Figure 4. Mutation in H95 and Y96 on KRAS as a potential mechanism for acquired resistance to MRTX1133.
A. KRASG12D Rasless MEFs were stably transduced with HA-tagged KRASG12D or KRASG12D/Y96D. Cells were treated with MRTX1133 for 3 days and cell viability was determined using CellTiter-Glo assay. Dose-response curves were fitted from three independent repeats (Error bars represent S.D. (Data in Figure 3A, 3D and 4A are from the same series of experiments, and they share the same “none” and “HA-KRAS_G12D” controls. Data from these two controls are therefore duplicated in these panels).
B. Cells used in panel A were treated with 1 μM MRTX1133 (+) or DMSO (−) for one day. Whole cell lysates were harvested and subjected to RBD pulldown assay to determine active Ras levels. The same whole cell lysates were also subjected to immunoblotting to determine the input levels (WCL). Shown is a representative of two independent experiments.
C. Cells used in panel A were treated with different concentrations of MRTX1133 for one day. Cell lysates were harvested and immunoblotted for pERK and total ERK. Changes in the level of pERK relative to untreated sample was quantified and shown under the pERK blots (a representative of two independent experiments is shown. Data in Figure 3C and 4C are from the same series of experiments, and they share the same “none” and “HA-KRAS_G12D” controls. Blots from these two controls are therefore duplicated in these panels).
D. Human pancreatic cancer cell line Panc10.05 were stably transduced with HA-tagged KRAS mutants as indicated. Cells were treated with MRTX1133 for 3 days and cell viability was determined using CellTiter-Glo assay. Dose-response curves were fitted from three independent repeats (error bars represent S.D.).
E, Cells used in panel D were treated with or without 1 μM MRTX1133 (+) or DMSO (−) for one day. Cell lysates were harvested and immunoblotted for pERK and total ERK (a representative of two independent experiments is shown).