Targeting β-catenin degradation with GSK3β inhibitors induces cell death in acute lymphoblastic leukemia

Cosgun, Kadriye Nehir; Jumaa, Huda; Robinson, Mark E.; Cheng, Zhangliang; Oulghazi, Salim; Kume, Kohei; Fonseca Arce, David; Agadzhanian, Nikol; Kistner, Klaus M.; Leveille, Etienne; Drivet, Elsa; Yu, Fang; Qian, Zhijian; Song, Joo Y.; Chan, Wing-Chung; Xu, Liang; Xiao, Gang; Taketo, M. Mark; Kothari, Shalin; Davids, Matthew S.; Schjerven, Hilde; Jellusova, Julia; Müschen, Markus

doi:10.1038/s43018-025-01093-z

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Published: 08 January 2026

Targeting β-catenin degradation with GSK3β inhibitors induces cell death in acute lymphoblastic leukemia

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Abstract

As part of canonical Wnt signaling, T cell factor (TCF)–β-catenin complexes promote MYC-dependent proliferation. Lesions of the β-catenin protein degradation machinery are common oncogenic drivers. Here, we show that B cell acute lymphoblastic leukemia (B-ALL) lacks these mutations and critically depends on unencumbered β-catenin protein degradation. Compared to solid tumors, we found that mouse and human B-ALL express β-catenin protein at much lower levels; β-catenin protein was constitutively phosphorylated by glycogen synthase kinase 3B (GSK3β) and poised for proteasomal degradation. Instead of TCF–β-catenin complexes to activate MYC, β-catenin paired with B lymphoid Ikaros and NuRD complex factors, resulting in MYC repression and acute cell death. To leverage β-catenin protein degradation as a previously unrecognized vulnerability in B-ALL, we validated GSK3β inhibition in patient-derived xenograft models in vivo. CRISPR screens confirmed β-catenin protein degradation as a central mechanistic target of established GSK3β inhibitors. As several GSK3β inhibitors achieved favorable safety profiles in clinical trials, our results provide a rationale for repurposing these compounds for persons with refractory B cell malignancies.

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Main

The canonical Wnt–β-catenin pathway regulates fundamental processes including embryonic development, organogenesis and tissue homeostasis¹. β-catenin protein degradation is initiated by phosphorylation of N-terminal β-catenin residues² (S33, S37, T41 and S45) by glycogen synthase kinase 3B (GSK3β) and casein kinase 1α (CK1α) and the scaffolding proteins Axin1/2 and APC^3,4, followed by ubiquitination⁵ and proteasomal degradation. Unphosphorylated β-catenin accumulates in nucleus⁶ and pairs with T cell factors (TCFs)^7,8 to promote transcriptional activation of Wnt targets including MYC^9,10. Transcriptional activation of MYC by TCF–β-catenin complexes represents a central oncogenic driver in multiple cancer types^9,10. In contrast to epithelial, neuronal and mesenchymal lineages¹, β-catenin deletion was dispensable for hematopoietic and B cell development^11,12,13,14. Instead, targeted removal of GSK3β and CK1α phosphorylation sites to stabilize β-catenin^(S33^;S45)+/fl mice² impaired hematopoietic multilineage differentiation and lymphoid development^15,16. Conversely, in colon cancer, melanoma and other epithelial cancers, defective β-catenin protein degradation accelerated MYC-dependent proliferation and malignant transformation^17,18,19,20. Genetic mouse models of myeloid malignancies demonstrated that β-catenin was required for the initiation of acute myeloid leukemia (AML)²¹ and chronic myeloid leukemia (CML)^22,23. However, the development of murine B cell acute lymphoblastic leukemia (B-ALL) was unperturbed by deletion of β-catenin²³. Two studies on the effects of Wnt3A-dependent β-catenin accumulation on proliferation of B-ALL cells reported conflicting results^24,25. B-ALL cells with TCF3–PBX1 fusion aberrantly overexpress the WNT16 ligand²⁶. However, subsequent work showed that WNT16 does not promote canonical β-catenin signaling²⁷. Here, we show that B-ALL cells have evolved and critically depend on high-efficiency mechanisms of β-catenin protein degradation (Extended Data Fig. 1a). GSK3β has a central role in mediating high-efficiency β-catenin protein degradation^3,4. Small-molecule inhibitors of GSK3β have been developed for the treatment of solid tumors and neurological conditions (Extended Data Fig. 1b)^{28,29,30,31,32,33}. While these small-molecule inhibitors achieved favorable safety profiles in early-phase clinical trials, our preclinical experiments provide a rationale to repurpose these existing GSK3β inhibitors to subvert β-catenin protein degradation to improve outcomes for persons with refractory B cell malignancies.

Results

Lack of β-catenin protein expression in B cell malignancies

Studying β-catenin protein levels by immunohistochemistry in normal lymphoid tissues (n = 30) in comparison to epithelial and mesenchymal tissues (n = 51) (Extended Data Fig. 2a,b)³⁴ (https://www.proteinatlas.org/) as well as lung cancer (n = 15), colon cancer (n = 25), malignant melanoma (n = 5) in comparison to mantle cell lymphoma (MCL; n = 26), follicular lymphoma (n = 38), diffuse large B cell lymphoma (DLBCL; n = 35) and Hodgkin’s disease (HD; n = 44), revealed a previously unrecognized lack of β-catenin protein expression in normal and malignant B cells (Fig. 1a and Extended Data Fig. 2c,d). In stark contrast to solid tumor cell lines, nuclear β-catenin was barely detectable in B cell malignancies, while four of 22 B lymphoid cell lines expressed low levels of cytoplasmic β-catenin (Fig. 1b and Extended Data Fig. 2e). Studying publicly accessible datasets, including RNA sequencing (RNA-seq) and reverse-phase protein arrays (RPPAs)³⁵ (https://depmap.org/), revealed an unexpected discordance between β-catenin mRNA and protein levels in B cell malignancies. β-catenin mRNA levels were similar in B cell malignancies and solid tumors. However, β-catenin protein (RPPA) was barely detectable in B-ALL and B cell lymphoma cells (Fig. 1c). To address this discordance, we engineered nine epithelial and ten B lymphoid cell lines with a dual-reporter construct for concurrent detection of β-catenin mRNA (mScarlet) and protein (GFP). As expected, epithelial cells showed a linear relationship between mRNA and protein levels. Compared to epithelial cells, B-ALL and B cell lymphoma cells showed ~32-fold lower protein-to-mRNA ratios (P = 2.2 × 10⁻⁸; Fig. 1d,e).

**Fig. 1: B lymphoid cells express β-catenin mRNA but lack β-catenin protein expression.**

β-catenin is poised for proteasomal degradation in B-ALL cells

β-catenin protein degradation is initiated by N-terminal phosphorylation by GSK3β and CK1α (refs. ^3,4), while nonphosphorylated β-catenin is active and stable⁶. Studying tissue sections and protein lysates with antibodies recognizing N-terminally phosphorylated, nonphosphorylated and total β-catenin revealed that β-catenin in solid tumors (n = 134) was mostly stable in the nonphosphorylated conformation (Fig. 1f,g and Extended Data Fig. 3). In contrast, B cell lymphomas (n = 142) only expressed N-terminally phosphorylated β-catenin, while stable nonphosphorylated β-catenin was not detectable (Fig. 1f,g and Extended Data Fig. 3). Treatment of patient-derived B-ALL xenograft (PDX) cells with the small-molecule GSK3β inhibitor LY2090314 for 8 h (10 nM) suppressed β-catenin degradation and induced accumulation of nonphosphorylated β-catenin (Extended Data Fig. 4a). Studying LY2090314 in B-ALL cells carrying the dual β-catenin mRNA (mScarlet) and protein (GFP) reporter showed that GSK3β inhibition induced a diagonal distribution between β-catenin mRNA and protein as in epithelial cells (Extended Data Fig. 4b,c). Treatment of B-ALL PDX cells with 400 ng ml⁻¹ Wnt3A ligand resulted in only weak and transient β-catenin accumulation and failed to affect MYC expression or proliferation of B-ALL cells (Extended Data Fig. 4d–f) highlighting that high-efficiency β-catenin protein degradation in B cells limits Wnt3A-induced β-catenin accumulation.

Lack of genetic lesions in the β-catenin protein degradation pathway in B cell malignancies

Loss-of-function lesions of GSK3β, APC, CK1α and other components of the β-catenin protein degradation machinery are common oncogenic drivers throughout the spectrum of cancer^17,18,19,20. Studying deleterious β-catenin protein degradation pathway lesions (filtered for coding, nonsilent) in 90,812 cancer samples encompassing 13 types of solid tumors and B cell malignancies³⁶, we found frequent mutations in solid tumors (n = 86,426), namely APC (14%), AXIN1 (1.7%), AXIN2 (1.5%), GSK3B (0.5%) and CSNK1A1 (CK1α; 0.4%), as well as GSK3β and CK1α phosphorylation sites in β-catenin (4.7%) (Fig. 2a). In contrast, among 4,386 B cell malignancies, lesions in the β-catenin protein degradation pathway were significantly underrepresented (expected: 1,119, observed: 21, χ² test P = 1.2 × 10⁻¹²; Fig. 2a). These results suggest that, unlike other cancers, lesions of the β-catenin degradation pathway are not oncogenic drivers in B cell malignancies.

**Fig. 2: B cell malignancies are uniquely dependent on efficient β-catenin protein degradation.**

B-ALL cells critically depend on GSK3β, CK1α and APC for efficient β-catenin protein degradation

To address the role of central components of the β-catenin protein degradation pathway in B-ALL cells, we developed genetic loss-of-function models of APC (Apc^+/fl), GSK3β (Gsk3b^+/fl) and CK1α (Csnk1a^+/fl). To model haploinsufficiency of APC, GSK3β and CK1α in B-ALL cells, we transduced B cell precursors isolated from Apc^+/fl, Gsk3b^+/fl and Csnk1a^+/fl mice with BCR–ABL1 or NRAS^G12D oncogenes and Cre (Cre-ER^T2) or empty vector (EV) controls (ER^T2) for tamoxifen-inducible deletion. Induced haploinsufficiency of APC, GSK3β and CK1α resulted in acute cell death and loss of colony formation in BCR–ABL1-driven and NRAS^G12D-driven B-ALL cells (CD19⁺CD43⁺IgM⁻), corroborating dependency on efficient β-catenin degradation (Fig. 2b–g and Supplementary Fig. 1a). Likewise, in normal pre-B cells, deletion of one allele of either APC, GSK3β or CK1α resulted in cell death and elimination of pre-B cells from cell culture (Extended Data Fig. 5a–c), highlighting the importance of intact β-catenin degradation for B cell survival. N-terminal β-catenin S33, S37, T41 and S45 residues are phosphorylated by the concerted activity of GSK3β, CK1α and APC^3,4. To determine whether the dependency of B-ALL cells on GSK3β, CK1α and APC (Fig. 2b–g) is mechanistically linked to phosphorylation of S33, S37, T41 and S45 residues, we studied a genetic model based on excision of S33, S37, T41 and S45 residues², leaving other functions of GSK3β, CK1α and APC intact. Excision of N-terminal phosphorylation sites in β-catenin^{(S33;S45)+/fl} pre-B cells induced β-catenin accumulation and acute cell death (Extended Data Fig. 5d). In β-catenin^{(S33;S45)+/fl} mice that were crossed with a B cell-specific Mb1-Cre deleter strain, B lymphopoiesis was arrested at pre-BCR⁺ stages in the bone marrow, spleen and lymph nodes (Extended Data Fig. 5e–h and Supplementary Fig. 1b). Together, these results show that B cell development depends on the activity of GSK3β, CK1α and APC, N-terminal phosphorylation and degradation of β-catenin.

B-ALL leukemia initiation depends on high efficiency of β-catenin protein degradation

To determine whether B-ALL cells depend on effective β-catenin protein degradation in vivo, we transduced β-catenin^{(S33;S45)+/fl} B-ALL cells with tamoxifen-inducible Cre (Cre-ER^T2) or EV controls (ER^T2). Compared to EV controls, activation of Cre substantially delayed leukemogenesis and prolonged survival of transplant recipient mice (Fig. 2h). Defective β-catenin protein degradation also impaired leukemia initiation in vivo as shown in extreme limiting dilution assays (ELDAs) based on 300, 6,000 and 120,000 injected β-catenin^{(S33;S45)+/fl} B-ALL cells carrying tamoxifen-inducible Cre-ER^T2 or ER^T2 EVs. ELDA analysis revealed a 38-fold reduction of leukemia-initiating cells compared to controls (P = 6.2 × 10⁻⁴; Fig. 2h,i).

Dependency on β-catenin protein degradation is predicated on B cell identity

To assess whether this vulnerability reflects B cell-intrinsic transcriptional programs that determine B cell identity, we reprogrammed β-catenin^{(S33;S45)+/fl} B-ALL cells into the myeloid lineage through inducible expression of the myeloid transcription factor CEBPα. Then, 2 days after doxycycline-induced expression of CEBPα, B-ALL cells expressed the myeloid cell antigen CD11b (Mac1) and downregulated CD19 and the B cell transcription factors Ikzf1 (Ikaros) and Ikzf3 (Aiolos; Fig. 2j,k). Activation of Cre in β-catenin^{(S33;S45)+/fl} B-ALL cells subverted β-catenin protein degradation, competitive fitness and MYC expression. However, CEBPα-mediated B cell-to-myeloid reprogramming rendered leukemia cells resistant to β-catenin accumulation and restored clonal fitness and MYC expression (Fig. 2k,l). These findings suggest the diverging roles of β-catenin in myeloid and B cells and raise the possibility that lineage-specific transcription factors predicate the unique dependency of B lymphoid cells on β-catenin protein degradation.

β-catenin protein degradation is required to maintain MYC expression in B lymphoid cells

To determine the underlying mechanism of B lymphoid-specific dependency on β-catenin protein degradation, we examined gene expression changes upon activation of Cre in β-catenin^{(S33;S45)+/fl} B-ALL cells. RNA-seq analysis revealed enrichment for two principal gene sets, namely activation of Wnt–β-catenin signaling and suppression of Myc targets (Fig. 3a). The most prominent gene expression changes, repression of Myc and upregulation of Prdm1, a central negative regulator of Myc, were confirmed by Western blot (Fig. 3b,c). In addition, a set of β-catenin targets and feedback regulators (Nkd1, Nkd2, Tcf7, Kremen1, Axin2 and Tle3) were strongly increased upon β-catenin stabilization. β-catenin-mediated repression of Myc in B-ALL cells contradicted previous studies in epithelial cells that identified MYC as a classical Wnt target and strongly activated by TCF7L2–β-catenin complexes^9,10. To directly visualize the unexpected dependency of MYC expression on efficient β-catenin protein degradation in B-ALL cells, we generated a dual-reporter knock-in model expressing mTq–β-catenin and eGFP–Myc fusions. B-ALL cells generated from dual-reporter knock-in mice expressed high levels of Myc at baseline and lacked β-catenin protein expression (Fig. 3d). Treatment with the GSK3β small-molecule inhibitor LY2090314 resulted in a dramatic shift after 10 h of treatment with de novo expression of mTq:β-catenin and subsequent loss of eGFP:Myc signal (Fig. 3d and Supplementary Video 1). Reconstitution of MYC expression rescued the deleterious effects of β-catenin accumulation and restored the colony formation and competitive fitness of B-ALL cells (Fig. 3e–g). Analogous to genetic deletion of GSK3β (Gsk3b^+/fl; Fig. 2d,e and Extended Data Fig. 5a), treatment with the GSK3β inhibitor LY2090314 reduced β-catenin protein degradation, resulting in repression of MYC in murine and human B-ALL cells (Fig. 3h,i). Importantly, genetic deletion of β-catenin was sufficient to relieve MYC suppression in response to GSK3β inhibition in mouse and human B-ALL cells (Fig. 3h,i). These results corroborate that the expression of MYC in B-ALL cells critically relies on GSK3β-mediated β-catenin degradation. Beyond suppression of MYC, GSK3β-inhibitor-induced gene expression changes broadly depend on β-catenin accumulation. Principal component analyses visualized that genetic deletion of β-catenin alone reversed the effects of LY2090314, indicating that gene expression changes, most notably repression of MYC, largely reflect subversion of GSK3β-mediated β-catenin protein degradation (Fig. 3j).

**Fig. 3: Impaired β-catenin protein degradation in B cells results in transcriptional repression of MYC.**

β-catenin forms repressive complexes with Ikaros factors and NuRD components in B-ALL cells

To elucidate how β-catenin accumulation repressed MYC in B cells, we identified β-catenin-interacting proteins by coimmunoprecipitation (Co-IP) and mass spectrometry. In addition to known β-catenin interaction partners (Apc, Axin1 and Gsk3β), the proteins with the highest enrichment included B lymphoid Ikaros transcription factors (Ikzf1 and Ikzf3) (Fig. 4a). Ikaros factors function as B cell-specific repressors to maintain B cell identity and recruit repressive NuRD components for transcriptional repression^{37,38,39,40,41}. In addition to Ikzf1 and Ikzf3, we identified multiple NuRD complex components (Chd4, Mta1, Mta2, Gatad2a, Gatad2b, Rbbp4, Mbd3, Hdac1 and Hdac2) (Fig. 4a) among β-catenin-interacting proteins. Prominent interactions between β-catenin and Ikzf1, Ikzf3 and the NuRD component Chd4 were validated by co-IP and western blot (Fig. 4b). To examine lineage-specific composition of β-catenin-interacting protein complexes, we identified β-catenin-bound proteins by co-IP and mass spectrometry in human B lymphoid (B-ALL and B cell lymphoma), myeloid (AML) and epithelial (colon and lung cancer) cells. Confirming results in murine B-ALL, Ikaros factors IKZF1 and IKZF3 were among the most prominent β-catenin interaction partners in human B-ALL and B cell lymphoma (Fig. 4c and Extended Data Fig. 6a). Co-IP and western blot experiments confirmed B lymphoid-selective interactions between β-catenin and IKZF1, IKZF3, MTA1, MTA2 and GATAD2A as opposed to colon cancer cells with preferential TCF7L2–β-catenin interactions (Fig. 4d and Extended Data Fig. 6b). Principal component analysis corroborated B lymphoid-specific β-catenin interactomes that were clustered together along the first principal component axis and separated from myeloid, colon and lung epithelial cells (Extended Data Fig. 6c).

**Fig. 4: β-catenin forms repressive complexes with B lymphoid transcription factors Ikzf1 and Ikzf3.**

Repressive β-catenin–NuRD complexes depend on B lymphoid Ikaros factors

To assess whether Ikaros factors are required for the recruitment of repressive NuRD components and their interactions with β-catenin, we deleted both Ikzf1 and Ikzf3 in murine B-ALL cells and repeated the co-IP and mass spectrometry identification of β-catenin-interacting proteins. Deletion of Ikzf1/3 in B-ALL cells (Ikzf1/3 double knockout) was achieved by electroporation-based delivery of Cas9 ribonucleoproteins (RNPs) containing Cas9 and guide RNAs (gRNAs) (gIkzf1/Ikzf3) or nontargeting control (gNT). Deletion of Ikaros factors resulted in the loss of interactions between β-catenin and the NuRD complex components Mta2, Mbd2, Mbd3 and Gatad2b (Fig. 4e), suggesting a central role of Ikaros factors in assembling repressive β-catenin–NuRD complexes. To elucidate the contribution of Ikaros factors to β-catenin-mediated transcriptional repression, we compared gene expression (RNA-seq) in Ikzf1/3 wild-type (WT) and Ikzf1/3 double-knockout B-ALL cells. Deletion of both Ikaros factors largely reversed β-catenin-mediated repression of Myc and Myc targets (Fig. 4f), restored Myc expression and Myc-dependent transcriptional programs and rescued colony formation and cell survival in competitive cell culture assays (Fig. 4g–i). Deletion of IKZF1 is a frequent lesion in B-ALL⁴², whereas IKZF3 mutations are detected in mature B cell malignancies⁴³. However, these lesions are monoallelic and cases with combined defects of IKZF1 and IKZF3 have not been reported to date. For this reason, we assessed whether deletion of one single Ikaros factor could reverse β-catenin-induced deleterious effects in B-ALL cells. Unlike Ikzf1/3 double knockout, deletion of one Ikaros factor had no notable effect on β-catenin-induced repression of MYC and cell death in B-ALL cells (Fig. 4h,i). This result suggests that one single Ikaros factor is required and sufficient for β-catenin-dependent repression of MYC and induction of cell death.

Ikaros–β-catenin complexes mediate transcriptional repression at MYC superenhancer regions

To determine how Ikaros factors and β-catenin interact at the chromatin level, we performed β-catenin and Ikzf1/3 chromatin IP and sequencing (ChIP-seq) analyses to study changes of β-catenin-binding and Ikaros-binding peaks upon deletion of Ikaros and accumulation of β-catenin. Highlighting the dominant role of Ikaros factors in controlling β-catenin functions in B cells, ChIP-seq analyses showed that nearly 75% of β-catenin peaks were shared with Ikaros (Fig. 5a). In most cell types, Myc superenhancer regions are found in a proximal cluster of peaks (50 kb to 450 kb downstream of Myc) that overlaps with the Pvt1 gene^44,45. Studying H3K27ac signals across multiple MYC superenhancer clusters in B-ALL cells revealed that most of the enhancer activity was concentrated in a different region, located 1.7 Mb downstream of Myc (Fig. 5b and Extended Data Fig. 7a). This region was previously identified as the hematopoietic-specific blood enhancer cluster (BENC) region⁴⁶. Ikaros and β-catenin strongly bound to element C of the BENC region (Fig. 5c and Extended Data Fig. 7a,b). In the presence of Ikaros factors, β-catenin accumulation suppressed H3K27ac signals at BENC-C regions. However, upon Ikaros deletion, β-catenin accumulation had the opposite effect and greatly increased H3K27ac signals at BENC-C enhancer regions and other enhancers that were cobound by both β-catenin and Ikaros factors (Fig. 5b–d and Extended Data Fig. 7a). Mirroring β-catenin-mediated repression at MYC BENC enhancer regions, accumulation of β-catenin strongly increased recruitment of repressive NuRD complex components MTA2 and CHD4 (Fig. 5e). Consistent with Ikaros-dependent formation of repressive β-catenin–NuRD complexes (Fig. 4e), deletion of Ikaros factors abrogated recruitment of NuRD factors to BENC-C (Fig. 5e and Extended Data Fig. 7c) and restored H3K27ac marks for enhancer activity (Fig. 5b–d and Extended Data Fig. 7a). While Ikaros–β-catenin complexes suppressed MYC expression in B-ALL cells, these observations suggest that deletion of Ikzf1/3 releases β-catenin from transcriptional repression and restores transcriptional activation of MYC.

**Fig. 5: Identification of an Ikaros motif in the MYC BENC enhancer region required for β-catenin-mediated repression of MYC.**

Ikaros and TCF7 factors compete for binding to β-catenin

Accumulation of β-catenin did not affect Ikaros binding and ~90% of Ikaros peaks remained unchanged. In contrast, loss of Ikaros factors affected about half of all β-catenin targets and generated 1,202 new β-catenin-binding peaks, while 1,675 β-catenin peaks were lost (Extended Data Fig. 8a). When β-catenin protein degradation was subverted in the presence of Ikaros factors, new β-catenin-binding peaks were mostly devoid of H3K27ac marks for active enhancers. However, upon Ikaros deletion, β-catenin peaks were enriched for H3K27ac binding, suggesting increased enhancer activity at these sites after Ikaros deletion (Extended Data Fig. 8a). This shift suggests that Ikaros deletion released β-catenin from repressive complexes to form activating complexes with other transcription factors. For instance, Ikaros factors would not only promote repressive β-catenin–NuRD complexes but also oppose canonical interactions between β-catenin and TCF7 factors. Motif enrichment analyses revealed that Ikaros factor deletion restored targeting of β-catenin to canonical TCF7, TCL7L1 and TCF7L2 motifs at the expense of Ikaros motifs (Extended Data Fig. 8a). These results suggest that Ikaros factors sequester β-catenin away from transcriptional activation at canonical TCF7 sites and redirect β-catenin to Ikaros-binding sites for transcriptional repression in concert with NuRD factors. Consistent with this scenario, co-IP and western blot analyses (Extended Data Fig. 8b) showed that Ikaros factor deletion restored canonical interactions between β-catenin and TCF7-family transcription factors (Tcf7, Tcf7l1 and Tcf7l2) (Extended Data Fig. 8a–c).

Identification and functional validation of an Ikaros motif required for MYC repression

The MYC superenhancer BENC-C region at 8q24.21 harbors three prominent risk alleles that predispose to B-ALL (rs4617118, rs75777619 and rs28665337) (Fig. 5b, Extended Data Fig. 7a)^47,48,49. Combining Ikzf1 and Ikzf3 ChIP-seq and sequence analysis of the MYC superenhancer BENC-C region identified three Ikaros-binding motifs (m1–m3) within a window of 100 bp of notable Ikzf1/3 ChIP-seq peaks, including m1 with an exact match to the Ikaros core motif GGGAA (Fig. 5c,f). To assess the functional importance of the Ikaros m1 motif within the BENC-C region, we engineered knock-in alleles to mutate Ikaros m1 to an EcoRI site. After Homology-directed repair (HDR)-based knock-in of mutant BENC-C Ikaros motifs in β-catenin^{(S33;S45)+/fl} B-ALL cells, leukemia cells were plated for colony formation. Colonies carrying biallelic knock-in mutations of the Ikaros motif were selected on the basis of EcoRI digestion and confirmed by Sanger sequencing (Fig. 5f). Upon induction of Cre in β-catenin^{(S33;S45)+/fl} B-ALL cells, BENC-C WT knock-in B-ALL clones rapidly lost Myc expression and underwent cell death (Fig. 5g,h). In contrast, B-ALL clones carrying BENC-C knock-in alleles with mutant Ikaros motif expressed Myc at higher baseline levels and were resistant to β-catenin accumulation. Despite defective β-catenin degradation, the Myc levels, cell viability and competitive fitness of B-ALL clones carrying the mutant Ikaros m1 motif remained intact (Fig. 5g,h). These findings underscore that β-catenin-induced repression of Myc depends on its interactions with Ikaros factors, particularly one Ikaros-binding motif within the Myc BENC-C enhancer region.

B cell-selective activity of GSK3β inhibitors to subvert β-catenin protein degradation

As B lymphoid cells were uniquely dependent on high-efficiency β-catenin protein degradation, we tested the hypothesis that small-molecule inhibitors of this pathway would induce cell death in a B cell-selective manner. We analyzed public drug sensitivity data (DepMap; https://depmap.org/)³⁵ based on 4,518 compounds and 172 B lymphoid (32 B-ALL, 140 B cell lymphoma) and 1,787 nonlymphoid (colon, lung, skin, breast, kidney, liver and glioma) cell lines. Comparing B lymphoid to other cell lines, we plotted the differential area above the dose–response curve (ΔAAC) for all compounds on the basis of Wilcoxon effect size and significance (Fig. 6a). Along with known B cell-selective drugs (for example, ibrutinib and dexamethasone), the compounds with the most B cell-selective profiles included GSK3β inhibitors (Fig. 6a and Extended Data Fig. 9a). Moreover, compound set enrichment analyses confirmed strong B cell-selective activity for GSK3β inhibitors (normalized enrichment score (NES) = 1.78, q = 0.008; Fig. 6b). Sensitivity to GSK3β inhibitors was inversely correlated with β-catenin protein levels (RPPA, CCLE2019, n = 646; Fig. 6c). In agreement with this correlation, the GSK3β inhibitor LY2090314 not only exerted B cell-selective activity in DepMap datasets (https://depmap.org/)³⁵ but also rapidly suppressed N-terminal β-catenin phosphorylation and protein degradation after 1 h (Fig. 6d). A combined analysis of responses to LY2090314 based on 343 epithelial and 42 B cell tumor cell lines (Prism drug-repurposing secondary screen; https://depmap.org/)³⁵ revealed that B cell malignancies, including B-ALL (176-fold lower half-maximal effective concentration (EC₅₀) values than solid tumors), MCL (147-fold) and CLL (218-fold) were substantially more sensitive than solid tumors (Fig. 6e). Comparing the effect of LY2090314 on β-catenin and MYC protein levels by western blot in B-ALL, MCL, colon cancer and lung cancer cell lines revealed that responses to LY2090314 were predicated on the expression of B lymphoid Ikaros factors (Fig. 6f). This was consistent with computational analyses of gene expression biomarkers for responses to the GSK3β inhibitor CHIR99021 (https://depmap.org/)³⁵ CTD2 screen). This analysis identified expression of IKZF1 and IKZF3 as the top-ranking positive correlation with sensitivity to CHIR99021, whereas TCF7L1 and TCF7L2 levels were negatively correlated (Fig. 6g).

**Fig. 6: Small-molecule inhibition of β-catenin protein degradation induces B cell-selective cell death.**

Chemogenomic screens identified β-catenin degradation as a mechanistic target of GSK3β inhibitors

To comprehensively identify mechanistic targets of GSK3β inhibition in human B-ALL cells, we performed chemogenomic CRISPR screens in human B-ALL cells (NALM6) bearing an integrated inducible Cas9 expression cassette. NALM6 cells were transduced with the genome-wide knockout EKO single gRNA (sgRNA) library (278,754 sgRNAs, targeting 22,956 genes, with 12 sgRNAs per gene and negative controls)⁵⁰ and treated with LY2090314 at 3.5 nM to induce partial GSK3β inhibition for 8 days. Context-dependent chemogenomic interaction scores were calculated using a modified version of the RANKS algorithm⁵⁰, which uses sgRNAs targeting similarly essential genes as controls to distinguish condition-specific chemogenomic interactions from nonspecific fitness and essentiality phenotypes. sgRNAs targeting CTNNB1 (β-catenin) had the single most prominent rescue effect in protecting B-ALL cells against LY2090314-mediated GSK3β-inhibition (Fig. 6h). Likewise, deletion of repressive NuRD complex components, including HDAC1, HDAC2, MBD3, GATAD2A and MTA2, provided a strong selective advantage. CRISPR sgRNAs targeting individual Ikaros factors did not have notable effects, presumably because concurrent deletion of IKZF1/3 was required to rescue cell death and MYC repression (Fig. 4h,i). The chemogenomic CRISPR screen also revealed synthetic lethal interactions, including deletion of AXIN1, APC and most prominently GSK3B (Fig. 6h). To independently validate β-catenin protein degradation as central mechanistic target of GSK3β inhibitors, we compared the effect of LY2090314 in the presence and absence of β-catenin deletion. Cre-mediated deletion of β-catenin in murine β-catenin^fl/fl B-ALL cells prevented repression of MYC (Fig. 3h) and conferred near-complete resistance to LY2090314 (Fig. 6i). Likewise, CRISPR-mediated deletion of β-catenin in human B-ALL cell lines (BV173) and B-ALL PDX (PDX2) prevented suppression of MYC (Fig. 3i) and resulted in near-complete resistance to LY2090314 (Fig. 6j).

Preclinical testing of GSK3β inhibitors for refractory B lymphoid malignancies

To determine B cell selectivity of GSK3β inhibition, we compared LY2090314 sensitivity in B cell malignancies (n = 18) to myeloid and epithelial tumors (n = 12) (Fig. 7a). In contrast to myeloid, colon and lung cancer cells, LY2090314 elicited strong responses in B-ALL, CLL and MCL cells at low-nanomolar concentrations (EC₅₀ = 4 nM) (Fig. 7a). DLBCL and other B cell lymphomas showed a bimodal distribution with three LY2090314-sensitive and four resistant cell lines carrying MYC translocations (RAMOS, RAJI, Daudi and JJN3) (Fig. 7a). To further examine uneven GSK3β inhibitor responses in this group, we studied CHIR99021 across 84 cell lines among lymphoid tumors, which confirmed two clusters of cell lines that substantially differed in sensitivity to GSK3β inhibition. The difference between the two groups tracked with MYC translocations. Lymphoid cell lines with MYC rearrangement (n = 31; 8q24⁺) were significantly less sensitive to GSK3β inhibition than cell lines without MYC translocation (n = 53, P = 1.3 × 10⁻⁵; Fig. 7b). Consistent with our finding that the MYC superenhancer region (BENC-C) is a critical target of repressive β-catenin–Ikaros complexes (Fig. 5), this observation suggests that chromosomal translocations in B cell lymphomas that remove MYC from its intrinsic control elements will confer resistance to GSK3β inhibition. As IKZF1 deletions are common in human B-ALL⁴², we assessed the impact of IKZF1 deletion on sensitivity to the GSK3β inhibitor LY2090314. Studying ten PDXs, including five with IKZF1 deletion, we found no significant differences in responses to LY2090314 (Fig. 7c). In addition, whether B-ALL PDXs were derived from participants who previously responded to standard chemotherapy or were refractory or relapsed did not affect responses to LY2090314 (Fig. 7c). This result suggests that GSK3β inhibition is orthogonal to conventional mechanisms of drug resistance and may represent a vulnerability that is retained in relapsed B cell malignancies.

**Fig. 7: Rationale for repurposing of GSK3β inhibitors for refractory B lymphoid malignancies.**

Five GSK3β inhibitors have achieved favorable safety and pharmacokinetic and pharmacodynamic (PK/PD) profiles in 20 clinical trials (Extended Data Fig. 1b)^{28,29,30,31,32,33}. To assess the efficacy of GSK3β inhibitors in human B-ALL and MCL PDX models in vivo, we treated immunodeficient NSG mice bearing PDX from refractory B-ALL cells or MCL with LY2090314 as a single agent (Fig. 7d–i). Sublethally irradiated (2 Gy) NSG mice bearing refractory B-ALL and MCL PDX were injected intraperitoneally with LY2090314 or vehicle control. In our treatment experiment, LY2090314-treated mice did not show notable weight loss or any indication of organ toxicity in the colon, lung, liver and kidneys (Extended Data Fig. 9b). In addition, B-ALL cells in treated mice had no notable phenotypic changes (Extended Data Fig. 9c). Compared to vehicle-treated mice, LY2090314 reduced tumor burden and significantly extended overall survival in primary and refractory B-ALL PDX, as well as refractory MCL PDX (Fig. 7d–i).

Discussion

A central finding of our study that B-ALL cells have evolved and critically depend on high-efficiency β-catenin protein degradation is seemingly in conflict with previous work suggesting a role of β-catenin as an oncogenic driver in B cell malignancies. In a common B-ALL subset, the E2A–PBX1 fusion oncogene induced aberrant overexpression of WNT16 (ref. ²⁶); however, subsequent work showed that WNT16 did not induce canonical β-catenin signaling²⁷. Two studies examined the effects of Wnt3A and β-catenin accumulation on proliferation of B-ALL cells with conflicting results^24,25. To reconcile the differences, we treated B-ALL PDX cells with 400 ng ml⁻¹ Wnt3A ligand. Treatment with Wnt3A resulted in weak and transient β-catenin accumulation and failed to suppress MYC expression or proliferation of B-ALL cells (Extended Data Fig. 4d–f). While B-ALL cells are responsive to Wnt3A, these results suggest that high-efficiency β-catenin protein degradation in B cells limits the amount and duration of β-catenin accumulation.

Previous studies demonstrated that deletion of FZD9 results in a defect in early B cell development⁵¹, whereas deletion of FZD6 delays disease onset in a mouse model of CLL⁵². Given that Frizzled receptors are widely known as mediators of canonical Wnt signaling to stabilize β-catenin, these findings seem to contradict previous studies demonstrating that β-catenin is dispensable for normal B cell development^11,12,13,14 and our work highlighting the dependency of B cells on β-catenin protein degradation. In addition to stabilizing β-catenin, Frizzled receptors can initiate β-catenin-independent, noncanonical Wnt signaling, which could explain the different outcomes of FZD6 and FZD9 deletion^51,52 and β-catenin deletion^11,12,13,14. Deletion of FZD6 in CLL cells resulted in a 1.8-fold reduction in β-catenin protein levels (measured by flow cytometry)⁵². However, β-catenin protein levels in B cell malignancies were 32-fold lower compared to solid tumors as reported by our study (Fig. 1 and Extended Data Figs. 1 and 2), indicating that the small effect of FZD6 deletion on β-catenin protein levels may not be functionally relevant.

The biological importance of high-efficiency β-catenin protein degradation is illustrated by genetic defects of this pathway. For instance, 5q− syndrome with heterozygous deletion and haploinsufficiency of APC (5q21) and CK1α (CSNK1A; 5q32)⁵³ represents the most frequent myelodysplastic syndrome (MDS) and results in reduced efficiency of β-catenin protein degradation^54,55. While 5q− MDS exhibits hyperplasia of myeloid and megakaryocytic lineages, persons suffer from anemia and defective B lymphopoiesis^53,54,55. Our genetic experiments showed that haploinsufficiency of APC or CK1α caused accumulation of β-catenin and B cell depletion (Extended Data Fig. 5b,c).

Five GSK3β inhibitors previously achieved favorable safety and PK/PD profiles at micromolar plasma concentrations (C_max). Despite potential concerns related to accumulation of β-catenin, even after 18 months of treatment at micromolar concentrations, only mild adverse effects, including diarrhea and lymphopenia, were observed^{28,29,30,31,32,33} (Extended Data Fig. 1b). Lymphopenia as a side effect is consistent with a rationale to repurpose GSK3β inhibition for B lymphoid malignancies and suggests that GSK3β inhibitors affect both normal and malignant B cells. Systemic B cell depletion as a side effect of treatment of B cell malignancies is generally well tolerated. For instance, antibodies to CD20 (for example, rituximab) are highly effective in the treatment of B cell lymphomas and induce profound B cell depletion⁵⁶. Likewise, inhibitors of B cell signaling, including Bruton’s tyrosine kinase (for example, ibrutinib)⁵⁷ are effective components of a therapeutic regimen for B cell lymphoma, induce B cell depletion and are well tolerated. In addition, cell therapeutics based on chimeric antigen receptor T cells against CD19 have revolutionized the treatment of persons with B-ALL and long-lasting B lymphopenia in these persons is considered an acceptable side effect⁵⁸. GSK3β inhibitors have demonstrated safety in more than 20 clinical trials with low-micromolar plasma concentrations (Extended Data Fig. 1b). Importantly, our preclinical studies show that GSK3β small-molecule inhibitors are effective at low-nanomolar (~100-fold lower) concentrations in B cell malignancies. Together, these results support a rationale for repurposing GSK3β inhibitors to inhibit β-catenin protein degradation as a previously unrecognized strategy that is amenable to near-term evaluation in persons with refractory B cell malignancies.

Methods

Ethical approvals

Our research complies with all relevant ethical guidelines. Details of the committees and institutions that approved the study protocol can be found in each specific section.

Participant samples and cell lines

Participant samples were obtained in compliance with the Institutional Review Boards of University of California, San Francisco, Yale University and the Dana-Farber Cancer Institute. Written informed consent was obtained from all participants. Leukemia and lymphoma cells (Supplementary Table 1) were cultured in RPMI-1640 (Gibco) with GlutaMAX containing 20% FBS (Gibco) and 100 IU per ml penicillin–streptomycin (P/S; Gibco). Solid tumor cell lines were cultured in RPMI-1640 with GlutaMAX containing 10% FBS and 100 IU per ml P/S. Breast epithelial cell lines were cultured using the mammary epithelial cell growth medium bullet kit (Lonza). Human B-ALL (Supplementary Table 2) and MCL (Supplementary Table 3) PDXs were cultured in MEMα (Life Technologies) with GlutaMAX containing 20% FBS and 100 IU per ml P/S. CLL samples (Supplementary Table 4) were cultured in RPMI-1640 with GlutaMAX containing 20% FBS, 100 IU per ml P/S, 50 ng ml⁻¹ interleukin 2 (IL-2; Peprotech) and 1 μg ml⁻¹ CpG. All cells were cultured at 37 °C in a humidified incubator with 5% CO₂; they were authenticated and tested negative for Mycoplasma (MycoAlert PLUS, Lonza).

Animal experiments

All experiments were approved by the regional council in Freiburg or Yale University and carried out in accordance with the German Animal Welfare Act (TVA: 35-9185.81/G-15/157) and Institutional Animal Care and Use Committee (protocol no. 20345). Mice were maintained in a pathogen-free environment at 18–23 °C with 40–60% humidity and a 14-h light and 10-h dark cycle. Ctnnb1^ex3fl (referred to as the β-catenin^{(S33;S45)+/fl})² strain was provided by M. Taketo. Apc^fl/+ mice⁵⁴ were provided by Z. Qian. Vav^Cre Csnk1a ^fl/+ mice⁵³ were provided by B. Ebert and crossed with B6 mice to generate Csnk1a1^fl/+ mice. To activate or delete β-catenin in B cell precursors, Mb1^Cre mice were crossed with β-catenin^{(S33-S45)+/fl} and β-catenin^fl/fl (ref. ¹¹) mice, respectively. For dual-reporter mice, eGFP–Myc and mTq–β-catenin mice were crossed. All other mice were purchased from Jackson Laboratory. Genotyping of the mice was performed using the primers described in Supplementary Table 5. Csnk1a1 mice were genotyped by Transnetyx. Both female and male mice were used and control and experimental groups were age and sex matched.

For transplantation experiments, 8–12-week-old sublethally irradiated NSG mice (200 cGy) were used. For limiting dilution of β-catenin^{(S33;S45)+/fl} cells, pre-B cells were transduced with MSCV-BCR-ABL1-IRES-LUC and Il-7 was removed to transform the cells. Cells were subsequently transduced with tamoxifen-inducible Cre (Cre-ER^T2) and GFP or GFP (ER^T2). GFP⁺ cells were sorted by fluorescence-activated cell sorting (FACS) and treated with 4-hydroxy-tamoxifen (4-OHT; 1 μM) for 2 days; 120,000, 6,000 or 300 cells were injected through the tail vein.

For GSK3β inhibitor treatment, cells were labeled with luciferase and selected with 25 μg ml⁻¹ blasticidin; 1 million cells were injected. Then, 3 days after transplantation, LY2090314 or vehicle control (dissolved in 5% DMSO, 45% PEG300 and PBS) was administered intraperitoneally at a dose of 12.5 mg kg⁻¹ twice per day for 4 days per week (every 12 h). Mice that displayed the signs of leukemia such as hunched back, weight loss and inability to move were killed. To measure leukemia burden, 2.5 mg of D-luciferin (Promega) was injected intraperitoneally and mice were anesthetized with 5% isoflurane for 15 min. Luminescent signal was measured by Lago X (Accela) and mice continued receiving 2% isoflurane through a nose cone during imaging. For histology analysis, tissues were fixed in Safe Fix II (Fischer Scientific) and embedded in paraffin. Before fixing, the mouse intestine was flushed with PBS to remove feces. Paraffin blocks were sectioned at 4 μm and stained with hematoxylin and eosin by the Yale Pathology Tissue Services. Images were acquired using Cytation5 (Biotek).

Murine pre-B and B-ALL cells

Bone marrow cells were obtained by flushing the femur and tibia with PBS and filtering through a 70-μm mesh. Cells from spleen and lymph nodes were extracted using a 40-μm strainer. Erythrocytes were lysed for bone marrow and spleen cells using red blood cell lysis buffer (Biolegend). Bone marrow cells were cultured in IMDM (Gibco) with GlutaMAX containing 20% FBS, 50 μmol ml⁻¹ 2-mercaptoethanol, 100 IU per ml P/S and 10 ng ml⁻¹ IL-7 (Peprotech) to generate pre-B cell cultures. For the BCR–ABL1-driven leukemia model, pre-B cells were transduced by BCR–ABL1 and IL-7 was removed to enrich transformed cells. For the NRAS^G12D model, pre-B cells were transduced by NRAS^G12D and cultured in the same conditions as pre-B cells.

Retroviral and lentiviral transduction

Lenti-X HEK293T cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions and cultured in high-glucose DMEM (Gibco) with GlutaMAX containing 10% FBS, 100 IU per ml P/S (Gibco), 1 mmol l⁻¹ sodium pyruvate (Gibco) and 0.1 mmol l⁻¹ nonessential amino acids (Gibco). The next day, cells were treated with 10 mmol l⁻¹ Sodium butyrate (Sigma-Aldrich) for 6–8 h and the medium was replaced. Then, 24 h later, supernatants were collected and filtered through a 0.45-μm filter. For all transductions, non-tissue-culture plates were coated with Retronectin (50 μg ml⁻¹; Takara). For retroviral transductions, viral supernatants were loaded onto plates by centrifugation at 2,000g for 90 min at 32 °C and 2–3 million cells were centrifuged at 600g for 30 min. For lentiviral transduction, 2–4 million cells per well were centrifuged at 600g for 30 min in the presence of lentiviral supernatant and the medium was changed 16 h later.

Fluorescent protein stability assay for β-catenin

Cells were transduced with vectors expressing β-catenin fused with GFP tag and mScarlet or EV with GFP and mScarlet expression. FACS analysis was performed for detecting frequencies of cells expressing WT β-catenin–GFP fusion within mScarlet⁺ cells. GFP to mScarlet signal was confirmed to be 1:1 in cells expressing EV.

Reprogramming of B-ALL into myeloid lineage

B-ALL cells were transduced with Tet3G-neomycin vector (Takara) and subjected to neomycin (1 mg ml⁻¹) selection and subsequently transduced with Tre-3G-driven C/EBPα or EV (puromycin resistance). Reprograming was induced by doxycycline (1 μg ml⁻¹) treatment and myeloid conversion was studied by FACS by analyzing CD19 and CD11b expression.

TOP-Flash (7TFC) assay

B-ALL cells were transduced with 7TFC lentiviral vector (Addgene, 24307) and mCherry⁺ cells were sorted by FACSAria III. A total of 100,000 cells were seeded in a 96-well plate in 80% of Wnt3A-conditioned medium from the L-Wnt3A cell line (American Type Culture Collection, CRL-2647) and 20% MEMα containing 20% fetal calf serum. Luciferase activity was measured by adding 100 µl of ONE-Glo buffer from Promega (E6120) using a GloMax plate reader (Promega).

Cell viability assay

In total, 10,000–40,000 cells were seeded per well in a 96-well plate (Eppendorf) in 80 μl of medium. Inhibitors were added at the indicated concentrations to achieve a total volume of 100 μl. After 3 days, Cell Titer Glo 2.0 assays (Promega) were performed according to the manufacturer’s instructions. Relative viabilities were calculated by measuring the luminescence value and normalizing to the values of untreated cells.

Western blotting

Cells were washed twice with ice-cold PBS and lysed in CelLytic buffer (Sigma-Aldrich) supplemented with 1% protease inhibitor (Roche Diagnostics) and 1% phosphatase inhibitor (EMD Millipore) and 1 mM PMSF (Cell Signaling Technologies) on ice for 15 min. Then, 10–20 µg of cell lysates were separated on precast gels (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with 2% BSA in Tris-buffered saline with Tween-20 for 1 h at room temperature and then probed with appropriate primary antibodies at 4 °C overnight. Membranes were incubated with AP-conjugated secondary antibodies (Invitrogen) for 1 h at room temperature and analyzed with Chemi Doc MP imaging system (Bio-Rad). Fractionation experiments were performed using nuclear and cytoplasmic extraction reagent (Thermo Scientific) according to the manufacturer’s instructions.

Flow cytometry

Cells were washed twice with PBS containing 2% FBS and blocked with Fc blocker (BD Biosciences) for 20 min followed by antibody or isotype control staining for 30 min at 4 °C. Cells were then washed and resuspended with DAPI (0.75 μg ml⁻¹) to exclude dead cells and analyzed on LSRFortessa X-20 or FACSSympony A3 (BD Biosciences). Cells were sorted using FACSAria III or FACSAria Fusion (BD Biosciences). FACS data were analyzed using FlowJo software (FlowJo).

Live-cell time-lapse imaging

For live-cell imaging, cells were cultured in phenol-red-free MEMα (Gibco), 1% P/S and 10% FBS at 37 °C with 5% CO₂. Imaging plates (µ-Slide eight-well high-glass-bottom plates) were coated with poly(L-lysine) for 2 h before loading cells to minimize cell movement. Images were acquired with an Andor Dragonfly spinning disk confocal microscope using a ×40 oil objective. Each individual frame was performed every 20 min over the course of a 24-h time course. Images were processed using ImageJ.

CRISPR-mediated gene deletion

CRISPR RNAs (crRNAs; Supplementary Table 5; 100 µmol l⁻¹) and trans-activating crRNAs (tracrRNAs; 100 µmol l⁻¹) were annealed by incubation at 95 °C for 5 min. Recombinant Cas9 (40 µmol l⁻¹) was incubated with gRNA mixture for 10–20 min at room temperature and Cas9–gRNA complexes were electroporated using the Neon transfection system (Invitrogen). For Ikzf1 and/or Ikzf3 deletion in mouse B-ALL cells and CTNNB1 deletion in BV173 cells, single-cell-derived knockout cell lines were generated.

HDR-mediated gene editing

For editing the BENC region, gRNA complex was formed by annealing crRNAs (Supplementary Table 5) and tracrRNAs at 95 °C for 5 min. Recombinant Cas9 (Integrated DNA Technologies (IDT); 125 pmol) was incubated with gRNA (150 pmol) to form RNP complexes. Mouse B-ALL cells were resuspended in Lonza P4 buffer and electroporated with the RNP complex, single-stranded DNA HDR donor oligos (Supplementary Table 5; IDT) and Alt-R Cas9 electroporation enhancer (IDT) using Nucleofector (Lonza, pulse code CV104). Following electroporation, cells were treated with Alt-R HDR enhancer V2 (1 µmol⁻¹; IDT) for 24 h. To assess HDR editing, gDNA was extracted using Nucleospin tissue (Macherey-Nagel) following the manufacturer’s protocol and PCR amplification using Q5 high-fidelity master mix (New England Biolabs). PCR amplicons were digested with EcoRI-HF (New England Biolabs) in 1× CutSmart buffer (New England Biolabs) at 37 °C for 4 h and analyzed on 2% agarose gel. Single-cell-derived cell lines were generated and Sanger sequencing was performed.

For β-catenin deletion in PDX2 cells, double-stranded DNA HDR template was amplified using TrueTag knockout enrichment donor template (puromycin–RFP; Invitrogen) and primers listed in Supplementary Table 5. crRNAs and tracrRNAs were annealed at 95 °C for 5 min, followed by incubating with Cas9 (125 pmol) for 15 min at room temperature. PDX2 cells were resuspended in Lonza SF buffer and electroporated with the RNP complex, double-stranded HDR template and Alt-R Cas9 electroporation enhancer (IDT) using Nucleofector (Lonza, pulse code ED-120). β-catenin knockout cells were enriched by sorting for RFP⁺ cells and selecting for puromycin resistance (0.6 μg ml⁻¹).

Colony formation assay

A total of 10,000 B-ALL cells were plated on MethoCult medium (Stem Cell Technologies) in 3-cm-diameter dishes and colonies were counted after 10–14 days. For BCR–ABL1 transformed cells, medium without cytokines (M3231) was used; for NRAS^G12D transformed cells, medium with IL-7 (M3630) was used.

mRNA-seq and analysis

RNA was isolated using Macherey-Nagel RNA extraction kit according to the manufacturer’s instructions. Sequencing was performed on an Illumina Hiseq 2500 instrument using the TruSeq paired-end cluster kit V4-cBot-HS (Illumina) to generate 101 bp paired-end reads for sequencing with v4 chemistry. Quality control of RNA-seq reads was performed using FastQC (version 0.11.9), SAMtools (version 1.7) and Picard (version 2.23.8). Transcripts were quantified with Salmon (version 1.4.0)⁵⁹ and reads were aligned using STAR (version 2.7.6)⁶⁰ to the mouse genome (mm10/GRCm38, gencode vM24). Downstream analysis was performed in R⁶¹; differential expression was analyzed with DESeq2 (version 1.30.1)⁶² with standard models and normal shrinkage estimators. Gene set enrichment analyses (GSEAs) were performed with fgsea (version 1.16.0) using log₂ fold changes from DESeq2 and gene sets from MSigDB or internal data as indicated.

Co-IP and proteomic analysis

Co-IP experiments were performed using the Pierce crosslink magnetic IP/co-IP kit according to the manufacturer’s instructions (Thermo Scientific). Anti-β-catenin (14; BD Biosciences) or isotype (107.3; BD Biosciences) antibody were coupled to protein A/G beads and covalently crosslinked with 20 µmol l⁻¹ DSS; beads were incubated with cell lysates at 4 °C overnight. Beads were washed twice, eluted in a low-pH elution buffer and subjected to mass spectrometry (Northwestern Proteomics Core Facility). Peptides were analyzed by liquid chromatography–tandem mass spectrometry using a Dionex UltiMate 3000 rapid separation liquid chromatography system and an Orbitrap mass spectrometer (Thermo Fisher Scientific).

Downstream analysis of proteomic datasets was performed in R⁶¹; protein values were quantile-normalized and mixed imputations were used to estimate missing values. Missing values were classified as missing not at random (MNAR) if proteins were detected in fewer than two replicates from a condition and missing at random (MAR) otherwise. MNAR values were imputed by minimum probability, whereas MAR values were estimated by maximum-likelihood imputation using the MSnbase and DEP packages⁶³. Normalized, imputed values were used in linear modeling and empirical Bayes testing for differentially enriched proteins between conditions using limma and DEP packages^63,64; differential enrichment results are given in pulldown experiments. For visualization, fold change over Ig background binding was further divided by average background binding detected in CRAPome to downweight common contaminants⁶⁵.

ChIP experiments

The ChIP-Rx method was applied to mapping histone marks among various cell conditions⁶⁶. In each experiment, B-ALL cells were fixed with 1% formaldehyde for 10 min at room temperature and quenched by 125 mmol l⁻¹ glycine. For ChIP-seq of IKZF1 and IKZF3, B-ALL cells were crosslinked by 2 mmol l⁻¹ disuccinimidyl glutarate for 45 min and 1% formaldehyde for 10 min at room temperature before chromatin enrichment and library construction. Nuclei were lysed with SDS lysis buffer and sonicated using Bioruptor (Diagenode). After sonication, cell debris was removed by centrifugation (13,000g, 10 min) and supernatants were diluted and precleared by incubation with protein A/G Dynabeads (Invitrogen) conjugated to an isotype control. ChIP reactions were performed using antibodies specifically recognizing H3K27ac (39133, Active Motif), H3K4me3 (MC315, Millipore), IKZF1 (GTX129438, GeneTex) and IKZF3 (D1C1E, CST). ChIPed DNA was reverse-crosslinked overnight at 65 °C and purified by a QIAquick PCR purification kit (Qiagen). ChIP-seq libraries were constructed using a SMARTer ThruPLEX DNA-seq kit (Takara) and subjected to Illumina deep sequencing. β-catenin ChIP was performed by Active Motif using the clone CAT-15 (Thermo). Quality control was performed using FastQC (version 0.11.9) and ChIPQC. Reads were aligned with BWA (version 0.7.17)⁶⁷ against the mouse genome (mm10/GRCm38, gencode vM24). Peak calling was performed with MACS2 (version 2.2.7.1). Downstream analysis was performed in R⁶¹; differential binding was analyzed with DiffBind (version 3.0.15)⁶⁸. Peaks with −log₁₀ q value > 10 in two or more conditions were retained after blacklisting and graylisting. For transcription factors (IKZF1, IKZF3 and CTNNB1), within-peak normalization was applied, whereas whole-genome normalization was applied to histone modification data. Annotation was performed with ChIPpeakAnno⁶⁹ to the closest transcription start site, except for the BENC enhancer region, which was manually annotated as described previously⁴⁶.

For ChIP–qPCR, protein A/G beads were incubated with antibodies recognizing MTA2 (8106, Abcam), CHD4 (72418, Abcam) or isotype control (910801, Biolegend) for 4 h at 4 °C. Precleared DNA fragments were incubated with the antibody-conjugated magnetic beads for 16 h at 4 °C. Crosslinking was reversed for 6 h at 65 °C and DNA was purified with the NucleoSpin gel and PCR cleanup kit (Macherey-Nagel). qPCR was performed with PowerUp SYBR green master mix (Applied Biosystems) using a ViiA7 real-time PCR system (Applied Biosystems). Data were analyzed using QuantStudio real-time PCR software (Thermo Fisher Scientific) and amplification values from ChIPed DNA were normalized to input DNA.

Tissue microarray (TMA) analysis

Participant biopsies were obtained in compliance with the internal review board of the Beckman Research Institute and TMAs were constructed. Formalin-fixed paraffin-embedded TMAs were cut at 4 μm. TMAs were processed on a Ventana Discovery Ultra IHC automated stainer (Ventana Medical Systems). This included deparaffinization, rehydration, endogenous peroxidase activity inhibition and antigen retrieval. The TMAs were stained with anti-human β-catenin antibody (clone 14, Roche) or β-catenin pS37 antibody (ab47335, Abcam), followed by anti-mouse HQ secondary antibody (Discovery) and an anti-HQ horseradish peroxidase detection system (Discovery). The stains were visualized with a ChromoMap DAB kit (DISCOVERY), counterstained with hematoxylin (Ventana).

Compound screen sensitivity analysis

Data from GDSC1, GDSC2 and CTD2 screens were downloaded from DepMap (https://depmap.org/)³⁵ and analyzed in R using the gdscic50 package for normalization⁷⁰. Four-parameter log-logistic dose–response curves were fit for all datasets using the drc package⁷¹. The ΔAAC and statistics were calculated between B cell and solid tumor cell lines for each drug screen separately and combined into a single differential score by weighted meta-analysis using the fraction of response curves within an EC window (defined as minima and maxima fitting of P < 0.05 and response > 50%) as a weighting parameter to downweight screens using ineffective concentration ranges. Final combined effect scores were used for volcano plots and as input to feature set enrichment analysis; feature sets used were any compound groups with common targets and n > 5 compounds.

Chemogenomic CRISPR–Cas9 knockout screen

CRISPR screen was performed by ChemoGenix (Institute for Research in Immunology and Cancer, Université de Montréal) as previously described⁷². Briefly, the NALM6 clone with inducible Cas9 expression (Addgene, 50661) was transduced with the genome-wide knockout EKO sgRNA library (278,754 different sgRNAs)⁵⁰ and knockouts were induced by doxycycline (2 μg ml⁻¹) for 7 days. Cells were treated with LY2090314 (3.5 nM) or DMSO for 8 days and gDNA was extracted using the Gentra Puregene kit (Qiagen) according to the manufacturer’s instructions. sgRNA sequences were PCR-amplified as described previously⁵⁰ and analyzed by next-generation sequencing (Illumina NextSeq 500). Reads were aligned using Bowtie 2.2.5 in the forward direction only (‘--norc’ option) and total read counts per sgRNA were tabulated. Context-dependent chemogenomic interaction scores were calculated using a modified version of the RANKS algorithm⁵⁰ to distinguish chemogenomic interactions from essentiality phenotypes. Raw read counts are available upon request from ChemoGenix.

Statistics and reproducibility

Data are shown as the mean ± s.d. unless stated otherwise. Data distribution was assumed to be normal but this was not formally tested. Statistical analysis was performed by Prism 9 (GraphPad) using an unpaired two-tailed t-test or log-rank test as indicated in the figure legends. P < 0.05 was considered significant. Kaplan–Meier survival analysis was used to estimate overall survival. No statistical methods were used to predetermine sample sizes but our sample sizes are comparable to those reported in previous publications. No data were excluded from the analysis. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Experiments were repeated to ensure reproducibility. Half-maximal inhibitory concentration values were estimated using default dose–response models in drc (version 3.0.1)⁷³ with an upper limit of 1.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The mutation data for CTNNB1, APC, AXIN1, AXIN2, CSNK1a and GSK3B genes (Fig. 2a) were acquired from COSMIC³⁶. The data for IHC staining of β-catenin in normal tissues (Extended Data Fig. 2a,b) were obtained from the Human Protein Atlas³⁴ (https://www.proteinatlas.org/). RNA-seq and RPPA data (Figs. 1c and 6c) and compound screen data (Fig. 6a–c,e and Extended Data Fig. 9a) were obtained from DepMap (https://depmap.org/). Hallmark Myc-Targets V1 and Hallmark Wnt–β-catenin signaling gene sets were acquired from MSigDB (https://www.gsea-msigdb.org/gsea/msigdb). ChIP-seq and RNA-seq data that support the findings of this study were deposited to the Gene Expression Omnibus under accession codes GSE305472, GSE196767, GSE245287 and GSE196745. Mass spectrometry data were deposited to the PRIDE repository under accession codes PXD067271, PXD067314, PXD067306 and PXD067318. All other data are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We would like to thank B. Ebert (Dana-Farber Cancer Institute) for CK1α-haploinsufficient leukemia models, as well as current and former members of the M.M. laboratory for their support and helpful discussions. Research in the M.M. laboratory is supported by the National Institutes of Health through a National Cancer Institute Outstanding Investigator Award (R35CA197628, R01CA157644, R01AI164692, R01AI192914, R01CA282877 and P01CA233412, to M.M.), the Howard Hughes Medical Institute (HHMI-55108547, to M.M.), Blood Cancer United through Translational Research Program Grant 6709-26 and SCOR grant 7026-21 (to M.M.) and the V Foundation for Cancer Research through All Stars Award AST2025-015 (to M.M.). S.O. is supported by a Mildred Scheel postdoctoral fellowship from the German Cancer Aid. Research in the J.J. laboratory is supported by the German Research Foundation (project numbers 419193696, 510573839 and TRR130-TP25).

Author information

Liang Xu & Gang Xiao
Present address: College of Life Sciences and School of Medicine, Zhejiang University, Hangzhou, China
These authors contributed equally: Kadriye Nehir Cosgun, Huda Jumaa.
These authors jointly supervised this work: Julia Jellusova, Markus Müschen.

Authors and Affiliations

Center of Molecular and Cellular Oncology, Yale University, New Haven, CT, USA
Kadriye Nehir Cosgun, Mark E. Robinson, Zhangliang Cheng, Salim Oulghazi, Kohei Kume, David Fonseca Arce, Nikol Agadzhanian, Etienne Leveille, Shalin Kothari & Markus Müschen
Signalling Research Centres BIOSS and CIBSS, Albert Ludwigs University of Freiburg, Freiburg, Germany
Huda Jumaa & Julia Jellusova
Department of Immunobiology, Yale University, New Haven, CT, USA
Zhangliang Cheng & Markus Müschen
Institute of Clinical Chemistry and Pathobiochemistry, School of Medicine and Health, Technical University Munich, Munich, Germany
Klaus M. Kistner & Julia Jellusova
TranslaTUM, Center for Translational Cancer Research, Technical University of Munich, Munich, Germany
Klaus M. Kistner & Julia Jellusova
Department of Immunology, Oslo University Hospital, Oslo, Norway
Elsa Drivet & Hilde Schjerven
KG Jebsen Center for B-Cell Malignancies, Oslo University, Oslo, Norway
Elsa Drivet & Hilde Schjerven
Department of Systems Biology, City of Hope Comprehensive Cancer Center Biomedical Research Center, Monrovia, CA, USA
Fang Yu, Zhijian Qian, Liang Xu & Gang Xiao
Department of Pathology, City of Hope Comprehensive Cancer Center, Duarte, CA, USA
Joo Y. Song & Wing-Chung Chan
Kyoto University Yoshida-Honmachi Sakyo, Kyoto, Japan
M. Mark Taketo
Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA
Matthew S. Davids
Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA
Hilde Schjerven
Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA
Hilde Schjerven

Authors

Kadriye Nehir Cosgun
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Huda Jumaa
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Mark E. Robinson
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Zhangliang Cheng
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Salim Oulghazi
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Kohei Kume
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David Fonseca Arce
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Nikol Agadzhanian
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Klaus M. Kistner
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Etienne Leveille
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Elsa Drivet
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Fang Yu
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Zhijian Qian
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Joo Y. Song
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Wing-Chung Chan
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Liang Xu
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Gang Xiao
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M. Mark Taketo
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Shalin Kothari
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Matthew S. Davids
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Julia Jellusova
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Contributions

K.N.C., H.J., Z.C., S.O. and E.D. designed and performed the experiments and analyzed the data. K.K., D.F.A, N.A., K.M.K., L.X. and G.X. performed the experiments. M.E.R. performed all bioinformatics analyses. F.Y., Z.Q. and M.M.T. provided the mouse models. J.Y.S. and W.-C.C. performed the TMA analysis and interpretation of the results. E.L. and H.S. provided critical discussions and editing of the manuscript. S.K. and M.S.D. provided expertise and characterized participant samples. M.M. coordinated the study, developed the concept, and secured funding. M.M. and J.J. provided mentorship. K.N.C. and M.M. wrote the manuscript with assistance from all coauthors.

Corresponding author

Correspondence to Markus Müschen.

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Competing interests

M.M. discloses filing of Yale University patent application PCT/US2023/067398. S.K. reports consulting or advisory roles for Eli Lilly, ADC Therapeutics, AbbVie, Genentech and Incyte, and research funding from Bristol Myers Squibb. M.S.D. reports consulting or advisory roles for AbbVie, Ascentage Pharma, AstraZeneca, BeOne Medicines, Bristol Myers Squibb, Eli Lilly, Genentech, Genmab, Janssen, MEI Pharma, Merck, Nuvalent and Schrӧdinger, and research funding from Ascentage, AstraZeneca, MEI Pharma and Novartis.

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Peer review information

Nature Cancer thanks Christian Steidl and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Targeting high-efficiency β-catenin protein degradation in B-cell leukemia.

(a) In epithelial cancers (left) efficiency of β-catenin protein degradation is low and further reduced by oncogenic mutations. β-catenin accumulates and pairs with TCF factors for transcriptional activation of MYC. In B-cell leukemia (middle) β-catenin is constitutively phosphorylated by GSK3B and targeted for B-cell-specific high-efficiency protein degradation. GSK3B-inhibitors impair β-catenin protein degradation (right) and β-catenin accumulates in the nucleus. Instead of TCF, β-catenin pairs with Ikaros and NuRD factors for MYC repression (right). b) Summary of Phase I and Phase II clinical trials with GSK3β inhibitors for a variety of clinical indications. In a total of 20 clinical trials, all tested small molecule inhibitors achieved favorable safety and PK/PD profiles at micromolar plasma concentrations (Cmax). None of the inhibitors achieved clinical responses. Moderate adverse effects included diarrhea, anemia and lymphopenia.

Extended Data Fig. 2 Lack of β-catenin expression and activity in B-lymphoid cells.

(a-b) Immunohistochemical staining for β-catenin expression (brown, hematoxylin counterstain) in (a) human lymphoid tissues (left), including bone marrow (n = 7 tissues, biological replicates), spleen (n = 6 tissues, biological replicates), lymph node (n = 8 tissues, biological replicates), tonsil (n = 9 tissues, biological replicates) and (b) epithelial tissues (right), including colon (n = 7 tissues, biological replicates), liver (n = 9 tissues, biological replicates), pancreas (n = 12 tissues, biological replicates), kidney (n = 8 tissues, biological replicates), lung (n = 8 tissues, biological replicates) and skin (n = 7 tissues, biological replicates). Representative images are shown. Data derived from: https://www.proteinatlas.org/ENSG00000168036-CTNNB1/tissue#expression_summary (c-d) β-catenin expression was assessed by immunohistochemistry (brown) and using hematoxylin as counterstain (blue) on tissue microarrays from (c) B-lymphoid malignancies, including mantle cell lymphoma (n = 12 tumors, biological replicates), follicular lymphoma (n = 24 tumors, biological replicates), DLBCL (n = 24 tumors, biological replicates) and Hodgkin’s lymphoma (n = 24 tumors, biological replicates) as well as (d) epithelial cancers, including colon cancer (n = 12 tumors, biological replicates), lung cancer (n = 12 tumors, biological replicates) and malignant melanoma (n = 3 tumors, biological replicates). (e) Western blot analysis of β-catenin expression in cytoplasmic fractions of epithelial cancers, including lung and colon cancer, malignant melanoma, as well as B-lymphoid malignancies, including B-ALL, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitt’s, Hodgkin’s disease (HD) and multiple myeloma cell lines. β-tubulin and TBP were used to verify purity of cytoplasmic fractions.

Source data

Extended Data Fig. 3 β-catenin is constitutively phosphorylated and poised for degradation in B-lymphoid cells.

Non-phosphorylated β-catenin and β-catenin phosphorylated on N-terminal by GSK3B (pS37) were visualized by immunohistochemistry (brown, counterstain hematoxylin) on tissue microarrays from epithelial cancers, including colon cancer (n = 13 tumors, biological replicates), lung cancer (n = 41 tumors, biological replicates), breast cancer (n = 58 tumors, biological replicates), as well as B-lymphoid malignancies, including mantle cell lymphoma (n = 21 tumors, biological replicates), follicular lymphoma (n = 54 tumors, biological replicates), DLBCL (n = 47 tumors, biological replicates) and Hodgkin’s lymphoma (n = 20 tumors, biological replicates). Representative images are shown.

Extended Data Fig. 4 GSK3B-inhibition impairs β-catenin protein degradation.

(a) Human B-ALL (PDX2) and colon cancer (LOVO) cells were treated with LY2090314 for 0-8 hours and Western blot was performed to measure total β-catenin, as well as active, non-phosphorylated β-catenin using β-actin as loading control (n = 3 independent experiments). (b-c) FACS plots are shown for dual reporter cells that were treated with the GSK3B-inhibitor LY2090314 (10 nM; red) or vehicle control (green) for 16 hours. Inhibition of β-catenin protein degradation by LY2090314 was compared in (b) human colon cancer (SW480, HT-29) cell lines and in (c) human B-ALL (NALM6, KOPN8) cell lines by measuring the changes in GFP- compared to mScarlet-signal (2 independent repeats). (d) β-catenin and Myc levels in B-ALL cells (PDX2, MXP2) following 0-6 h WNT3A (400 ng/ml) treatment in comparison to levels achieved following GSK3β inhibition (LY2090314, 20 nM) and colon cancer (SW620) (n = 2 independent experiments). (e) B-ALL xenografts (MXP2, PDX2) were stimulated with WNT3A conditioned media for indicated time points and activation of Wnt/β-catenin pathway was assessed by measurement of luminescence signal by TOP-Flash reporter. Data is representative of 2 independent experiments and shown as mean±s.d (n = 3 technical replicates). (f) Effect of WNT3a (grey circle) stimulation on growth of B-ALL cells (PDX2) that are wild type (CTNNB1 + /+, green line) and knock-out (CTNNB1-/-, red line) for β-catenin compared to vehicle control (white circle). Data shows mean values from 2 independent experiments each with n = 3 technical replicates.

Source data

Extended Data Fig. 5 Genetic attenuation of β-catenin protein degradation induces acute cell death in pre-B cells in vitro and in vivo.

(a) Pre-B cells from Gsk3b^fl/+ mice were transduced with 4-OHT-inducible Cre-ER^T2 or ER^T2 along with GFP. Western blot analysis was performed to visualize Gsk3b deletion and β-catenin accumulation in GFP⁺ cells 2 days after 4-OHT mediated deletion of one allele of Gsk3b. Effects of Gsk3b haploinsufficiency and β-catenin accumulation were assessed in competitive growth assays monitoring changes in percentages of GFP⁺ cells for 0-8 days after 4-OHT treatment. Mean values from 2 independent experiments each with n = 2 technical replicates are shown. (b) Apc^fl/+ pre-B cells were transduced with GFP-tagged Cre-ER^T2 or ER^T2 constructs, and Western blot analysis was performed in GFP⁺ cells to confirm Apc levels and accumulation of β-catenin 2 days after 4-OHT treatment. Following loss of one allele of APC, competitive fitness of pre-B cells was assayed by measuring changes in percentages of GFP⁺ cells. Data presents mean values from 2 independent experiments with each n = 3 technical replicates. (c) Ck1a^fl/+ pre-B cells were transduced with Cre-ER^T2 or ER^T2 and GFP. Western blot analysis was performed for Ck1α, β-catenin and β-actin 2 days after 4-OHT mediated CK1α deletion. Growth kinetics of cells with monoallelic deletion of Ck1α (Cre-ER^T2) was compared to cells with wild type Ck1α (ER^T2) by flow cytometry of changes in GFP⁺ cells 0-8 days post 4-OHT treatment. Data shows mean values from 2 independent experiments each with n = 2 technical replicates. (d) Pre-B cells from β-catenin^{(S33-S45)+/fl} mice expressing Cre-ER^T2 or ER^T2 along with GFP were treated with 4-OHT to induce Cre-mediated excision of GSK3B-phosphorylation sites. Accumulation of β-catenin was confirmed by Western blot. Removal of exon-3 results in a shorter form of β-catenin. Competitive fitness of pre-B cells was assessed by measuring the frequencies of GFP⁺ cells by FACS. Data are presented as the mean±s.d of 3 independent experiments each with n = 3 technical replicates. (e-h) β-catenin^{(S33-S45)+/fl} mice were crossed with Mb1^Cre/+ mice for B-cell-specific excision of GSK3B-phosphorylation sites and B-cell development in the bone marrow and peripheral lymphoid organs was studied by FACS. (e) Absolute numbers (f) percentages of B-cell precursor subsets in the bone marrow of the mice are shown for Mb1^Cre/+ β-catenin^{(S33-S45)+/fl} (red, n = 6 mice, biological replicates) and Mb1^Cre/+ β-catenin^(S33-S45)+/+ mice (green, n = 6 mice, biological replicates). Bone marrow B-cell precursors were distinguished as pro-B cells (CD43⁺ B220^low IgM⁻ BP1⁻), pre-BI cells (CD43⁺ B220^low IgM⁻ BP1⁺), pre-BII cells (CD43⁻ B220^low, IgM⁻), immature B cells (CD43⁻ B220^low IgM⁺) and mature B cells (CD43⁻ B220^high IgM⁺). (g) Representative FACS plots and absolute numbers and frequencies of B220⁺ B-cells in spleens of Mb1^Cre/+ β-catenin^{(S33-S45)+/fl} (red, n = 5 mice, biological replicates) and Mb1^Cre/+ β-catenin^(S33-S45)+/+ (green, n = 5 mice, biological replicates) mice are shown. (h) Representative FACS plots, absolute numbers, and fractions [%] of B-cells in the peripheral lymph nodes of Mb1^Cre/+ β-catenin^{(S33-S45)+/fl} (red, n = 4 mice, biological replicates) and Mb1^Cre/+ β-catenin^(S33-S45)+/+ (green, n = 4 mice, biological replicates) mice are shown. Throughout (e-h) two tailed unpaired t-tests were used to calculate P values and data are presented as mean±s.d.

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Extended Data Fig. 6 β-catenin forms repressive complexes with Ikaros and NuRD factors in B-cells.

(a-c) Human B-ALL (MXP2), B-cell lymphoma (JEKO), AML (MOLM13), colon cancer (SW480) and lung cancer (H446) cell lines were engineered with β-catenin mutations impairing GSK3B and CK1a-mediated phosphorylation. (a) Proteins bound to β-catenin were identified by mass-spectrometry following Co-IP with antibodies against β-catenin or control Ig. β-catenin binding proteins in each cell type were plotted as a function of background binding (x-axis, non-specific binding defined by CRAPOME database) and log₂-fold enrichment over control Ig (y-axis). (b) Western blot was performed to validate β-catenin-binding of Ikaros factors IKZF1, IKZF2, IKZF3 and NuRD complex component MTA2, as well as AXIN1 (positive control) using input lysates and elutes from the Co-IP experiments with antibodies against β-catenin or control Ig (n = 2 biological replicates). (c) Principal component analysis corroborated B-lymphoid-specific (B-ALL, B-cell lymphoma) β-catenin-interactomes that were clustered together along PC1 axis and separated from myeloid, colon and lung epithelial cells.

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Extended Data Fig. 7 β-catenin and Ikaros factors mediate recruitment of NuRD complex at BENC enhancer.

(a) ChIP-seq analysis for β-catenin, Ikaros factors Ikzf1 and Ikzf3, histone marks H3K27ac and H3K4me3 shown for the Myc locus, including upstream Myc promoter regions and long-range transcriptional enhancers of Myc in B-ALL cells from β-catenin^{(S33-S45)+/fl} mice. Peak density plots show colocalization of β-catenin, Ikzf1 and Ikzf3 peaks and their concentration at BENC enhancer regions. Comparison of changes in H3K27ac active enhancer and H3K4me3 active promoter marks following accumulation of β-catenin (red boxes) or β-catenin baseline (light green boxes), upon Ikaros factor deletion (empty boxes) or Ikaros baseline (dark green boxes) conditions. Ikaros factors and β-catenin show marked enrichment at BENC regions. While accumulation of β-catenin depleted H3K27ac marks at BENC enhancer regions, β-catenin had the opposite effect and increased BENC enhancer activity and H3K27ac signals when Ikaros factors (Ikzf1 and Ikzf3) were deleted. (b) β-catenin binding (mean value of two technical replicates) to BENC enhancer region and other β-catenin targets was measured by ChIP-seq in β-catenin^{(S33-S45)+/fl} B-ALL cells (Ikzf1/3 WT) 1 day after β-catenin stabilization. β-catenin is strongly enriched at BENC enhancer (input n = 11863, Other n = 11857, BENC n = 6 peaks; two-sided unpaired t-test P = 0.0005). Boxplots display boxes at first and third quartile with line at median and whiskers from minimum to maximum datapoints within 1.5x interquartile range. (c) ChIP-qPCR was performed for NuRD complex components (MTA2 and CHD4) at the epithelial Myc enhancer regions as well as lymphoid BENC Myc enhancer region (BENC-D). For each region, MTA2 and CHD4 ChIP were performed for B-ALL cells with induced β-catenin accumulation (red boxes), β-catenin deletion (empty boxes) or intact β-catenin (light green boxes), as well as deletion of Ikaros factors (empty boxes) or intact Ikaros factors (dark green boxes). Data shown represents mean values from 3 independent experiments (n = 3 technical replicates). Two-sided unpaired t-test was used to calculate P values.

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Extended Data Fig. 8 Ikaros factors compete with TCF family transcription factors for binding to β-catenin.

a) Changes of β-catenin peaks as well as H3K4me3 and H3K27ac histone marks were assessed in the presence (green boxes) and absence of Ikaros factors (empty boxes) and inducible accumulation of β-catenin (red boxes). Heatmaps to illustrate genome-wide distribution of β-catenin peaks, H3K27ac and H3K4me3 histone marks. Ikzf1 and Ikzf3 deletion enabled binding of β-catenin to inactive enhancers and their subsequent activation (gained H3K27ac). Upon loss of Ikzf1 and Ikzf3 regions that have decreased (n = 1,020) or lost (n = 1,675) β-catenin binding are strongly enriched for Ikaros motifs (P = 6.5E-65). Deletion of Ikaros factors resulted in redistribution of β-catenin to canonical Tcf7 (P = 1.0E-42), Tcf7l2 (P = 1.0E-17) and Tcf7l1 (P = 1.0E-16) motifs and depletion from Ikaros motif (P = 6.5E-65), Fisher’s Exact test. b) Changes in β-catenin interactomes in mouse B-ALL cells upon Ikaros factor deletion (gIkzf1/3) were analyzed by co-IP and Western blot analysis in whole cell lysates (input), proteins bound (elute) and flow through (FT) after Co-IP with antibodies against β-catenin or control antibodies. Co-IP was performed under conditions of β-catenin accumulation (red box) and in the presence of Ikaros factor deletion (empty box) or Ikaros baseline levels (dark green box). Deletion of Ikaros factors enabled binding of β-catenin to Tcf7 and Tcf7l2 (n = 2 biological replicates). (c) Scenario of transcription factor complexes with β-catenin in B-lymphoid vs other cell types: TCF factors pair with β-catenin for transcriptional activation of Myc at Wnt responsive elements (WRE) and epithelial enhancer regions (left). In B-cells, Ikaros factors outcompete TCF to bind to β-catenin. Ikaros factors and β-catenin recruit of repressive NuRD complexes to B-lymphoid BENC enhancer regions of Myc, resulting in transcriptional repression of Myc (middle). Loss of Ikaros factors enables interactions between β-catenin and TCF factors to restore transcriptional activation of Myc (right).

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Extended Data Fig. 9 Repurposing clinically approved inhibitors targeting β-catenin degradation.

(a) Drug responses (AUC) to the GSK3β-inhibitor (CHIR99021) were plotted for lymphoid (n = 98 cell lines, biological replicates) and solid (n = 298 cell lines, biological replicates) tumors (CTD2 screen). Data are represented as mean±s.d and two-sided unpaired t-test was used to calculate significance. (b) To assess whether LY2090314 induced any changes in epithelial tissues, histopathological evaluation was performed in colon, lung, liver and kidneys of the mice that received vehicle control or LY2090314 (n = 4 biological replicates/group) and representative images of H&E staining are shown. (c) To characterize phenotypic changes induced by LY2090314 treatment, B-ALL cells harvested from LY2090314 (red, n = 9 mice, biological replicates), or vehicle (green, n = 10 mice, biological replicates) treated mice were analyzed by FACS for surface expression of CD20, CD19, CD10, CD34, IL7R, TSLPR, VPREB1 and CD117. Fold-changes in mean fluorescence intensities (MFI [FC], y-axis) following LY2090314 treatment compared vehicle control are shown. No major differences were observed (two-sided unpaired t-test). Data are presented as mean±s.d.

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Supplementary information

Supplementary Information

Gating strategy for FACS analysis.

Reporting Summary

Supplementary Video 1

Time-lapse imaging of β-catenin Myc dual reporter B-ALL cells following LY2090314 treatment

Supplementary Table 1–5

Supplementary Tables 1–5.

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Cosgun, K.N., Jumaa, H., Robinson, M.E. et al. Targeting β-catenin degradation with GSK3β inhibitors induces cell death in acute lymphoblastic leukemia. Nat Cancer (2026). https://doi.org/10.1038/s43018-025-01093-z

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Received: 22 February 2025
Accepted: 10 November 2025
Published: 08 January 2026
Version of record: 08 January 2026
DOI: https://doi.org/10.1038/s43018-025-01093-z

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Abstract

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Main

Results

Lack of β-catenin protein expression in B cell malignancies

β-catenin is poised for proteasomal degradation in B-ALL cells

Lack of genetic lesions in the β-catenin protein degradation pathway in B cell malignancies

B-ALL cells critically depend on GSK3β, CK1α and APC for efficient β-catenin protein degradation

B-ALL leukemia initiation depends on high efficiency of β-catenin protein degradation

Dependency on β-catenin protein degradation is predicated on B cell identity

β-catenin protein degradation is required to maintain MYC expression in B lymphoid cells

β-catenin forms repressive complexes with Ikaros factors and NuRD components in B-ALL cells

Repressive β-catenin–NuRD complexes depend on B lymphoid Ikaros factors

Ikaros–β-catenin complexes mediate transcriptional repression at MYC superenhancer regions

Ikaros and TCF7 factors compete for binding to β-catenin

Identification and functional validation of an Ikaros motif required for MYC repression

B cell-selective activity of GSK3β inhibitors to subvert β-catenin protein degradation

Chemogenomic screens identified β-catenin degradation as a mechanistic target of GSK3β inhibitors

Preclinical testing of GSK3β inhibitors for refractory B lymphoid malignancies

Discussion

Methods

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Animal experiments

Murine pre-B and B-ALL cells

Retroviral and lentiviral transduction

Fluorescent protein stability assay for β-catenin

Reprogramming of B-ALL into myeloid lineage

TOP-Flash (7TFC) assay

Cell viability assay

Western blotting

Flow cytometry

Live-cell time-lapse imaging

CRISPR-mediated gene deletion

HDR-mediated gene editing

Colony formation assay

mRNA-seq and analysis

Co-IP and proteomic analysis

ChIP experiments

Tissue microarray (TMA) analysis

Compound screen sensitivity analysis

Chemogenomic CRISPR–Cas9 knockout screen

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Reporting summary

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