Yan Dai

KRASG12D is the most prevalent subtype of KRAS mutation across solid tumors, but no drug is available in the clinic. RNK08954 is a potent and selective KRASG12D inhibitor that inhibits proliferation of KRASG12D-mutant cells and demonstrates significant tumor regressions in mouse xenograft models while inhibiting KRAS-mediated signaling. The in vivo effects of RNK08954 are explained by its unique pharmacokinetic (PK) profile and significantly prolonged retention time in tumor tissues. RNK08954 shows synergy with immune checkpoint blockade (ICB). In a phase Ia study, the median follow-up was 4.85 months for 36 evaluable patients. In patients with non–small cell lung cancer (NSCLC), the objective response rate (ORR; unconfirmed) was 58.33%, and in patients with pancreatic ductal adenocarcinoma (PDAC), the ORR (unconfirmed) was 33.33% in the 1,000- to 1,200-mg cohort. This study supports the clinical potential of RNK08954 in patients with KRASG12D mutation either as a single agent or in combination.Significance:RNK08954 is potentially the first orally bioavailable KRASG12D inhibitor, distinguished by unique PK properties, preclinical efficacy, preliminary clinical activity with encouraging results in patients with NSCLC and PDAC, and synergistic potential with ICB. RNK08954 may address a critical unmet need by targeting the most prevalent KRAS mutation across solid tumors.

Supplementary Figure S1. Effect of RNK08954 on KRAS signaling pathway and cell cycle. Supplementary Figure S2. RNK08954 inhibits tumor growth in various human tumor xenograft in vivo (body weight). Supplementary Figure S3. RNK08954 inhibits tumor growth in GP2d tumor xenograft in vivo. Supplementary Figure S4. KRAS G12D inhibition reprograms the immune microenvironment. Supplementary Figure S5. KRAS G12D inhibition increased CD8+ T cell population. Supplementary Figure S6. Exposure at C1D8 of RNK08954 following multiple oral doses (GeoMean). Supplementary Figure S7. ctDNA clearance in NSCLC patients with KRAS G12D mutation treated with RNK08954 from Phase 1a dose escalation.

Supplementary Table S1 Single Intravenous or Oral Dose PK of RNK08954 in the Mice. Supplementary Table S2 Summary of Baseline Characteristics - Phase 1a of RNK08954. Supplementary Table S3 Pharmacokinetic Parameters of RNK08954 Following Multiple Oral Doses of Phase 1a

Additional information for materials and Methods

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.