Targeting immunoproteasome-dependent β-catenin degradation as a therapeutic strategy in Relapsed/Refractory acute lymphoblastic leukemia

Abstract

Abstract Background and Significance: As central component of canonical WNT-signaling, β-catenin functions as oncogenic driver that promotes expression of MYC across a broad range of solid tumors and myeloid leukemia. Genetic defects of the β-catenin protein degradation machinery stabilize β-catenin and result in formation of TCF7/β-catenin complexes for transcriptional activation of MYC-dependent proliferation. Results: Paradoxically, we discovered that B- and T-cell acute lymphoblastic leukemia (ALL) cells not only lacked activating β-catenin lesions but instead evolved and critically depend on high-efficiency β-catenin protein degradation mechanisms. Upon inhibition of β-catenin protein degradation, accumulating β-catenin in ALL cells formed repressive LEF1/β-catenin complexes. In contrast to activating TCF7/β-catenin complexes in solid tumors and myeloid leukemias, repressive LEF1/β-catenin complexes in B- and T-ALL cells suppress MYC, resulting in acute cell death. The unique dependency of ALL cells on high-efficiency β-catenin degradation presents a novel therapeutic opportunity that can be exploited with multiple existing drugs that are in late stages of clinical development. The immunoproteasome is a specialized form of the proteasome that is primarily expressed in B- and T-cells. Three catalytic subunits of the standard proteasome (PSMB5, PSMB6, PSMB7) are replaced with immunoproteasome-specific subunits PSMB8, PSMB9 and PSMB10. Beyond its well-established role in antigen processing, the immunoproteasome regulates cellular stress responses, cytokine signaling, and protein homeostasis in B- and T-lymphocytes. Selective inhibitors of the immunoproteasome including Zetomipzomib (KZR-616), ONX-0914, and M3258 were initially developed for patients with refractory autoimmune diseases, and have shown safety in early-phase clinical trials. Here we evaluate the role of the immunoproteasome as a key mediator of β-catenin degradation in lymphoid leukemias. To investigate the role of the immunoproteasome 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 leukemia cell lines and patient-derived xenografts measuring luminescence 3 days after treatment. Pharmacologic inhibition of PSMB8 and PSMB9 (Zetomipzomib and ONX-0914) led to marked b-catenin accumulation and subsequent cell death at low nanomolar doses in both primary and patient-derived B-ALL and T-ALL cells. Interestingly, treatment with M3258 (a selective PSMB8 inhibitor) did not interfere with β-catenin protein degradation, suggesting that combined inhibition of both PSMB8 and PSMB9 is required for efficient inhibition. Genetic deletion of β-catenin in mouse B-ALL cells largely rescued the effects of Zetomipzomib and ONX-0914, providing genetic evidence for β-catenin protein accumulation as the mechanism of action of immunoproteasome inhibition. Likewise, genetic deletion of LEF1 rescued cell viability and caused resistance to Zetomipzomib and ONX-0914, highlighting the importance of repressive LEF1/β-catenin complexes in B- and T-ALL cells to suppress MYC. Conclusions: Our findings uncovered a previously unrecognized dependency of B- and T-ALL cells on immunoproteasome-mediated β-catenin degradation to sustain MYC expression and survival. Targeting the immunoproteasome, rather than the generic proteasome, offers a more selective strategy for lymphoid malignancies, with the potential for reduced off-target toxicity compared to pan-proteasome inhibitors such as Bortezomib. Recent Phase 1/2 trials have demonstrated safety and feasibility of immunoproteasome inhibition in patients with autoimmune diseases. Here we propose targeting the immunoproteasome subunits PSMB8 and PSMB9 to leverage inhibition of β-catenin degradation mechanisms in relapsed/refractory B- and T-ALL. Given that several of our candidate compounds have already undergone clinical development, we anticipate that this approach can rapidly be developed to benefit patients with relapsed or refractory B- and T-ALL.