Targeting the GCK pathway: a novel and selective therapeutic strategy against RAS-mutated multiple myeloma

Abstract

In multiple myeloma (MM), frequent mutations of NRAS, KRAS, or BRAF are found in up to 50% of newly diagnosed patients. The majority of the NRAS, KRAS, and BRAF mutations occur in hotspots causing constitutive activation of the corresponding proteins. Thus, targeting RAS mutation in MM will increase therapeutic efficiency and potentially overcome drug resistance. We identified germinal center kinase (GCK) as a novel therapeutic target in MM with RAS mutation. GCK knockdown (KD) in MM cells demonstrated in vitro and in vivo that silencing of GCK induces MM cell growth inhibition, associated with blocked MKK4/7-JNK phosphorylation and impaired degradation of IKZF1/3, BCL-6, and c-MYC. These effects were rescued by overexpression of a short hairpin RNA (shRNA)-resistant GCK, thereby excluding the potential off-target effects of GCK KD. In contrast, overexpression of shRNA-resistant GCK kinase-dead mutant (K45A) inhibited MM cell proliferation and failed to rescue the effects of GCK KD on MM growth inhibition, indicating that GCK kinase activity is critical for regulating MM cell proliferation and survival. Importantly, the higher sensitivity to GCK KD in RASMut cells suggests that targeting GCK is effective in MM, which harbors RAS mutations. In accordance with the effects of GCK KD, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 and led to MM cell proliferation inhibition accompanied by induction of apoptosis. Here, our data identify GCK as a novel target in RASMut MM cells, providing a rationale to treat RAS mutations in MM. Furthermore, GCK inhibitors might represent an alternative therapy to overcome immunomodulatory drug resistance in MM.

Graphical abstract

Figure 1.

GCK expression is elevated in MM. (A) The GCK gene and transcript expression values were obtained from the publically available genotype-tissue expression portal on 30 June 2019 and shown as reads per kilobase transcript per million reads (RPKM). (B) The GCK protein expression values were obtained from the Human Integrated Protein Expression Database (HIPED) on 30 June 2019. Parts per million (PPM): each protein entity is enumerated relative to all other protein molecules in the sample. (C) mRNA from a panel of MM cell lines (MM.1S, H929, RPMI-8226, U266), primary MM cell (MM 1 and MM 2), PBMC#1 and PBMC #2, BMSC #1 and BMSC#2, and HEK-293 cells was extracted and GCK mRNA expression was analyzed by quantitative PCR with β-actin as control. GCK mRNA level was calculated relative to its level in HEK-293. (D) RAS^Mut MM cell lines (MM.1S, RPMI-8226, JJN3, H929, JIM3, and MOLP-8) and RAS^WT cell lines (U266, OPM2, LP-1, KMS12-PE, and SKMM2) were analyzed for GCK protein levels by western blotting of whole cell extracts using β-actin as a loading control. GCK protein levels were quantified using ImageJ software. (E) Immunohistochemistry of paraffin-embedded bone marrow biopsy sections for GCK expression (Brown staining) of normal donor (n = 26) and myeloma patients (n = 26). The slides were scanned using a high-resolution scanner (Leica SCN400 Slide Scanner) at ×40 magnification. Images were analyzed using Aperio ImageScope software (Aperio Technologies, Inc., Vista, CA). Statistical analysis was performed using the Aperio Positive Pixel Count algorithm in the ImageScope viewing software.

Figure 2.

GCK is required for RAS^Mut MM cells survival. (A) MM.1S (K-RAS^G12A), RPMI-8266 (K-RAS^G12A), and H929 (N-RAS^G13D) MM cells were infected by pLKO-Tet-On scramble control (shCNTL) or shGCK lentivirus and selected by puromycin (3 ug/μL) for 1 week. Knockdown of GCK by doxycycline (Dox) treatment (400 ng/mL) for 3 days was confirmed by western blotting. (B-D) Transduced and selected MM.1S, RPMI-8266, and H929 cells were cultured in the presence Dox (400 ng/mL) for 5 days. Cell proliferation was detected by (B) MTS assay, and cells were stained with PI for (C) cell-cycle analysis, or (D) with Annexin V and 7-AAD for apoptosis analysis. (E) U266 and LP-1 RAS^WT cells were infected by pLKO-Tet-On scramble control (shCNTL) or shGCK lentivirus and selected by puromycin (3 μg/μL) for 1 week. Knockdown of GCK by Dox treatment (400 ng/mL) for 3 days was confirmed by western blotting. (F-G) Transduced and selected U266 and LP-1 RAS^WT cells were cultured in the presence of Dox (400 ng/mL) to induce shRNA for 5 days. (F) Cell proliferation was detected by MTS assay, and (G) cells were stained with Annexin V and 7-AAD for apoptosis analysis.

Figure 3.

Knock-down of GCK decreases c-MYC, IKZF1, IKZF3, and BCL6 expression in RAS^Mut MM cells. (A-C) MM.1S (K-RAS^G12D), RPMI-8266 (K-RAS^G12A), H929 (N-RAS^G13D), and U266 (RAS^WT) MM cells were infected by pPLKO-Tet-On scramble control (Tet-On-shCNTL) or sh-GCK lentivirus (Tet-On-shGCK), and selected by puromycin (3 μg/μL) for 1 week. To induce the shRNA, selected cells were treated with Dox 400 ng/mL for 3 days, and analyzed for GCK, IKZF1, IKZF3, c-MYC, and BCL-6 expression by western blotting. (A,B) β-actin was detected as loading control. For mRNA expression of GCK, IKZF1, and c-MYC by quantitative real-time PCR. Data were analyzed according to the ΔΔCt method. Results are shown as mRNA expression relative to control. (C) mRNA levels were normalized with β-actin mRNA expression as control. (D) Tet-On-shCNTL or Tet-On-shGCK MM.1S or (E) LP-1 cells were treated with Dox 400 ng/mL for 36 hours, starved in RPMI-1640 FBS free medium for 12 hours, and treated with IL-6 for 15 minutes. GCK, p-MKK4, p-MKK7, and p-JNK was detected by western blot and β-actin was detected as loading control. (F) Transduced and selected MM.1S (Tet-on-shGCK) cells were cultured with Dox (400 ng/mL) for 2 days, then treated with IL-6 and Dox for 3 days. Cell proliferation was detected by MTS assay.

Figure 4.

Rescue experiment excluded the possible off-target for GCK shRNA. (A) C-terminal Myc-tagged GCK WT or GCK shRNA-resistant allele M5 expression construct was generated based on PCDH-MCS-EF1-eGFP lentiviral vector as stated in the Methods section. (B) Tet-On-shGCK MM.1S cells were infected with PCDH-EV, PCDH-GCK-myc, or PCDH-GCK(M5)-myc lentiviral particles. Cells were selected by GFP and treated with Dox 400 ng/mL for 2 days to turn on shRNA. Cell lysates were analyzed by western blot for GCK, IKZF1, and c-MYC expression. Selected cells from panel B were cultured with Dox 400 ng/mL for 5 days and cell proliferation was measured by (C) MTS assay, (D) Annexin V and 7-AAD staining for apoptosis analysis, and (E) PI staining for cell-cycle analysis.

Figure 5.

Inhibition of GCK in MM cells abrogates tumor growth in vivo. Tet-on-shCNTL-MM.1S or Tet-On-shGCK-MM.1S cells were injected subcutaneously into SCID/bg mice. Sixteen days after implantation, all mice developed a subcutaneous tumor and were randomized to receive either vehicle (5% sucrose) or Dox (1 mg/mL in 5% sucrose) via drinking water for the duration of study. (A) Subcutaneous tumor growth was measured by using calipers and calculated with the volume formula: 0.5 × long diameter × short diameter. Each bar represents the mean ± standard error of the mean (SEM; n = 5). **Indicates significance with P < .01. (B) Mice were euthanized after 38 days. Tumors were excised and weighed. Tumor weights are reported as mean ± SEM (n = 5). **Indicates significance with P < .01. (C) Tumors harvested at the end of the study were fixed in 10% formalin and subsequently processed for immunohistochemical staining for GCK and IKZF1. The slides were scanned using a high-resolution scanner (Leica SCN400 Slide Scanner) at ×40 magnification. (D) SCID/bg mice were injected with Tet-On-shCNTL-MM.1S or Tet-On-shGCK-MM.1S cells expressing luciferase (n = 5). After 1, 2, and 3 weeks, mice received intraperitoneal (3 mg/mouse) d-luciferin 10 minutes before BLI. Bioluminescent signal and grayscale photographic images were acquired using the IVIS Spectrum Bioluminescence and Fluorescence Optical Imaging System and Living Image software.

Figure 6.

GCK kinase activity is required for MM cell growth. (A) GCK consists of 820 amino acid residues. The kinase domain (in orange) is located at its N terminus. Lysine 45 is critical for GCK kinase activity. Lysine 45 was replaced by alanine for kinase dead mutation. (B-F) MM.1S cells were transduced with EV (PCDH-EV), shRNA-resistant GCK-M5 (PCDH-GCK(M5)-myc), or shRNA-resistant GCK and GCK kinase dead mutation K45A-M5 (PCDH-GCK-K45A(M5)-myc). GFP sorted cells were cultured in the presence of Dox and analyzed for (B) GCK expression by western blot, (C) proliferation by MTS assay, and (D) for GCK and c-MYC expression by western blot. Selected cells from panel D were cultured in the presence of Dox and analyzed for (E) cell proliferation by MTS assay (F) apoptosis by Annexin V and 7-AAD staining.

Figure 7.

Pharmacological blockage of GCK inhibits MM cell proliferation and induces cell death. (A) RAS^WT MM cell lines (KMS12-PE, OPM2, LP-1, SKMM-2, and U266) and RAS^Mut MM cell lines (MOLP-8, RPMI-8226, H929, MM.1S, and JJN3) were incubated with TL4-12 for 4 days. Cell proliferation was analyzed by MTS assay. Data represent the mean ± SD of n = 3 experiments. (B) RAS^WT LP-1 and K-RAS^G12A MM.1S cells were incubated with TL4-12 for 24 hours to analyze IKZF1, c-MYC, BCL-6, and P53 expression by western blotting. β-actin was detected as loading control. (C) Cells were treated by TL4-12 for 4 days to detect cell apoptosis with Annexin V and 7-AAD staining by flow cytometry assay. (D) MM.1S cells were treated with TL4-12 at the indicated concentrations with or without PS341 (1μM) for 24h. Lysates were analyzed by western blotting to compare the levels of IKZF1, c-MYC, and IRF-4. β-actin expression was probed for loading control. (E-F) N-RAS^G13D H929 cells were transduced using a lentivirus with control shRNA (shCNTL) or CRBN-shRNA (shCRBN). GFP-sorted cells were cultured in the presence of LEN or TL4-12 for 24 hours to analyze IKZF1, CRBN expression by western blotting (E); and for 4 days for cell proliferation detected by MTS assay (F). (G) Transduced and selected Tet-On-shGCK-MM.1S and Tet-On-shGCK-RPMI-8266 cells were cultured with LEN at the indicated concentrations with or without Dox 400 ng/mL for 3 days and cell proliferation was measured by MTS assay,