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Integrated study of the role of B-cell receptor signaling in the development of Follicular Lymphoma

Periodic Reporting for period 2 - LYMPHOSIGN (Integrated study of the role of B-cell receptor signaling in the development of Follicular Lymphoma)

Reporting period: 2017-03-01 to 2018-02-28

Signaling through the B cell receptor (BCR) is central to the development of normal B-lymphocytes. In light of the numerous proliferative and survival pathways activated downstream of the BCR, it comes as no surprise that malignant B cells would co-opt this receptor to promote their own growth and survival. Direct evidence for a role of aberrant BCR signaling in an aggressive lymphoma subtype has only come to light recently showing a constitutive BCR activity that keeps cells alive. B cell receptor (BCR) signaling has emerged as a therapeutic target in B cell lymphomas and are now in clinical trials, but the precise deployment of inhibitors to target oncogenic BCR signaling requires detailed knowledge of the signaling cascades that the BCR triggers in individual tumors. In this project, we aim to better understand the role and function of BCR signaling as a pathogenic mechanism in Germinal center B-cell lymphomas, notably Follicular lymphoma (FL). FL is a relatively indolent disease with actual first-line treatment often providing extended survival over 15 years. Yet, ~20% progress within 2 years of diagnosis or eventually transform into an aggressive and fatal disease with a poor prognosis. Treatment of follicular lymphoma that has transformed into an aggressive lymphoma (tFL) remains an unmet medical need since therapies that are successful in treating other aggressive lymphomas such as diffuse large B cell lymphoma (DLBCL) are often ineffective in tFL. We do not currently have clear strategies for therapeutic development in tFL since we do not understand the molecular mechanisms that sustain the abnormal proliferation and survival of these malignancies. To identify new therapeutic opportunities in tFL, I have devised genetic screens using tFL cell line models derived from patients with this disease to identify genes that control essential cellular processes in these cells. In addition, I have used genetic screens to suggest ways to improve the efficacy of drugs that have shown activity or are approved in other lymphomas but do not work in tFL adequately.
During the last 3 years, I have implemented and used an innovative technology called CRISPR/Cas9. This new technology exploits a bacterial system called CRISPR to inactivate genes in human cells revealing unsuspected genes and pathways required for the survival of tumor cells that we were not able to study before the CRISPR/Cas9 area. During the first report period, I have set-up and adapt tools to carry-out genome-wide CRISPR/Cas9 screens on t-FL cell lines. This includes briefly the engineering of a collection of single-cell GCB/t-FL- clonal cell lines carrying an inducible Cas9 expression, the functional testing of the efficiency of Cas9 editing in these cells and the completion of the genome-wide screens per se. By comparing CRISPR from multiple lymphoma types studied in parallel in the lab, we discovered genes that are essential only to the t-FL subtype. In particular, our initial CRISPR screen survey revealed that the BCR is, in fact, essential in several tFL and GC-derived B lymphoma lines, but in a fundamentally different fashion than in agressive Diffuse large B cell lymphoma of the ABC type. Indeed, tFL and GBC DLBCL subtypes are more akin to a tonic form of BCR signaling, engaging the BCR itself, its co-receptor CD19 and SYK and LYN kinase to trigger downstream activation of the PI3K/AKT survival pathway, without engaging BTK/NF-kB. Based on these compelling data, we propose that differences in oncogenic BCR signaling in the tFL vs. aggressive DLBCL subtype setting will likely necessitate a different suite of therapeutic approaches that we are currently investigating. Selected elements of this work have been recently included in a paper published in Nature on which I'm a co-author (Phelan et al. 2018).
Using an unbiased approach, we uncovered fundamentally different BCR signaling modalities in t-FL cell lines as compared to aggressive diffuse B-cell lymphomas. From a clinical point of view, this raises the important possibility that distinct kinase inhibitors will be required to interrupt BCR signaling in those different molecular subtypes of B-cell lymphomas. This work is a first step that should help to elucidate and predict the response of these tumor subtypes to targeted therapies and thus improve the clinical behavior of t-FL patients with a poor prognosis. We expect that in a near future the outcomes from these studies will end-up in drug repurposing opportunities to biomarker development to the discovery of new targets for therapeutic development.
Schematics of an essential genome-wide CRISPR screen in lymphoma cell line