Figure 3. Histidine 95 on KRAS determines the paralog selectivity of MRTX1133.

A. KRAS^G12D Rasless MEFs were stably transduced with cDNAs expressing HA-tagged KRAS^G12D, KRAS^G12D/H95Q or KRAS^G12D/H95L. Resulting cells were treated with MRTX1133 for 3 days. Cell viability was determined using the CellTiter-Glo assay. Data shown as average of 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. Cell lines used in panel A were treated with 1 μM MRTX1133 (+) or DMSO (−) for one day. Whole cell lysates were harvested and the level of active KRAS was measured using RBD pulldown assay. Same whole cell lysates were immunoblotted to determine the input levels (WCL). Untagged KRAS^G12D and HA-tagged KRAS were detected with a pan-Ras antibody (a representative experiment from two independent repeats is shown).

C. Cell lines used in panel A were treated with MRTX1133 for one day. Cell lysates were immunoblotted for pERK and total ERK. Changes in the level of pERK relative to untreated sample was quantified and shown under the pERK blot (a representative experiment from two independent repeats 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-F. Analogous experiments as in panels A-C were performed in KRAS^G12D Rasless MEFs transduced with cDNAs expressing HA-tagged HRAS^G12D, HRAS^G12D/Q95H, NRAS^G12D, or NRAS^G12D/L95H mutants (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).