Repressive β-Catenin-Ikaros Complexes Are Essential to Prevent Clonal Evolution of Human Lymphopoiesis

Introduction β-catenin signaling represents a central driver of proliferation and survival in multiple epithelial, neuronal, and mesenchymal lineages, but is dispensable for hematopoietic development. Illegitimate activation of Wnt/β-catenin signaling induce development arrest at progenitor stages and underlies development of myeloid dysplastic syndrome (MDS). Moreover, β-catenin is required for emergence and self-renewal of leukemia stem cells (LSC), which are responsible for initiation and relapse of acute myeloid leukemia (AML).

Experimental approach and results Our global interactome and gene editing experiments revealed that β-catenin engages lymphoid-specific Ikaros zinc finger (IKZF) factors to repress MYC and restrict lymphoid cell proliferation. Therefore, we reasoned that genetic deletion of β-catenin in human hematopoietic stem/progenitor cells (HSPC) could selectively benefit lymphoid development, which could be useful under lymphopenic conditions, immunosenescence and myeloid skewing in elderly. To this end, we introduced Crispr/Cas9 mediated deletion of β-catenin in CD34⁺ HSPCs from human cord blood and injected them into immune-deficient recipients for assessing multi-lineage reconstitution of human hematopoiesis. Compared to the controls, mice that received β-catenin-deleted progenitor cells showed increased human chimerism in the peripheral blood at 10 and 15 weeks after transplantation. Comparing absolute cell numbers, β-catenin-deficient progenitors gave rise to markedly increased human B-cell and T-cell production in the bone marrow and spleen, while myeloid populations were largely unchanged. Mice receiving β-catenin deficient cells contained significantly more human thymocytes compared to the control mice. These findings suggest that genetic deletion of β-catenin does not adversely impact human hematopoiesis and instead relieves lymphoid-intrinsic repression of B- and T-lymphopoiesis by β-catenin:IKZF complexes. Therefore, we hypothesized that deletion of β-catenin in pre-leukemic cells from MDS patients would favor generation of normal lymphocytes and prevent transformation into AML. To test this hypothesis, we deleted β-catenin in CD34⁺ HSPCs derived from bone marrow of MDS patients and injected them into immune-deficient recipients. 16 weeks after transplantation, we detected human B cells only in the blood of the recipients that received β-catenin deficient HSPCs but the mice that were injected with control cells only had myeloid cells in their blood. Interestingly, β-catenin deletion increased lymphoid cell generation by 5-and 2-fold in the bone marrow and spleen, respectively. Pseudo-bulk analysis of single-cell RNA sequencing of MDS-derived bone marrow cells revealed that upon β-catenin deletion, genes that define leukemic stem cell signature in AML (LSC17) such as CD34, SOCS2, EMP1 etc. were repressed. Strikingly, β-catenin deleted bone marrow progenitors from MDS patients could migrate to the thymus and give rise to de-novo thymopoeisis, while none of the mice that received control cells could generate human thymocytes. Although β-catenin deletion improved human lymphopoiesis from pre-leukemic HSPCs, we observed evolution into T-ALL and CD8⁺ TCR γδ T-cell lymphoma in two cases studied. In the first case human thymocytes generated from HSPCs with 5q- deletion, were arrested at CD4CD8 double positive stage (CD7⁺ CD3^-), resembling a subgroup of T-cell acute lymphoblastic leukemia (T-ALL) with 5q- deletions. In the second case, deleting β-catenin in HSPCs carrying EZH2^G660V mutation led to clonal expansion of CD8⁺ γδ-T cells in spleen of the mice.

Conclusion In summary, deletion of β-catenin in human HSPCs improved lymphoid output in humanized mice. However, in the absence of β-catenin-IKZF mediated tumor-suppression, HSPCs carrying 5q- deletion and EZH2^G660V mutation evolved into T-ALL and γδ-T cell lymphoma, respectively. These results suggest, although targeting Wnt/β-catenin pathway may be a promising strategy to treat MDS and AML and improve lymphopoiesis, interference with β-catenin-IKZF mediated restraint of lymphoid expansion may unleash pathological lymphocytes and lead to development of lymphoid malignancies.

Halene:Forma Therapeutics: Consultancy.