TBL1 Inhibition Eradicates Drug Resistant Leukemia Initiating Cells in T-ALL

BACKGROUND: Relapse in T-cell acute lymphoblastic leukemia (T-ALL) following chemotherapy remains a therapeutic challenge, necessitating novel treatment approaches. Aberrant activation of the Wnt pathway, particularly through β-catenin, is commonly observed in T-ALL, and plays a crucial role in the self-renewal of drug-resistant ALL. Targeting Wnt signaling in T-ALL is challenging due to the presence of a subset of leukemia-initiating cells (LICs) that are responsible for disease relapse. These LICs exhibit elevated levels of β-catenin expression, underscoring the importance of β-catenin signaling in T-ALL. Here, we focus on inhibiting the Wnt pathway by targeting transducin β-like protein 1 (TBL1), a protein required for Wnt- β-catenin-mediated transcription, with tegavivint, a novel small molecule TBL1 inhibitor. We propose that by disrupting the interaction between TBL1 and β-catenin, we can reduce the self-renewal capacity and survival of T-ALL cells. This approach aims to sensitize T-ALL cells to standard chemotherapy agents (vincristine, dexamethasone, L-asparaginase, referred to as VDL) in both in vitro and in vivo settings, as part of the preclinical evaluation of tegavivint in T-ALL treatment.

METHODS: We used three relapse T-ALL cell lines (MOLT-4, JURKAT, and CCRF-CEM) and one patient-derived T-ALL case relapsed after chemotherapy (332x) for in vitro studies. Cell viability was assessed using cytotoxicity studies, which involved Annexin-V/DAPI staining followed by flow cytometry analysis. β-catenin-mediated signaling was investigated by using western blot and co-immunoprecipitation assays (co-IP). Cell cycle analysis was performed using the BrdU cell proliferation assay. Additionally, we used flow cytometry to evaluate the expression of the CD7+CD1a- LIC phenotype. For in vivo studies, we utilized NSG mice engrafted with 332x relapse T-ALL cells and a serial transplantation assay to confirm the eradication of T-ALL in vivo.

RESULTS: Our study found that both β-catenin and TBL1 were uniformly expressed in all T-ALL cell lines and the patient sample, although at varying levels, as determined by western blotting. TBL1 inhibition using tegavivint treatment resulted in a reduction of β-catenin protein expression and decreased the binding of TBL1 to β-catenin, as shown by co-IP. The study demonstrated that tegavivint, used alone, significantly decreased the survival of all T-ALL cells, even at a low dose of 20 nM after 24 hours of incubation. Cell cycle analysis indicated that tegavivint causes T-ALL cells to arrest in the G2 phase. Furthermore, tegavivint increased the sensitivity of the tested cases to chemotherapy (VDL) within 24 hours of treatment as cell survival was reduced markedly when tegavivint was combined with VDL compared to VDL alone. The p-values for the comparison between VDL alone and the combination of tegavivint with VDL (TV+VDL) were significant across four different cases: MOLT-4 (p=0.0175), JURKAT (p=0.0004), CCRF-CEM (p=0.0049), and 332x (p=0.019), indicating that tegavivint enhances the efficacy of the chemotherapy regimen within 24 hours. Importantly, we showed that tegavivint reduced CD7+CD1a- leukemia-initiating cell subset in the patient-derived 332x relapse T-ALL patient case. In vivo studies using NSG mice engrafted with patient-derived 332x relapse T-ALL cells demonstrated that mice treated with tegavivint alone (n=6, Median Survival time (MST) =62.0 Days) had extended survival compared to the untreated control (n=6, MST=32 days, p=0.0006) and chemotherapy alone treated group (n=6, MST=37.5 Days, p=0.0006). All mice treated with tegavivint in combination with chemotherapy survived until the end of experiment (n=6, MST=160 days). Serial transplantation of bone marrow from surviving mice treated with tegavivint and VDL resulted in 100% survival of recipient NSG mice compared to 100% death of mice engrafted with BM of VDL only treated mice.

CONCLUSION: Our preliminary data suggests a promising new therapeutic approach for T-ALL and LICs by targeting TBL1-β-catenin with tegavivint. Tegavivint induces apoptosis as a single agent in T-ALL cells, sensitizes the leukemic cells to chemotherapy, and reduces the subset of LICs. These findings support our hypothesis that inhibiting TBL1-β-catenin is a viable target in T-ALL. Further preclinical studies are ongoing to evaluate the potential of this approach for clinical use.