RNA interference screening identifies lenalidomide sensitizers in multiple myeloma, including RSK2

Abstract

• High-throughput RNAi screening identified lenalidomide sensitizer genes, including RSK2, RAB, peroxisome, and potassium channel family members.• Knockdown or inhibition of RSK2 synergized with lenalidomide to induce myeloma cytotoxicity and downregulation of interferon regulatory factor 4 and MYC.To identify molecular targets that modify sensitivity to lenalidomide, we measured proliferation in multiple myeloma (MM) cells transfected with 27 968 small interfering RNAs in the presence of increasing concentrations of drug and identified 63 genes that enhance activity of lenalidomide upon silencing. Ribosomal protein S6 kinase (RPS6KA3 or RSK2) was the most potent sensitizer. Other notable gene targets included 5 RAB family members, 3 potassium channel proteins, and 2 peroxisome family members. Single genes of interest included I-k-B kinase-a (CHUK), and a phosphorylation dependent transcription factor (CREB1), which associate with RSK2 to regulate several signaling pathways. RSK2 knockdown induced cytotoxicity across a panel of MM cell lines and consistently increased sensitivity to lenalidomide. Accordingly, 3 small molecular inhibitors of RSK2 demonstrated synergy with lenalidomide cytotoxicity in MM cells even in the presence of stromal contact. Both RSK2 knockdown and small molecule inhibition downregulate interferon regulatory factor 4 and MYC, and provides an explanation for the synergy between lenalidomide and RSK2 inhibition. Interestingly, RSK2 inhibition also sensitized MM cells to bortezomib, melphalan, and dexamethasone, but did not downregulate Ikaros or influence lenalidomide-mediated downregulation of tumor necrosis factor-a or increase lenalidomide-induced IL-2 upregulation. In summary, inhibition of RSK2 may prove a broadly useful adjunct to MM therapy. (Blood. 2015;125(3): [483][484][485][486][487][488][489][490][491] IntroductionThe immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide are broadly used in the treatment of patients with multiple myeloma (MM) and have produced significant clinical advances in both newly diagnosed and advanced disease.1,2 However, only 30% of relapsed MM patients respond to single agent IMiD therapy and most patients eventually develop drug resistance. The underlying mechanisms defining this nonresponsiveness are largely unknown. Recently, Cereblon (CRBN) and Ikaros/Aiolos (IZKF1 and IZKF3) were identified as the molecular targets of IMiDs in MM 3-6 via their ability to induce cytotoxicity by inhibiting interferon regulatory factor 4 (IRF4) and MYC. The action of IMiDs appears to be mediated via binding to CRBN with activation of its associated E3 ubiquitin ligase and subsequent proteasomal degradation of Ikaros. 5,6 Furthermore, low CRBN and IZKF1 levels are associated with IMiD resistance, 3,4,7,8 but other parallel pathways or downstream events that enhance or preclude drug responsiveness are unknown.In the present study, a druggable genome short interfering RNA (siRNA) screen was used to identify 63 genes whose loss of expression enhance...

Bortezomib induces methylation changes in neuroblastoma cells that appear to play a significant role in resistance development to this compound

“…CREB1 TFs was shown to associate with RSK2 to regulate several signalling pathways and inhibition of RSK2 sensitized MM cells to BTZ. This is an example of the opposite effect to that observed in our cell proliferation experiment, nevertheless, we can speculate that methylation changes at this TF binding sites may be involved in this phenomenon 52 . In the performed literature searches, we did not identify studies describing a direct link between BTZ activity and NF1-halfsite (CTF), TEAD3 (TEA), Egr2 (Zf), Smad4 (MAD) and PROP1 transcription factors identified in these analyses.…”

Caveolin‑1 facilitated KCNA5 expression, promoting breast cancer viability

“…In HEK293 cells, IGF-1 induces the expression of several K v channels in response to mitogenic signals ( 10 ). The mitogenic stimulation of G 0 phase activates K + channels, drives the cells into G 1 phase, and then initiates proliferation ( 3 , 11 ). K v channels play a major role in advancing the cell cycle when activated by mitogenic factors ( 12 , 13 ).…”

Epigenetic repression of miR‐375 is the dominant mechanism for constitutive activation of the PDPK1/RPS6KA3 signalling axis in multiple myeloma

“…We have recently identified 3‐phosphoinositide‐dependent protein kinase 1 (PDPK1), a serine threonine kinase, and its major downstream substrate RPS6KA3 (also termed RSK2), a member of the 90 kDa ribosomal S6 kinase family of serine threonine kinases, as universally active molecules in MM, regardless of the type of cytogenetic abnormality and the mutation status of RAS , RAF and fibroblast growth factor receptor 3 ( FGFR3 ) genes. Importantly, PDPK1/RPS6KA3 signalling was found to play pivotal roles in myeloma pathophysiology by regulating a series of myeloma‐promoting molecules, such as MYC, IRF4, D‐type cyclins, and PLK1, while inactivation of PDPK1 or the N‐terminal domain of RPS6KA3 resulted in induction of apoptosis in myeloma cells, accompanied by activation of BH3‐only proteins BCL2L11 (BIM) and BAD (Shimura et al , ; Chinen et al , ), while RPS6KA3 activation has been shown to be one of the leading causes of resistance to immunomodulatory agents, such as lenalidomide (LEN) (Zhu et al , ). Autophosphorylation is the dominant mechanism for PDPK1 activation, and therefore, the kinetics of PDPK1 are largely determined by its protein expression level.…”