Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma.

Expression of the serine/threonine kinase never in mitosis gene A (NIMA)-related kinase 2 (NEK2) is essential for entry into mitosis via its role in facilitating centrosome separation. Its overactivity can lead to tumorigenesis and drug resistance through the activation of several oncogenic pathways, including AKT. Although the cancer-enabling activities of NEK2 are documented in many malignancies, including correlations with poor survival in myeloma, breast, and non-small cell lung cancer, little is known about the role of NEK2 in lymphoma. Here, in tumors from patients with diffuse large B-cell lymphoma (DLBCL), the most common, aggressive non-Hodgkin lymphoma, we found a high abundance of NEK2 mRNA and protein associated with an inferior overall survival. Using our recently developed NEK2 inhibitor, NBI-961, we discovered that DLBCL cell lines and patient-derived cells exhibit a dependency on NEK2 for their viability. This compromised cell fitness was directly attributable to efficient NEK2 inhibition and proteasomal degradation by NBI-961. In a subset of particularly sensitive DLBCL cells, NBI-961 induced G2/mitosis arrest and apoptosis. In contrast, an existing indirect NEK2 inhibitor, INH154, did not prevent NEK2 autophosphorylation, induce NEK2 proteasomal degradation, or affect cell viability. Global proteomics and phospho-proteomics revealed that NEK2 orchestrates cell-cycle and apoptotic pathways through regulation of both known and new signaling molecules. We show the loss of NEK2-sensitized DLBCL to the chemotherapy agents, doxorubicin and vincristine, and effectively suppressed tumor growth in mice. These studies establish the oncogenic activity of NEK2 in DLBCL and set the foundation for development of anti-NEK2 therapeutic strategies in this frequently refractory and relapse-prone cancer.

Conservation of the insert-2 motif confers Rev1 from different species with an ability to disrupt G-quadruplexes and stimulate translesion DNA synthesis.

In some organisms, the replication of G-quadruplex (G4) structures is supported by the Rev1 DNA polymerase. We previously showed that residues in the insert-2 motif of human Rev1 (hRev1) increased the affinity of the enzyme for G4 DNA and mediated suppression of mutagenic replication near G4 motifs. We have now investigated the conservation of G4-selective properties in Rev1 from other species. We compared Rev1 from Danio rerio (zRev1), Saccharomyces cerevisiae (yRev1), and Leishmania donovani (lRev1) with hRev1, including an insert-2 mutant form of hRev1 (E466A/Y470A or EY). We found that zRev1 retained all of the G4-selective prowess of the human enzyme, but there was a marked attenuation of G4 binding affinity for the EY hRev1 mutant and the two Rev1 proteins lacking insert-2 (yRev1 and lRev1). Perhaps most strikingly, we found that insert-2 was important for disruption of the G4 structure and optimal stimulation of processive DNA synthesis across the guanine-rich motif by DNA polymerase kappa (pol κ). Our findings have implications for how Rev1 might contribute to G4 replication in different species spanning the evolutionary tree - signaling the importance of selection for enzymes with robust G4-selective properties in organisms where these non-B DNA structures may fulfill taxa-specific physiological functions.

Targeting a Novel G-Quadruplex in the CARD11 Oncogene Promoter with Naptho(2,1-b)furan-1-ethanol,2-nitro- Requires the Nitro Group.

The aggressive nature of the activated B cell such as (ABC) subtype of diffuse large B cell (DLBCL) is frequently associated with altered B cell Receptor (BCR) signaling through the activation of key components including the scaffolding protein, CARD11. Most inhibitors, such as ibrutinib, target downstream BCR kinases with often modest and temporary responses for DLBCL patients. Here, we pursue an alternative strategy to target the BCR pathway by leveraging a novel DNA secondary structure to repress transcription. We discovered that a highly guanine (G)-rich element within the CARD11 promoter forms a stable G-quadruplex (G4) using circular dichroism and polymerase stop biophysical techniques. We then identified a small molecule, naptho(2,1-b)furan-1-ethanol,2-nitro- (NSC373981), from a fluorescence-resonance energy transfer-based screen that stabilized CARD11 G4 and inhibited CARD11 transcription in DLBCL cells. In generating and testing analogs of NSC373981, we determined that the nitro group is likely essential for the downregulation of CARD11 and interaction with CARD11 G4, and the removal of the ethanol side chain enhanced this activity. Of note, the expression of BCL2 and MYC, two other key oncogenes in DLBCL pathology with known promoter G4 structures, were often concurrently repressed with NSC373981 and the highly potent R158 analog. Our findings highlight a novel approach to treat aggressive DLBCL by silencing CARD11 gene expression that warrants further investigation.