Interrogation Of Signaling Networks In B Cell Malignancies To Improve Classification Of Patient Subsets For Targeted Therapy

Targeted therapies have demonstrated promising advances in treatment of B cell malignancies. The premise of this project is that patients with closely related signaling “fingerprints” will have similar biological and clinical features. The objectives were to identify signaling profiles that are associated with clinical features and classify patients based on a signaling signature. 19 DLBCL and CLL cell lines with different molecular features were assessed in this study to establish relevant signaling features. We examined chronic BCR signaling, which leads to the activation of several downstream signaling pathways such as MAPK, AKT and NFκB pathways, in DLBCL and CLL cell lines. Phosphoflow cytometry was used to profile cell lines; a panel of 2 stimulation conditions was combined with 3 phospho-protein readouts. Basal level signal activities in cells were compared to enhance activation with PMA/IONO and F(ab’)₂ IgG/IgM. We observed that AKT signaling in cell lines contrasted significantly with signaling in Normal B cells (CD20⁺ PBMCs). Constitutive activation of the AKT signaling pathways at baseline was observed in DLBCL cell lines; very high basal phosphorylated Ribosomal Protein S6 was observed in ABC (activated B-cell) phenotype DLBCL cell lines. In addition, upon ex vivo stimulation 30% of the cell lines demonstrated high level of pathway activity when compared to normal B cells. In this study we have identified 2 distinct signaling profiles based on BCR signaling in DLBCL cell lines; 7 of 12 DLBCL cell lines demonstrated impaired BCR signaling and 4 of 12 cell lines were hyper responsive to BCR stimulation. In the case of NFκB signaling, ex vivo stimulation revealed an absence of functional NFκB activity in the several cell lines. However, the lack of NFκB pathway activity observed in these cell lines was restored with the use of a phosphatase inhibitor, indicating that impaired signaling is not through loss of kinase function but is due to an increase in negative regulation. We also explored inhibition of pathway activities in response to small molecule inhibitors targeting BCR signaling. The inconsistency in MAPK, AKT, and NFκB pathway activities observed could aid in defining a signaling profile applicable to patient stratification. Overall, our results suggest that profiling of signaling network activities represents a promising approach to molecularly classifying DLBCL and CLL subtypes.