Non-chemotherapy adjuvant agents in TP53 mutant Ewing sarcoma

Ewing sarcoma (EWS) is a malignant tumor arising in bone or soft tissue that occurs in adolescent and young adult patients as well as adults later in life. Although non-metastatic EWS is typically responsive to treatment when newly diagnosed, relapsed cases have an unmet need for which no standard treatment approach exists. Recent phase III clinical trials for EWS comparing 7 vs 5 chemotherapy drugs have failed to improve survival. To extend the durability of remission for EWS, we investigated 3 non-chemotherapy adjuvant therapy drug candidates to be combined with chemotherapy. The efficacy of these adjuvant drugs was investigated via anchorage-dependent growth assays, anchorage-independent soft-agar colony formation assays and EWS xenograft mouse models. Enoxacin and entinostat were the most effective adjuvant drug in both long-term in vitro and in vivo adjuvant studies. In the context that enoxacin is an FDA-approved antibiotic, and that entinostat is an investigational agent not yet FDA-approved, we propose enoxacin as an adjuvant drug for further preclinical and clinical investigation in EWS patients.

Here we investigated 3 preclinically-and clinically-investigated non-chemotherapy adjuvant drug candidates for their potential to improve chemotherapy effectiveness, using etoposide as a topoisomerase II inhibitor causing double-stranded DNA breaks 14,15 as our chemotherapy model drug.TK216 (also known as, ONCT-216) is an analog of YK-4-279 that has recently been studied in EWS with vincristine in a phase 2 clinical trial together (NCT05046314) 16 , but has been since deprioritized by Oncternal Therapeutics and is now also thought to be a microtubule inhibitor 17 .Mechanistically, YK-4-279 (and presumably TK-216) binds directly with the pathognomonic fusion-mediate chimeric transcription factor and tumor maintenance driver EWS-FLI1 [18][19][20][21] to inhibit its interaction with RNA helicase A (RHA) 22 .YK-4-279 has been shown to induce apoptosis and growth inhibition of EWS tumors 22,23 .Entinostat is a histone deacetylase inhibitor that reveals high potency against Type I HDACs 24 and has preclinical activity against EWS 17 .A Phase 1 clinical study for entinostat was performed in patients with advanced solid tumors or lymphoma (NCT00020579), and single agent activity was demonstrated in an EWS patient 25 .Enoxacin is an oral fluoroquinolone antibacterial agent that was shown to enhance TRBP/ DICER dependent miRNA maturation 26 , leading to tumor regression 27,28 and EWS tumor stem cell depletion 29,30 .The miRNA maturation led by enoxacin inhibited cancer stem cell self-renewal and tumor maintenance 30 .
These 3 adjuvant drugs were tested with the chemotherapy etoposide in short-term and long-term twodimensional (2D) cell inhibition assays, soft-agar colony formation assays and in vivo EWS mouse models.In these studies, enoxacin and entinostat were found to be the most effective drug candidates in both long-term in vitro and in vivo adjuvant studies.We propose enoxacin as a potential adjuvant drug for further preclinical and clinical EWS studies based on the basis of its FDA approval status.

Results
Long-term effects of adjuvant drugs in 2D cell culture.To investigate the long-term effect of the adjuvant drug candidates, an anchorage-dependent 2D cell culture cell growth/viability assay was performed over 24 days.The design of the assay was to "debulk" tumor cell mass over 72 hr with a EWS-specific chemotherapy (etoposide) followed by application of the adjuvant drugs.EWS cell lines A673, SK-N-MC, SK-ES-1 and RDES (Table 1) were treated with etoposide using 1.6 µM at a lethal dose (LD) 90-95% for 3 days.Thereafter, candidate adjuvant drugs were added at concentrations of either the IC 20 (990 nM for entinostat) or a clinically relevant concentration (C steady-state 3.9 µM for TK-216 (J.Toretsky, p.c.), C max 9.2 µM for enoxacin 31,32 ) for Effect of adjuvant drugs for in vivo mouse study.Next we tested entinostat, TK216, and enoxacin for their adjuvant efficacy using in vivo mouse models (Figs. 4 and 5).Drug doses were selected to be most similar between humans and mice (Tables 2 and 3).For engraftment, SK-N-MC EWS cells were injected in the right leg of 6 week-old male (n = 4) and female (n = 8) NOD/SCID/IL2gr-null mice in each treatment group.When the tumor size reached 0.1-0.15cc, mice were treated with 10 mg/kg etoposide for 5 days followed by adjuvant drugs for 37 days dosed at the estimated human drug exposures described in "Materials and methods".Tumor growth rates were then measured.Two mice studies were performed to evaluate the adjuvant drugs.First, entinostat and TK216 were used as adjuvant drugs (Fig. 4A-G).Next, enoxacin was tested as the adjuvant drug (Fig. 5A-F).Entinostat treatment following etoposide was effective for tumor regression compared with the vehicle group (p < 0.001), but not when compared with the etoposide-only treated group (p = 0.57) (Fig. 4B,C,G).TK216 caused toxicity >10 % (weight loss) for both doses of 25 mg/kg QD and 50 mg/kg QD with 50 % treatment-related mortality.The efficacy of TK216 was similar to but less sustained than entinostat (Fig. 4G).Overall p-value was < 0.001 in the mouse study (Fig. 5E-G).Enoxacin inhibited the tumor progression most efficiently compared with both the vehicle group (p < 0.05) and etoposide-only treated group (p = 0.4) showing the overall p-value as 0.017 (Fig. 5B-E).For tumors harvested either at day 84 or when tumor volume reached 1.4 cc, enoxacin treatment following etoposide inhibited expression of a cancer stem cell marker CD133 in EWS 34 , and increased cleavedcaspase 3 which indicates the elevated apoptosis compared with that of the vehicle or etoposide-only groups (Fig. 4).All regimens were well-tolerated by body weight monitoring (Supplementary Figs. 1 and 2).

Discussion
Although newly diagnosed EWS patients may expect long-term benefit from upfront chemotherapy, metastatic and relapsed EWS patients have consistently poor outcomes.Here we evaluated 3 drug candidates via long-term in vitro and in vivo assays for effectiveness as adjuvants to chemotherapy in EWS.While in vitro assays were useful first evaluations, the most informative results came from EWS xenograft mouse model studies.
In some animals and overall, entinostat suppressed tumor growth in a subset of animals and extended overall xenograft animal survival after etoposide compared to etoposide alone.Enoxacin had a more consistent ability to suppress tumor growth and extend overall xenograft animal survival after etoposide compared to etoposide alone -albeit with a similar effect on time to event compared to entinostat.As described in the Introduction, the mechanism underlying enoxacin activity as a chemotherapy adjuvant and tumor repopulating cell depletion has been well-described previously 29,30 .Given that enoxacin is an FDA-approved antibiotic that might be repurposed, and entinostat is an agent not yet FDA approved, we have set future sights on this agent for further preclinical investigation.
Like many pilot studies, our investigation has potential limitations and notable future directions.For example, increasing cohort size may improve power and statistical significance.Studying additional patient-derived xenograft models (e.g. more independent biological replicates) would also extend the overall value of these preclinical studies in clinical trial concept development.The expression of CD133 and cleaved-caspase 3 in mouse tumors treated with etoposide and enoxacin suggests that the sequential treatment of the drugs suppress stem cell formation inducing cell death.Further investigation in the expression of CD133 and cleaved-caspase 3 with other treatments such as etoposide with entinostat will also be of interest in future studies.Assessment In conclusion, enoxacin was identified as an efficient adjuvant drug by long-term 2D cell culture, soft agar colony formation and in vivo studies.Taken altogether with its FDA approval status, we propose enoxacin or a fluoroquinolone with fewer drug interacts 35 as an adjuvant agent for additional preclinical animal studies then possible clinical trial investigation in EWS.

Soft agar colony formation assay.
Five thousand EWS cells were suspended in growth medium containing 0.35 % SeaPlaque agarose and then plated on 0.7 % base agar in 6-well plate.After 24 hours, EWS cells were treated with 1.6 µM etoposide (LD 90-95 ) for 3 days, then adjuvant drug candidates were added at concentrations of either the IC 20 or clinical Cmax for 8 weeks (see "Results").EWS cells were treated with TK216, entinostat,    Mouse studies.Host animals for xenografts were 6 week-old male and female NOD/SCID/IL2gr-null mice purchased from The Jackson Laboratory (Bar Harbor, MA, stock # 005557).Mice were inoculated with 5x10 5 of SK-N-MC cells to the gastrocnemius after gastrocnemius injury was induced by 50 µl of 2.5 µM cardiotoxin for 24 hr.SK-N-MC was chosen as a representative EWS cell culture as it harbors the typical Type I fusion of EWSR1 to FLI1 as well as a TP53 mutation.When tumor volume reached 0.1-0.15cc, 10 mg/kg of etoposide (Selleckchem, cat# S1225; Table 2) or vehicle was administered for 5 days via daily i.p. injection.After 1 day rest, mice were treated with the adjuvant drug candidates entinostat (Selleckchem, cat# S1053, 5.5 mg/kg, QD, oral gavage), enoxacin (MedChemExpress, cat# HY-B0268A, 100 mg/kg, QD, oral gavage; Table 3) and TK216 (Selleckchem, cat# S9718, 25 mg/kg or 50 mg/kg, QD, i.p injection; Table 3).The drugs were dissolved in 5% DMSO, 40% PEG300, 5% Tween 80 and 50% of ddH 2 O that were used as a vehicle in all mice studies.The adjuvant drugs were dosed until day 42, and primary tumor size and weight of the mouse were measured twice a week with digital calipers during treatment.Tumor progression was defined as a 25% increase in volume.Animals were grossly examined for post-treatment recurrence or metastasis at the end of the experiment on day 84, or when tumor volume reached the humane endpoint of 1.4 cc.Table 3 summarizes etoposide and adjuvant drug doses used and references for dose selection.

Animal research.
Children's Cancer Therapy Development Institute (cc-TDI) Institutional Animal Care and Use Committee (IACUC) approved the investigated animal study.The IACUC Federal wide Assurance number is 14-5406A.In animal studies, anesthesia was performed using inhaled isoflurane by means of an anesthesia machine with a scavenger system.For euthanasia, CO 2 was delivered from a compressed gas cylinder to a chamber that had not been pre-filled.Animals were asphyxiated by CO 2 inhalation, then cervical dislocation was subsequently performed.All methods were carried out in accordance with relevant guidelines and regulations.All methods are reported in accordance with ARRIVE guidelines (https:// arriv eguid elines.org).

Figure 1 .
Figure 1.Working model and rationale for an adjuvant therapy approach to prevent progression of Ewing sarcoma.

Figure 2 .
Figure 2. Anchorage-dependent adjuvant therapy assay in Ewing sarcoma cells.(A) Schematic design of assay.(B) Adjuvant assay results in cell lines A673 and SK-N-MC (type 1 EWS) as well as SK-ES-1 and RDES (type 2 EWS).The cells were treated with adjuvant drug candidates for 21 days after 3-day etoposide treatment.The surviving cells were measured using CTG.Statistical analysis was performed using two sided t test.***p < 0.001; **p < 0.01; *p <0.05 versus chemo-only.Chemo, chemotherapy (etoposide).All experiments included at least three replicates.

Figure 3 .
Figure 3. Anchorage-independent soft agar colony formation assay in Ewing sarcoma cells.Soft agar assay was performed in A673, SK-N-MC, SK-ES-1, and RDES EWS cell lines.Cells were treated with 1.6 µM of etoposide for 3 days, then followed by adjuvant drug candidate treatment.(A) Diagram of 8 week soft agar colony formation assay.(B) Cell colonies were counted from soft agar assay in four Ewing sarcoma cell lines after adjuvant drug treatments for 8 weeks.Statistical analysis was performed using two sided t test.***p < 0.001; **p < 0.01; *p <0.05 versus no chemo.Chemo, chemotherapy.3-BP, 3-bromopyruvate.All experiments included at least three replicates.

Figure 4 .
Figure 4. Effect of entinostat and TK216 for adjuvant therapy in vivo mouse study.SK-N-MC EWS cells harboring TP53 and STAG2 mutations were injected in the right leg of 6-week old male (n = 4) and female (n = 8) NOD/SCID/IL2gr-null mice in each treatment group.When tumor size reached to 0.1-0.5 cc, mice were treated with 10 mg/kg etoposide for 5 days followed by the adjuvant drugs for 37 days dosed at the estimated human drug exposures described in "Materials and methods".(A) Diagram of mice study.Individual tumor volumes in (B) vehicle, (C) etoposide-only, (D) entinostat following etoposide, (E) TK216 (50 mg/kg) after etoposide treatment, and (F) TK216 (25mg/kg) following etoposide.(G) Kaplan-Meier plot represents eventfree survival.The event was defined as when tumor reached to 1.4 cc.Overall p-values were calculated by the log-rank test.P < 0.001 for each treatment group's comparison to vehicle only.

Figure 5 .
Figure 5.Effect of enoxacin for adjuvant therapy in vivo mouse study.Mouse study was performed as described in Fig. 5. (A) Diagram of mice study.Individual tumor volumes in (B) vehicle, (C) etoposide-only and (D) enoxacin following etoposide treatment.(E) Kaplan-Meier plot represents event-free survival.The event was counted when tumor reached 1.4 cc.Overall p-values were calculated using the log-rank test.P = 0.014 for etoposide then enoxacin group compared to vehicle only.(F) The expression of CD133 and cleaved (c)-caspase 3 in EWS xenograft tumors from vehicle, etoposide-only, or enoxacin after etoposide treated groups.Eight protein samples in each group were used for western blot.To compare the protein intensity among immunoblots, one vehicle sample was applied in the first lane of each blot.The relative expression of CD133 and c-caspase 3 was shown in the graph of densitometry analysis.

Table 3 .
Etoposide and adjuvant doses, route, interval and literature references.