Structural Elements of the BCR Determine Permissiveness to Oncogenic RAS-Driven B-Cell Transformation

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Background: RAS-pathway activating mutations (e.g. NRAS, KRAS, BRAF) invariably promote constitutive ERK-activation and are oncogenic drivers in B-cell acute lymphoblastic leukemia (B-ALL; Irving et al., 2014). Conversely, persistent ERK-activation can induce plasma cell differentiation (Yasuda et al., 2011), negative B-cell selection and cell death (Limnander et al., 2011) during B-cell development. In RAS-associated autoimmune leukoproliferative disorder (RALD), RAS-activating germline mutations cause lymphoproliferative disorder but not leukemia. The molecular determinants driving these divergent outcomes of RAS-activation have yet to be characterized. For these reasons, we here examined the mechanisms that enable permissiveness to RAS-driven transformation of B-cells. Results: Studying RAS-pathway activating mutations, we found that these genetic lesions are very common in B-cell malignancies derived from the earliest (e.g. B-ALL) and the latest (e.g. multiple myeloma) stages of B-cell development. In contrast, RAS-pathway lesions are exceedingly rare in B-cell receptor (BCR)-expressing B-cell malignancies, including mantle cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, Burkitt's lymphoma and diffuse large B-cell lymphoma (Figure 1). We also studied the consequence of oncogenic RAS-activation in mature B-cells, which express BCR. Consistent with previous findings that ERK-activation results in negative B-cell selection and cell death of B-cells (Limnander al., 2011), we found that expression of NRASG12D in mature B-cells resulted in upregulation of Prdm1, a tumor suppressor and mediator of negative B-cell selection and cell death (Hug et al., 2014; Setz et al., 2018). Altogether, our results suggest that mature B-cells are not permissive to RAS-driven transformation and prompted us to investigate whether expression of BCR counteracts constitutive ERK-activation resulting from oncogenic RAS signaling in B-cells. Reflecting incompatibility between oncogenic RAS-ERK activation and BCR-expressing malignant B-cells, expression of NRASG12D in BCR-positive mantle cell lymphoma cells transiently decreased surface expression of BCR but eventually led to depletion of cells from culture in growth competition assays. The non-receptor protein tyrosine kinase SYK is a key mediator of BCR signaling. Notably, treatment with small molecule inhibitor of SYK (entospletinib) largely reversed cell death caused by oncogenic RAS-signaling. Taken together, our results suggest that expression of an active BCR prevents permissiveness to constitutive ERK-activation. BCR consists of µ heavy chain (µHC) and conventional light chains (LCs; either kappa LC or lambda LC). Given that expression of a functional BCR opposes constitutive ERK-activation, this raised the question of whether structural elements of BCR are involved in determining outcome of RAS-activation. To this end, we performed CRISPR/Cas9-based gene editing to disrupt the constant region of immunoglobulin heavy chain (IGHM) or the constant region of the immunoglobulin kappa LC (IGKC) in kappa LC-expressing mantle cell lymphoma cells. While CRISPR-mediated deletion of IGHM reduced surface expression of IgM (μHC), this did not rescue cells from call death caused by oncogenic RAS-ERK activation. On the contrary, genetic ablation of IGKC enabled permissiveness to oncogenic RAS-signaling and prevented cell death resulting from constitutive ERK-activation. Hence, these findings showed that expression of kappa LC opposes constitutive RAS-ERK signaling in B-cell lymphoma cells. Conclusions: Surface expression of BCR is conserved in most mature B-cell lymphomas. Recent studies suggest that constitutive RAS-ERK signaling can override BCR requirement in B-cell lymphoma and that B-cell lymphoma cells acquiring RAS mutations may lose selective pressure to express a functional BCR (Varano et al., 2017). Here, our results demonstrated that conventional LCs of the BCR determine permissiveness to RAS-driven malignant transformation of B-cells. Conventional LCs were previously shown to inhibit the autonomous signaling capacity of BCR (Meixlsperger et al., 2007). We here showed that conventional LCs of BCR counteract oncogenic RAS signaling, providing a mechanistic basis of the low frequency of RAS-pathway activating mutations observed in BCR-expressing B-cell malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal