(P 90) TARGETING HIGH RISK MYC- AMPLIFIED OSTEOSARCOMA BY AURORA KINASE INHIBITOR--RESULT FROM A NGS BASED UMBRELLA TRIAL

Objective: Osteosarcoma (OS) is the most common bone tumor in pediatric patients, whose effective therapies are of great unmet medical needs, particularly for those of high-risk features. C-Myc is commonly amplified in OS patients, contributes to dismal prognosis and associates with lower 5-year overall survival rate, which is confirmed by several previous studies including ours. However, there is little effective strategies for this subset of patients with MYC amplification. TT-00420, a small molecule targeting Aurora A/B in clinical trial has demonstrated down-regulation on c-Myc expression in TNBC studies. Moreover, Aurora kinases and c-Myc are reciprocally regulated, triggering maintenance of the malignant state. We designed an umbrella trial based on NGS test to subtype OS patients with advanced disease. We further examined the efficacy of the novel new drug TT-00420 in Myc-amplified OS.

Methods: In pre-clinical assay, we treated human osteosarcoma cell line with TT-00420 in vitro and vivo. Western blot was used to verify Myc degradation. Using a MYC-amplified Patient-Derived Xenografts (PDX) to validate in vivo (15mg/kg) efficacy. We used murine-derived lm-8 cells for subcutaneous tumor implantation in C3H mice to evaluate the effect of TT-00420(8mg/kg) in combination with PD1(10mg/kg).
Our previous study classified OS into four subtypes with putative therapeutic targets. We conducted an investigator-initiated umbrella trial (Chinese Clinical Trial Registry: CTR2000036618). The study allows patients to be assigned to the appropriate precision treatment arm by NGS sequencing and immunohistochemistry. In the Myc-amplified arm, we evaluated the safety and tolerability of TT-00420. The study was divided into 2 cohorts for 3+3 dose escalation according to body surface area (BSA), Cohort A (BSA ≤ 1.5m2): 5mg, 8mg, 10mg, Cohort B (BSA > 1.5m2): 8mg, 10mg, 12mg. The primary endpoints were safety and tolerability. The second endpoints included evaluating the efficacy of TT-00420 by investigators per RECIST 1.1.

Results: 1)TT-00420 significantly inhibited the colony formation in Myc-amplified OS cell lines in a dose-dependent manner. Western blot results show that TT-00420 dramatically suppressed Aurora kinase pathway and blunted c-Myc protein expression level. In pre-clinical in vivo efficacy studies, treatment with TT-00420 clearly inhibited tumor growth compared to the vehicle group in mice xenograft model. Moreover, in a Myc-amplified PDX model, TT-00420 dramatically blunted the tumor growth with a TGI of 87.6%. In addition, in the synergistic LM-8 mice model, TT-00420 sensitized the efficacy of immune checkpoint inhibitor. Immunohistochemistry showed that TT-00420 in combination with PD1 increased CD4 and CD8 cell infiltration.
2) We identified 11 patients with osteosarcoma with Myc amplification by NGS. These patients were enrolled in a once-daily oral dose of TT-00420 tablets to evaluate safety and preliminary efficacy. No dose-limiting toxicity (DLT) events occurred in all dose groups of TT-00420, good safety profile in patients with advanced osteosarcoma. The major adverse events include: low lymphocytes(n=5, 45.5%), low platelets(n=2, 18.2%), fever(n=4, 36.4%), hand-foot syndrome(n=2, 18.2%). The median treatment duration 57 days, median OS is 99 days, median PFS is 57 days.

Conclusion: Patients with MYC amplification are very aggressive and have an extremely poor prognosis. TT-00420 depicted robust anti-tumor activity in Myc-amplified OS both in vitro and in vivo with downregulation of c-Myc expression, indicating its potential as active drug for OS patients with c-Myc amplification. In addition, TT-00420 may increase the immune infiltration in microenvironment and enhance efficacy of PD-1 immunotherapy.