Non-chemotherapy adjuvant agents in TP53 mutant Ewing sarcoma

Abstract

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.

Figure 1

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

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

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

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 event-free 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

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.