Kerenyi et al., 2013). Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 18 of 58
Figure 5: Diagram of LSD1 catalytic activity
A. Structure of LSD1 in complex with FAD-cofactor. AOD: Amine Oxidase domain (catalytic activity), TOWER domain (protein binding), SWIRM: Swi3/Rsc8/Moira domain (chromatin binding) FAD: Flavin adenine dinucleotide (enzyme cofactor). Inset illustrates coordination of FAD, and inhibitor TCA in the active site, substrate K4M is the catalytic substrate od H3K4me1/2, Adapted from Dong et al., 2014. B. Mechanism of LSD1 catalysis, Adapted from Zheng et al., 2016. In addition to its enzymatic function, LSD1 possesses both a SWIRM domain and a tower domain which mediate protein-protein interactions essential for its function as a scaffolding protein, assisting with the maintenance and stability of transcriptional regulatory and chromatin modifying complexes. SP-2577 interacts with a different region of LSD1, inhibiting LSD1’s Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 19 of 58 scaffolding functions in addition to its enzymatic activity. Recent data suggests that SP-2577’s differentiated mechanism of action may play a significant role in its therapeutic activity in Ewing sarcoma and desmoplastic round cell tumors (Figure 9), (Romo-Morales et al., 2019).
4 Nonclinical Studies in Ewing Sarcoma Models
4.1 Background There is substantial preclinical proof-of-concept for efficacy of Salarius’ LSD1 inhibitor SP- 2577 in Ewing sarcoma, generated both internally and by third parties with SP-2509 (a non- proprietary analog compound to SP-2577). SP-2577 was identified as the lead candidate due to its improved solubility and oral bioavailability. However, the two compounds are nearly identical, with an oxygen on SP-2509 being replaced by a nitrogen and a methyl group on SP- 2577. Comparative studies between SP-2509 and SP-2577 demonstrate comparable in vitro 50% growth inhibitory concentrations (GI50) across 30 cell lines (SP-2509 = 0.74 M, SP- 2577 = 1 M) (Figure 6). Both compounds inhibit LSD1’s catalytic and scaffolding properties such that results in SP-2509 should readily translate to results with SP-2577. The following is a review of non-clinical data generated with both SP-2577 and SP-2509, demonstrating the ability of Salarius’ LSD1 inhibitors to deplete LSD1 stability, reverse gene expression patterns, and reduce tumor cell viability in both in vitro and in vivo models of Ewing sarcoma.
Figure 6: SP-2509 and SP-2577 demonstrate similar efficacy in vitro
A panel of 33 cell lines was treated with a 6-point 10-fold dilution dose response curve of SP-2577 or SP-2509 for 72 hours. Cell viability was assessed using the Sulforhodamin B assay. Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 20 of 58
4.2 SP-2577 and SP-2509 inhibit LSD1 activity LSD1’s oncogenic function is mediated through its ability to promote a pro-tumorigenic gene expression pattern through its interaction with other oncogenic driver transcription factors and complexes. LSD1 inhibition with both SP-2577 and SP-2509 decreases stability of LSD1 and associated protein complex members. In a study aimed at investigating the effect of epigenetic drugs on the activity of the EWS-FLI1 activity, SP-2509 was assessed in a range of Ewing sarcoma cell lines and showed destabilization of EWS-FL1 protein (Garcia-Dominguez et al., 2018). Internal results show that 24-hour treatment of SP-2577 in SK-N-MC, Ewing sarcoma cell lines, reduces protein levels of LSD1 and cMYC, an oncogenic driver, target and co-regulatory protein of LSD1 (Figure 7, SP-2577-RS-013). Taken together these results indicate the ability of SP-2577 to inhibit and destabilize LSD1 and associated proteins.
Figure 7: SP-2577 destabilizes LSD1 and cMyc protein
Densitometry quantification of Western Blots of LSD1 or cMYc, normalized to GAPDH in SK-N-MC Ewing sarcoma cells following 24-hour SP-2577 treatment at 1 µM or 5 µM. Multiple in vitro studies in Ewing sarcoma cell lines exhibit SP-2577 and SP-2509’s ability to reverses LSD1/EWS/FLI1-driven gene expression. After 24 or 48 hours of SP-2509 treatment at both the IC50 (0.247 µM) and IC90 (1.2 µM), Garcia-Dominguez and colleagues noted a reversal in the gene expression pattern of EWS-FLI1 target genes in models of Ewing sarcoma (Garcia- Dominguez et al., 2018). Studies by Sankar et al revealed that SP-2509 de-repressed EWS/FLI repressed genes and silences EWS/FLI activated genes including HMOX, IGFBP3, CDH1, and RUNX2. Moreover, SP-2509 was effective in EWS/ERG fusion containing Ewing cell lines as well (Sankar et al., 2014). In more recent studies comparing the efficacy of SP-2509 to other clinical-stage LSD1 inhibitors in Ewing sarcoma, SP-2509 treatment resulted in more robust reversal of EWS-FLI1 target gene expression when compared to irreversible LSD1 inhibitors Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 21 of 58
(Figure 8), (Romo-Morales et al., 2019). These data suggest that inhibition of the catalytic activity of LSD1 alone is insufficient to affect the oncogenic phenotype of EWS-FLI1 translocation-containing cells. This makes a strong argument for the use of SP-2577, which disrupts both demethylation (i.e. catalytic) and scaffolding capacity of LSD1, in Ewing sarcoma.
Figure 8: SP-2509 reverses LSD1/EWS/FLI1-driven gene expression more robustly than irreversible LSD1 inhibitors
Effect of LSD1 inhibitors on EWF-FLI1 target gene expression. qRT-PCR analysis of LOX and HMOX in A673 cell lines treated with vehicle or irreversible LSD1 inhibitors ORY-1001, GSK2879552, or reversible inhibitor SP- 2509, normalized to RPLPO endogenous control. Adapted from Romo-Morales et al., 2019 Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 22 of 58
4.3 SP-2577 and SP-2509 reduce tumor cell viability in vitro In a Salarius sponsored study, treatment with SP-2509 induced apoptosis via the endoplasmic reticulum stress pathway and caused a dramatic reversal of both the up- and down-regulated transcriptional profiles of EWS/FLI1 and EWS/ERG accompanied by the induction of apoptosis and disruption of morphologic and oncogenic phenotypes modulated by EWS/FLI (Pishas et al., 2018). Additional peer-reviewed studies make evident the capacity for SP-2509 mediated LSD1 inhibition to reduce cell viability, induce apoptosis, reduce anchorage-independent growth, and reduce cell migration more potently than the irreversible inhibitors GSK2879552 and ORY-1001 (Figure 9), (Garcia-Dominguez et al., 2018; Romo-Morales, 2019; Pishas et al., 2018; Sankar et al., 2014). Intriguingly, in experiments in which EWS /FLI was knocked down with shRNA, those cells appear to be less sensitive to SP-2509 treatment, demonstrating that 2509-mediated cell death was dependent on EWS/FLI presence in Ewing sarcoma models (Sankar et al., 2014). Internal data demonstrate that 72-hour treatment with SP-2577 in A673, SK-N-MC, TC32,
TTC466, 6647 and TC71 Ewing sarcoma cell lines significantly reduce cell viability with IC50 ranging from 185 nM to 1269 nM. The Ewing sarcoma cell lines, SK-ES-1 demonstrated accumulation of H3K4me2 after 16 hours of SP-2577 treatment at 3 µM. Taken together, these studies establish the efficacy of SP-2509 and SP-2577 treatment to effectively reverse the pro- oncogenic gene expression pattern of LSD1 in Ewing sarcoma and ultimately lead to tumor cell death.
Figure 9: SP-2509 LSD1 inhibition reduces cell viability more robustly than irreversible inhibitors and impedes anchorage-independent growth
A. Ewing sarcoma cell lines A673 and TC71 were exposes to a range of concentrations of LSD1 inhibitor for 96 hours. Cell viability was assessed by MTS assay. B. Representative images and quantification of soft-agar colonies of A673 and TTC-466 cells stably transduced with iKDM1A or iLuc control shRNA constructs following puromycin selection. Adapted from Romo-Morales et al., 2019 Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 23 of 58
4.4 SP-2577 and SP-2509 reduce tumor burden in vivo A number of in vivo studies conducted with SP-2577 are detailed in the following section. SP- 2577 is efficacious in these models at intraperitoneal (i.p.) doses ranging from 30 mg/kg to 100 mg/kg daily (Table 3). Pharmacokinetic (PK) profiling in mice at 100 mg/kg i.p. (Figure 10) demonstrate representative plasma concentrations after a single dose.
Figure 10: SP-2577 in vivo pharmacokinetics
Animals were a single dose of 100 mg/kg SP-2577 i.p. plasma collected over at 1, 2.5, 4, 8 and 24 hours after dosing and SP-2577 concentration measured.
A673 and SK-N-MC Ewing sarcoma cell lines are well established models for both in vitro and in vivo applications which contain the EWS-FLI translocation and express the resultant EWS- FLI oncoprotein. A673 and SK-N-MC cells grown as a human tumor xenograft in mice have proven to be useful in vivo models to evaluate tumor growth and to identify therapeutic targets for sarcomas (Teicher et al., 2011). Salarius has taken advantage of these xenograft models to assess the ability of various doses, formulations, and routes of administrations of SP-2577 to inhibit tumorigenesis in Ewing sarcoma. The results of those studies are summarized in Table 3 and detailed below. Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 24 of 58
Table 3: Summary of in vivo efficacy studies
Model Total Daily Route N Percent Response Treatment Dose SP-2577 Duration (mg/kg) (days)
SKNMC 30 i.p. 10 80% 54
SKNMC 40 i.p. 10 83% 24
80 i.p. 98%
80 Oral 98%
A673 30 i.p. 10 50% 41
7 CDX 100 i.p. 10 See graphs in 28 Models Figure 12
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Effect of i.p. SP-2577 on SK-N-MC (Ewing sarcoma cell line) cells in a mouse xenograft model (study RES-004-2577 i.p. EFF-1) The aim of this study was to assess the efficacy of a 30 mg/kg dose of SP-2577 in the SK-N-MC mouse xenograft model of Ewing sarcoma. Animals were treated for 54 days or until they reached maximum allowable tumor burden. Vehicle control animals died or had to be sacrificed by Day 29 due to rapid tumor progression. In the SP-2577 treatment group, mouse #1 did not respond to treatment and was sacrificed at Day 25 due to non-healing skin lesions and mouse #7 responded well to SP-2577 treatment, but had to be sacrificed due to malocclusion, overgrowth of teeth, at Day 33. The remaining eight animals treated with SP-2577 survived until Day 54. In these mice, tumors initially progressed, but continued drug treatment resulted in tumor shrinkage to the point that tumors were not observed upon palpable examination. By Day 54 all eight of these animals showed complete tumor remission (Figure 11A), and no signs of tumor relapse during the 6-month observation period following treatment completion. As shown in Figure 11B, the survival rate of the SP-2577 group was significantly better than that of sham-treated controls (p<0.0001, by Log-rank [Mantel-Cox] test).
Figure 11: In vivo efficacy of 30 mg/kg treatment of SP-2577 in SK-N-MC xenograft model of Ewing sarcoma
Ten animals per cohort were treated with 30 mg/kg SP-2577 via i.p. injection daily for 54 days. Tumor volume and survival was measured. Study RES-004-2577 i.p. EFF-1.
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Effect of i.p. SP-2577 on A673 (Ewing sarcoma cell line) cells in a mouse xenograft model (Salarius study number: RES-005-2577-i.p.-EFF-2) The aim of this study was to assess the efficacy of a 30 mg/kg dose of SP-2577 in the A673 mouse xenograft model of Ewing sarcoma. All vehicle treated animals had rapid tumor progression and either died or had to be sacrificed by Day 24, as shown in Figure 12. The measured average tumor volume increased from 118.5+11.2 mm3 on Day 1 to 175.5+ 275.7 mm3 on Day 21, a 14.8-fold average increase in three weeks. In contrast, for mice in the SP-2577 treated group, four animals (#4, #6, #7, #10) showed moderate initial tumor growth followed by tumor regression with continued treatment of SP-2577 and by 41 days of treatment, these four animals achieved complete or nearly complete remission. One animal (#9) showed a less than complete inhibition of tumor growth but survived until Day 41. The rest of the mice in the SP- 2577 group showed tumor growth similar to that of the vehicle control group and either died or were sacrificed during the 41-day experimental period. SP-2577 administered i.p. at 30 mg/kg once per day was shown to be effective in 5/10 of the treated animals in this A673 xenograft model.
Figure 12: In vivo efficacy of 30 mg/kg treatment of SP-2577 in A673 xenograft model of Ewing sarcoma
Ten animals per cohort were treated with 30 mg/kg SP-2577 via i.p. injection daily for 41 days. Tumor volume was calculated. RES-005-2577-i.p.-EFF-2.
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Effect of oral SP-2577 on SK-N-MC (Ewing sarcoma cell line) cells in a mouse xenograft model (Study RES-006-2577-PO-eff) The aim of this study was to assess the efficacy of oral versus i.p. administration of SP-2577 in the SK-N-MC mouse xenograft model of Ewing sarcoma. As shown in Figure 13, all 10 animals in the vehicle control group showed rapid tumor growth with a more than 44-fold average increase at Day 24. In each SP-2577 dose group, all mice responded to treatment. In 40 mg/kg i.p. group all treated mice showed significantly less tumor progression, with an average tumor volume increase of about 7-fold at Day 24. In both 80 mg/kg i.p. and oral (PO) groups, all tumor volumes decreased at the end of the 24-day experimental period. This study demonstrated that maximum antitumor efficacy of SP-2577 can be achieved by intraperitoneal and oral route of administration at 80 mg/kg in this model.
Figure 13: In vivo efficacy of i.p. and oral-administered SP-2577 in SK-N-MC xenograft mouse model
Ten animals per cohort were administered vehicle, SP-2577 at 40 mg/kg or 80 mg/kg via intraperitoneal injection, or 80 mg/kg via oral gavage, daily for 28 days. Tumor volume was calculated. In summary, treatment with SP-2577 as a single agent in Ewing sarcoma xenograft mouse models (using cell lines SK-N-MC and A673) was effective and in some cases resulted in complete tumor regression and relapse-free survival of up to six months. Also, maximum in vivo efficacy was achieved by oral administration of SP-2577 at a non-toxic dose of 80 mg/kg (human equivalent dose = 6.4 mg/kg) in mice. Taken together, these studies demonstrate the potential for SP-2577 to significantly reduce the morbidity and mortality associated with Ewing sarcoma. Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 28 of 58
SALA Pharm 015 (PPTC Study 17-05-01) This clinical study was conducted by the NCI Pediatric Preclinical Testing Consortium (PPTC) and primary test facility was the PPTC Greehey Children’s Research Institute (San Antonio, TX). The objective of this study was to evaluate the efficacy of SP-2577 as a single agent against an array of pediatric sarcoma xenografts, including seven Ewing sarcomas, four alveolar rhabdomyosarcoma and one embryonal rhabdomyosarcoma. Mice bearing subcutaneous tumors received SP-2577 when tumors reached 200-500 mm3. Mice were treated with SP-2577 at a concentration of 100 mg/kg i.p.. In all in vivo studies, no evidence of a detrimental effect on health, as determined by changes in body weight, was seen at dose levels of SP-2577 tested in mice. As a single agent, SP-2577 significantly (p<0.05) prolonged time to event in four of eight Ewing sarcoma models. Event-free survival across the tumor models is shown in Figure 14. Complete study results are summarized in Table 4. Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 29 of 58
Figure 14: Responses of Ewing sarcoma xenografts to SP-2577
Ten mice per cohort received SP-2577 (100 mg/kg daily for 28 days when tumors were 200-400 mm3). Kaplan- Meier probability plots for event-free survival (EFS) Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 30 of 58
Table 4: Study results PPTC study PPTC-1705-01; SALA Pharm 015
Tumor Group KM EFS EFS P-value PD PD1 PD2 Median (Med) T/C T/C (G/W) Response (days) (days)
A673 A 6.3 10
B 8.7 2.4 1.37 0.037 9 8 1 PD1
ES-1 A 9.7 10
B 13.3 3.6 1.37 <0.001 8 5 3 PD1
ES-4 A 6.0 10
B 7.9 2.0 1.33 <0.001 10 8 2 PD1
ES-7 A 9.9 10
B 12.6 2.7 1.28 0.173 10 8 2 PD1
EW-5 A 9.5 10
B 9.6 0.1 1.02 0.327 10 10 0 PD1
EW-8 A 11.8 10
B 12.6 0.9 1.07 0.148 10 10 0 PD1
NCH-EWS-1 A 14.6 10
B 16.1 1.5 1.10 0.935 9 9 0 PD1
SK-NEP A 13.5 9
B 18.5 5.0 1.37 0.002 9 7 0 PD1 Group A –Vehicle control, Group B – SP-2577 treated, KM – Kaplan-Meier, EFS – event-free survival, PD - <50% tumor regression throughout study and >25% tumor growth at end of study, PD1 when PD and the mouse’s time to event ≤ 200% the KM median time-to-event in control group, PD2 when PD but, additionally, time-to-event is > 200% of the KM median time-to-event in control group.
4.5 SP-2577 and SP-2509 are effective in combination with epigenetic and chemotherapeutic agents In addition to single agent efficacy, SP-2509 and SP-2577 act synergistically with other epigenetic agents and chemotherapy. In a study by Garcia-Dominguez and colleagues, SP-2509 demonstrated single agent activity as well as synergy when combined with the HDAC inhibitor, SAHA, both in in vitro models of Ewing sarcoma as well as in in vivo patient-derived xenograft models of Ewing sarcoma. Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 31 of 58
Of particular interest is a study of SP-2509 and SP-2577 treatment in four Ewing sarcoma cell lines in combination with second-line chemotherapeutic agents. When cells were treated with conventional treatment used in relapsed Ewing sarcoma (vincristine, irinotecan, and temozolomide), along with SP-2509 or SP-2577 (62.5 ng/ml to 2,500 ng/ml), cell viability was reduced by up to 90% (Figure 15). Additionally, when combined with romidepsin, an inhibitor of class I and class II HDACs, also members of the NuRD complex, viability was significantly reduced (Figure 15c) Full results may be found in Welch et al., 2019.
Figure 15: Combination of SP-2509 and SP-2577 with chemotherapy
Order of addition experiments in (A) A-673 and (B) TC32 cells and varying concentrations (Top – maximum, top/2 – half maximum, and top/4 – one quarter maximum) of relevant drugs. Cells were plated on Day 0 and incubated for 24 hours. On Day 1 they were treated with the first round of drugs (single drug, doublet or triplet). On Day 2, cells were read using RT-Glo. After Day 2 reading, cells were treated with the final drug and incubated for 72 hours after which they were read again on Day 5. (C) 5X5 checkerboard matrixes assessing combination activity of SP-2577 and vincristine or romidepsin. FA – fraction of cells affected CI – combination index. Adapted from Welch et al., 2019 and unpublished data. Preliminary (unpublished) results from in vivo studies with SP-2577 in combination with irinotecan and temozolomide suggest increased efficacy in Ewing sarcoma xenografts when compared to single agent therapy or chemotherapy alone.
Non-Ewing sarcomas In addition to Ewing sarcoma, numerous sarcomas are driven by EWS fusion genes (Nakano et al., 2018) and/or demonstrate heightened levels of LSD1 protein expression (Bennani-Baiti et al., 2012). LSD1 is a gatekeeper of mesenchymal cell stemness, and depletion of LSD1 in Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 32 of 58 mesenchymal stem cells (MSCs) result in differentiation. Sarcomas originate from mesenchymal cells and may highjack aberrantly expressed LSD1 to promote a stem cell-like phenotype. A screen of 462 soft tissue tumors identified over-expression of LSD1 in malignant tumors including synovial sarcomas, rhabdomyosarcomas, desmoplastic small round cell tumors and malignant peripheral nerve sheath tumors (Schildhaus et al., 2011). An additional study of 50 sarcomas, confirmed the increase of expression of LSD1 in an array of sarcomas, including chondrosarcoma, Ewing sarcoma, synovial sarcoma, osteosarcoma and rhabdomyosarcoma (Figure 16, adapted from Bennani-Baiti et al., 2012). A third party investigator demonstrated SP- 2509 induced apoptotic morphology in the desmoplastic round cell tumor cell line, JN-DSRCT- 1, while no morphological change was observed with irreversible inhibitors (Romo-Morales et al., 2020). In addition to Ewing sarcoma, Salarius has investigated the effect of SP-2577- mediated LSD1 inhibition in rhabdomyosarcoma, osteosarcoma and clear cell sarcoma.
Figure 16: LSD1 is over-expressed in various sarcomas
The efficacy of SP-2577 as a single agent was evaluated against four alveolar rhabdomyosarcoma (Rh10, Rh28, Rh30 and Rh36), one embryonal rhabdomyosarcoma (Rh65), and six osteosarcoma (OS2, OS9, OS31, OS33, OS36 and OS60) xenograft models. Female mice were inoculated with the different tumors and treatment was initiated when tumors reached 200- 500 mm3. Mice were randomized to groups of 10 for vehicle control or 100 mg/kg SP-2577. Mice were dosed by the i.p. route once daily for 28 consecutive days. As a single agent, SP-2577 significantly prolonged time to event in four of five alveolar rhabdomyosarcoma models (Rh10, Rh28, Rh36, Rh65) and six of six osteosarcoma models (Table 5). The extent of prolongation of event was small for most of those models for which time to event was significantly prolonged, with tumor event-free survival treated/control (EFS T/C) values ranging from 1.9 to 14 days and with EFS T/C values ranging from 1.15 to 2.79. None of the models tested showed objective responses indicative of tumor regression (where minimum Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 33 of 58
RTC was less than 1.0), but rather showed progressive disease (PD1 or PD2) as the best response. Event-free survival across the tumor models is shown in Figure 17. Significant differences in time to event were observed for Rh10, Rh28 and Rh65. Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 34 of 58
Table 5: Study results PPTC-1705-01
Tumor Group KM EFS EFS P-value PD PD1 PD2 Median (Med) T/C T/C (G/W) Response (days) (days)
Rhabdomyosarcoma (RMS)
Rh10 A 7.8 10 B 21.8 14.0 2.79 <0.001 10 0 10 PD2
Rh28 A 12.3 10 B 17.6 5.2 1.43 0.007 6 4 0 PD1
Rh30 A 14.3 10 B 19.7 5.3 1.37 0.111 8 7 1 PD1
Rh36 A 20.2 10 B 23.8 3.6 1.18 0.040 9 8 0 PD1
Rh65 A 12.7 10 B 14.5 1.9 1.18 0.030 9 9 0 PD1
Osteosarcoma (OS)
OS-2 A 31.1 10 B 39.2 8.1 1.26 0.003 10 10 0 PD1
OS-9 A 15.3 10 B 19.0 3.6 1.24 0.003 10 10 0 PD1
OS-31 A 17.4 10 B 19.5 2.2 1.12 0.008 10 0 0 PD1
OS-33 A 19.8 10 B 26.4 6.7 1.34 0.004 10 10 0 PD1
OS-36 A 14.5 10 B 16.4 1.8 1.13 0.015 10 10 0 PD1
OS-60 A 30.1 10 B 35.8 5.6 1.19 0.036 10 0 0 PD1
Group A –Vehicle control, Group B – SP-2577 treated, KM – Kaplan-Meier, EFS – event-free survival, PD - <50% tumor regression throughout study and >25% tumor growth at end of study, PD1 when PD and the mouse’s time to event ≤ 200% the KM median time-to-event in control group, PD2 when PD but, additionally, time-to-event is > 200% of the KM median time-to-event in control group. Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 35 of 58
Figure 17: Event-free survival, study PPTC-1705-01
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Figure 17 (continued)
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SP-2577 in hematologic malignancies LSD1 has an important role in healthy hematopoietic stem cell and progenitor maintenance, as well as helping drive progenitor differentiation to various lineages. However, in hematological malignancies LSD1 is often over-expressed and is aberrantly recruited to incorrectly repress differentiation and self-renewal, resulting in myeloproliferative neoplasms. Consequently, LSD1 has been a target of interest for several hematological malignancies and is currently being explored through ongoing studies. In preclinical studies, SP-2577 shows activity in cell lines of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) (Figure 18). SP-2577 (2 μM) has anti-proliferative properties across several AML cell lines, such as OCI-AML 5 (Figure 18a). In addition, SP-2577 (1 μM) is able to remove the differentiation block evident by an increase in differentiation markers including CD86 and CD11b (Figure 18b). In an MDS cell lines (MDS1), treatment with SP-2577 significantly reduced colony growth when compared to the control group (Figure 18c). Interestingly, SP-2577 showed synergy (Combination Index = 0.6) with another epigenetic inhibitor, 5-azcitidine, which is commonly used in myeloproliferative neoplasms (Figure 18d).
Figure 18: SP-2577 in hematological malignancies
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Potential for SP-2577 in combination with checkpoint inhibition To investigate whether therapeutic inhibition of LSD1 with SP-2577 resulted in anti-tumor activity in an immunologically inactive tumor, we evaluated the combined anti-tumor efficacy of SP-2577 and anti-PD-1 therapy in the sub-cutaneous B16F10 melanoma syngeneic model in female C57BL/6 mice. Mice, 6–8 weeks of age, were inoculated into the flanks of animals and randomly assigned to one of the following groups when tumors reached 80–150 mm3: • vehicle control • 25 mg/kg SP-2577 daily • 75 mg/kg SP-2577 daily • 10 mg/kg anti-PD-1 every three days • 25 mg/kg SP-2577 daily + 10 mg/kg anti-PD-1 every three days • 75 mg/kg SP-2577 daily + 10 mg/kg anti-PD-1 every three days The B16F10 model is an aggressive tumor model and animals reached humane endpoint by Day 11. This model is also known to be non-responsive to anti-PD-1 therapy and that resistance is illustrated in this study. This preliminary study suggests SP-2577 sensitizes B16-F10 tumors to anti-PD-1 therapy (Figure 19). No toxicities were observed in combination groups that were not also observed in the vehicle control group. Further evaluation and optimization of this combination is ongoing.
Figure 19: SP-2577 in combination with checkpoint blockade inhibition
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4.6 Summary of nonclinical studies LSD1 is required for EWS/FLI1 mediated oncogenesis and inhibition of LSD1 affords a unique opportunity to target the immediate molecular machinery on which the EWS/FLI transcription factor relies. The nonclinical data in in vitro and in vivo models of Ewing sarcoma demonstrate the ability of SP-2577 and SP-2509 to effectively disrupt the LSD1/EWS/FLI1 molecular machinery and reverse the oncogenic program they drive, supporting the scientific rationale for initiating a first-in-human Phase 1 study in Ewing sarcoma. Given that LSD1 is a key driver in Ewing and other sarcomas, SP-2577, a specific and reversible inhibitor of LSD1, may provide a strong benefit/risk in the treatment of these patients who inevitably relapse and die where current cytotoxic agents have limited efficacy and high frequency of toxicities, which can be severe. Further data from in vitro and preliminary, unpublished in vivo models of Ewing sarcoma support the combination of SP-2577 with standard chemotherapy used to treat Ewing sarcoma. Additionally, the potential for use of SP-2577 in combination with immune checkpoint blockade therapy is supported by a strong scientific rationale and confirmatory in vivo studies are underway to support this potential development.
5 SP-2577: IND Enabling Toxicology and Pharmacology Studies
5.1 SP-2577 GLP toxicology studies in rats and dogs
Nonclinical safety pharmacology studies did not raise concern related to either neutropenia or thrombocytopenia, common adverse events with irreversible LSD1 inhibitors. Toxicology studies in dogs and rats indicated the kidney was the target organ at the highest doses tested. Toxicity in other organs were only present in one species. Findings in target organs were reversible after two weeks. There were no findings of clinical significance noted during any ophthalmic examinations or effects of test article administration on dog electrocardiograms (ECGs). No significant changes in body weight were observed in rats, or in the low- and mid- dose dog groups. There were no clinical pathology findings.
5.2 Nonclinical pharmacokinetics and rationale for starting dose Pharmacokinetic studies demonstrated 33% oral bioavailability following administration of the capsule formulation in dogs. A cross-over study of the tablet and capsule formulations in dogs showed that the two formulations are equivalent in terms of systemic exposures following oral administration. The PK of multiple doses of SP-2577 in dogs was consistent with the single-dose PK, which indicates no unusual accumulation or induction of metabolism in this species. The solubility, permeability, and animal PK data indicate that SP-2577 will be effectively delivered into systemic circulation following oral administration of the drug in humans. SP-2577 distributes into tissues of dogs with a volume of distribution (350-1000 mL/kg) that is greater Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 40 of 58 than total body water. The drug is highly bound to plasma proteins with bound fractions exceeding 99% in dogs. A cross-over study of the tablet and capsule formulations administered orally to fasted or fed dogs found that systemic exposures were considerably greater when the SP-2577 was administered to fasted compared to fed animals. Based on International Council for Harmonisation (ICH) S9 Guidance the recommended starting clinical dose derived from nonclinical toxicology studies is 200 mg (for 70 kg person). Given a 75 mg tablet we proposed a starting clinical dose of 150 mg (75 mg twice daily [BID]) that would also cover individuals lighter than 70 kg. In the preclinical setting, SP-2577 has been well characterized to support clinical development and the draft plan for future proposed studies. To date, it has shown promising efficacy by decreasing metastatic growth in previously untreated tumor models and has displayed a favorable PK profile in animal studies.
6 Seclidemstat: Clinical Human Experience to Date
6.1 Compassionate use study In 2016, Salarius conducted a compassionate use study with seclidemstat, A Single Patient Compassionate Use Study of Seclidemstat in a Patient with Metastatic Ewing Sarcoma (Protocol SALA-CP01-16-001-CC), before the current active IND was filed. The patient was exposed to seclidemstat at an initial dose of 90 mg by mouth daily and was dose escalated to a maximum dose of 420 mg by mouth BID (total daily dose of 840 mg) over approximately three months and was well tolerated. The 25-year-old male was heavily pre-treated with multiple standard-of-care treatment regimens and had previously been treated on other investigational protocols. Prior to study entry, he completed palliative radiation and had metastatic lesions in both lungs, mediastin.al masses, and a lytic L4 lesion. Minimal adverse events were observed during the study and none of the adverse events were attributed to seclidemstat. Anemia, anorexia, pain, fatigue, fever, chills, and diarrhea were present at baseline and were not exacerbated by administration of seclidemstat. Dose dependent systemic exposures were observed with increasing doses of seclidemstat which did not correlate with frequency or severity of reported adverse events. Overall seclidemstat was well tolerated in this heavily pretreated subject and offered this subject an additional treatment option despite not producing an objective response. Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 41 of 58
6.2 Seclidemstat: Phase 1 studies 6.2.1 Patients with relapsed or refractory Ewing sarcoma The first-in-human Phase I study of seclidemstat is currently being conducted at eight investigational sites in patients with relapsed or refractory Ewing sarcoma (SALA-002-EW16). This study is an open-label, non-randomized dose-finding study of seclidemstat given twice- daily as oral tablets of 75 mg strength in the fasted state in patients with refractory or recurrent Ewing sarcoma in 28-day cycles. The primary objective of the Phase 1 study is to evaluate the safety and tolerability of seclidemstat. Additional objectives include to determine the maximum tolerated dose (MTD), to characterize the PK of seclidemstat, to evaluate the effect of food on the PK of seclidemstat (dose escalation only) and to assess preliminary anti-tumor activity of seclidemstat in this patient population. Exploratory objectives include to explore the use of circulating tumor cells (CTCs), cell-free deoxyribonucleic acid (cfDNA) and hemoglobin F as pharmacodynamic markers of disease burden, drug effect, and tumor response. In addition, at the expansion, patients, if willing, will have pre- and post- and end-of-treatment biopsies, which is optional for patients with ages < 18 years, to assess for ribonucleic acid (RNA)-seq testing to evaluate genome wide expression patterns. The study design is based on a Simon’s 4B design without intra-patient dose escalation. This study utilizes an accelerated dose escalation phase in order to minimize sub-optimal drug exposures, followed by a conventional 3+3 dose escalation phase to determine MTD. Initially, one patient per dose cohort will be recruited (accelerated dose escalation phase) until the first instance of a Common Terminology Criteria for Adverse Events (CTCAE) ≥ Grade 2 drug- related toxicity or dose-limiting toxicity (DLT). Further cohorts will be recruited in blocks of three patients (3+3 dose escalation phase). All dose escalation decisions are made through consensus of the safety review committee (SRC) comprised of study investigators and the Salarius study team. In addition, the study allows for patients to be enrolled on a dose that had been previously cleared for safety by the SRC (backfill patients) and any patient who did not meet the criteria for eligibility for DLT consideration in Cycle 1 is replaced. Once the MTD is defined, the study will expand to treat a total of 20 patients at this dose to further characterize the safety profile of seclidemstat. Key eligibility criteria include: • histologic confirmed diagnosis of Ewing sarcoma that is relapsed or refractory and must have received at least one prior course of therapy for Ewing sarcoma (refractory disease is defined as metastatic or unresectable disease that has either progressed or is stable at completion of planned therapy) • prior camptothecin-based regimen, have a contraindication to camptothecin-based regimen, or declined treatment with a camptothecin-based regimen
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For the 16 patients, demographics include: 10 (62.5%) male; median age (range) of 25.2 years (15.2-67.6) with 14 (87.5%) > 18 years of age. Karnofsky score in 14 pts of 90-100 in 10 (71.4%) and 70 to 80 in 4 (28.6%) and Lansky score in two patients of 90-100 and 70-80 in one patient each. Study disease characteristics include: 100% with positive local assessment of EWSR1 gene arrangement; 13 (81.3%) with measurable disease; 10 (62.5%) with at least pleuropulmonary metastases and 11 (68.8%) with at least bone metastases. The median time (range) from initial diagnosis of Ewing sarcoma to Cycle 1 Day 1 of treatment is 4.2 years (1.9- 14.6) with 14 patients (87.5%) ≥ 2 years. Study disease history includes: 11 (68.8%) with prior surgery; 11 (68.8%) with prior radiation therapy; and 13 (81.3%) with prior camptothecin- containing regimen; median number of prior lines of chemotherapy of three (range 2-12).
Table 6: SALA-002-EW16 patient demographics
All patients N=16
Age at treatment start, years 25.2 (15.2–67.6) Median (range) 2 ≤ 18 9 > 18 to ≤ 29 5 > 29
Male, n (%) 10 (62.5)
Karnofsky score, n (%) n=14 90 to 100 10 (71.4) 70 to 80 4 (28.6)
Lansky score, n (%) n=2 90 to 100 1 (50.0) 70 to 80 1 (50.0)
Table 7: SALA-002-EW16 disease characteristics
All Patients N=16
EWSR1 gene re-arrangement, n (%) 16 (100)
Measurable disease, n (%) 13 (81.3)
Sites of metastatic disease, n (%) 11 (68.8) Any bone 10 (62.5) Any pleuropulmonary Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 44 of 58
Bone only 5 (31.3) Pleuropulmonary only 3 (18.8)
Time from initial diagnosis of Ewing sarcoma to C1D1, n (%)
Median, years (range) 4.2 (1.9–14.6) < 1 year 0 1 to < 2 years 2 (12.5) 2 to < 3 years 3 (18.8) 3 to < 4 years 2 (12.5) 4 to < 5 years 2 (12.5) ≥ 5 years 7 (43.8)
Prior therapy, n (%)
Surgery 11 (68.8) Radiation therapy 11 (68.8) Chemotherapy Camptothecin-containing regimen Non-camptothecin-containing regimen 13 (81.3) Number of prior lines of chemotherapy 3 (18.8) Median (range) 2 3 (2–12) 3 5 (31.3) 4 6 (37.5) > 4 3 (18.8) 2 (12.5)
Cycle 1 single-dose PK have been assessed in 13 evaluable patients treated at doses of 75 mg, 150 mg, 300 mg, 600 mg, and 900 mg PO BID as of 4 February 2020. Cycle 1, fasted PK by dose level in SALA-002-EW16 is presented in Table 8 and Figure 20. Under fasting conditions, a proportional and linear increase in the area under the plasma concentration over time curve (AUC) by dose level has been observed. The half-life is approximately 5-8 hours with peak plasma concentrations (Cmax) reaching 3230 mg/mL at the 900 mg PO BID dose level.
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Based on Cycle 1, Day 1 PK in the fasted state from both trials a two-compartment nonlinear mixed effect model was constructed. The model simulates both daily and twice-daily administration of seclidemstat over six days at the 600 mg, 900 mg, and 1200 mg dose levels, see Figure 23. The model demonstrates at all three dose levels once-daily administration results in minimal accumulation with similar trough and peaks concentrations across the dosing period. However, twice-daily administration, as administered in both trials, should result in significant accumulation producing increased trough and peak concentrations at steady-state, which is expected to occur in approximately 35 hours. In Figure 23, a dashed line indicating an SP-2577 concentration of 1000 ng/mL is denoted, which is the hypothesized minimal concentration needed for efficacy in humans based on in vitro (Section 4.3) and in vivo studies (Section 4.4). With twice-daily administration, the time above 1000 ng/mL at steady state increases per dose level. At 1200 mg PO BID it is anticipated SP-2577 steady state concentrations will be above 1000 ng/mL the entire day for the average patient.
Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 51 of 58 predictable absorption under fasting conditions with proportional increases in exposure per dose level and elimination half-life estimates supporting twice daily dosing. Based on PK modeling of clinical PK data, at 1200 mg PO BID patients are expected to have SP-2577 drug concentrations above the hypothesized minimal concentration needed for efficacy (1000 ng/mL) the entire day once steady-state is reached. The 16 patients enrolled onto the SALA-002-16 study are representative of an advanced Ewing sarcoma patient population who have been heavily pretreated with multiple lines of treatment (median of 3) who received a camptothecin-based chemotherapy (87.5%) or refused or were intolerant to such therapy, and have extensive pleuropulmonary and bone disease. As additional data on the Phase 1 program become known for this patient population, Salarius requests the opportunity to closely develop seclidemstat’s clinical trial program with the Agency via Fast Track Designation as outlined in the proposed scenario development designs for further clinical development.
7 Future Clinical Development of Seclidemstat Following the dose escalation and declaration of an MTD and recommended Phase 2 dose (RP2D), the SALA-002-EW16 study will expand for an additional 14 Ewing sarcoma patients to complete treatment of 20 patients at that dose. As suggested by the older literature discussion above, new agents, especially those with an underlying targeted mechanism suggesting that inhibition of that target could lead to efficacy, have been either tested in Phase 2 trials or were retrospective analyses. Overall response rates of at least 10-15% in the heavily pre-treated, relapsed setting have been considered as reasonable single-agent activity to further pursue investigation of that agent in clinical trials, including in combination regimens. In 2019, COG recently reported on the treatment outcomes of 128 patients with relapsed or progressive Ewing sarcoma patients enrolled from January 1997 until October 2007 on seven Phase 2 COG trials utilizing single agent treatments (median number of patients per trial 18 pts, range 9-26) (Collier et al., 2019). Of the seven reported trials, six treatments were with single-agent cytotoxic agents. Treatment response outcomes included EFS and OS, using the method of Kaplan and Meier. For all studies combined, for a total of 124 events (including 106 relapses and 18 deaths), the 6-month EFS was 12.7% (95% CI 7.6-19%). These results support the poor treatment outcomes of patients with relapsed or progressive Ewing sarcoma treated with available therapies. The authors suggest that the pooled 6-month EFS of 12.7% from the seven trials could be utilized as the EFS benchmark in future trials of new single agents conducted in recurrent Ewing sarcoma. In the ongoing rEECur Phase 2/3 study, a prospective randomized, multi-arm multi-stage international trial, assessing four accepted standard of care chemotherapy treatments of recurrent and refractory Ewing sarcoma recently reported on the first interim results of the first 220 randomized patients, 71% with primary refractory disease or first recurrence less than two years. Patients were randomized to either 1) gemcitabine/docetaxel, 2) cyclophosphamide/topotecan, 3) Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 52 of 58 high dose ifosfamide and 4) temozolomide/irinotecan. Efficacy data from the stopped gemcitabine/docetaxel arm, combined three remaining arms and the combined four arms, demonstrate an ORR of 11% (7/62), 24% (29/123) and 20% (36/185), respectively, for patients with evaluable measurable disease mainly receiving combination chemotherapy. The preliminary data from the rEECur Phase 2/3 study in predominantly refractory and first relapsed Ewing sarcoma patients demonstrate modest results even when the most active doublet chemotherapy is chosen. In addition, safety data from that study reports that following multimodality treatment for newly diagnosed Ewing sarcoma, there is considerable toxicity with approximately 60% of all randomized patients experiencing Grade 3 or higher adverse events, yet only a 24% combined response rate, at best. Taken together, these data in relapsed/refractory Ewing sarcoma support that novel targeted agents in Ewing sarcoma following standard-of-care, are needed. Appropriate response endpoints and innovative trials designs are needed to shorten the time to bring new agents to regulatory approval. Given the low incidence of patients with relapsed/refractory Ewing sarcoma, randomized, controlled clinical trials with sufficient statistical power are challenging to conduct in a reasonable timeframe. Upon completion of the dose expansion portion of the Phase 1 study, if seclidemstat demonstrates a modest and durable ORR (PR and CR) and/or a 6-month EFS rate > 12.7% with a tolerable safety profile in the 20 patients treated at the MTD/RP2D, Salarius would approach the FDA for advice and consideration of potentially designing a single arm, single-agent study in Ewing sarcoma patients age >12 years, who have been treated with at least two and no more than four prior lines of therapy, including standard of care, as a potential registration path. Dependent on the treatment response endpoint (magnitude and duration of the response, and/or 6-month EFS rate) and given the rare incidence of this relapsed/refractory disease, it could be estimated that up to 50 patients would be treated. Details of the study protocol design, along with a robust statistical analysis plan, would be agreed upon with FDA. If the ORR and duration of response (DoR) are modest, approximately 10-15% ORR with DoR at least one cycle past determination of confirmed response or good stabilization of disease (median PFS of three months), Salarius would propose combining seclidemstat with one of the chemotherapies identified from the two remaining arms of the rEECur Phase 2/3 study to assess safety and preliminary efficacy of the combination. This information from the rEECur study that identifies which of the two treatment regimens are moving forward into Phase 3 should be available in 2020. The combination Phase 1 study(s) with seclidemstat would be in preparation for conducting a registration trial of standard-of-care chemotherapy with and without seclidemstat in a patient population similar to the ongoing rEECur study described above, patients with refractory/relapsed Ewing sarcoma, predominantly represented by those who are either primary refractory or at first recurrence. The type of randomized study design and endpoints to support demonstration of enhanced therapeutic benefit for seclidemstat in Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 53 of 58 combination with chemotherapy could be discussed at an end of Phase 1 meeting between Salarius and the FDA. Based on the data from the expansion of the Phase 1 study in Ewing sarcoma, it is possible that an initial randomized Phase 2 study of chemotherapy with or without seclidemstat based on primary endpoint of EFS rate could be proposed. At every step in this clinical development plan, Salarius would seek advice from the FDA in order to better design the proposed clinical trials in this rare patient population that could accelerate research in order to bring novel therapies sooner to this patient population. There is an urgent need in this disease to: 1) identify novel targeted agents to enhance efficacy outcomes in the refractory/relapsed patient population, 2) identify agents with an improved therapeutic benefit over traditional chemotherapy, and 3) identify agents with better toxicity profiles. These factors all contribute to the need to collaborate with the FDA on study designs that optimize the number of patients and the time to endpoints for registration pathways for relapsed or refractory Ewing sarcoma patients.
8 Summary In summary, Ewing sarcoma is an aggressive disease with no standardized treatment for the relapsed/refractory population. Although advancements have been made with multimodality treatment options for newly diagnosed patients with local disease, the five-year survival rate for patients that present with metastasis or who relapse resides at devastatingly low rates of less than ~20%. The relapsed/refractory Ewing sarcoma makes up a significant proportion of patients, around 40%. For these patients, current therapy options have shown limited activity and/or tolerability with high discontinuation rates, high toxicity and low cure rates, defining an unmet medical need for these patients. There is a dire need for novel therapeutic agents to help treat this devastating disease. Salarius has developed SP-2577, seclidemstat, a novel, oral, selective and reversible LSD1 inhibitor to help address the clear unmet need of relapsed/refractory Ewing sarcoma patients. The drug is administered orally, an advantage over IV therapy, and convenient to patients and caregivers. Seclidemstat selectively targets LSD1, thereby targeting the underlying cause of Ewing sarcoma to treat the disease and improve patient outcomes. Preliminary Phase 1 clinical data shows that seclidemstat is well-tolerated with a promising PK profile. A plan to continue evaluating seclidemstat, either as a single agent or as an addition to a polychemotherapy approach to relapsed, metastatic disease is warranted. We seek advice from the committee on a plan for a Proposed Pediatric Study Request to FDA for granting of a future Written Request. Salarius Pharmaceuticals ODAC Pediatric Subcommittee Meeting Seclidemstat (SP-2577) Page 54 of 58
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