Triple targeting of RSK, AKT, and S6K as pivotal downstream effectors of PDPK1 by TAS0612 in B-cell lymphomas

2.1 Immunohistochemical (IHC) staining of phosphorylated (p-) PDPK1^Ser241, p-RSK2^Ser227, and p-AKT^Thr473 in biopsied specimens from patients with BCLs

According to the Declaration of Helsinki, biopsied tumor specimens and medical records of patients with DLBCL (n = 5) and FL (n = 5) were obtained with informed consent. Formalin-fixed and paraffin-embedded tissue samples were subjected to IHC staining with anti-p-PDPK1^Ser241 MoAb, anti-p-RSK2^Ser227 MoAb, and p-AKT^Ser473 MoAb (Cell Signaling Technology). These analyses were approved by our institution's Institutional Review Board (IRB) (RBMR-G-124-13).

2.2 Cells and reagents

Eight human BCL-derived cell lines, including two BL-derived cell lines (Daudi and Raji), two activated B-cell-like DLBCL-derived cell lines (HBL1 and DLBCL2), two FL-derived cell lines (FL518 and FL18), and two double-expressor DLBCL-derived cell lines (KPUM-UH1 and KPUM-MS3) were used.¹⁵ Cells were maintained in RPMI-1640 (Wako) containing 10% fetal calf serum (Life Technologies), 2-mM L-glutamate, and penicillin/streptomycin at 37°C in humidified 95% air and 5% CO₂. The IRB approved the study using patient samples (RBMR-G-124-13) with written informed consent per the Declaration of Helsinki. Briefly, the biopsied tumor cells or bone marrow mononuclear cells were labeled with anti-CD19 MicroBeads and positively isolated using the Mini-MACS separator (Miltenyi Biotec) or EasySep Human CD19 Positive Selection Kit II (VERITAS Corporation). After confirming that more than 95% of the isolated cells were lymphoma cells by flow cytometric analysis, the isolated CD19-positive primary lymphoma cells were resuspended in conventional culture media supplemented with the CellXVivo Human B Cell Expansion Kit (R&D SYSTEMS). An RSK NTKD-specific inhibitor BI-D1870 (Cayman Chemical Company),¹⁶ an AKT inhibitor ipatasertib, a p70S6K (S6K) inhibitor PF-4708671 (Selleck Biotech Ltd), and a PDPK1 inhibitor BX-912 (Med Chem Express) were used. TAS0612 was provided by Taiho Pharmaceutical Co., Ltd.¹⁷

2.3 Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted using an RNeasy Mini Kit (Qiagen). Primers were purchased from Hokkaido Systems Science Co., Ltd. (Table S1). qRT-PCR was performed using Fast SYBR Green Master Mix with a StepOnePlus instrument (Applied Biosystems). Transcriptional levels of target genes were adjusted based on that of β-actin (ACTB) using Taqman ACTB control reagents. Three independent experiments were performed for each target gene, and the results are expressed as means ± standard deviations (SDs).

2.4 Analysis of cell proliferation, cell cycle, and apoptosis

Cells were seeded at 2.0 × 10⁵ cells/mL and treated with various concentrations of BX-912, BI-D1870, ipatasertib, PF-4708671, or TAS0612 for various periods up to 72 h. Growth inhibition was analyzed by a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using a Cell Counting Kit-8 (Dojindo Molecular Technologies). For cell cycle distribution analysis, cells were treated with phosphate-buffered saline containing 0.1% Triton X-100, stained with propidium iodide (PI), and analyzed using flow cytometry. For apoptosis analysis, cells were counterstained with Annexin V–fluorescein isothiocyanate and PI using the Annexin V FITC Assay Kit (Cayman Chemical Company) and subjected to flow cytometric analysis. Data were analyzed using FlowJo (version X; Tomy Digital Biotechnology).

2.5 Western blotting

Western blotting was performed as described previously.^10-14 The primary antibodies used were those against total RSK (RSK1/RSK2/RSK3), p-total RSK^Ser380, RSK2, p-RSK2^Ser227, AKT, p-AKT^Ser473, p-AKT^Thr308, ERK, p-ERK^{Thr202/Tyr204}, PDPK1, p-PDPK1^Ser241, PKCβII, p-PKCβII^Ser660, PLK1, p-PLK1^Thr210, PRAS40, p-PRAS40^Thr246, S6, p-S6^Ser235/236, S6K, p-S6K^Thr389, SGK, S6, p-S6^Ser235/236, TP53, β-Actin (ACTB) (Cell Signaling Technology), p-SGK^Ser422 (Assay Biotechnology Company, Inc.), TP53INP1 (Abcam), and p27Kip1 (Santa Cruz Biotechnology, Inc.).

2.6 RNA interference (RNAi)

RNA interference of TP53INP1 was performed by transfecting the shRNA plasmid into KPUM-UH1 cells. 1.0 × 10⁶ cells were resuspended in 100 μL of transfection buffer and transfected with shRNA plasmid for TP53INP1 (#sc-76,715-SH) or with control shRNA plasmid (#sc-108060) (Santa Cruz Biotechnology, Inc.) using the Amaxa™ SF Cell Line 4D-Nucleofector™ X Kit (Lonza AG). The shRNA plasmid for TP53INP1 was a pool of three target-specific plasmids designed to knock down TP53INP1 gene expression.

2.7 Analysis of gene expression profile (GEP) and gene set enrichment analysis (GSEA)

Cells were treated with either BX-912 for 12 h or TAS0612 for 6 h at IC₈₀ concentrations, and total RNA was isolated using an RNeasy Mini Kit. The GEP was analyzed using a Clarion S array (Affymetrix), a GeneChip WT Plus Reagent Kit (Thermo Fisher Scientific), and a GeneChip Scanner 7G (Affymetrix). Data were analyzed using the Affymetrix Transcriptome Analysis Console (TAC) software (ver. 4.0.0.25), and robust multiarray (RMA) normalization was performed with default parameters using TAC programming. GSEA was performed using the GSEA software (https://www.gsea-msigdb.org/gsea/index.jsp).¹⁸

2.8 Assay for the drug combinatory effect

Cells sensitive to the agents of interest were treated with eight concentrations (i.e., 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, and 2.0 × IC₅₀) of either BI-D1870 or combinatory agent or with both agents for 48 h and were subjected to a modified MTT assay. Fractional effect concentrations (i.e., a fractional effect of 0.25 equals a 25% growth inhibitory effect) and the combination index (CI) were calculated using CalcuSyn (Biosoft). This method facilitates the quantification of synergism (CI <1) and antagonism (CI >1) at different doses and effect levels. CI calculations were performed assuming that drug mechanisms were not mutually exclusive.

2.9 Statistical analysis

Statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University) (The R Foundation for Statistical Computing).¹⁹ Student's t-test was used to compare continuous variables between groups, and p-values <0.05 denote statistical significance.

3.1 The activation status of PDPK1^Ser241 and downstream effector molecules in various types of BCL

We first evaluated the activation status of PDPK1, RSK2, and AKT by investigating the phosphorylation status of PDPK1^Ser241, RSK2^Ser227, and AKT^Thr473 in patient-derived tumor tissues of DLBCL and FL. Of five specimens of DLBCL, p-PDPK1^Ser241 was strongly positive in two (#1 and #2), moderately positive in one (#3), and weakly positive in two (#4 and #5). p-RSK2^Ser227 at RSK2-NTKD was moderately to strongly positive in all specimens, and p-AKT^Thr473 was moderately positive in one (#1) and weakly positive in two (#2 and #5) but was negative in the remaining specimens (#3 and #4). In five FL specimens, p-PDPK1^Ser241 was weakly to moderately positive in four (#6–9) and negative in one (duodenum FL) (#10). p-RSK2^Ser227 was weakly to strongly positive in three specimens (#6, 8, and 9), and p-AKT^Thr473 was weakly to moderately positive in three specimens (#6, 7, and 9) but was negative in the remaining specimens. Thus, PDPK1^Ser241, RSK2^Ser227, and AKT^Thr473 are frequently active in DLBCL and FL. Furthermore, at least one of PDPK1^Ser241, RSK2^Ser227, and AKT^Thr473 was active in most patient-derived samples (Figure 1A).

Then, we investigated the activation status of PDPK1 and its major downstream molecules, that is, RSK2, AKT, S6K, SGK, PKC, and PLK1, in eight cell lines derived from four subtypes of BCLs. As shown in Figure 1B, PDPK1, RSK2-NTKD, and S6K were commonly active in all eight cell lines examined, whereas the activation status of AKT and PKC varied among cell lines and was cellular context dependent. PLK was faintly active, and SGK was inactive in all cell lines examined.

3.2 Cellular and molecular effects of the inactivation of PDPK1 in BCL-derived cell lines

Next, we investigated the cellular effect of PDPK1 inactivation in eight BCL-derived cell lines using BX-912 as an inhibitor of PDPK1. BX-912 treatment dose-dependently induced growth inhibition in all eight cell lines examined (Figure 2A), accompanied by the blockade of G2/M and the induction of apoptosis, regardless of the disease subtype (Figures 2B,C).

Then, we investigated the molecular effects of PDPK1 blockade on four cell lines of diverse disease subtypes, that is, KPUM-UH1, Raji, HBL1, and FL518 cells. At the gene expression level, GEP analysis with microarray revealed that 27 and 18 genes were commonly upregulated >1.5 folds (Table S2) and downregulated <0.67 folds (Table S3), respectively, by PDPK1 blockade at the transcription level. The commonly upregulated genes included several tumor suppressor genes, such as YPEL3, BTG2, and TP53INP1, which encode a bona fide stress-induced protein (Figure 3A). qRT-PCR validated the results of these microarray analyses, except for YPEL3 in FL518 cells (Figure 3B).

3.3 RSK2 is the central effector, and AKT and S6K are the subsidiary effectors of PDPK1 downstream in the proliferation of BCL-derived cell lines

Next, we attempted to identify functionally responsible downstream molecules of PDPK1 in the survival and proliferation of BCL-derived cell lines. For this purpose, we first examined the impact of PDPK1 inactivation on the activation status of its primary target molecules in BCL-derived cell lines of different subtypes. Treatment with BX-912 resulted in the dephosphorylation of RSK2 in all four cell lines examined. Moreover, treatment with BX-912 resulted in AKT and S6K dephosphorylation in cell lines with active AKT and/or S6K at the basal level. In contrast, BX-912 did not significantly or consistently reduce the phosphorylation status of SGK, PKC, or PLK1 (Figure 4A). These results reveal RSK2, AKT, and S6K as the relevant effector molecules of PDPK1 in BCLs. Moreover, the inactivation of RSK2-NTKD by its specific inhibitor BI-D1870 exerted universal and robust dose-dependent antiproliferative effects on all eight BCL-derived cell lines examined. In contrast, the growth inhibitory effects of exposure to ipatasertib, an inhibitor for AKT, and PF-4708671, an inhibitor for S6K, for 48 h were largely modest and varied among cell lines (Figure 4B).

Based on these findings, we next examined the antiproliferative effects of the combinatory blockade RSK2-NTKD and either AKT or S6K on four cell lines with various sensitivities to ipatasertib and PF-4708671. As a result, the combinatory blockade of RSK2 and either AKT or S6K showed at least additive and synergistic effects in most settings on various types of BCL-derived cell lines (Figure 4C,D). These collectively indicate that RSK2, AKT, and S6K are the major downstream target molecules of PDPK1 and that RSK2-NTKD plays a central role and AKT and S6K play subsidiary functional roles as the downstream effectors of PDPK1 in the proliferation of BCL-derived cell lines.

3.4 Antitumor effect of TAS0612 in BCL-derived cell lines and patient-derived primary lymphoma cells

Based on the robust antilymphoma efficacy of the simultaneous blockade of RSK2 and AKT or S6K, we next examined the antitumor effect of TAS0612, the triple inhibitor against total RSK, including RSK2, AKT, and S6K.¹⁷ As shown in Figure 5A, TAS0612 treatment showed dose-dependent growth inhibitory effects on all eight BCL-derived cell lines, regardless of the disease subtype, with IC₅₀ concentrations ranging from 0.41 μM to 6.73 μM. This growth inhibitory effect of TAS0612 was accompanied by the accumulation of its direct substrates p-total RSK^Ser380, p-AKT^Ser473, and p-S6K^Thr389 in most cell lines examined and the subsequent dephosphorylation of their respective target substrates in all cell lines examined. Furthermore, no feedback reactivation of ERK was observed following TAS0612 treatment (Figure 5B). In the process of growth inhibition, TAS0612 induced G1 cell cycle blockade in KPUM-UH1 cells accompanied by the accumulation of p-AKT^Ser473 and upregulation of p27Kip1; G2/M cell cycle blockade in Daudi, HBL1, and FL18 cell lines accompanied by the accumulation of phosphorylated of total RSK^Ser380; and the induction of apoptosis in all cell lines (Figure 5B–D, and Table S4).

We also investigated the ex vivo antiproliferative effect of treatment with 2.5 and 5.0 μM TAS0612 for 72 h in patient-derived lymphoma cells of various disease subtypes. As a result, TAS0612 treatment showed an antitumor effect on all 25 patient-derived lymphoma cells examined, including nine DLBCLs, seven FLs, three MCLs, one mucosa-associated lymphoid tissue lymphoma, one splenic marginal-zone BCL, one chronic lymphocytic leukemia, and three B-lymphoblastic lymphomas (Figure 6).

3.5 Molecular effects induced by TAS0612 on BCL-derived cell lines

We examined the global molecular effects of TAS0612 using GEP on two cell lines derived from DEL, that is, KPUM-UH1 and KPUM-MS3 cells. TAS0612 treatment with IC₈₀ concentrations for 6 h commonly resulted in a >2.0-fold increase in 222 genes (Table S5) and a <0.5-fold decrease in 296 genes (Table S6) between KPUM-UH1 and KPUM-MS3 cells (Figure 7A). Among several tumor suppressor genes upregulated by TAS0612 in both KPUM-UH1 and KPUM-MS3 cells, the increased expression of tumor protein 53-inducible nuclear protein 1 (TP53INP1) by treatment with TAS0612 was also confirmed at the transcriptional (Figure 7B) and protein (Figure 7C) levels in cell lines of other disease subtypes, that is, Daudi, HBL1, and FL18 cells. Furthermore, the upregulation of TP53INP1 was not accompanied by TP53 accumulation (Figure 7C).

Next, we investigated functionally relevant changes in gene sets caused by the treatment of KPUM-UH1 and KPUM-MS3 cells using GSEA. As judged by the normalized enrichment score, TAS0612 significantly downregulated the gene sets driven by the upregulation of MYC and mTORC1, reactive oxygen species, and the unfolded protein response (Figure 7D,E). Simultaneously, significant gene sets upregulated by TAS0612 treatment included those for interleukin (IL)-6/JAK–STAT and WNT/βcatenin signaling (Figure 7F). We finally examined the functional role of TP53INP1 in the antitumor effect of TAS0612 using RNAi technique. As a result, the antitumor effect of TAS0612 was not significantly different between control cells and TP53INP1 knockdown KPUM-UH1 cells (Figure S1).