The Universal Antitumor Activity of TAS0612, a Triple Inhibitor for RSK, AKT, and S6K, in B-Cell Lymphomas Regardless of Disease Subtype

Abstract

B-cell lymphomas (BCLs) are the most common disease entity among hematological malignancies and have various genetically and molecularly distinct subtypes. Although the prognosis of most BCL subtypes has been greatly improved by the advancements of disease subtype-oriented molecular-targeted therapeutics, there remain patients who are incurable with currently available therapeutics. Therefore, further development of new therapies that are effective regardless of disease subtypes is still awaited. 3-phosphoinositide dependent protein kinase 1 (PDPK1) is the master serine-threonine kinase of >20 AGC kinases, including RSK family proteins, AKT, S6K, and several non-AGC kinases. By regulating a series of downstream molecules and mediating the RAS/ERK/RSK2 and PI3K/AKT pathways, PDPK1 plays a pivotal role in disease development and progression in various malignancies. We have also shown that PDPK1/RSK2 axis plays pivotal roles in the pathophysiology of multiple myeloma and mantle cell lymphoma (MCL) (Chinen Y, Cancer Res 2014; Shimura Y, Mol Cancer Ther 2012; Matsumura-Kimoto Y, Cancer Med 2020). In the search for novel molecular targeted therapy for BCLs, these provoked us to investigate the role of PDPK1 and its effector molecules as the therapeutic targets in a broad range of BCLs. We first revealed that the blockade of phosphoinositide-dependent kinase-1 (PDPK1) using its specific inhibitor BX-912 induces a growth inhibition via G2/M cell cycle arrest and the induction of apoptosis in all 8 BCL-derived cell lines examined, including two from activated B-cell-like diffuse large B cell lymphoma (DLBCL), two from double expressor DLBCL, two from Burkitt lymphoma, and two from follicular lymphoma (FL). By using specific inhibitors for the downstream kinases of PDPK1, we also demonstrated that RSK2, AKT, and S6K, but not other PDPK1 substrates, such as PLK1, SGK, or PKC, plays critical roles for cell survival and proliferation as target molecules of PDPK1 in these cell lines examined. In addition, RSK2 plays a central role and AKT and S6K play subsidiary functional roles as the downstream effectors of PDPK1. Following these results, we next investigated the anti-BCL efficacy of TAS0612 which is the triple inhibitor for total RSK, including RSK2, AKT, and S6K, by inhibiting the binding of its target RSK, AKT, and S6K with their substrate molecules (Shibata T, Mol Cancer Ther 2020). As the results, TAS0612 showed the antiproliferative effect in all 8 BCL-derived cell lines examined with IC80s ranging from 1.18 to 8.46 µM, leading to G1 or G2/M cell cycle blockade, and induced apoptosis. At the protein level, TAS0612 caused the accumulation of its direct substrates p-total RSK Ser380, p-AKT Ser473, and p-S6K Thr389 in most cell lines examined as the results of the inhibition of binding to their respective substrates, and the subsequent inactivation through dephosphorylation of their respective target substrates in all cell lines examined. The 72-hour treatment with TAS0612 at 2.5 and 5.0 µM also showed the ex vivo antiproliferative effect in 25 patients-derived lymphoma cells of various disease subtypes, including nine DLBCLs, seven FLs, three MCLs, one mucosa-associated lymphoid tissue lymphoma, one splenic marginal zone B cell lymphoma, one chronic lymphocytic leukemia, and three B-lymphoblastic lymphomas. We finally examined the global molecular effects of TAS0612 using gene expression profiling. Among several tumor suppressor genes upregulated by TAS0612, we found the increased expression of tumor protein 53 inducible nuclear protein 1 (TP53INP1) in cell lines regardless of disease subtypes. Moreover, gene set enrichment analysis identified that TAS0612 significantly downregulated the gene sets driven by the upregulation of MYC and mTORC1, reactive oxygen species, and the unfolded protein response. Thus, this study revealed that the concomitant blockade of RSK2, AKT, and S6K, which are the pivotal downstream substrates of PDPK1, is a novel universally active therapeutic target for the various disease subtypes of BCLs, and line up TAS0612 as an attractive candidate agent for BCLs for future clinical development.