Supplementary Figure 8: Responses to combinatorial drug treatments in BRAFV600E melanoma cells validate HT-KAM-generated phospho-catalytic signatures, and initiate the translation of kinase hits into novel therapeutic opportunities.
We tested whether some of the kinases found as differentially active by HT-KAM screen in patient tumors (Fig. 7, Supplementary Fig. 7e–h) revealed druggable vulnerabilities capable of potentiating the response of BRAFV600E melanoma cell models to BRAF-targeted therapy. (a) Maps of cell growth responses (left) and combination indices (right) for A375 and Sk-Mel-28 maintained in 0.25% FBS. See Fig. 8a legend for details. (n≥3). (b-d) Evaluating drug combination effects on melanoma cell death. Fluorescence-Activated Cell Sorting (FACS) was used to measure cell death, as exemplified in (b) (n=1 per treatment condition). Graphs in (c-d) show percentages of apoptosis for Sk-Mel-28 and A375 (averages measured in 5% or 0.25% FBS conditions at GI50 concentrations of drugs). Numbers above grey bars indicate the percentage gain (+) or loss (-) in apoptosis when combining VEM with a 2nd kinase-targeting drug. (e) Response to VEM + 2nd kinase-targeting drug in six melanoma cell models maintained in 0.25% FBS media and measured at their respective GI50, GI50x2, and GI50x0.5 concentrations. See Fig. 8d for details. (n≥3). (f-h) Evaluating whether A375 cells expressing a constitutively active AKT1 oncogenic kinase remain sensitive to RPS6KB- or PIM-targeting. Control for myrAKT1 expression is shown in (f) (n=2). A375 myrAKT1 cell growth responses to drug treatments in both 5% and 0.25% FBS culture conditions are shown in (g), and cell death in (h). A375 myrAKT1 cells are less sensitive to VEM than their control counterpart (65% increase in GI50; p=2.77E-06, two-sided paired Student t-test). (i-k) The phospho-RPS6KB profiles of two patient-derived xenografts (PDXs) tumors and derived primary cell lines, as well as their sensitivity to RPS6KB-inhibitor PF-4708671 in 3-week colony formation assays are respectively shown in panels (i-k) (n=1). The PDX tumor M032R6.X1 and derived M032R6.X1.CL line display high levels of RPS6KB1 and is sensitive to RPS6KB1 targeting. Conversely, the PDX tumor M061R.X1 and derived M061R.X1.CL line display low levels of RPS6KB1 and don’t respond to RPS6KB1 targeting. Hence, targeting RPS6KB can be a successful therapeutic intervention in BRAFV600E melanoma tumors/tumor cells where RPS6KB1 is elevated. Such vulnerability may be valuable to restore therapeutic sensitivity in patients who do not respond to, or relapse from, current therapies.
