Deletion of Bak and Bax in Tie2+ BM Hematopoietic Stem Cells Induces a B Cell Lymphoproliferative Disorder.
Authors: Doan Phuong L. , Russell J. Lauren , Meadows Sarah K. , Himburg Heather A. , Daher Pamela , Chao Nelson J. , Kirsch David G. , Chute John P. 
Published: October 2019

Abstract
Abstract 1247
Poster Board I-269
Chronic lymphocytic leukemia (CLL) is characterized by a monoclonal, mature B-cell lymphocytosis, lymphadenopathy and splenomegaly. The pathogenesis of CLL is thought to be related to abnormal programmed cell death mechanisms but the precise contribution of several pro-apoptotic genes remains unknown. Elucidation of the pathogenesis of CLL is also limited by the lack of animal models which reproduce the CLL phenotype. We present a transgenic mouse model in which conditional, tissue-specific deletion of Bax is achieved in Tie2+ hematopoietic stem cells (HSCs) via Cre-LoxP recombination. In this model, Tie2Cre;Bak-/-;BaxFl/- mice carried constitutive deletion of Bak and targeted deletion of Bax in Tie2+ BM HSCs. Surprisingly, the combined deletion of Bak and Bax in Tie2+ cells caused a pronounced leukocytosis with total WBCs>100,000 in 8-week old mice. In contrast, Tie2Cre;Bak-/-;BaxFl/+ mice, which retained one Bax allele, had normal peripheral blood WBCs comparable to wild type C57Bl6 mice (range 4-10,000;p<0.01 and p<0.01). Tie2Cre;Bak-/-;BaxFl/- mice had massive splenomegaly with a 6-fold greater spleen weight compared to Tie2Cre;Bak-/-; BaxFl/+ controls. Review of PB smears demonstrated a preponderance of “smudge” cells (70% of total cells per HPF) in the Tie2Cre;Bak-/-;BaxFl/- mice, whereas smudge cells were not observed in Tie2Cre;Bak-/-;BaxFl/+ mice. Flow cytometric analysis of PB cell differential revealed a significant increase in B cells (B220+, p=0.0001), a decrease in T cells (Thy 1.2+, p=0.0002), decreased myeloid cells (Mac1/Gr1+ cells, p=0.02), and decreased erythroid cells (Ter119+, p=0.01) compared to Tie2Cre;Bak-/-;BaxFl/- mice. The B cell-to-T cell ratio was also aberrantly increased (p=0.0007) suggesting a predominant B-cell lymphocytosis. Interestingly, while Tie2Cre;Bak-/-;BaxFl/- mice demonstrated no increase in committed BM colony forming cells (CFCs) or colony forming unit-spleen day 12 (CFU-S12), these mice contained significantly increased numbers of BM long term culture-initiating cells (LTC-ICs, p=0.04), suggesting that the combined deletion of Bak and Bax in Tie2+ cells yielded an expansion of primitive BM HSCs. Lastly, in order to confirm whether the development of the lymphoproliferative disorder was autonomous to deletion of Bak and Bax in Tie2+ HSCs, we transplanted 4 × 106 BM cells from Tie2Cre;Bak-/-;BaxFl/- mice into lethally irradiated (950 cGy) wild type B6.SJL mice. At 12 weeks post-transplant, the recipient mice were chimeric with Bak and Bax deletion in BM hematopoietic cells and Bak and Bax retained in the BM microenvironment. Remarkably, the chimeric recipient mice demonstrated a significant reduction in PB WBCs (range 23-60,000, p=0.001) and a 2.4-fold decrease in spleen size compared to Tie2Cre;Bak-/-;BaxFl/- mice (p=0.02), suggesting that the development of the lymphoproliferative disorder was modulated by Tie2+ BM endothelial cells. Taken together, these data demonstrate that the targeted deletion of both Bak and Bax, but not Bak deletion alone, in Tie2+ HSCs produces a profound lymphoproliferative disorder in mice. While it has been previously shown that constitutive deletion of Bak and Bax caused a lymphoproliferative disorder in mice (Lindsten T et al. Mol Cell 2000;6:1389), the data presented here suggest that deletion of Bak and Bax in BM HSCs may be sufficient to induce a lymphoproliferative disorder and this phenotype may be significantly modulated by the BM microenvironment. We are currently testing whether mice bearing deletion of Bak and Bax in Tie2+ cells have a monoclonal B cell leukemia and are utilizing Cre-LoxP recombination methods to determine at which point in HSC differentiation that the Bak and Bax deletions are required in order for the lymphoproliferative disorder to occur. Given the profound phenotype of the Tie2Cre;Bak-/-;BaxFl/- mice, this may be a useful animal model for the study of the pathogenesis of lymphoproliferative disorders.

Disclosures
No relevant conflicts of interest to declare.