The Cyclin-G Associated Kinase (GAK) Is a Critical Targetable Dependency for Successful Mitosis By Diffuse Large B-Cell Lymphomas Linked to Retinoblastoma Loss of Function

Background:

Diffuse large B-cell lymphoma (DLBCL) is an aggressive non-Hodgkin lymphoma and the most common lymphoma type in the United States. Despite significant molecular and clinical heterogeneity among DLBCL patients, standard frontline chemoimmunotherapy is uniformly administered, yet at least a third of patients remain uncured. This underscores a critical need for novel targeted therapies. Recurrent genomic alterations in specific cancers reveal tumor drivers that inform the development of therapeutic approaches. However, in DLBCL, oncoprotein-focused drug development has achieved limited success. By an emerging alternative, phenotypic screening, we reveal a novel kinase target, the cyclin-G associated kinase (GAK). GAK, involved in clathrin-mediated endocytosis and mitotic-spindle alignment, has not been previously explored in cancer therapeutics.

Methods and Results:

We conducted a phenotypic screen of kinase inhibitors against DLBCL followed by machine learning-based target deconvolution, revealing GAK as a tumor-selective target. GAK siRNA knockdown was active against both activated B-cell (ABC) and germinal center B-cell (GCB) subgroups, without significantly affecting non-malignant blood cells. Chemical inhibition with the highly selective GAK inhibitor SGC-GAK-1 further validated our results, showing reduced viability in DLBCL but not in normal blood cells. Broad assessment of GAK inhibition revealed ≤1 µM GI50 across DLBCL and enhanced sensitivity in the ABC subtype.

Mechanistic studies found no impact of GAK inhibition on clathrin-mediated signaling events such as B-cell receptor recycling to the cell surface or its downstream pathways. Instead, we revealed GAK as a critical cell-cycle kinase in DLBCL progression: GAK inhibition activated the spindle-assembly checkpoint, followed by G2/M arrest and cell death. Moreover, immunofluorescent confocal microscopy showed widespread chromosome misalignment and spindle distortion in DLBCL cells following GAK inhibition, preventing successful progression through mitosis. RNA-seq analysis from DLBCL cell lines treated with SGC-GAK-1 strongly validated GAK's essential role in cell-cycle progression. Gene Set Enrichment Analysis (GSEA) revealed that gene sets related to spindle organization, mitotic nuclear division, and chromosome segregation were enriched at q<7.7841e-10. GAK inhibition led to significant upregulation of key cell-cycle kinase genes, including polo-like kinase and aurora kinase families. Additionally, genes involved in checkpoint control, such as BUB1, were markedly upregulated. These cellular responses highlight GAK's critical role in mitotic progression, as the cells attempt to bypass and manage mitotic arrest induced by GAK inhibition.

Additionally, RNA-seq data analysis from clinical samples showed increased GAK expression was correlated with RB1 deficiency in DLBCL cases, suggesting a dependency on GAK for proper mitotic progression linked to this common biomarker. In cell-cycle analyses and immunofluorescent confocal microscopy, RB-deficient cells treated with SGC-GAK-1 showed pronounced arrest at G2/M. Multiple lines of evidence indicate RB-deficient tumors are particularly dependent on GAK for mitotic progression. Unbiased phosphoproteomics studies identified specific participants in mitotic spindle alignment as high-probability GAK phosphosubstrates, which remain under active investigation to determine GAK's mechanisms as a cell-cycle kinase. Finally, in vivo studies of DLBCL xenograft NSG mice treated with SGC-GAK-1 demonstrated dramatic tumor volume reduction. These results underscore the potential of GAK as a therapeutic target in DLBCL, particularly in RB-deficient contexts, paving the way for precision medicine approaches.

Conclusions:

We reveal GAK as a novel targetable dependency in DLBCL. Our findings highlight GAK's role in cell-cycle regulation and its particular importance in RB-deficient DLBCL, presenting a significant therapeutic vulnerability. There is potential for rapid clinical translation emphasized by frequent off-target inhibition of GAK by existing kinase inhibitors available for clinical use. Ongoing research is focused on defining the molecular mechanisms and specific phospho-substrates of GAK to further elucidate its role in DLBCL pathogenesis.

Maura:Sanofi: Consultancy, Honoraria; Medidata: Consultancy, Honoraria.