p90RSK2 as a Therapeutic Target in Treatment of FGFR3-Expressing t(4;14) Multiple Myeloma.

p90 Ribosomal S6 Kinase 2 (p90RSK2) is a substrate of ERK and plays an active role in anti-apoptosis signaling and cell cycle regulation. Our proteomics studies revealed that p90RSK2 is not only activated but also tyrosine-phosphorylated in hematopoietic cells expressing constitutively activated leukemogenic FGFR3 mutants. FGFR3 is a receptor-tyrosine kinase that responds to fibroblast growth factor (FGF). Recent studies have suggested that FGFR3 may play a significant role in the pathogenesis and disease progression of some hematopoietic malignancies including t(4;14)(p16.3;q32.3) multiple myeloma, which is associated with dysregulation of FGFR3. We found that the MEK1 inhibitor U0126 effectively inhibited RSK2 activation induced by FGFR3, suggesting that FGFR3 activates RSK2 through the MEK/ERK pathway. Interestingly, FGFR3-dependent tyrosine phosphorylation at Y529 was required for phosphorylation and activation of the Ser/Thr kinase RSK2 by ERK. By using a specific phospho-RSK2 (Y529) antibody, we identified FGFR3 as the direct tyrosine kinase that phosphorylates RSK2 at Y529 in an in vitro kinase assay, which only contains purified recombinant active FGFR3 and RSK2 proteins. Substitution of Y529 in RSK2 attenuates inactive ERK binding to RSK2, which is prior to and required for ERK-dependent phosphorylation and activation of RSK2. In addition, reconstitution of RSK2 phosphorylation at Y529 consequently enhanced inactive ERK binding by a GST pull down, suggesting that phosphorylation at Y529 by FGFR3 facilitates ERK binding to RSK2, which is a sequential event and precedes inactive ERK binding. These observations support a novel two-step model that FGFR3 activates RSK2 by both assisting inactive ERK binding via tyrosine phosphorylation of RSK2 at Y529 and activating the MEK/ERK pathway. Moreover, we observed that RSK2 is specifically phosphorylated at Y529 and activated in FGFR3 expressing various human t(4;14) positive MM cell lines. Targeted down-regulation of RSK2 by siRNA induced significant apoptosis in FGFR3-expressing human myeloma cell lines including OPM1 and KMS11. In addition, a specific RSK inhibitor fmk induced significant apoptosis in t(4;14) positive human myeloma cells including KMS11, OPM1, LP1 and KMS18, but not in control t(4;14) negative myeloma cell lines including ANBL6 and U266. Similar results were obtained by using another RSK specific and potent inhibitor BI-D1870. Furthermore, we observed that fmk induced significant apoptosis in primary CD138-positive, FGFR3-expressing myeloma cells from a t(4;14) positive multiple myeloma patient, but not in the control CD138-negative cells from the same patient, nor primary samples from a t(4;14)-negative patient as a control. Together, these data not only support our model that RSK2 is a critical signaling effector of FGFR3 in hematopoietic transformation, but also provide “proof of principle” for the therapeutic potential of RSK2 inhibitors such as fmk in treatment of t(4;14) positive, FGFR3-expressing multiple myeloma with minimal non-specific cytotoxicity.