Role of Fc gamma RIIb signaling in CML stem cell biology and therapy resistance

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We and others have previously demonstrated that CML stem cells survive despite complete inhibition of the oncogenic driver Bcr-Abl (Schemionek et al., BLOOD 2010; Hamilton*, Helgason*, Schemionek* et al., BLOOD 2012). Therefore we are now aiming to identify new and potentially druggable targets that induce leukemic stem cell (LSC) persistence. We previously performed expression profiling of CML stem cells and identified significant upregulation of the ITIM (immunoreceptor tyrosine-based inhibition motif) receptor FcgRIIB (Fc gamma receptor 2b). This upregulation was not abrogated by state of the art therapy with tyrosine kinase inhibitors (TKIs) suggesting that the receptor may contribute to Bcr-Abl independent resistance. ITIM receptors have recently been described to support leukemogenesis also in Bcr-Abl positive ALL as well as AML suggesting a key role in malignant cell biology. Our preliminary results show that FcgRIIB inactivation affects leukemogenesis of Bcr-Abl transduced lineage negative bone marrow cells in vitro as well as in vivo. Our first aim is to determine the impact of FcgRIIB depletion on CML stem cell function. To achieve this we want to infect primary FcgRIIB-/- or wt HSC using Bcr-Abl retrovirus and analyze the effect on CML potency, self-renewal, growth, homing, ROS (reactive oxygen species) levels, apoptosis and survival. Our second aim is to validate the potential of FcgRIIB to act as a therapeutic target. Therefore we want to generate a mouse model that we can use to distinguish CML and normal stem cells that do express or do not-express the receptor within the same animal. We will then treat these mice using first and second generation TKIs, approved for first-line therapy in CML and monitor LSC persistence in vivo. Finally, we want to analyze the mechanism of FcgRIIB mediated oncogenic signaling using previously established leukemic cell lines overexpressing the receptor as well as knock-out mouse models and primary human CML stem cells. Here we want to study the mechanism of Bcr-Abl induced FcgRIIB activation and subsequently study the downstream signaling cascades. We then want test for effects of therapeutic FcgRIIB inhibition by peptide-binding. These data will show if FcgRIIB activation allows for LSC persistence besides current therapy and can be specifically targeted in vivo to eradicate the disease driving cell population.