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

Targeting a Novel G-Quadruplex in the <i>CARD11</i> 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

Table S11 from Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma

Differentially expressed proteins identified from the phospho-proteomic analysis in RIVA treated with NBI-961 relative to DMSO vehicle control.</p

Table S3 from Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma

DLBCL patient characteristics.</p

Figure S10 from Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma

NBI-961 does not alter FLT3 expression or its downstream substrate with greater cytotoxicity than sorafenib. (A) Western blot analysis of FLT3 in NBI-961 treated SUDHL5 in lysates from the same three independent experiments showing NEK2 degradation in Figure 4A. (B) Western blot analysis of phosphorylated mTOR (a known downstream substrate of FLT3) in newly NBI-961 treated SUDHL5 lysates from three independent experiments. NEK2 degradation was confirmed. Quantification of p-mTOR band intensity normalized to ACTIN is shown below. (C) Cell viability curves after 96 h NBI-961 treatment of RIVA (red dashed line) and SUDHL5 (blue dashed line) DLBCL cells compared to sorafenib treatment (solid lines) at a higher serial dilution (40 – 0.039 M; left panel) and at the same serial dilution as NBI-961 (2 – 0.00195 M; right panel). Sorafenib GI50 (95% C.I.): RIVA, 4.15 M (3.24 – 5.31); SUDHL5, 3.32 M (2.43 – 4.54).</p

Table S5 from Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma

Growth inhibitory concentrations (GI50) of NBI-961, doxorubicin and vincristine.</p

Figure S8 from Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma

Doxorubicin alone as a positive control induces apoptosis. Stacked bar graph of cell lines untreated, treated with DMSO or doxorubicin (DOX) shows percent viable cells and cells in early or late apoptosis. Data are shown as mean ± SEM from three independent experiments. Adjusted P-values as determined by a two-way ANOVA analysis with Tukey’s multiple test correction for comparisons to DMSO treated control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001</p

Figure S6 from Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma

Original blot images from Figure 5H in the main text. Uncropped blot images of Figure 5H. Samples were run on duplicate gels and each duplicate blot was immunoblotted with either a mixture of anti-NEK2 and anti-ACTIN primary antibodies (left panels) or a mixture of anti-p-AKT, anti-AKT, and anti-ACTIN primary antibodies (right panels). The blots were imaged in both the Dylight 650 and 800 channels and shown as the respective image (NEK2 or p-AKT from 650 in top panels, and AKT and ACTIN from 800 in lower panels). All blots were resolved with a mixture of secondary antibodies Dylight 650 and Dylight 800 and imaged using the BioRad ChemiDoc MP as described in the methods.</p

Table S1 from Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma

Short Tandem Repeat (STR) analysis of the DLBCL and benign B cell lines.</p

Supplementary Data - Tables from Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma

supplemental tables S1, 4, and 6-12</p