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4125 Identification of High-Efficiency β-Catenin Protein Degradation As Critical Vulnerability in B-Cell Malignancies

Program: Oral and Poster Abstracts
Session: 603. Lymphoid Oncogenesis: Basic: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Diseases, Lymphoid Malignancies, Biological Processes, Pathogenesis
Monday, December 9, 2024, 6:00 PM-8:00 PM

Kadriye Nehir Cosgun, PhD1*, Mark E Robinson, PhD1*, Nikol Agadzhanian, MSc1*, Zhangliang Cheng, BSc1*, Salim Oulghazi, MD1*, Philipp Berning, MD1*, David Fonseca-Arce1*, Kohei Kume, PhD1*, Jake Fontaine1*, Lai N Chan, PhD1, Jaewoong Lee, PhD1,2*, Fang Yu, PhD3*, Zhijian Qian4, Joo Y. Song, MD5, Wing Chung Chan, MD5, Jianjun Chen, PhD6, Mark M Taketo7*, Hilde Schjerven, PhD8* and Markus Müschen, MD1

1Center of Molecular and Cellular Oncology, Yale University, New Haven, CT
2School of Biosystems and Biomedical Sciences, Korea University, Seoul, Korea, Republic of (South)
3Division of Hematology/Oncology, Department of Medicine and Department of Biochemistry and Molecular Biology, The University of Florida, Gainesville, FL
4Division of Hematology/Oncology, Department of Medicine and Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL
5Department of Systems Biology, City of Hope Comprehensive Cancer Center, Duarte, CA
6City of Hope Comprehensive Cancer Center, Duarte, CA
7Graduate School of Medicine, Kyoto University, Kyoto, JPN
8Department of Laboratory Medicine and Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, CA

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.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH