Diffuse large B‐cell lymphoma cell‐cycle progression requires the cyclin‐G associated kinase (GAK), a novel drug target from machine learning‐enabled phenotypic screening

Background: Recurrent genomic alterations in specific cancers reveal tumor drivers that inform the development of therapeutic approaches. In diffuse large B-cell lymphoma (DLBCL), oncoprotein-focused drug development has achieved limited success. Phenotypic screening is an emerging alternative that can be leveraged through machine-learning analysis. Under this paradigm, we reveal strong therapeutic potential in DLBCL of inhibiting the cyclin-G associated kinase (GAK). GAK participates in clathrin-mediated endocytosis and regulates mitotic-spindle alignment. GAK has been minimally explored in cancer therapeutics and is a novel target in DLBCL. Methods: We performed a first-of-its-kind screen of kinase inhibitors in DLBCL followed by machine learning-based analysis using our in-house target identification platform, idTRAX. We analyzed gene-expression data for 414 previously untreated DLBCL patients (Lenz cohort). Immunofluorescent microscopy was performed to visualize the mitotic effect of GAK inhibition. Results: Our screen of kinase inhibitors revealed GAK as a strong target for both activated B-cell (ABC) and germinal center B-cell (GCB) derived cell lines with complete sparing of peripheral-blood mononuclear cells. Analysis of gene-expression data found tumors with high GAK expression had dramatically worse overall survival compared to those with low expression (p = 0.0002). We found that GAK inhibition in DLBCL results in profound disruption of progression through mitosis, potently triggering the spindle assembly checkpoint and causing accumulation in G2 before onset of apoptosis. Additionally, gene-expression data showed correlation between high GAK expression and low expression of RB1, encoding the tumor suppressive master cell-cycle regulator retinoblastoma-associated protein (RB, p < 0.0001). The DLBCL cell lines RIVA and U2932 completely lack RB expression and show high sensitivity and accumulation in G2 in response to GAK inhibition. Pooled analyses of DLBCL cell lines show increased sensitivity to GAK inhibition in ABC-derived lines (mean EC50 0.2817 µM) compared to GCB (mean EC50 0.6517 µM, p = 0.0078). Immunofluorescent microscopy revealed profound disruption of mitotic spindles and shattering of chromosomes in these cells. Multiple participant molecules in mitotic progression are implicated as candidate GAK substrates and are under investigation to define key mechanisms. Conclusion: GAK is a novel kinase therapeutic target in DLBCL revealed by our target identification platform, idTRAX. GAK's role in mitotic-spindle alignment represents a critical kinase dependency for DLBCL tumors, in particular with cell-cycle deregulation due to loss of RB function. Our synthesis of novel inhibitors of GAK and synergistic targets may reveal new therapeutic strategies for translation to clinical trials for DLBCL patients with unmet clinical needs. The research was funded by: Florida Department of Health Bankhead-Coley Cancer Research Program Keywords: Aggressive B-cell non-Hodgkin lymphoma, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. V. Lemmon Employment or leadership position: Truvitech LLC H. A. Ali Employment or leadership position: Truvitech LLC J. H. Schatz Consultant or advisory role: WCG - ACI Clinical

Topic: Ubiquitin and proteasome pathways
Subfield: Molecular Biology
Field: Biochemistry, Genetics and Molecular Biology
Domain: Life Sciences