G12D Mutation Research Articles

Association of KRAS G12D vs. G12V circulating tumor DNA variant allele fraction and real-world overall survival in metastatic non-small cell lung and colorectal cancers.

3041 Background: We previously reported KRAS G12D but not G12V variant allele fraction (VAF), as measured in circulating tumor DNA (ctDNA), was associated with overall survival (OS) in metastatic treatment-naïve pancreatic cancer (mPDAC). However, this has not been examined in other RAS-driven cancers. Here we assessed association of real-world OS (rwOS) with KRAS G12D and G12V VAF in metastatic colorectal cancer (mCRC) and non-small cell lung cancer (mNSCLC). Methods: We queriedGuardantINFORM, a real-world evidence database of aggregated commercial payer health claims and de-identified results from Guardant360 (G360) ctDNA testing from 2014 to 2023, to identify mNSCLC/mCRC patients who were treatment-naive and had KRAS G12D or G12V mutations identified < 30 days prior to first-line therapy initiation. Those with co-occurring KRAS alterations, KRAS amplifications, and/or MSI-H were excluded. Outcomes were assessed as above vs. below median VAF and stratified by treatment type. The Cox regression model was run for continuous VAF, adjusting for age, gender, summary comorbidity score [Elixhauser Comorbidity Index (ECI)], and year of G360. Results: For mCRC, KRAS VAF above median was significantly associated with shorter rwOS for both G12D and G12V (Table 1); subgroup analysis of the predominant standard-of-care chemotherapy, FOLFOX, showed similar findings. For mNSCLC, KRAS VAF above median was associated with shorter rwOS for G12V, but not G12D (Table). Subgroup analysis by first-line treatment type revealed this association to be similar for chemotherapy-containing regimens but not immunotherapy (IO) alone. Conclusions: While a variant-specific association of KRAS VAF with rwOS was observed in mNSCLC overall (G12V associated, G12D not), above vs below median VAF was not associated with rwOS for the subset of patients receiving IO only. In contrast, for mCRC both G12D and G12V VAF were associated with rwOS, including for the subset of patients who received chemotherapy. These data suggest a complex interplay between KRAS variant, ctDNA shedding, first-line treatment, and OS that will be important in the interpretation of the prognostic implications of ctDNA-identified KRAS alterations. [Table: see text]

TTF-1 negativity as a biomarker of outcomes to immunotherapy, chemoimmunotherapy, and KRAS G12C inhibitors in lung adenocarcinoma.

8608 Background: Thyroid transcription factor 1 (TTF-1) expression is routinely assessed in lung adenocarcinomas (LUAD) and is negative (TTF-1Neg) in 15-20% of cases. Whether these tumors differ from LUAD expressing TTF-1 (TTF-1Pos) in terms of biologic features and outcomes is unclear. Methods: Patients (pts) with LUAD and available TTF-1 expression by IHC at five institutions were included. Clinicopathologic, genomic, and outcomes data were compared according to TTF-1 expression. Results: Among 3,315 pts with LUAD, TTF-1 was negative in 15% (N=499). Compared to TTF-1Pos (N=2,816), pts with TTF-1Neg LUAD were more likely male (44% vs 38%, P=0.01), smokers (82% vs 74%, P<0.001), with stage IV disease at diagnosis (76% vs 71%, P<0.001), and lower median PD-L1 tumor proportion score (TPS, 1% vs 10%, P<0.001). At stage IV diagnosis, TTF-1Neg cases less commonly had brain metastasis than TTF-1Pos cases (35% vs 44%, P=0.04), but more frequently had bone metastasis (53% vs 45%, P=0.03). TTF-1Neg LUAD was enriched for KRAS, STK11, KEAP1, SMARCA4, and CDKN2A mutations (q<0.05), while TTF-1Pos LUAD was enriched for EGFR and MET mutations (q<0.05). Compared to TTF-1Pos LUAD, TTF-1Neg LUAD had higher prevalence of KRAS G12D (12% vs 4%, P<0.001) and G12V mutations (11% vs 7%, P=0.001), but similar frequency of G12C mutations (14% vs 15%, P=0.82). The association between TTF-1 and treatment outcomes was next investigated. Pts with stage III unresectable TTF-1Neg LUAD who received durvalumab after chemo-radiation (N=20), compared to TTF-1Pos cases (N=174), had shorter median progression-free survival (mPFS, 7.6 vs 24.7 months, P=0.01) and overall survival (mOS, 22.7 months vs not reached, P=0.01). Among pts with stage IV LUAD, TTF-1Neg cases treated with immune checkpoint inhibitors (N=237), compared to TTF-1Pos (N=1,161), had worse objective response rate (ORR, 17% vs 28%, P<0.001), mPFS (2.5 vs 4.7 months, P<0.001) and mOS (9.8 vs 20.9 months, P<0.001). Similarly, pts with TTF-1Neg LUAD (N=297) treated with chemoimmunotherapy, compared to TTF-1Pos cases (N=920), had worse ORR (27% vs 42%, P<0.001), mPFS (4.6 vs 8.1 months, P<0.001) and mOS (11.2 vs 23.4 months, P<0.001). TTF-1 negativity retained its association with worse outcomes after adjusting for treatment line, ECOG performance status, smoking status, PD-L1 TPS, STK11/ KEAP1 mutation status in multivariable cox regression models. Lastly, pts with KRAS G12C mutated TTF-1Neg LUAD treated with KRAS inhibitors (N=25), compared to TTF-1Pos (N=138), had worse ORR (12% vs 35%, P=0.03), mPFS (2.7 vs 5.9 months, P<0.001), and mOS (4.4 vs 12.1 months, P<0.001). After excluding mucinous LUAD cases, which are more commonly TTF-1Neg, similar results were observed. Conclusions: TTF-1 negativity identifies a unique clinicopathologic and genomic subset of LUAD with worse outcomes to diverse systemic therapies.

Survey of actionable driver mutations KRAS and EGFR in non–small cell lung cancer to evaluate coexisting splicing gene and pathway mutations: A CBioportal database analysis.

8561 Background: Molecularly targeted therapies have advanced treatment for certain cancers, such as lung cancer with specific EGFR or KRAS mutations. However, oftentimes the tumor becomes eventually treatment resistant. This may be partly because cancers driven by EGFR/KRAS mutations may also harbor additional co-occurring genetic alterations. In non-small cell lung cancer (NSCLC), aberrations in RNA splicing pathways have shown substantial implications for tumor behavior and therapeutic responsiveness. Our study focuses on elucidating the interplay between RNA splicing defects and prominent NSCLC mutations. Methods: In this retrospective, cross-sectional study, we analyzed data from 20,719 NSCLC patients enrolled in participating institutions between 2017 and 2022. The data were sourced from the American Association for Cancer Research Genomics Evidence Neoplasia Information Exchange (version 13.0). EGFR L858R, EGFR E746_750del, KRAS G12C, KRAS G12D and other KRAS G12 mutations cases were identified and individually analyzed to abstract co-occurring RNA splicing genomic alterations. Results: Among 20,719 patients with NSCLC, we identified 1309 patients with EGFR L 858R mutation, 1121 patients with EGFR E746_750del mutation, 2446 with KRAS 12C, 852 with KRAS G12D, 1107 with KRAS G12V, 452 with KRAS G12A, 173 with KRAS G12F, and 109 with KRAS G12S. RBM 10 was the most common co-occurring RNA splicing gene for EGFR L 858R (178, 13.6%), followed by CDK12 (31, 2.3%) and SPEN (27, 2 %). Similarly, the most prevalent co-occurring RNA splicing genes with EGFR E746_750del were RBM 10 (38, 3.4%) and SPEN (23, 2 %). RBM 10 was the most prevalent co-existing RNA splicing gene for KRAS G12C (211, 8.6 %), KRAS G12D (63, 7.4 %), KRAS G12V (80, 7.2%), KRAS G12A (59, 13.1%), KRAS G12F (12, 7.0%), KRAS G12S (11, 10.1%) and KRAS G12R (6, 8%). Conclusions: Our study uncovers a crucial link between EGFR and KRAS mutations in NSCLC and RNA splicing gene dysregulation, particularly involving RBM10. Further research in this field is warranted as targeting RNA splicing, alone or alongside existing targeted inhibitors, could substantially enhance treatment efficacy for EGFR+ or KRAS+ NSCLC patients. [Table: see text]

A potent and selective pan-RAS inhibitor, ADT-1004, targeting complex KRAS mutations for pancreatic cancer.

e15085 Background: Pancreatic ductal adenocarcinoma (PDAC) is a challenging cancer with a 5-year survival rate of under 12%. More than 90% of PDAC cases involve KRAS mutations. KRASG12C inhibitors recently developed have limited use because only 3% of PDAC express this mutation, highlighting the urgent need for pan-RAS inhibitors to address the complex mutation landscape in PDAC while avoiding resistance mechanisms. We synthesized and evaluated a novel pan-RAS inhibitor, ADT-1004, in mouse tumor models transplanted with mouse PDAC cell lines or patient-derived xenografts. ADT-1004 is an orally bioavailable prodrug of ADT-007, a highly potent and selective pan-RAS inhibitor (doi.org/10.1101/2023.05.17.541233). Methods: Human and mouse PDAC cell lines were utilized to evaluate in vitro growth inhibitory potency and selectivity of ADT-007. For in vivo experiments, KPC-Luc (1 × 105) and 2838C3-Luc (1.5 × 105) PDAC cells harboring the KRASG12D mutation were injected into the pancreas of C57BL/6J mice. After one week, the mice were randomly divided into treatment groups (n = 7 per group) and received oral ADT-1004 at a dose of 40mg/kg body weight five times a week for 4 weeks. Tumor burden was assessed weekly by monitoring bioluminescence signals using the IVIS Xenogen imaging system. Four patient-derived xenografts from PDAC patients with KRASG12C, KRASG12D, KRASG12V, KRASG13Q mutations were subcutaneously implanted in NSG mice by a small incision in the right flank. A week later, when the tumors reached a size of 100mm3 mice were randomized (n = 7 per group) and treated orally with 40mg/kg of ADT-1004 five times a week for 6 weeks. Body weight and tumor size were measured twice weekly. Results: ADT-007 potently and selectively inhibited the growth of human and mouse PDAC cell lines. For example, the IC50 value of ADT-007 for KRASG12C MIA PaCA-2 PDAC cells was as low as 2 nM compared to 2500 nM for RASWT BxPC3 PDAC cells. Growth inhibition was dependent on activated RAS and associated with reduced GTP-RAS levels and MAPK/AKT signaling. ADT-1004 was well tolerated in mice at a dose up to 175 mg/kg bid orally with sustained plasma levels of ADT-007 far exceeding growth IC50 values. When administered orally at 40 mg/kg body weight, ADT-1004 displayed robust antitumor activity in mouse tumor models using patient or mouse derived PDAC cell lines with G12D, G12V, G12C, or G13Q KRAS mutations, causing significant inhibitory effects on ERK phosphorylation. Conclusions: ADT-1004 inhibited tumor growth of KRAS mutant PDA tumors by targeting activated RAS to disrupt downstream MAPK/AKT signaling. The results highlight the promise of ADT-1004 as a novel pan-RAS inhibitor and open new strategies for addressing the complex genetic landscape of PDAC and other RAS driven cancers.

Synergistic inhibition of CXCL8 following MRTX1133 treatment with ONC212 or 5-FU in KRAS G12D and G12V mutant CRC and PDAC cell lines.

e15196 Background: CXCL8, serves as a crucial multifunctional cytokine that plays a significant role in regulating tumor growth, invasion, and migration. Although in some cancers the critical role of CXCL8 in patient prognosis and disease progression is stablished but its role in CRC & PDAC patients is under investigation. High levels of CXCL8 in tumor microenvironment (TME) and association with cancer stem cell survival, immune surveillance escape & metastasis make it an interesting therapeutic target. Both CRC & PDAC are among the deadliest cancers with dismal prognosis. The need for novel therapeutics with a focus on TME is emerging. MRTX1133 was identified as an inhibitor of KRASG12D. ONC212 is a fluorinated imipridone with strong anti-cancer activity in nM range and preclinical efficacy against pancreatic and other malignancies. Here we report the synergistic inhibition of CXCL8 in both KRAS G12D & KRAS G12V cell lines with use of KRAS G12D inhibitor in combination with 5-FU or ONC212. Methods: Different CRC & PDAC cells were treated with MRTX1133 with 5-FU or ONC212 cytokine profiling at 48h time point was done. Human protein atlas was used for protein and RNA data &TCGA data for survival was used. Results: We previously have shown the synergistic inhibition of pERK and cytokine alteration between MRTX1133 KRAS G12D inhibitor with 5FU or ONC212 in both cell lines with KRAS G12D & KRAS G12V mutation (Tajiknia V et al. AACR 2023 annual meeting). In LS513 KRAS G12D CRC cell line, treatment of ONC212, MRTX1133 and combination of both showed a decrease of 52.36%,58.83% and 71.65% respectively in CXCL8 levels compared to control at 48h time point. In HPAF-II KRAS G12V PDAC cell line, treatment of ONC212, MRTX1133 and combination of both resulted in 38.69%, 64.44%, 57% inhibition of CXCL8 levels respectively compared to control. In SW480 KRAS G12V CRC cell line, treatment of ONC212, MRTX1133 and combination of both demonstrated 25.7%,47.3% and 66.5% inhibition in CXCL8 level respectively compared to control at 48h time point. Single treatment of MRTX1133 caused a decline in CXCL8/IL-8 levels by 30% while single treatment of 5-FU showed a 36% decrease, the combination of both resulted in more than 55% decrease compared to control in LS513 KRAS G12D cell line. In SW480 KRAS G12V cell line, combination of 5-FU & MRTX1133 showed 30% inhibition while in Capan-2 PDAC KRAS G12V cells the same combination showed a 48.2% reduction in CXCL8 compared to control at 48h time point. TCGA data for RNA shows low specificity of CXCL8 for cancer type but it is significantly high in CRC and PDAC. Protein concentration in the Pan-cancer cohort shows significant high level of CXCL8 in CRC. Conclusions: Considering the important role of KRAS mutations and CXCL8 in pathogenesis of CRC & PDAC, the effective inhibition of CXCL8 with the use of targeted therapy in combination could enhance treatment response and patient survival.

An open-label, phase 1, multicenter study to evaluate the safety and preliminary anti-tumor activity of NT-112 in human leukocyte antigen-C*08:02–positive adult patients with unresectable, advanced, and/or metastatic solid tumors that are positive for the KRAS G12D mutation.

TPS2677 Background: The landscape of cancer treatment is evolving with the emergence of innovative immunotherapies. Among these, T cell receptor-engineered T cell (TCR-T) therapy has shown promise for patients with solid tumor malignancies. Targeting driver mutations, such as KRAS, may offer novel therapeutic options for patients with limited treatment options. Oncogenic driver mutations in KRAS typically involve single-base mutations at genomic hotspot locations. The KRAS isoform is mutated in 84% of RAS-driven cancers and in nearly 100% of pancreatic ductal adenocarcinoma (PDAC). The KRAS G12D mutation is the most frequent mutation in PDAC (28%), followed by colorectal cancers (12%). Despite its well-established target role in tumorigenesis, there is no FDA-approved therapy targeting the G12D variant. NT-112 is an autologous TCR-T cell therapy targeting the KRAS G12D mutation presented by HLA-C*08:02, armored by disruption of the gene encoding the transforming growth factor beta receptor type 2 (TGFBR2) in order to reduce the immunosuppressive effect of TGF-β in the tumor microenvironment. Methods: This first-in-human, open label, multicenter, Phase 1, dose escalation study aims to assess the safety, identify the MTD and evaluate potential anti-tumor activities of NT-112 in subjects with solid tumors. The study will enroll up to 24 adult subjects harboring the KRAS G12D mutation that are HLA-C*08:02 positive, who exhibit progressive or recurrent disease after at least one line of standard of care treatment. Enrolled patients will undergo leukapheresis, from which, CD4 and CD8 T cells will be enriched and activated ex vivo. The genes encoding the TGFBR2 and the endogenous TCR α and β chains are knocked out from the activated T cells utilizing clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). The HLA-C*08:02-restricted TCR specific to the KRAS G12D mutation is knocked-in in the TCR α locus. Subjects will undergo standard lymphodepletion followed by a single infusion of NT-112 and concurrent, daily, subcutaneous, recombinant IL-2 for up to 8 days. Dose escalation will occur across 3 ascending dose levels following the BOIN design. Subjects will be monitored for dose-limiting toxicities for 28 days following NT-112 infusion. Disease response will be assessed according to the RECIST v1.1 criteria. Comprehensive translational analysis will be conducted to gain insights into the mechanism of action, biomarkers associated with treatment response, and potential mechanisms of resistance. All subjects will be followed for up to 15 years. Clinical trial information: NCT06218914 .

Non-clinical evaluation of NT-112, an autologous T cell product engineered to express an HLA-C*08:02-restricted TCR targeting KRAS G12D and resistant to TGF-b inhibition.

e14533 Background: Adoptive transfer of T-cell receptor (TCR)-engineered T-cells to target shared cancer neoantigens is a promising new immunotherapy approach for patients harboring mutations in oncogenes such as KRAS. Activating mutations in KRAS are among the most prevalent oncogenic driver mutations in human cancers, with KRAS G12D being the most frequent KRAS mutation in colorectal carcinoma and pancreatic cancer. NT-112 is an autologous engineered T-cell product expressing an HLA-C*08:02-restricted TCR that specifically targets the KRAS G12D mutation. NT-112 is additionally engineered to lack TGF-β receptor II (TGFBR2) expression, rendering T-cells insensitive to TGF-b-mediated inhibition in the tumor microenvironment. Methods: For manufacturing of NT-112, CD4 and CD8 T-cells are enriched from leukapheresis material, activated in vitro and gene-edited to knock-out both endogenous TGFBR2 and TCR β constant 1/2 (TRBC1/2) and knock-in an HLA-C*08:02-restricted KRS G12D neoantigen-specific TCR at the TCR α constant (TRAC) locus using CRISPR-Cas9 technology. NT-112 product functionality was evaluated in both in vivo models and in vitro co-culture experiments with a panel of cell lines including those endogenously expressing the KRAS G12D mutation. T-cell reactivity was assessed by measuring cytotoxicity, proliferation, and cytokine production. For determination of NT-112 product safety, cross-reactivity and allo-reactivity assessments were carried out. A comprehensive analysis of potential off-target editing by CRISPR/Cas9 was performed to assess potential risk of genotoxicity in clinical grade NT-112. Results: High reactivity of the NT-112 TCR against KRAS G12D and HLA-C*08:02-expressing target cells was observed. T-cell activation and functionality was highly specific, as demonstrated by a lack of reactivity against KRAS WT, minimal cross-reactivity in Alanine and Glycine scans, and lack of T-cell activation in the absence of HLA-C*08:02. In vivo, NT-112 T-cells were able to induce tumor clearance in two independent models. Low frequency chromosomal translocation events (<0.1%) between on-target and off-target Cas9 cleavage sites were detectable in NT-112 T-cells. However importantly, these did not result in autonomous cytokine-independent growth. Conclusions: Non-clinical studies revealed a favorable safety and efficacy profile for NT-112 and supported further clinical development of a TGF-β-resistant TCR-edited T-cell product for mutant KRAS-targeted cancer immunotherapy.

Effect of targeting FAK by ifebemtinib and immunogenic cell death of Kras G12C/G12D inhibition on the efficacy of PD1 blockade.

e15102 Background: Focal adhesion kinase (FAK) is one major component of focal adhesion complex and is important in regulating cancer cell survival. Previously, we found that a clinical-stage FAK inhibitor, ifebemtinib (aka IN10018) can overcome drug resistance of Kras G12C inhibitors by down regulating the FAK-YAP signaling. Combination of Kras G12Ci and FAKi has demonstrated encouraging efficacy in NSCLC. Here, we reported that targeting Kras G12D by selective Kras G12Di can also be enhanced by ifebemtinib. Further, Kras G12C inhibition plus PD1 blockade has demonstrated clinical benefits in PD-L1 high expression patient population, but ≈30% of patients experienced early disease progression; this might be attributed to loss-of-function of LKB1 and others. Immunogenic cell death (ICD) of Kras inhibition is enhanced by ifebemtinib, ICD reportedly enhances anti-PD1 activity. Thus, Kras inhibition and PD1 blockade plus ifebemtinib may be an ideal combination for patients with Kras G12D. Methods: Cell killing and apoptosis assays were performed to test the combination of Kras G12D inhibition and ifebemtinib. FACs analysis evaluated exposure levels vs. ICD biomarkers. The mice inoculated with CRC GP2D and PDAC PC-07-0013 were used for evaluating the combinational effects of Kras G12Di and ifebemtinib. The tumor bearing mice with Kras G12D mutant/LKB1 loss, NSCLC KPL, and CRC CT26 with knock-in Kras G12C were used for testing the effects of Kras inhibition, ifebemtinib, and PD1 blockade. Results: Ifebemtinib enhances the drug effects of Kras G12D inhibition in vitro and in vivo. The dual regimen of ifebemtinib and Kras inhibition outperformed either monotherapy with significant ICD induction in vitro (calreticulin/GRP94 exposure). The triple regimen of Kras inhibition, FAK inhibition and PD1 blockade exhibited stronger anti-cancer effects compared to the other related therapies, especially in a Kras G12D mutant, LKB1 loss animal model which is intrinsically resistant to PD1 blockade/Kras G12D inhibition. Conclusions: We discovered that targeting FAK by ifebemtinib enhances the anti-cancer effects of Kras G12D inhibition. It promotes ICD by targeting Kras G12D or G12C, further primes the dual regimen of Kras inhibition and ifebemtinib towards a response to anti PD1 therapy. The preclinical results warrant a further evaluation in the clinic.

Development of an arenavirus-based immunotherapy for treatment of KRAS mutant cancer.

e14672 Background: Oncogenic mutations in KRAS are prevalent in more than 80% of pancreatic ductal adenocarcinomas (PDAC), approximately 30% of colorectal cancer (CRC) and 15-20% of lung adenocarcinoma (LUAD). It has been demonstrated previously that CD8+ T cell responses specific for KRAS mutations can lead to tumor control and regression of metastasis. Replicating arenavirus vectors are currently evaluated in multiple clinical trials in infectious diseases and oncology and have demonstrated potent induction of target antigen specific CD8+ T cell responses of up to almost 50% of the total CD8+ T cell compartment in peripheral blood. To augment KRAS mutation specific CD8+ T cell responses in patients, we are using replicating arenavirus vectors targeting five prevalent KRAS mutations which are found in approximately 95% (PDAC), 74% (CRC), and 83% (LUAD) of the KRAS mutated tumors, representing a promising off-the-shelf immunotherapy for these indications. Here, we systematically investigate immune responses induced by mutant KRAS targeted arenavirus-vector-based cancer vaccines in HLA transgenic mice and characterize the efficiency of target cell killing of activated KRAS mutation specific CD8+ T cells. Methods: A transgene cassette consisting of peptide stretches including KRAS mutations G12D, G12V, G12C, G12R and G13D was generated by in silico aided antigen design. Vectors encoding peptide stretches of single KRAS mutations as well as the corresponding wildtype KRAS sequence served as controls in preclinical experiments. KRAS mutation specific CD8+ T cell expansion was evaluated in HLA transgenic mice and functionality of induced CD8+ T cells was evaluated by assessing CD8+ T cell mediated killing of mutant KRAS peptide loaded target cells in vivo. Results: All treatment regimens were well tolerated, and no mortalities or major adverse events were observed. Following vector administration, HLA I restricted antigen-specific CD8+ T cell responses were detected against 4 encoded mutant KRAS epitopes in HLA-A*11:01 (KRAS G12D and G12V), or HLA-B*07:02 (KRAS G12C and G12R) transgenic mice. These cells did not cross-react with wildtype KRAS epitopes presented in the same HLA I molecules. Specific cytotoxicity against KRAS G12D/V or KRAS G12C/R pulsed target cells was observed in vivo in HLA transgenic animals, indicating functionality of the induced KRAS mutation specific T cells. No specific cytotoxicity towards target cells pulsed with KRAS WT peptides was observed in any of the groups. Conclusions: This study indicates efficient induction of KRAS mutation specific T cell responses in HLA transgenic mice using an arenavirus vector based vaccine. Expanded CD8+ T cells were capable of killing cells loaded with KRAS mutation specific epitopes in vivo indicating functionality of the induced T cell responses. Based on these results, initiation of a Phase I study for the treatment of KRAS mutated cancers is planned.

Identification of Gα12-vs-Gα13-coupling determinants and development of a Gα12/13-coupled designer GPCR

G-protein-coupled receptors (GPCRs) transduce diverse signals into the cell by coupling to one or several Gα subtypes. Of the 16 Gα subtypes in human cells, Gα12 and Gα13 belong to the G12 subfamily and are reported to be functionally different. Notably, certain GPCRs display selective coupling to either Gα12 or Gα13, highlighting their significance in various cellular contexts. However, the structural basis underlying this selectivity remains unclear. Here, using a Gα12-coupled designer receptor exclusively activated by designer drugs (DREADD; G12D) as a model system, we identified residues in the α5 helix and the receptor that collaboratively determine Gα12-vs-Gα13 selectivity. Residue-swapping experiments showed that G12D distinguishes differences between Gα12 and Gα13 in the positions G.H5.09 and G.H5.23 in the α5 helix. Molecular dynamics simulations observed that I378G.H5.23 in Gα12 interacts with N1032.39, S1693.53 and Y17634.53 in G12D, while H364G.H5.09 in Gα12 interact with Q2645.71 in G12D. Screening of mutations at these positions in G12D identified G12D mutants that enhanced coupling with Gα12 and to an even greater extent with Gα13. Combined mutations, most notably the dual Y17634.53H and Q2645.71R mutant, further enhanced Gα12/13 coupling, thereby serving as a potential Gα12/13-DREADD. Such novel Gα12/13-DREADD may be useful in future efforts to develop drugs that target Gα12/13 signaling as well as to identify their therapeutic indications.

Exploring the Relationship Between KRAS, NRAS, and BRAF Mutations and Clinical Characteristics in Iranian Colorectal Cancer Patients.

Patients with colorectal cancer can benefit from anti-EGFR (epidermal growth factor receptor) therapy. However, this therapy is not effective for treating colorectal cancers with constitutive activating mutations in the KRAS, NRAS, and BRAF genes. Molecular analysis of tumor tissue frequently informs treatment decisions for colorectal cancer. This study aims to identify KRAS, NRAS, and BRAF mutations in Iranian patients diagnosed with colorectal cancer and to assess the prevalence of these mutations relative to the tumor differentiation stage within these populations. From April 2018 to December 2022, 2000 specimens from patients with colorectal cancer were collected. Data on sex, age, and tumor differentiation stage were recorded for all samples. For mutation detection, the KRAS and NRAS exons (2, 3, and 4) were amplified using the Diatech kit, and a specific primer was used to amplify BRAF exon 15. Pyrosequencing was then performed. Analysis of samples revealed that 1105 specimens (55.3%) contained mutations in at least one of the screened genes. Among the genes studied, the highest occurrence was the KRAS mutation at 47.4%, followed by NRAS at 5.3% and BRAF at 2.7%. Most KRAS mutations were found in exon 2 (89.7%), with the G12D mutation being the most prevalent at 32% of cases. There was a significant difference in the rate of KRAS mutations in women (52.5%) compared to men (43.5%) (P = 0.02). For NRAS, the majority mutations were observed in exon 3 (76.2%), with the Q61H mutation being the most prevalent at 28.5% of cases. There were no significant associations between the clinicopathological parameters and mutations. The study's findings indicate a rising frequency of mutations in these genes in Iran, highlighting the need to screening mutations in the main exons of all three genes for effective colorectal cancer treatment strategies.

In Silico Prediction of New Inhibitors for Kirsten Rat Sarcoma G12D Cancer Drug Target Using Machine Learning-Based Virtual Screening, Molecular Docking, and Molecular Dynamic Simulation Approaches.

Single-point mutations in the Kirsten rat sarcoma (KRAS) viral proto-oncogene are the most common cause of human cancer. In humans, oncogenic KRAS mutations are responsible for about 30% of lung, pancreatic, and colon cancers. One of the predominant mutant KRAS G12D variants is responsible for pancreatic cancer and is an attractive drug target. At the time of writing, no Food and Drug Administration (FDA) approved drugs are available for the KRAS G12D mutant. So, there is a need to develop an effective drug for KRAS G12D. The process of finding new drugs is expensive and time-consuming. On the other hand, in silico drug designing methodologies are cost-effective and less time-consuming. Herein, we employed machine learning algorithms such as K-nearest neighbor (KNN), support vector machine (SVM), and random forest (RF) for the identification of new inhibitors against the KRAS G12D mutant. A total of 82 hits were predicted as active against the KRAS G12D mutant. The active hits were docked into the active site of the KRAS G12D mutant. Furthermore, to evaluate the stability of the compounds with a good docking score, the top two complexes and the standard complex (MRTX-1133) were subjected to 200 ns MD simulation. The top two hits revealed high stability as compared to the standard compound. The binding energy of the top two hits was good as compared to the standard compound. Our identified hits have the potential to inhibit the KRAS G12D mutation and can help combat cancer. To the best of our knowledge, this is the first study in which machine-learning-based virtual screening, molecular docking, and molecular dynamics simulation were carried out for the identification of new promising inhibitors for the KRAS G12D mutant.

In situ quantitative mapping of coding single nucleotide polymorphism on mRNA inside cells by SERS-fluorescence dual-mode probe

Surface Enhanced Raman Scattering (SERS) has potential in nucleic acid detection, but its ability on single nucleotide polymorphisms (SNPs) detection in the early diagnosis of diseases is still limited. In fact, there is few research about in situ detection of coding SNPs which are carried on mRNA. In this paper, we developed SERS-Fluorescence Dual-mode probes to achieve in situ quantitative mapping of intracellular cSNP. The probes were based on Au@Ag core-shell structure that possessed both excellent biocompatibility and localized surface plasmon resonance. Fluorescence can provide a rapid visualization of intracellular cSNP, while SERS can achieve its in situ quantification. The quantitation was conducted by comparing the SERS signals of the reporting molecule p-mercaptobenzoic acid and fluorescent molecule FAM, the detecting limitation of which can be achieved as low as 1.4 fM. This ratiometric detection is more accurate than traditional analysis based on signal intensity directly. It successfully achieved intracellular imaging of the G12D mutation site of KRAS gene in A549 cells, which can help to get the distribution of cSNP in single cell more clearly. And it also demonstrated the good mapping ability for biomolecules (1003 cm−1 for phenylalanine) simultaneously. In conclusion, this kind of probe could be used as a potential tool to monitor cSNP as well as biomolecules in multiple biological processes, which could provide reference for disease prediction.

Flavonoids as potential KRAS inhibitors: DFT, molecular docking, molecular dynamics simulation and ADMET analyses

KRAS mutations linked with cancer. Flavonoids were docked against KRAS G12C and G12D receptors. Abyssinone III, alpha naphthoflavone, beta naphthoflavone, abyssinone I, abyssinone II and beta naphthoflavone, genistin, daidzin showed good docking scores against KRAS G12C and G12D receptors, respectively. The MD simulation data revealed that Rg, RMSD, RMSF, and SASA values were within acceptable limits. Alpha and beta naphthoflavone showed good binding energies with KRAS G12C and G12D receptors. DFT and MEP analysis highlighted the nucleophilic and electrophilic zones of best-docked flavonoids. A novel avenue for the control of KRAS G12C and G12D mutations is made possible by flavonoids.

Efficacy of Regorafenib and Trifluridine/Tipiracil According to Extended RAS Evaluation in Advanced Metastatic Colorectal Cancer Patients: A Multicenter Retrospective Analysis.

There are few molecular markers driving treatment selection in later lines of treatment for advanced colorectal cancer patients. The vast majority of patients who progress after first- and second-line therapy undergo chemotherapy regardless of molecular data. We aimed to assess the prognostic and predictive effects of specific RAS mutations on overall survival of patients receiving regorafenib (rego), trifluridine/tipiracil (TFD/TPI), or both. This was a retrospective observational study based on data from a previous study of our research network, involving nine Italian institutions over a 10-year timeframe (2012-2022). Extended RAS analysis, involving KRAS exon 2-4 and NRAS exon 2-4, and BRAF were the main criteria for inclusion in this retrospective evaluation. Patients with BRAF mutation were excluded. Patients were classified according to treatment (rego- or TFD/TPI-treated) and RAS mutational status (wild-type [WT], KRAS codon 12 mutations, KRAS codon 13 mutations, KRAS rare mutations and NRAS mutations, KRAS G12C mutation and KRAS G12D mutation). Overall, 582 patients were included in the present analysis. Overall survival did not significantly differ in rego-treated patients according to RAS extended analysis, although a trend toward a better median survival in patients carrying G12D mutation (12.0 months), Codon 13 mutation (8.0 months), and Codon 12 mutation (7.0 months) has been observed, when compared with WT patients (6.0 months). Overall survival did not significantly differ in TFD/TPI-treated patients according to RAS extended analysis, although a trend toward a better median survival in WT patients had been observed (9.0 months) in comparison with the entire population (7.0 months). Patients receiving both drugs displayed a longer survival when compared with the population of patients receiving rego alone (p=0.005) as well as the population receiving TFD/TPI alone (p<0.001), suggesting a group enriched for favorable prognostic factors. However, when each group was analyzed separately, the addition of TFD/TPI therapy to the rego-treated group improved survival only in all-RAS WT patients (p=0.003). Differently, the addition of rego therapy to TFD/TPI-treated patients significantly improved OS in the Codon 12 group (p=0.0004), G12D group (p=0.003), and the rare mutations group (p=0.02), in addition to all-RAS WT patients (p=0.002). The rego-TFD/TPI sequence, compared with the reverse sequence, significantly improved OS only in the KRAS codon 12 group (p=0.003). Our data demonstrate that RAS mutations do not affect outcome in rego-treated patients as well as TFD/TPI-treated patients. Nevertheless, a trend toward a higher efficacy of rego in RAS-mutated (in particular codon 12, rare RAS mutations, and G12D) patients has been recorded. The rego-TFD/TPI sequence seems to be superior to the reverse sequence in patients carrying an RAS codon 12 mutation, although the impact of other factors as disease burden or performance status cannot be excluded.

Abstract LB031: Discovery and Characterization of HH100937, a potent and selective SOS1 inhibitor demonstrates synergistic efficacy in combination with KRAS/MAPK therapies

Abstract Besides marketed inhibitors targeting KRAS G12C, novel therapies are needed to effectively treat cancer patients harboring other KRAS mutations. SOS1, a guanine nucleotide-exchange factors (GEFs), plays a critical role in catalyzing the conversion of KRAS from its GDP- to GTP-bound form, regardless of KRAS mutation status and represents a promising therapeutic target for all KRAS-driven tumors. Up to date, several SOS1 inhibitors have entered early phase clinical trials. But a more potent SOS1 inhibitor with improved drug-like properties is still needed to prove efficacy as monotherapy or in combination with other drugs targeting the KRAS/MAPK pathway.We discovered a potent SOS1 inhibitor, HH100937 that effectively disrupted the interaction between SOS1 and wild-type KRAS or with G12C, G12D or G12V mutations. HH100937 exhibited lower IC50s than BI-3406 and MTRX0902 in blocking ERK phosphorylation and cancer cell proliferation, leading to better anti-tumor efficacies in xenografts than reference compounds at same doses. HH100937 also showed synergistic effect with AMG510 in cancer cells with KRAS G12C mutation but refractory to AMG510. The combination of HH100937 and AMG510 in xenografts led to tumor regression in all mice, while some tumors did not response well to AMG510 monotherapy. Our SOS1 inhibitor also exhibited synergistic effect with HH2710, a clinical-stage ERK inhibitor, in a broad panel of cancer cells with KRAS mutations.The pharmacological potency of HH100937 was highly selective over SOS2, other kinases tested or other targets in a safety panel of 47 targets. HH100937 was well tolerated in 28-day DRF study in rats and dogs up to 250 mg/kg QD and 30 mg/kg QD, respectively. The AUC of drug exposure in DRF studies was approximately three to six times higher than that at the efficacious dose. HH100937 also showed favorable DMPK properties in mice, rats and dogs, and acceptable safety profiles in vitro.Taken together, HH100937 is a highly potent and selective SOS1 inhibitor with better anti-tumor activities than some SOS1 inhibitors at clinical stage. It can be developed as mono therapy or in combination with other KRAS/MAPK targeting therapies. Citation Format: Qing Xie, Dongsheng Li, Shujuan Jiang, Jie Zhu, Songda Yu, Maozhi Yang, Yalei Cai, Lei Liu, Bing Yu, Wangyang Tu, Yixiang Zhang, Leping Li. Discovery and Characterization of HH100937, a potent and selective SOS1 inhibitor demonstrates synergistic efficacy in combination with KRAS/MAPK therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB031.

Abstract LB053: A novel BRAF-specific allosteric inhibitor that effectively targets acquired RAFi-resistant BRAF(V600E) cancers and RAS-mutated cancers in vitro and in vivo

Abstract Targeting hyperactive RAS-RAF-MEK-ERK signaling for treating cancers has achieved a promising outcomes, and a number of RAS/RAF inhibitors have been applied to clinical cancer treatment. However, these inhibitors have only a short-term efficacy and their effectiveness is abrogated by acquired drug resistance in less than one year. For those resistant/refractory RAS/RAF-mutated cancers, there’s no effective therapeutics available in clinic. In this study, we developed a novel BRAF-specific inhibitor that docks on an allosteric site on BRAF kinase domain. This inhibitor effectively killed both primary and drug-resistant cancer cell lines with RAS mutation or BRAF(V600E) mutation in vitro and inhibited the growth of xenografted cancers derived from these cell lines in vivo without apparent toxicity. Mechanistically, we found that although this inhibitor blocked the catalytic activity of BRAF in vitro kinase assay, it triggered a super high ERK signaling through improving BRAF/CRAF heterodimerization and hence induced cellular apoptosis of cancer cells with RAS mutation or BRAF(V600E) mutation. Surprisingly, there’s no drug resistance occurring when treating cancer cells with RAS mutation or BRAF(V600E) mutation at a low concentration. To further evaluate the efficacy of this BRAF against RAS/RAF-mutated cancers in vivo, we constructed a series of pancreatic patient-derived xenograft cancer models with Kras G12D or G12V mutations, and found that our BRAF inhibitor exhibited a comparable or much better efficacy than Kras G12D-specific inhibitor on theses PDX models. Altogether, our data indicated that our BRAF-specific allosteric inhibitor may serve as a good therapeutic drug for treating BRAF(V600E)/RAS- cancers, particularly those resistant/refractory cancers, though it still need further evaluations via clinical trials. Citation Format: Jiancheng Hu. A novel BRAF-specific allosteric inhibitor that effectively targets acquired RAFi-resistant BRAF(V600E) cancers and RAS-mutated cancers in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB053.

Design, synthesis, and evaluation of purine and pyrimidine-based KRAS G12D inhibitors: Towards potential anticancer therapy

Oncogenic RAS mutations, commonly observed in human tumors, affect approximately 30% of cancer cases and pose a significant challenge for effective cancer treatment. Current strategies to inhibit the KRAS G12D mutation have shown limited success, emphasizing the urgent need for new therapeutic approaches. In this study, we designed and synthesized several purine and pyrimidine analogs as inhibitors for the KRAS G12D mutation. Our synthesized compounds demonstrated potent anticancer activity against cell lines with the KRAS G12D mutation, effectively impeding their growth. They also exhibited low toxicity in normal cells, indicating their selective action against cancer cells harboring the KRAS G12D mutation. Notably, the lead compound, PU1-1 induced the programmed cell death of KRAS G12D-mutated cells and reduced the levels of active KRAS and its downstream signaling proteins. Moreover, PU1-1 significantly shrunk the tumor size in a pancreatic xenograft model induced by the KRAS G12D mutation, further validating its potential as a therapeutic agent. These findings highlight the potential of purine-based KRAS G12D inhibitors as candidates for targeted cancer therapy. However, further exploration and optimization of these compounds are essential to meet the unmet clinical needs of patients with KRAS-mutant cancers.

IL-2 promotes expansion and intratumoral accumulation of tumor infiltrating dendritic cells in pancreatic cancer

This study aims to investigate the diagnostic potential of IL-2 for PDAC and develop a method to improve the dendritic cell (DC) based vaccine against PDAC. The gene expression data and clinical characteristics information for 178 patients with PDAC were obtained from The Cancer Genome Atlas (TCGA). DCs were isolated from Human peripheral blood mononuclear cells (PBMCs) and were cultured in 4 different conditions. DCs were pulsed by tumor cell lysates or KRAS G12D1 − 23 peptide, and then used to activate T cells. The mixture of DCs and T cells were administered to xenograft mouse model through the tail vein. The infiltration of DCs and T cells were detected by immunohistochemistry. The generation of KRAS G12D mutation specific cytotoxic T cells was determined by in vitro killing assay. We observed that PDAC patients with higher IL-2 mRNA levels exhibited improved overall survival and increased infiltration of CD8 + T cells, NK cells, naïve B cells, and resting myeloid DCs in the tumor microenvironment. IL-2 alone did not enhance DC proliferation, antigen uptake, or apoptosis inhibition unless co-cultured with PBMCs. DCs co-cultured with PBMCs in IL-2-containing medium demonstrated the strongest tumor repression effect in vitro and in vivo. Compared to DCs obtained through the traditional method (cultured in medium containing GM-CSF and IL-4), DCs cultured with PBMCs, and IL-2 exhibited increased tumor infiltration capacity, potentially facilitating sustained T cell immunity. DCs cultured in the PBMCs-IL-2 condition could promote the generation of cytotoxic T cells targeting tumor cells carrying KRAS G12D mutation.

Abstract 5900: INCB161734: A novel, potent, and orally bioavailable KRAS G12D selective inhibitor demonstrates antitumor activity in KRAS G12D mutant tumors

Abstract KRAS G12D (G12D) is one of the most frequent oncogenic driver mutations, and is especially common in pancreatic (PDAC) and colorectal (CRC) cancers. Patients with G12D-mutated disease experience poor treatment outcomes, representing a significant unmet medical need. G12D mutation results in constitutively active signaling, including hyperactivation of the ERK and PI3K pathways, which drive cell proliferation and survival. Here we describe INCB161734, a novel, potent, selective, and orally bioavailable small molecule G12D inhibitor that demonstrates in vivo efficacy in G12D-bearing tumor models. INCB161734 binds to both the GDP and GTP forms of the G12D mutant at the switch II pocket with picomolar affinity (KD), and exhibits &amp;gt;80-fold selectivity over wildtype (WT) KRAS. INCB161734 potently inhibits SOS1-dependent GDP/GTP exchange activity in cell-free assays (IC50 &amp;lt;3 nM), and exhibits &amp;gt;40-fold selectivity for G12D versus WT KRAS. Additionally, INCB161734 demonstrates high selectivity for G12D over WT in various cellular assays using G12D mutant versus WT cancer derived cell lines. INCB161734 potently inhibits ERK phosphorylation (a correlate for KRAS activity), with a mean IC50 of 14.3 nM (range: 1.9-45.2 nM) across 7 human and 3 mouse G12D cell lines; mean 21.5% inhibition was observed at 1 μM (maximum tested concentration) across 14 WT cell lines. Likewise, INCB161734 inhibits proliferation of G12D mutant cell lines, with a mean IC50 of 154 nM (range: 8.3-318 nM) across the same 7 human G12D cell lines; &amp;lt;30% inhibition (mean 13%) was observed at 1 μM across the same 14 WT cell lines. Treatment with INCB161734 induces caspase 3/7 cleavage in a PDAC cell line (G12D HPAC), with EC50 &amp;lt;100 nM; caspase 3/7 cleavage in a WT cell line (NCI-H838) was not induced at doses as high as 5 μM. In addition, INCB161734 induced cell cycle arrest (S-phase inhibition) in the G12D HPAC cell line, with IC50 12.8 nM; S-phase inhibition in the WT NCI-H838 cell line only occurred at much higher doses (IC50 &amp;gt;3.3 μM). INCB161734 demonstrates excellent oral bioavailability with good absorption, low clearance, and low metabolic turnover. Orally dosed INCB161734 clearly shows target engagement, generating continuous near-maximal KRAS inhibition in G12D HPAC mouse xenograft tumors for almost the entire dosing interval. INCB161734 is efficacious against multiple types of G12D-mutated tumors and xenografts, resulting in significant tumor growth inhibition, growth arrest and/or regression in multiple PDAC (HPAC, Panc0403, and 2838c3) and CRC (CT26, GP2D, and LS513) mouse tumor models. These preclinical results demonstrate that INCB161734 is a potent, selective, and orally bioavailable KRAS G12D inhibitor, strongly efficacious against KRAS G12D mutant tumors. The potential benefit of INCB161734 for patients with KRAS G12D mutant disease is under investigation in ongoing clinical trials. Citation Format: Matthew R. Farren, Valerie Roman, Alexandra Gallion, Abdellah Allali-Hassani, Alexander Sokolsky, Weixi Kong, Amanda Smith, Hui Wang, Gina Correa, Marc Deller, Leslie B. Epling, Jessica Procak, Guofeng Zhang, Katherine Pecko, Keith Kennedy, Jason Boer, Kerri Kurzeja-Lipinski, Maryanne Covington, Kwang-Jong Chen, Renee Wallower, Jennifer Rocha, Rina Pan, Anthony Perry, Brad Yuska, Xiaozhao Wang, Ricardo Macarron, Sunkyu Kim. INCB161734: A novel, potent, and orally bioavailable KRAS G12D selective inhibitor demonstrates antitumor activity in KRAS G12D mutant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5900.