Alireza Rahnama

Supplementary Figure S3 shows that dual inhibitors simultaneously target KRAS G12D and HSP90. Specifically, it demonstrates (A) that DI-1 markedly reduces pERK, pAKT, and total AKT levels in G12D-mutated cell lines; (B) that DI-1 decreases GTP-bound active KRAS and total RAS levels; and (C) that DI-1 more effectively inhibits PANC-1 cell proliferation compared to MRTX1133, onalespib, or their combination.

Supplementary Materials and Methods

Supplementary Figure S4 shows the in vivo efficacy of dual inhibitors against KRAS G12D–mutated models. Specifically, it demonstrates (A) that DI-2 treatment reduces cell viability in G12D-mutated PDOs compared to MRTX1133, as shown by live/dead staining, and (B) that DI-2 broadly suppresses receptor tyrosine kinase (RTK) activation in MRTX1133-resistant PANC-1 cells relative to vehicle, MRTX1133, or PROTAC treatment.

Supplementary Figure S1 shows differential sensitivity to MRTX1133 in G12D-mutated cell lines and patient-derived organoids (PDOs). Specifically, it demonstrates (A–B) that MRTX1133 inhibits pERK, which subsequently reactivates at different times across cell lines and PDOs; (C) that KRAS knockdown decreases pERK only in PANC-1 cells but induces apoptosis in AsPC-1 and SK-LU-1 cells; and (D) that KRAS degradation by a G12D-targeted PROTAC does not reduce pERK or pAKT levels, consistent with cell viability results.

KRASG12D is a common oncogenic driver mutation in diverse cancers, including non–small cell lung cancer, colorectal cancer, and pancreatic cancer. KRASG12D inhibitors have recently progressed into clinical trials but will likely face innate or acquired drug resistance similar to that which has been observed for KRASG12C inhibitors, such as activation of receptor tyrosine kinases, KRAS independence, and reactivation of RAS–MAPK signaling. This study investigates heterobifunctional small-molecule dual inhibitors that simultaneously target both KRASG12D and protein chaperone HSP90 in KRASG12D-mutated cancer cell lines and patient-derived organoids. Our findings reveal that the efficacy of the clinical-stage KRASG12D inhibitor MRTX1133 varies, with notable resistance being observed in some cell line and organoid models. In contrast, KRASG12D–HSP90 dual inhibitors were found to broadly display superior effectiveness in inducing apoptosis, reducing cell viability, and suppressing key downstream signaling pathways such as AKT and ERK1/2 in MRTX1133-resistant models. The rationale for targeting HSP90, which is preferentially activated in cancer cells, alongside KRASG12D, arises from the ability of HSP90 inhibition to destabilize substrate client proteins that are essential for cancer cell survival and have also been implicated in resistance to KRAS inhibitors. This dual-inhibitor approach presents a promising new strategy to combat de novo and acquired drug resistance in KRASG12D-mutated cancers and potentially paves the way for improved clinical outcomes by addressing the complex molecular mechanisms underlying cancer cell evolution that enables resistance to conventional inhibitors.

Supplementary Figure S2 shows that MRTX1133 treatment activates receptor tyrosine kinases (RTKs) to overcome KRAS G12D inhibition. Specifically, it demonstrates (A) increased phosphorylation and activation of EGFR family members and other RTKs in a model-dependent manner after 72 hours of MRTX1133 treatment, and (B) unchanged KRAS, HRAS, and NRAS mRNA levels in treated G12D-mutated PDOs.

Compound Synthesis Information

KRAS G12D is a common oncogenic driver mutation in diverse cancers, including non–small cell lung cancer, colorectal cancer, and pancreatic cancer. KRASG12D inhibitors have recently progressed into clinical trials but will likely face innate or acquired drug resistance similar to that which has been observed for KRASG12C inhibitors, such as activation of receptor tyrosine kinases, KRAS independence, and reactivation of RAS–MAPK signaling. This study investigates heterobifunctional small-molecule dual inhibitors that simultaneously target both KRASG12D and protein chaperone HSP90 in KRASG12D-mutated cancer cell lines and patient-derived organoids. Our findings reveal that the efficacy of the clinical-stage KRASG12D inhibitor MRTX1133 varies, with notable resistance being observed in some cell line and organoid models. In contrast, KRASG12D–HSP90 dual inhibitors were found to broadly display superior effectiveness in inducing apoptosis, reducing cell viability, and suppressing key downstream signaling pathways such as AKT and ERK1/2 in MRTX1133-resistant models. The rationale for targeting HSP90, which is preferentially activated in cancer cells, alongside KRASG12D, arises from the ability of HSP90 inhibition to destabilize substrate client proteins that are essential for cancer cell survival and have also been implicated in resistance to KRAS inhibitors. This dual-inhibitor approach presents a promising new strategy to combat de novo and acquired drug resistance in KRASG12D-mutated cancers and potentially paves the way for improved clinical outcomes by addressing the complex molecular mechanisms underlying cancer cell evolution that enables resistance to conventional inhibitors.

Cell-penetrating peptides (CPPs) have emerged as a powerful tool for the delivery of otherwise impermeable cargoes into intact cells. Recent efforts to improve the delivery capability of peptides have mainly focused on the identity of the CPP; however, there is evidence that the identity of the cargo itself affects the uptake. The goal of this work was to investigate how the characteristics of a peptide cargo, including net charge and length, either enhance or diminish the internalization efficiency of the CPP/cargo complex. A small library of CPP/cargo complexes were synthesized consisting of structured and unstructured CPPs with cargoes of net positive, negative, or neutral charge and lengths of 4 or 8 amino acids. Cargoes with a net positive charge were found to enhance the overall uptake of the complexes while net neutral and negatively charged cargoes diminished uptake. Conversely, the net length of the cargo had no significant effect on uptake of the CPP/cargo complexes. Microcopy images confirmed the increased uptake of the positively charged cargoes; however, an increase in punctate regions with the addition of a cargo was also observed. The effects of the net positively charged cargoes were confirmed with both structured and unstructured CPPs, which demonstrated similar trends of an increase in uptake with the addition of positively charged residues. These findings demonstrate that the net charge of cargoes impacts the uptake of the complex, which can be considered in the future when designing peptide-based reporters or therapeutics.