Targeting β-catenin nuclear export and protein degradation in high-risk acute lymphoblastic leukemia

Abstract Background and Significance: A recurrent, gain-of-function mutation in the nuclear export molecule XPO1 (E571K) has been identified in several lymphoid malignancies including B-cell acute lymphoblastic leukemia (ALL). Selinexor, a selective inhibitor of XPO1, has demonstrated clinical efficacy in hematologic malignancies, including relapsed or refractory B- and T-ALL. However, responses to selinexor monotherapy are often incomplete or transient, highlighting the need for rational combination strategies that improve therapeutic efficacy and durability of response. We recently discovered that ALL cells uniquely evolved and critically depend on high-efficiency β-catenin protein degradation mechanisms to prevent formation of transcriptionally repressive LEF1/β-catenin complexes that suppress MYC expression (Cosgun et al., Biorxiv 2023). Inhibition of this degradation pathway results in accumulation of β-catenin and leukemia cell death, suggesting this pathway is essential for leukemia cell survival. This dependency highlights a unique vulnerability in ALL that can be therapeutically exploited with existing drugs. Here we discovered β-catenin as a central cargo protein of XPO1-mediated nuclear export in B- and T-ALL cells. Based on these findings, we hypothesized that combining selinexor with agents that interfere with β-catenin degradation would synergistically promote β-catenin nuclear accumulation and potentiate cell death. We therefore investigated whether combining Selinexor with inhibitors of β-catenin degradation, including GSK3B- and immunoproteasome inhibitors, could represent a mechanistically grounded and therapeutically promising approach for relapsed/refractory ALL. Results Studying NALM6 B-ALL cells with and without XPO1 (E571K) CRISPR-knockin mutation and with the XPO1-inhibitor selinexor we identified b-catenin as a XPO1 cargo protein by immunofluorescence and cellular fractionation and Western blot. To investigate a role of XPO1-mediated nuclear export in regulating β-catenin protein degradation, we engineered NALM6 B-ALL cells and two B-ALL patient-derived xenografts with β-catenin protein expression reporters. To this end, B-ALL cells were CRISPR-engineered with N-terminal β-catenin fusions by homology-directed repair to knock-in a BFP-T2A-mScarlet-β-catenin sequence in place of exon 2. β-catenin accumulation was monitored by flow cytometry and validated by Western blot. Cell viability assays were performed to assess the sensitivity of each compound in a panel of lymphoid cancer cell lines and patient-derived xenografts measuring luminescence 3 days after treatment. Selinexor monotherapy had no effect on β-catenin degradation in ALL cells. The combination of Selinexor with GSK3B inhibition (LY2090314) or PSMB8/PSMB9 inhibition (Zetomipzomib/ONX0914) resulted in synergistic nuclear β-catenin accumulation in reporter cells. Notably, the co-treatment of GSK3B inhibition with Selinexor not only increased nuclear localization of β-catenin but also significantly extended its half-life, presumably, because nuclear β-catenin is sheltered from its degradation complex, which is restricted to the cytosol. The identification of β-catenin as an XPO1 cargo protein and the nucleus as a shelter from protein degradation provide a mechanistic rationale for combining XPO1 inhibition (selinexor, eltanexor) with GSK3B or immunoproteasome inhibition, which will support the design of forthcoming in vivo studies, including xenograft-based preclinical efficacy models in patient-derived B- and T-ALL xenografts. Conclusions These findings identify β-catenin as a previously unrecognized and functionally significant cargo protein of XPO1 in B- and T-ALL. Our finding supports a promising combinatorial strategy for the treatment of r/r ALL by co-targeting nuclear export and β-catenin protein degradation. Given the high-efficiency β-catenin degradation observed in ALL, it is not surprising that targeting XPO1 alone does not lead to β-catenin accumulation. However, the combination of XPO1 inhibition with established inhibitors of β-catenin degradation produces strikingly synergistic effects, resulting in robust β-catenin stabilization and leukemia cell death. Given selinexor's FDA-approved use in combination with proteasome inhibitors for multiple myeloma, and the advanced clinical development of immunoproteasome-targeted agents, this strategy is poised for rapid translation into clinical trials for relapsed or refractory ALL.