This proposal aims to understand mechanisms underlying B cell activation and dysregulation. It will explore the role of Rab7, a small GTPase that localizes on endosomes, in mediating NF-?B activation in normal B cells upon receptor engagement and in malignant B cells. It will also analyze the effect of Rab7 inhibition, through genetic ablation or small molecule inhibitors, in the prevention of B lymphomagenesis. Knowledge gained from the proposed study will be highly relevant to the development of vaccines to pathogens as well as therapeutics for B cell lymphoma, which is diagnosed annually in 70,000 new patients with poor ten-year survival rate under current therapies. As we have recently shown, Rab7 mediates induction of activation-induced cytidine deaminase (AID) in normal mouse B cells by engagement of CD40 or Toll-like receptors (TLRs) for immunoglobulin class switching and effective antibody responses. AID expression requires NF-?B. Reflecting a central role of NF-?B in B cell activation, differentiation and functions, subversion of the (canonical and non-canonical) NF-activation pathways leads to neoplastic transformation of cells primarily of a B cell origin. NF-?B subunits, however, are themselves poor drug targets due to their largely interchangeable roles in regulating gene transcription, prompting us to search for factors responsible for NF-?B activation as potential new targets. One candidate is Rab7, as our preliminary data suggest that Rab7-deficient B cells are defective in canonical NF-?B activation. As also suggested by our new data, Rab7 is upregulated in activated mouse B cells in vivo and in vitro and in B lymphoma cell lines. Importantly, we found that a Rab7-targeting small molecule CID 1067700 could arrest the growth of these tumor cells in vitro. Here we hypothesize that Rab7, upon B cell stimulation, is upregulated and promotes NF-?B activation, thereby mediating B cell activation and, when dysregulated, lymphomagenesis. To test this hypothesis, we will analyze Rab7 expression and NF-?B activation in normal human primary B cells stimulated with CD40 and in human primary B cell lymphomas (Aim 1.1). We will also address the impact of CRISPR-based Rab7 knockout (KO) on NF-?B activity and NF-?B-dependent gene expression in human B lymphoma cell lines (Aim 1.2). Further, we will address the role of Rab7 in exaggerated B cell activation and antibody responses as well as AID-dependent B cell lymphomagenesis using a murine model with Rab7 KO specifically in B cells (Aim 2.1). Finally, we will address the potential of Rab7 as a therapeutic target by analyzing the impact of Rab7- specific inhibitor CID 1067700 on the tumor engraftment in C57BL/6 mice by monoclonal lymphoma cells isolated from E-Myc transgenic mice (Aim 2.2). The proposed study is immediately feasible, has high clinical relevance, and will likely spur further studies fo deep mechanistic understanding of signaling transduction pathways central to B cell functions.
This proposal explore core mechanisms underlying underlying B cell lymphomagenesis, a cancer condition diagnosed annually in 70,000 new patients, less than 60% of whom would survive for ten or more years under current therapies. Our cross-disciplinary research will have sustained impact on the fields of immune-regulation and signaling, as it will analyze the role of the Rab7 protein in NF-?B hyper-activation, thereby driving B cell lymphomagenesis by using genetically modified mouse strains, small molecule pharmacological compounds and cutting-edge molecular biology, cell biology and imaging approaches. Our proposal is also highly translational, as it will address the potential of Rab7 as a novel therapeutic target by analyzing the effect of Rab7 inhibition in the prevention of B lymphomagenesis and killing of pre-formed tumors.
| Lam, Tonika; Kulp, Dennis V; Wang, Rui et al. (2016) Small Molecule Inhibition of Rab7 Impairs B Cell Class Switching and Plasma Cell Survival To Dampen the Autoantibody Response in Murine Lupus. J Immunol 197:3792-3805 |
