Background: Greatwall (GWL), also known as MASTL (microtubule-associated serine/threonine kinase-like), is an enzyme active during mitosis that promotes cell division and clonal expansion. Because of this role, it is considered a potential target for cancer therapy. Previous research in solid tumors—including breast, gastric, and pancreatic cancers—has shown that higher GWL levels in malignant cells are associated with more aggressive tumor growth and resistance to chemotherapy. Conversely, reducing GWL expression can increase tumor cells' sensitivity to chemotherapy. GWL regulates the cell cycle by inactivating the PP2A:B55 complex. It does this through phosphorylation of two upstream proteins, cAMP-regulated phosphoprotein 19 (Arpp19) and alpha-endosulfine (ENSA). These phosphorylated proteins then bind to and inhibit protein phosphatase 2A-B55 (PP2A-B55), causing cells to enter mitosis with low PP2A:B55 activity thereby stabilising mitotic Cdk1 dependent phosphor-sites.

To date, the role of GWL has not been explored in hematological malignancies.

Methods: We utilized a novel Greatwall kinase inhibitor (GWLi) developed by the Hocheggar lab (University of Sussex), which has been shown to selectively inhibit GWL and arrest cells in the G2/M phase of the cell cycle. We investigated its effects on the MEC-1 chronic lymphocytic leukemia (CLL) cell line as well as primary CLL cells co-cultured with CD40 ligand-transfected 3T3 fibroblasts, which simulate the activating and proliferative microenvironment that CLL cells experience within lymph nodes. Additionally, the impact of GWLi was evaluated on diffuse large B-cell lymphoma (DLBCL) cell lines (SUDHL6, SUDHL8, RIVA, and U2932). Cells were treated with varying concentrations of GWLi (0.5 µM to 4 µM) for 48 hours, after which cell cycle progression and apoptosis were assessed by flow cytometry. It's effect was compared between IGHV mutated CLL (M-CLL) and unmutated CLL(U-CLL) cells. Synergistic effect with venetoclax was also studied.

Immunofluorescence assays were also conducted to visually determine the effects of GWLi on the cells.

Results: GWLi inhibited cell cycling in all cell lines tested. Using 4uM it was most effective in MEC-1 cells (G1 reduced from 51.7% to 37.5% and G2/M increased from 8.1% to 23.7%). Additionally, a dose-dependent increase in apoptosis was seen with maximum effect in MEC-1 cells (11% in the untreated cells to 26.9% at 4uM). Co-cultured primary CLL cells from 9/10 patients tested were also blocked in G2/M phase using GWLi. When the effect was then compared between M-CLL and U-CLL cells, a similar rise was noted in G2/M phase, but a greater decrease noted in S phase in M-CLL cells (34.8% to 12% in M-CLL and 22% to 10.5% in U-CLL). A dose dependent increase in apoptosis was also seen with mean apoptosis increasing from 19.16% in the untreated cells to 43.04% using the 4uM of drug. Preliminary synergy experiments combining GWLi with the BCL2 inhibitor Venetoclax (ABT-199) indicated that the drugs have an additive effect (Bliss score 2.898).

Immunofluorescence analysis of MEC1 cells showed an increasing mean cell area, nuclear material and doublets/multiple nuclei with increasing drug concentration; supporting the finding that GWLi blocks cells in G2/M.

Conclusion: This data indicates that inhibition of GWL has potential as a future therapeutic strategy for CLL and DLBCL. Early western blot experiments are underway to determine mechanism of action. Further experiments to quantify and track the various kinases and proteins within the cell cycle would be required to provide more definitive information about the mechanism of action. Additionally, segregation of CLL cells based on other prognostic indicators and genetic characterization besides IGHV mutational status would help provide more data on which patients are likely to respond best to GWL inhibition.

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2025
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