Claire Doshier

Synthesis of NBI-961. Reagents and Conditions: a) NaOMe, MeOH 0 °C – rt. b) PPh3, DIAD, DCM 0 °C - rt. c) LDA, dry THF, -78 °C – rt. d) Na2CO3, Pd(dppf)Cl2.DCM, 4:1 THF:H2O. e) 7M NH3 in MeOH, NH4OH, 78 °C. f) K2CO3, Pd(dppf)Cl2.DCM, DMF, 80 °C.

KEA3 analysis that used the Cheng kinase-substrate interaction network in SUDHL5 and RIVA treated with NBI-961 relative to DMSO vehicle control using the top 25 proteins from each with a signficant loss of phosphorylation (FDR < 0.05; log2 fold change less than or equal to -1).

Differentially expressed proteins identified from the phospho-proteomic analysis in SUDHL5 treated with NBI-961 relative to DMSO vehicle control.

Raw data for kinase binding affinity assay with NBI-961.

1H NMR spectra of NBI-961. Purity of NBI-961 was determined by 1H NMR after dissolved in methanol. Spectra was recorded using an Agilent HPLC with a run time of 15.01 mins and a flow rate of 0.8 mL/min.

NBI-961 causes loss of NEK2 and NEK2 phosphorylation. A, B, Western blot analysis of NEK2 and phosphorylated NEK2 (p-NEK2) protein expression in NBI-961 treated RIVA (A) and U2932 (B) cell lines after 24 h. Quantification of band intensity normalized to ACTIN as mean ± SEM from three independent experiments shown as a bar graph below each blot. Adjusted P-values as determined by a one-way ANOVA with Dunnett’s multiple test correction for comparisons to DMSO vehicle treated control (VTC). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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

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

Supplemental Methods: Gene Expression by Quantitative PCR

Differentially expressed proteins identified from the phospho-proteomic analysis in SUDHL5 treated with NBI-961 relative to DMSO vehicle control.