Immunoglobulin Light Chains Control Permissiveness to Malignant B-Cell Transformation By RAS-Pathway Lesions

Background: RAS-pathway activating mutations (e.g. NRAS, KRAS, BRAF) cause constitutive ERK-activation which can lead to divergent outcomes. ERK-activation not only drives early stages of pre-B cell differentiation (Yasuda et al., 2008) but also induces negative selection and cell death to eliminate aberrantly activated B-cells at later stages of development (Limnander et al., 2011). Germline mutations in this pathway cause lymphoproliferative disorder and autoimmunity but not leukemia. Furthermore, activating 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, but they are rarely found in B-cell receptor (BCR)-expressing mature B-cell derived tumors ( Figure 1). These findings are consistent with previous observations that transgenic mouse models for RAS-oncogene were able to transform BCR-negative B-cell precursors to develop B-cell acute lymphoblastic leukemia (Chan et al., 2020). In contrast, transgenic expression of these oncogenes fails to transform BCR-positive mature B-cells (Mullins et al., 2013; Chung et al., 2014). Signals from the BCR, consisting of immunoglobulin (Ig) heavy chains (μ HC) and conventional light chains (kappa LC or lambda LC), and its precursor the pre-BCR, consisting of μ HC and surrogate light chains (SLC; λ5 and VpreB), regulate the development and function of B-cells. Here, we investigated whether structural elements of the (pre-) BCR determine the outcome of oncogenic RAS activation and how oncogenic RAS in B-ALL avoids cell death to promote leukemogenesis. Results: We first studied the impact of oncogenic RAS-activation in BCR-positive mantle cell lymphoma cells. Expression of NRAS G12D in BCR-positive mantle cell lymphoma (JeKo-1) cells transiently reduced the surface expression of μ HC and eventually resulted in depletion of cells from culture in growth competition assays. We then examined whether CRISPR/Cas9-based genetic ablation of BCR components will enable permissiveness to oncogenic RAS signaling. Despite loss of μ HC function, oncogenic RAS caused depletion of JeKo-1 cells from culture. In contrast, this effect was partially reversed by genetic ablation of the constant region of immunoglobulin kappa LC ( IGKC). We next examined whether reconstitution of surrogate light chain (SLC) components of the pre-BCR will confer permissiveness to oncogenic RAS signaling in JeKo-1 cells. We engineered JeKo-1 cells to express