Targeting glycogen synthase kinase 3 for therapeutic benefit in lymphoma

Abstract

Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and β are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3β compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3β genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3β binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3β is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3β correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.

Conflict of interest statement

Conflict-of-interest disclosure: F.G. and D.M.S. are employees and stockholders of Actuate Therapeutics Inc. D.D.B. is a member of Actuate Therapeutics Scientific Advisory Board and is a stockholder. The remaining authors declare no competing financial interests.

Figures

Graphical abstract

Figure 1.

GSK3α and GSK3β mRNA and proteins are overexpressed in lymphomas. (A) Real-time PCR quantitation showing that GSK3α and GSK3β mRNAs are overexpressed in lymphoma lines in comparison with low expression in normal B or T lymphocytes. (B) Western blot images demonstrating that GSK3α and GSK3β proteins are also abundantly expressed in various lymphoma lines in comparison with purified normal B or T lymphocytes.

Figure 2.

GSK3 is essential for lymphoma cell proliferation and survival. Unstimulated peripheral blood B and T lymphocytes isolated from a healthy donor were used as normal control. Proapoptotic effect of the GSK3 inhibitor 9-ING-41 in various MCL and TCL lines (A) and in DLBCL lines (B). (C) Cell-proliferation profile of various lymphoma cell lines upon treatment with 9-ING-41. All results are from 3 independent experiments.

Figure 3.

GSK3 inhibition or deletion in lymphoma cells leads to cell cycle arrest in G2/M. (A) Cell cycle profile of 3 representative cell lines Jeko, Mino, and OCI-Ly3 after a 24-hour treatment with 0, 1.0, or 2.0 μM 9-ING-41. (B) Cell cycle profiles of parental Ly-1 cells and GSK3α-, GSK3β-, and GSK3αβ-knockout subclones. Inset: western blot image showing the depletion of GSK3α and GSK3β protein in the knockout Ly-1 subclones.

Figure 4.

Inhibition of GSK3 by 9-ING-41 leads to mitotic prophase arrest. (A) Cartoon depiction of the sequential steps (M1-M5) during mitosis (purchased from Shutterstock and modified). (B) Representative images of Wright’s stained Jeko cells that were left untreated or treated with 1.0 μM 9-ING-41 for 24 hours. Various mitotic stage cells (M1-M5) are readily identified in untreated cells (left panel), whereas large numbers of only prophase cells (M1) are seen in 9-ING-41–treated cells (right panel); original magnification ×600. (C) Bar chart showing the number of mitotic M1 to M5 cells identified when 100 untreated or 9-ING-41–treated Jeko cells were counted. Similar results (data not shown) were observed in ≥4 lymphoma cell lines.

Figure 5.

GSK3β localized to centrosomes. (A) Immunofluorescence image showing GSK3β is localized to the nucleus and centrosome pairs in interphase Jeko cells. (B) Enlarged image of the boxed area in panel A. Centrosome pairs are marked by arrows. (C-F) Single- or multichannel images of coimmunostaining of GSK3β and pericentrin in wild-type Ly-1 cells showing their colocalization to the centrosomes. (G-J) Single- or multichannel images of coimmunostaining of GSK3β and pericentrin in GSK3β-null Ly-1 cells showing that staining is specific to GSK3β. (K) Immunofluorescence image showing GSK3β (green) localized to firework-like structures resembling mitotic spindles in mitotic Jeko cells. (L) Enlarged image of the mitotic cell shown in panel K. (M) Overlay image showing the microtubule structure by α-tubulin (red) staining and DNA (blue). (N) Enlarged image of the boxed area in panel M. (O-P) Images showing that spindle structure staining of GSK3β is absent in GSK3β-null Ly-1 cells. (Q) Immunofluorescence image showing GSK3β localized to mitotic spindle structure and polarized centrosomes in 9-ING-41–treated Jeko cells. (R) Enlarged image of the representative mitotic cell shown in panel Q. Original magnification ×600.

Figure 6.

Aberrant expression of GSK3 proteins in primary lymphoma patient (P) cells and their proliferative response to 9-ING-41. (A) Immunoblot of GSK3α and GSK3β proteins showing overexpression in patient samples vs normal B-cell control. P1, MCL; P2, high-grade B-cell lymphoma; P3, follicular large B-cell lymphoma 3B; P4, DLBCL; and P5, angioimmunoblastic T-cell lymphoma. (B) 9-ING-41 inhibited proliferation in all 5 patient samples. (C) Immunohistochemistry staining of GSK3β on paraffin tissue sections from patients with various lymphomas (original magnification ×40). Representative images show the spectrum of GSK3β (brown) overexpression in different lymphoma samples. Methylene blue counterstaining (blue) reveals cells negative for GSK3β in the background and in the antibody negative-control panel.

Figure 7.

Antilymphoma effect of 9-ING-41 in vivo in Jeko-derived xenograft mouse model. (A) Experimental design showing 9-ING-41 treatment schedule and dosage. (B) Bioluminescence images of xenograft-bearing mice that were left untreated or treated with 9-ING-41. The images were taken at the end of the experiment (day 17). The experiment was performed twice; both showed similar results.