Periodic Reporting for period 1 - BCRlossToBWinner (Genetic determinants promoting resistance to BCR-loss in B-cell lymphomas)
Reporting period: 2020-12-01 to 2022-11-30
BCRlossToBWinner aims at disclosing novel molecular, genetic and biomechanical mechanisms promoting the survival of B cells in supra-physiologic conditions (i.e. loss of BCR expression), leading to abnormal B cell expansion and B cell transformation. Specifically, we studied the contribution of FOXO1 mutations to promote cellular fitness in BCR-deficient Myc-transformed lymphoma B cells (Aim #1), and we screened for molecular determinants supporting the growth of BCR-negative lymphoma B cells ( Aim #2). Using genetic, molecular and cellular biology approaches to establish mouse-derived MYC-driven lymphoma cellular models carrying FOXO1 mutations frequently observed in human B-cell malignancies, we unveiled an unrecognized function for mutant FOXO1 proteins in constitutively activating signaling pathways controlled by the B cell receptor (BCR) to sustain lymphoma growth and respond to nutrient starvation conditions, thus providing fitness advantage to MYC-driven lymphoma B cells. Indeed, experimental observations (cellular behavior, signaling features, cellular fitness in response to BCR-deletion) confirmed the relevance of the generated cellular models as an invaluable tool to effectively address a number of yet unsolved questions, in part represented by unanticipated hypothesis inspired by this project, opening novel perspectives in the field of B-cell lymphomagenesis. Moreover, through a large-scale pharmacological screening we identified compounds interfering with the growth of MYC-driven B cell lymphomas, disclosing potentially unexplored synthetically lethal interactions between the loss of BCR-expression and specific cellular pathways. During this action, we finally identified a crosstalk between BCR signaling and the regulation of immune-modulatory molecules, thus intertwining the expression of a functional Ig-receptor with effective immune-evasion, possibly expanding the therapeutic landscape for the treatment of aggressive lymphomas according to their BCR-status and (FOXO1) mutational profile. Accordingly, experimental data and observations derived from the Action provide a solid conceptual infrastructure supporting the development of novel hypothesis proposing the involvement of FOXO1 mutations to predispose normal B cells to malignant transformation, as well as their cooperation with defined oncogenic events shaping evolutionary trajectories underlying the transformation of B cell lymphomas into aggressive and still uncurable B cell malignancies (i.e. high-grade B cell lymphomas).
Experimental activities carried out over the course of the action produced a significant amount of data coherently pointing towards the ability of FOXO1 mutations to rewire signaling activities in MYC-driven B cell lymphomas, to ultimately favor the acquisition of improved fitness, particularly in BCR-less cells, as demonstrated combining biochemical and cellular assays. Cellular mechanisms underlying the competitive advantage promoted by FOXO1 mutations in BCR-dependent lymphomas have been in part disclosed and reconducted to a reduced sensitivity to stress-induced apoptosis. It is also relevant to highlight that this effect was not strictly associated with BCR-deprived lymphomas, as it was also observed independently of BCR-expression, potentially allowing to extend these findings to a broader spectrum of B-cell malignancies (and not only those whose pathogenesis relates to the biology of the BCR). Results generated during the action are currently being further confirmed in a) at least 3 independent mouse lymphoma lines, retrovirally engineered to express FOXO1 mutations; b) CRISPR-Cas9 genetically engineered mouse MYC-driven lymphoma lines carrying FOXO1 Gain-of-Function (GOF) or Loss-of-function (LOF) mutations, and c) in human primary tonsil-derived germinal center B cells, immortalized upon enforced expression of specific oncogenic combinations (i.e. MYC/BCL2/BCL6). These cellular models have been generated within the timeframe of the action, and will represent an invaluable platform to address biologically relevant questions in the field of B-cell lymphomagenesis. Data generated during the action using these cellular models suggested a dual contribution of the BCR in the pathogenesis of B cell lymphoma, highlighting a cell autonomous regulation of lymphoma growth ensuring cellular fitness, while favoring tumor expansion in vivo hampering immune-recognition of lymphoma B cells, through the regulation of immune-related receptors, providing a roadmap to disclose mechanistic insights explaining differential in vivo dynamics of expansion and growth rates between BCR+ and BCR-deficient lymphomas. These observations become particularly relevant given the increasing genetic/genomic data on primary B-cell malignancies confirming the presence of FOXO1 mutations in a variety of B-cell lymphomas, characterized by different cell-of-origin, BCR-status and/or genetic/molecular features. As part of our dissemination plan, a manuscript describing these results is currently in preparation. Moreover, findings with some commercial potential will be intellectually protected and economically exploited thanks to the support of the Technology Transfer Office at IFOM-ETS. Finally, results stemming from this project have already been inspirational for the generation of novel hypothesis-driven research activities, which have been exploited for the preparation of competitive grant applications, one of which (American Society of Hematology, Global Research Award) has been already funded. Dissemination and outreach activities towards both scientific (through the attendance to national and international workshops, meetings and conferences to share our most recent advancements) and non-scientific communities (public outreach in schools, social media, in-person interactive events involving local communities with diverse social and educational backgrounds) have been extensively carried out throughout the action.
Based on extensive publicly available literature and preliminary data, we believe that interference with BCR expression/signaling delivers functional and mechanical cues that are interpreted by lymphoma cells as “death signals”, particularly in highly competitive and physically-contained microenvironments, such as the germinal center. We propose that FOXO1 mutant lymphomas resist better to stress-induced apoptotic signals by tuning signaling activities and cellular machinery to efficiently adapt to both cell-intrinsic (i.e. metabolic response to nutrient starvation) and cell-extrinsic components, with the latter contributed by solicitations imposed on the malignant B cells by the tumor microenvironment and immune recognition, eventually favoring the selection of mechanisms to escape from immune surveillance. The identification of vulnerabilities in aggressive B cell lymphomas and of synthetically lethal interactions between BCR-deficiency and specific cellular pathways, paves the way for the development of innovative tailored-designed therapeutic strategies, endowed with high efficacy and specificity, avoiding/reducing the off-target effects derived from intensive chemotherapeutic regimes, and potentially overcoming the acquisition of resistance to therapies, facilitating clinical management of lymphoma patients, and overall improving their quality of life.