Targeting β-Catenin Protein Degradation in Refractory B-Cell Malignancies

Background and Significance: Approaches for targeted elimination of B-cells as the root-cause of disease have been highly effective in the treatment of B-cell malignancies (rituximab, ibrutinib). To expand the portfolio of B-cell-selective drugs, we developed an interactive computational tool (lymphoblasts.org) to identify previously unrecognized B-cell selective vulnerabilities.

Results: Compound screening data (CTD, GDSC), together with gene-dependency scores from CRISPR and RNAi screens, were re-analyzed and integrated for B-ALL/mature B-cell lymphomas compared to myeloid leukemia and solid tumors. Most notably, among 4,518 compounds screened, we identified 9 top-ranking compounds that converge on targeting the β-catenin protein degradation pathway: GSK3B-inhibitors (AZD7969, LY2090314, GSK3i IV, CHIR99021, ML320), the neddylation-activating enzyme (NAE)-inhibitor pevonedistat and proteasome inhibitors (MG-132, Bortezomib, Ixazomib) showed high B-cell-selectivity along with known B-cell-selective drugs such as dexamethasone and ibrutinib. GSK3B phosphorylates β-catenin on N-terminal residues, the neddylation-activating enzyme (NAE1) induces β-catenin-neddylation followed by proteasome-dependent β-catenin proteolysis.

GSK3B-, NAE1- and proteasome-inhibitors decreased β-catenin protein degradation efficiency, induced dose-dependent accumulation of β-catenin and selectively induced cell death in B-lymphoid cells. CRISPR/Cas9 chemogenomic screens and genetic deletion of CTNNB1 confirmed β-catenin accumulation as central mechanism of action. Unlike GSK3B-inhibitors, proteasome inhibitors and the NAE1-inhibitor pevonedistat have been successfully used in clinical trials for B-cell malignancies, although β-catenin was not considered as mechanistic target.

As part of canonical WNT signaling, TCF7-family factors pair with β-catenin to activate MYC and promote survival and proliferation in other cell lineages. Compared to other cell types, we found that B-cells express 80-200-fold lower levels of β-catenin protein and critically depend on efficient β-catenin protein degradation: β-catenin-protein was constitutively phosphorylated by GSK3B, neddylated by NAE1 and poised for proteasomal degradation in B-cells. Interestingly, inhibition of GSK3B, NAE1 and the proteasome resulted in rapid β-catenin accumulation and induced toxicity in B-cells. While GSK3B, NAE1 and proteosome inhibitors interfere with β-catenin protein degradation at three distinct levels, the GSK3B-inhibitor LY2090314 was highly synergistic with both the NAE1-inhibitor pevonedistat and the proteasome inhibitor bortezomib at low nanomolar concentrations.

Mechanistically, our interactome studies in B-cell tumors revealed that, instead of interacting with TCF7, β-catenin formed repressive complexes with B-lymphoid Ikaros factors for transcriptional repression of MYC. While β-catenin/TCF7 complexes activate Myc and promote survival and proliferation in other cell types, we found that B-cells critically depend on high-efficiency β-catenin protein degradation. In B-cells, β-catenin assembles B-lymphoid-specific complexes with repressive Ikaros and NuRD factors for transcriptional repression of Myc.

To leverage β-catenin-protein degradation as a previously unrecognized vulnerability in refractory B-cell malignancies, we validated the GSK3B-inhibitor LY2090314 in preclinical treatment studies of patient-derived B-ALL and mantle cell lymphoma xenografts in vivo. Compared to control mice, 10 mg/kg LY2090314 i.p. substantially reduced leukemia burden and significantly extended overall survival for three refractory B-ALL PDX (P=6.5E-05, n=9 per group) as well as for mantle cell lymphoma (P=6.5E-04, n=7 per group). In line with highly B-cell-selective effects of GSK3B-inhibition, treated mice did not show dose-limiting toxicities.

Conclusion: This study reveals a previously unrecognized targetable dependency of B-cell malignancies on β-catenin protein degradation that is amenable to near-term evaluation in patients with refractory disease. The discovery of GSK3B-dependent β-catenin protein degradation as unique B-lymphoid vulnerability provides a rationale to repurpose clinically approved GSK3B-inhibitors for the treatment of refractory B-cell malignancies.

Davids:Adaptive Biosciences: Consultancy; Genmab: Consultancy; Merck: Consultancy; Ascentage Pharma: Consultancy, Research Funding; BeiGene: Consultancy; MEI Pharma: Research Funding; Genentech: Consultancy, Research Funding; Surface Technology: Research Funding; BMS: Consultancy; Janssen: Consultancy; Eli Lilly: Consultancy; Novartis: Research Funding; AbbVie: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding.