Identification of High-Efficiency β-Catenin Protein Degradation As Critical Vulnerability in B-Cell Malignancies
Authors: Cosgun Kadriye Nehir , Robinson Mark E , Agadzhanian Nikol , Cheng Zhangliang , Oulghazi Salim , Berning Philipp , Fonseca-Arce David , Kume Kohei , Fontaine Jake , Chan Lai N , Lee Jaewoong , Yu Fang , Qian Zhijian , Song Joo Y. , Chan Wing Chung , Chen Jianjun , Taketo Mark M , Schjerven Hilde , Müschen Markus 
Published: December 2024

Background and Significance
As part of canonical WNT signaling, TCF7-family factors pair with β-catenin to activate MYC and promote survival and proliferation. β-catenin protein degradation is initiated by GSK3B- and CK1a-dependent phosphorylation. Genetic lesions of the β-catenin protein degradation pathway are frequent throughout the spectrum of cancer, but conspicuously absent in B-ALL, CLL and mantle cell lymphoma. Compared to other cell lineages, we found that B-cells express 80-200-fold lower levels of β-catenin protein and critically depend on efficient β-catenin protein degradation.
Results
Gene expression and proteomic analyses of solid tumor and myeloid leukemia cancer cell lines (n=779; DepMap) and B-ALL and B-cell lymphoma (n=131) revealed that β-catenin protein levels were markedly reduced and barely detectable in the latter despite comparable mRNA expression levels. To address this discrepancy, we engineered 10 B-lymphoid and 9 non-lymphoid cell lines with a dual fluorescence protein stability reporter for simultaneous measurement of β-catenin mRNA and protein levels, which corroborated a B-cell-specific acceleration of β-catenin protein turnover. Unlike other cell types, in B-cells, β-catenin-protein was constitutively phosphorylated by GSK3B and CK1a, neddylated by NAE1 and poised for proteasomal degradation.
Combining computational and genetic approaches, we identified constitutive lack of N-cadherin (CDH2) and other adherens junction proteins as the underlying reason for accelerated β-catenin turnover in B-cells (r=0.65; P=3.78E-14). Consistent with their migratory behavior, B-cells lack adherens junctions, which shield β-catenin protein from degradation and promote cell adhesion in tissues.
Interestingly, doxycycline induced expression of CDH2 in B-ALL cells enabled rapid accumulation of β-catenin at the cell membrane, while removal of doxycycline caused depletion of both CDH2 and β-catenin protein degradation.
To investigate the significance of constitutive β-catenin degradation in B-cell malignancies, we developed genetic models of APC, GSK3B and CK1a haploinsufficiency in B-ALL cells. Induced haploinsufficiency resulted in a moderate accumulation of β-catenin protein, acute cell death and loss of colony formation in B-ALL cells. β-catenin stabilization in B-ALL cells subverted clonal fitness and leukemia initiation capacity. We engineered human B-ALL (n=6), mantle cell lymphoma PDX (n=4) as well as AML (n=4) for inducible expression of degradation resistant β-catenin, which induced acute cell death in B-lymphoid but accelerated proliferation in myeloid cells.
In non-lymphoid cells, β-catenin interacts with TCF7 factors to drive transcriptional activation of Myc. Surprisingly, defective protein degradation and accumulation of β-catenin resulted in repression of Myc in B-cell tumors. Flow-cytometry and time-lapse microscopy experiments using mTurqouise-β-catenin and eGFP-Myc double reporter B-ALL cells revealed that 12 hours after disruption of β-catenin protein degradation, B-ALL cells accumulated b-catenin protein and abruptly lost Myc expression. Mass spectrometry-based protein-interactome studies identified that instead of TCF7, β-catenin formed unique with B-lymphoid Ikaros factors (Ikzf1/Ikzf3) and repressive nucleosome remodeling and deacetylase (NuRD) components. ChIP-seq analysis highlighted that binding of β-catenin/Ikaros/NuRD complexes at lymphoid-specific Myc super-enhancer regions led to loss of H3K27Ac active enhancer marks and transcriptional repression of Myc. CRISPR-mediated engineering of a point mutation in a prominent Ikaros binding motif within the Myc-BENC superenhancer region subverted β-catenin-dependent Myc repression and enabled B-ALL to survive defective β-catenin protein degradation.
Conclusion
Here, we show that B-cell malignancies, including B-ALL and mantle cell lymphoma uniquely depend on constitutive β-catenin degradation. Genetic defects of the β-catenin protein degradation pathway that promote cancer in other lineages are not compatible with B-cell identity. The deleterious outcome of β-catenin accumulation in B-lymphoid cells is predicated on B-cell-specific Ikaros transcription factors and explains the unique dependency of B-lymphoid cells on highly efficient mechanisms of β-catenin protein degradation.