Genetic depletion of FcγRIIb affects CML stem cell biology

• Die genetische Depletion des Fc gamma Rezeptors 2b beeinflusst die CML Stammzellbiologie

Parting, Oliver; Huber, Michael (Thesis advisor); Koschmieder, Steffen (Thesis advisor); Panstruga, Ralph (Thesis advisor)

Aachen (2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2019

Abstract

Chronic myeloid leukemia (CML) is provoked by the chromosomal translocation t(9;22) that gives rise to the oncogenic tyrosine kinase BCR-ABL1. Despite the clinical success of tyrosine kinase inhibitor (TKI) therapy, the majority of patients require lifelong therapy due to persisting leukemic stem cells (LSCs) that lack oncogene addiction. By using a transgenic CML mouse model, the immunoreceptor tyrosine-based inhibition (ITIM) carrying Fc gamma receptor 2b(Fcgr2b) was identified to be up regulated within the therapy resistant disease initiating stem cell population.Fcgr2b up regulation was confirmed in primary leukemic murine cells, leukemic cell lines as well as primary patient derived CML stem cells. By using murine leukemic Fcgr2b knout-out cells, reduced clonogenic potential, metabolic activity, cell proliferation accompanied by a reduction in S and G2/M phase along with increased expression of negative cell cycle regulators was observed. Moreover, transplantation of BCR-ABL1 retroviral transduced Fcgr2b-/- BM cells impaired the leukemic phenotype in recipient mice. Similar results were obtained using shRNA targeting Fcgr2b in transgenic SCLtTA/BCR-ABL1 cells. The shRNA induced knock-down of Fcgr2b affected leukemia development in vitro and in vivo. Analysis of downstream signaling pathways revealed decreased activation of ERK1/2, p38 and BTK upon receptor knock-out in leukemic cells. To target malignant FCGR2B signaling the SH2 domain of SH-2containing inositol 5' polyphosphatase 1 (SHIP1), which preferably binds to the ITIM motif, was applied. Impaired cell proliferation and clonogenic potential were observed in leukemic cells. Moreover, affected downstream bruton´s tyrosine kinase (BTK) was targeted by the food and drug administration (FDA) approved drug Ibrutinib. Surprisingly, Ibrutinib in combination with TKI Imatinib statistical significantly reduced proliferation and clonogenic potential of CML cells. Furthermore, combination of both drugs increased apoptosis in quiescent patient derived CD34+ CML stem cells and statistical significantly reduced the proliferation of these cells. Taken together, the results demonstrate that FCGR2B is critical in malignant CML cells making it an attractive novel therapeutic target contributing to the eradication of CML LSCs.