Identifying and overcoming mechanisms of resistance to effectively target the TBL1/β-catenin signaling axis for high-risk osteosarcoma

Osteosarcoma (OS) is the most common bone malignancy in the pediatric population. Overall survival rates remain about 65-75% and are significantly worse for patients with metastatic or refractory/relapsed disease, often with long-term outcomes of less than 30%. The Wnt-signaling pathway and its downstream transcriptional

activator β-catenin are involved in a myriad of cellular processes including proliferation, differentiation, and oncogenesis. In OS, aberrant Wnt-pathway signaling results in uncontrolled transcription of Wnt target genes that promote growth and metastatic spread. When the Wnt pathway is activated, β-catenin is free to interact with

transduction beta-like protein 1 (TBL1) and/or its highly related family member TBLXR1. TBL1 protects beta- catenin from nuclear degradation and forms a TBL1/β-catenin complex that mediates transcription of Wnt target genes. Overexpression of TBL1XR1 and nuclear beta-catenin are positively related to adverse

clinicopathological features and poor prognosis for patients with OS. Tegavivint is a first-in-class small molecule inhibitor that selectively binds TBL1, thereby inhibiting TBL1/β-catenin complex formation. Importantly, this occurs without inhibiting normal physiological roles of β-catenin in the cell membrane. We previously

demonstrated that single agent Tegavivint inhibited tumor growth and suppressed lung metastasis, which directly led to the development of a Phase 1/2 clinical trial for the treatment of relapsed pediatric solid tumors (NCT04851119). Subsequently, using our OS syngeneic models, we have evidence that Tegavivint alters the

OS immunosuppressive microenvironment by increasing cytotoxic T cell and NK cell infiltration. Finally, we have recently completed a combination drug screen with Tegavivint and identified clinically applicable and molecularly rationale synergistic probe compounds, including multi-tyrosine kinase inhibitors and HDACs. We will execute

three independent, yet complementary aims with our overall objectives to (1) identify and transcriptionally characterize the clonal evolution of Tegavivint-resistant OS cells, (2) assess rationale combinatorial therapeutic regimens that can quickly be translated to the care of high-risk sarcoma patients, and (3) identify the effects of

targeting TBL1/β-catenin on the evolution of the tumor immune microenvironment (TiME). Aim1. Identification of subpopulations and molecular profiling of Tegavivint resistant OS cells. Use of cellular barcoding and scRNA- Seq profiling of longitudinally Tegavivint treated in vivo models to define specific transcriptional states and

emergence of cell states. Aim 2. In vitro, ex vivo and in vivo to assess the efficacy, and toxicities of mono- and combination therapy on high-risk OS. Aim3. Effects of genetic and pharmacologic targeting of TBL1/β-catenin on the tumor microenvironment. We will dissect the longitudinal effects of targeting β-catenin/TBL1 signaling on

TiME, immune cell constituents and gain insights into mechanisms of immune exclusion. The results will provide insights into mechanisms of resistance to Tegavivint and pre-clinical justification for rational combinatorial therapeutic approaches for future trial development to treat high-risk pediatric cancer patients.