Journal: bioRxiv

Article Title: Integrative Computational Framework, Dyscovr , Links Mutated Driver Genes to Expression Dysregulation Across 19 Cancer Types

doi: 10.1101/2024.11.20.624509

Figure Lengend Snippet: A. Schematic of proposed mechanism-of-action of synthetic lethality between mutated PIK3CA and dysregulated expression of KBTBD2 kinase. Dyscovr identifies a correlation between PIK3CA nonsynonymous mutation status and KBTBD2 downregulation, both pan-cancer ( q = 6.53E-04) and in breast cancer ( q = 4.80E-02). This pair is also identified as candidate synthetic lethal via cell viability analysis in DepMap cell lines ( q = 6.17E-02, see Methods VIII). KBTBD2 has been previously identified as being a regulator of insulin signaling in mouse models via its regulation of p85α protein abundance, with low levels of KBTBD2 corresponding to suppressed PI3K-AKT signaling . This proposed system lends itself to the hypothesis that inhibition of mutant PIK3CA would result in recovery of KBTBD2 expression; therefore, joint inhibition of mutant PIK3CA and KBTBD2 should result in synergistic effects on cell growth. B. The per-drug Spearman’s rank correlation between reported drug sensitivity score from DepMap (AUC) and cell viability upon CRISPR knockout of KBTBD2 ( x -axis) against the significance, i.e. -log10( q -value), of that correlation. Correlations were computed for pan-cancer cell lines and each of 4659 drugs from DepMap. Drugs with statistically significant correlations to cell viability upon KBTBD2 knockout ( q < 0.2) are colored, with positive correlations in red and negative correlations in blue. Labels with an asterisk (*) indicate drugs that target insulin like growth factor 1 receptor, IGF1R , suggesting that those cell lines most sensitive to KBTBD2 knockout also tend to be most sensitive to inhibitors of insulin signaling. C-D. Survival of pan-cancer (C) or breast cancer (D) TCGA patients significantly differs when stratified by “low” or “high” KBTBD2 expression ( p = 2.26E-07 and hazard ratio of 2.61 pan-cancer, p = 4.51E-02 and hazard ratio of 2.99 in BRCA). Survival rate ( y -axis) by time in days ( x -axis) was calculated using a Cox proportional hazards model that accounts for the nonsynonymous mutation status of PIK3CA and other clinical and molecular confounders (see Methods IX). E. qPCR-based quantification of KBTBD2 mRNA expression in MCF7 cells treated for 48hr with an siRNA control or siRNA against KBTBD2 . Data are presented as mean ± SD, and statistical significance is indicated (**p < 0.01). F. Relative cell growth in response to alpelisib treatment in siControl and siKBTBD2 treated cells. MCF7 cells were transfected with siControl or siKBTBD2 for 48hr. followed by alpelisib treatment at indicated doses for an additional 2 days. Cell growth was quantified by SRB staining. Data are presented as mean ± SD. G. Western blot analysis of PI3K signal transduction following alpelisib treatment and KBTBD2 knockdown. MCF7 cells were transfected with siControl or siKBTBD2 for 48Hr. followed by treatment with 1 µM alpelisib for time t (hrs).

Article Snippet: Probes and primers were obtained from ThermoFisher as follows: KBTBD2 (Assay ID: Hs01556149_m1, FAM-MGB and GAPDH (Assay ID: Hs02786624_g1, VIC-MGB). qPCR was performed in 20uL reaction volumes with KBTBD2 assay together with GAPDH and amplified using iTaq universal probes supermix (Bio-Rad Laboratories, #1725134).

Techniques: Expressing, Mutagenesis, Inhibition, CRISPR, Knock-Out, Control, Transfection, Staining, Western Blot, Transduction, Knockdown

Journal: bioRxiv

Article Title: Integrative Computational Framework, Dyscovr , Links Mutated Driver Genes to Expression Dysregulation Across 19 Cancer Types

doi: 10.1101/2024.11.20.624509

Figure Lengend Snippet: A. Relative cell growth in response to RLY-2608 treatment in siControl and siKBTBD2 treated cells. MCF7 cells were transfected with siControl or siKBTBD2 for 48hr. followed by RLY-2608 treatment at indicated doses for an additional 2 days. Cell growth was quantified by SRB staining. Data are presented as mean ± SD. B. Knockdown of KBTBD2 and western blot analysis of PI3K pathway components. MCF7 were transfected with control or siRNA targeting KBTBD2 for 48Hr. C. Western blot analysis of PI3K signal transduction following RLY-2608 treatment and KBTBD2 knockdown. MCF7 cells were transfected with siControl or siKBTBD2 for 48Hr. followed by treatment with 1 µM RLY-2608 for time t (hrs).

Article Snippet: Probes and primers were obtained from ThermoFisher as follows: KBTBD2 (Assay ID: Hs01556149_m1, FAM-MGB and GAPDH (Assay ID: Hs02786624_g1, VIC-MGB). qPCR was performed in 20uL reaction volumes with KBTBD2 assay together with GAPDH and amplified using iTaq universal probes supermix (Bio-Rad Laboratories, #1725134).

Techniques: Transfection, Staining, Knockdown, Western Blot, Control, Transduction