KRAS G13D Mutation Research Articles

Abstract 3315: Preclinical studies of TSN1611, a potent, selective, and orally bioavailable KRASG12D inhibitor

Abstract Background: KRAS mutations are the most frequently encountered driver oncogene, involved in ~25% of all human cancers [1,2]. KRASG12D is the predominant KRAS mutation isoform, detected in approximately 35% of pancreatic cancer, 13% of colorectal cancer, and 5% of NSCLC [3]. Compared to KRASG12C, targeting KRASG12D has proven to be more challenging since the target protein lacks a reactive amino acid residue for irreversible inhibitory modification by a ligand. Herein, we disclose TSN1611, a potent and selective KRASG12D inhibitor, which possesses favorable oral PK profiles and demonstrates significant in vitro and in vivo anti-tumor activity in various KRASG12D-mutant models. Method: Biochemical HTRF assay was used to measure the inhibition of TSN1611 to both GDP-bound and GTP-bound state of KRASG12D. Biophysical SPR method was used to directly measure the binding of TSN1611 to GDP-bound KRASG12D and KRASWT. Cell based activities were evaluated in a series of in vitro cell proliferation assay utilizing Ba/F3 cells engineered with KRASG12D or non-KRASG12D mutations and tumor cell lines harboring KRASG12D mutation. Human cancer cell-derived xenograft models of HPAC (pancreatic) and GP2D (colorectal) were used to evaluate its in vivo antitumor effect. in vitro and in vivo PK studies were performed in mouse, rat, and dog. Systematic nonclinical safety evaluations, including safety panel screen testing, safety pharmacology studies, and repeat-dose toxicity studies were carried out to assess its preliminary toxicity profile. Results: TSN1611 inhibited both active (GTP-bound) and inactive (GDP-bound) forms of KRASG12D protein at IC50 1.23 and 1.49 nM, respectively; the KD value of its direct binding to KRASG12D protein is 1.93 pM in SPR assay. TSN1611 demonstrated potent anti-proliferation activity against several tumor cell lines harboring KRASG12D mutation, and excellent selectivity over cells of NRAS, HRAS, and other KRAS isoforms. It also showed dose-dependent anti-tumor efficacy in GP2D and HPAC models. Mechanism of action studies concluded that the antitumor effect of TSN1611 is resulted from its effective inhibition of KRAS signaling pathway. Oral bioavailability and safety profile across multiple species supported its further development. Conclusion: TSN1611 is a selective KRASG12D inhibitor. It exhibited excellent selectivity and activity both in vitro and in vivo; it demonstrated favorable physicochemical properties, oral PK profiles, and brain penetration potential; it also showed acceptable margins of safety. The preclinical data supports further development. Pending regulatory submission and review, the phase I/II study is planned to start in H1 of 2024.

Abstract 6841: Mutation specific mechanisms of resistance to oncogenic KRAS inhibition

Abstract Mechanisms of resistance to inhibitors against mutant KRAS are linked to the remodeling of the tumor microenvironment (TME). Understanding this remodeling process during intervention and tumor recurrence will likely guide the development of future therapeutic treatment paradigms with long-term responses. 56% of never-smokers with non-small cell lung cancer have KRASG12D mutant cancer and respond little to immune checkpoint therapy (ICI) compared to smokers, who often have mutations in KRASG12C. Our analysis of the TME in a unique KrasG12D inducible and reversible mouse model of NSCLC using single cell RNA sequencing data shows KrasG12D-dependent control of the TME, particularly of the expression of PDL1 in a subset of myeloid cells. PDL1 expression was found to be KrasG12D dependent as it was reduced significantly when KrasG12D was turned ON in mouse lungs. Interestingly, when KrasG12D was turned OFF, the expression of PDL1 increased, indicating mechanisms of resistance. Furthermore, the cytokine expression and secretion of IL6, a known regulator of PDL1 expression, increased in lung tissue and lung cancer cells derived from this model upon genetic and chemical (MRTX1133) Kras inhibition. Moreover, the mechanisms of resistance to oncogenic Kras inhibition appear to be mutant specific. Transcriptome and secretome analyses in human and murine KrasG12D or KrasG12C mutant lung cancer cells show distinct cytokine regulation upon KRAS inhibition. Identification of mechanisms of resistance like the induction of PDL1 expression upon KrasG12D inhibition may provide a strong rationale for co-treatment of KRAS inhibitors with ICI for KrasG12D never-smokers with NSCLC. Citation Format: Ivana Barravecchia, Emily Lasse-Opsahl, Sophia Cavanaugh, Lily Rober, Rachael Baliira, Marzia Robotti, Rachael Hinshaw, Jennifer M. Lee, Yaqing Zhang, Marina Pasca di Magliano, Stefanie Galbán. Mutation specific mechanisms of resistance to oncogenic KRAS inhibition [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 6841.

Abstract 3392: Generation and characterization of transgenic oncopig model for lung cancer

Abstract Lung cancer (LC) is the leading cause of cancer-associated death in the United States. Preclinical findings in small animal models do not translate into human clinics. There is an urgent need to develop relevant preclinical large animal models for LC. We describe a novel method to induce LC in transgenic pigs carrying Cre-inducible KrasG12D and TrP53R167H mutations (Oncopigs). The Oncopigs (n=12; 9 females, and 3 males) at 9 weeks of age were anesthetized and adenovirus (1 × 1011 PFU) carrying Cre recombinase gene (Ad-Cre) ± polybrene (1:100) ± IL-8 (5 ng/ml) was injected via flexible bronchoscopy with two techniques: (1) Bronchial lavage (n=6) and (2) Transbronchial needle injection (n=6) into different lung lobes. As controls, a combination of polybrene ± IL-8 was injected without AdCre into the contralateral lobes in the same pigs (n=8). Oncopigs were monitored via clinical monitoring, blood counts, basic metabolic panel, and contrast-enhanced computed tomography (CT) imaging for LC progression and metastasis at 3-, 4-, 7-, 10-, 16-, and 26 weeks post-intervention. All animals tolerated the injections with expected weight gains. Blood counts and metabolic panels remained normal during LC progression. At the site of injection/lavage, lung masses were detected on CT imaging at 3 weeks post-injections. However, a decrease in masses was observed on CT at 7 weeks post-intervention. The decrease in size of the lung masses coincided with increasing attenuation. No distant metastases were observed on CT imaging. Lavage-based lesions appeared to be more heterogeneous with surrounding inflammatory changes. Transbronchial-injection-based lesions appeared more consolidated and coin-shaped without surrounding inflammatory changes. Control injection sites did not show any mass growth on CT imaging. Immunohistochemistry analysis on lung tumors revealed increased pancytokeratin, trichrome, E-cad, Ki-67, IBA, and CD3 expression. Further, immunofluorescence revealed decreased E-Cad, CK-19, and Zo-1 expression, whereas increased expression of N-Cad, FN1, snail, CD44, α-SMA, and γ-catenin, was observed in the LC and metastatic tissue compared to normal. We successfully generated and characterized a novel transgenic Oncopig LC model. There is robust tumor production status post AdCre delivery. One of four Oncopigs had carcinomatous metastatic skin growth. The remaining 7 Oncopigs are monitored longitudinally for tumor growth and development of metastasis. Citation Format: Kirtan Joshi, Nagabhishek Sirpu, Amanda Schmelzle, Ravikanth Poonooru, Benjamin Nelson, Pradeep Subramanyam, Colleen Garrett, Mariana Sponchiado, Sarah Schlink, Samantha Gerb, Jesse Porter, Dae Kim, Jeffrey Kunin, Jeffrey Bryan, Timothy Hoffman, Bhanu Telugu, Jussuf Kaifi, Satyanarayana Rachagani. Generation and characterization of transgenic oncopig model for lung cancer [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 3392.

Abstract 7080: Regulation of caveolin-1 by RAS/RAF oncogenic signaling in cancer cells

Abstract Background and Purpose: Caveolin-1 (Cav-1), a scaffolding protein implicated in cellular signaling and membrane dynamics, undergoes intricate regulation at multiple levels. Recent evidence has suggested a potential association between oncogenic mutations and alterations in Cav-1 expression. Our study aimed to comprehensively explore how RAS/RAF mutations influence Cav-1 expression in cancer cells, as well as the underlying molecular mechanisms, using tetracycline (tet)-inducible cell lines and cancer cell lines harboring endogenous BRAF or KRAS mutations. Experimental Design: We assessed the regulation of Cav-1 expression using tet-inducible cell lines with BRAFV600E, KRASG12D, or KRASG12C mutations. We measured the half-life of Cav-1 at protein levels by using cycloheximide. We monitored Cav-1 localization and degradation dynamics by SNAP-based pulse-chase assay. Additionally, we performed protein and gene expression analyses by immunoblotting and qRT-PCR, luciferase reporter assay, and immunofluorescence to dissect the impact of BRAF and KRAS mutations on Cav-1 regulation at various stages of its life cycle. Results: We found a significant downregulation of Cav-1 at both mRNA and protein levels upon induction of BRAF or KRAS mutations. The use of KRASG12C, BRAFV600E, ERK, and/or MEK inhibitors in tet-inducible cell lines rescued the effects observed upon induction. The rescue effects have also been observed in H23, MiaPaCa-2 and 8505-C cancer cell lines bearing either BRAF or KRAS mutations. Notably, BRAFV600E exhibited a more pronounced reduction in Cav-1 expression than other mutations. We observed shortened half-life of Cav-1 at protein levels after induction of RAS/RAF mutations by using cycloheximide, and SNAP-based pulse-chase. These mutations accelerated lysosomal-mediated degradation of Cav-1 as determined by immunoblotting and immunofluorescence, which was reversed by lysosomal inhibitors. Conclusions: Our study establishes a novel regulatory paradigm wherein RAS/RAF mutations exert a multi-faceted influence on Cav-1 dynamics in cancer cells. The transcriptional and translational downregulation, coupled with accelerated lysosomal degradation, collectively contributes to the decreased half-life of Cav-1 following the induction of BRAF or KRAS mutations. These findings provide insights into the intricate molecular mechanisms underpinning the dysregulation of Cav-1 in the context of RAS/RAF mutations, offering potential avenues for therapeutic interventions. Citation Format: Veronica Castro Aceituno, Tiantian Cui, Haihua Feng, Linlin Yang, Sindhu Nair, Terence M. Williams. Regulation of caveolin-1 by RAS/RAF oncogenic signaling in cancer cells [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 7080.

Abstract 4101: Immune inhibition of murine colorectal cancer by a p53 peptide mucosal vaccine

Abstract Colorectal cancer (CRC) is among the most common cancers with high morbidity and mortality for patients despite multi-modality treatment options. Immune checkpoint inhibitors often have impact in the 15% of CRC patients with microsatellite instability-high status CRCs but have limited utility in the remaining 85% of CRC patients. Given the presence of p53 mutations in CRC, we pursued a vaccine approach against tumor cells expressing murine CRC Trp53 codon 270 missense alleles (codon 273 mutation in human cancer) using a combination of R270H p53 peptide, wild type p53 recombinant protein and used a mucosal adjuvant (MA) to drive epithelial immune responses. The vaccine (p53/MA) was studied in a CDX2-CreERT2 (tamoxifen-induced Cre-Lox) transgenic mouse CRC model where tumors arise in the cecum and proximal colon epithelium following combined ApcΔ580, KrasG12D, and Trp53R270H mutations (AKP model). The p53/MA vaccine treatment was administered intranasally to the mice at defined time points after tamoxifen tumor induction. p53/MA vaccinated animals had increased anti-p53 IgG (p<0.01), IgG2a (p<0.01), and IgG2b (p<0.001) in sera compared to control mice. The treatment enhanced antigen-specific Th1 and Th17 cellular immune responses, as shown by increasing of IFN-ɤ (p<0.05), IL-17a (p<0.01) and IL-2 (p<0.01) both in splenocytes and draining lymph nodes measured by Luminex assay. Vaccinated mice also showed significant more cells producing IFN-ɤ (p<0.001) and IL-17a (p<0.001) as analyzed by ELISpot. Importantly, the immunized animals had decreased tumor size/volume (p<0.001) and prolonged survival (p<0.05) compared to control animals that received only saline or MA respectively. Tumors from vaccinated mice also showed more mononuclear cell infiltrates than tumors from the control animals. In conclusion, novel p53/MA vaccinations can induce significant p53-specific immune responses and anti-tumor effects in the mouse AKP CRC model. Citation Format: Suhe Wang, Zhengyi Cao, Katarzyna W. Janczak, Ying Feng, Maranne Green, Eric R. Fearon, James R. Baker Jr. Immune inhibition of murine colorectal cancer by a p53 peptide mucosal vaccine [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 4101.

Abstract 3319: The MTA-cooperative PRMT5 inhibitor, MRTX1719, demonstrates increased anti-tumor activity in combination with KRAS mutant-selective inhibitors in MTAP del,KRAS-mutant cancers

Abstract MRTX1719 is an MTA-cooperative PRMT5 inhibitor that leverages the increased concentration of the metabolite MTA in cancer cells harboring a homozygous deletion of the MTAP gene (MTAP del) under investigation in clincial trials. MRTX1719 preferentially binds to the PRMT5•MTA complex to selectively inhibit PRMT5, an essential gene for all cells, in MTAP del cancer cells while sparing PRMT5 activity in normal MTAP-wildtype cells. MTAP del occurs in ~10% of all cancers, and these patients exhibit remarkably poor survival. Among pancreatic cancer, MTAP del occurs in ~ 25% of patients and of these ~30% have co-occurring KRAS G12D mutations. In addition, ~15% of lung adenocarcinoma patients harbor MTAP del and of these ~13% harbor KRAS G12C. While recently approved KRAS G12C mutant selective inhibitors demonstrate anti-tumor activity, strategies to address drug resistance and maximize clinical benefit are warranted. Combination of MRTX1719 with adagrasib or the KRAS G12D inhibitor MRTX1133 in CDX models harboring both a KRAS mutation (G12C or G12D) and homozygous MTAP deletion resulted in increased anti-tumor activity compared to either single agent. This includes the pancreatic KP4 model in which initial tumor regression in response to single agent MRTX1133 was observed, followed by adaptative resistance and tumor progression with monotherapy while co-administration with MRTX1719 continued to result in marked tumor regression. Mechanistically, combination treated animals demonstrated strong inhibition of both PRMT5 and RAS pathway signaling and combinatorial targeting of these pathways converged on enhanced inhibition of RB-1 phosphorylation. We also characterized this combination treatment in the context of resistance to KRAS selective inhibitors or MRTX1719 to evaluate the benefit of combination over sequential treatment. Finally, an NF1 mutant, MTAP del malignant peripheral nerve sheath tumor (MPNST) PDX model was treated with MRTX1719, the SOS1 inhibitor MRTX0902, or the combination, and a similar increase in anti-tumor activity was observed in the combination treatment compared to either monotherapy alone. These results suggest that the combination of an MTA-cooperative PRMT5 inhibitor with a KRAS mutant selective or KRAS pathway inhibitor may lead to deeper and more durable responses in MTAP del cancer patients with co-alterations in the KRAS pathway. Citation Format: Laura Waters, Ruth Aranda, Xousaen Helu, Laura Vegar, Krystal Moya, Andrew Calinisan, Allan Hebbert, Jill Hallin, Darin Vanderpool, David M. Briere, James G. Christensen, Peter A. Olson, Lars D. Engstrom. The MTA-cooperative PRMT5 inhibitor, MRTX1719, demonstrates increased anti-tumor activity in combination with KRAS mutant-selective inhibitors in MTAP del,KRAS-mutant cancers [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 3319.

Abstract 7279: Discovery of SHR1127, an orally bioavailable, highly potent and selective KRAS G12D inhibitor

Abstract Mutant KRAS is the most frequent oncogenic driver in nearly 23% of human cancers. As the most common KRAS alteration, KRAS G12D mutation occurs in approximately 35%, 13%, and 4% of pancreatic, colorectal, and non-small cell lung cancers, respectively. It was worth noting that the clinical PoC of KRAS G12D inhibitor has been preliminarily validated by intravenous administration of HRS-4642 weekly. However, there still represents an extraordinary unmet clinical need requiring an oral KRAS G12D inhibitor featuring higher potency and favorable pharmacokinetic profiles. We disclose herein the development of an orally bioavailable, highly potent and selective KRAS G12D inhibitor, SHR1127.SHR1127 demonstrated picomolar binding affinity, with a KD value of 0.06 nM in a SPR binding assay with GDP-loaded KRAS G12D protein. SHR1127 significantly inhibited ERK phosphorylation with IC50s of 0.05 nM and 0.19 nM in GP2d and AGS cell lines, respectively. More importantly, 3D viability assay showed that SHR1127 displayed extremely high anti-proliferative activity against GP2d, AGS and AsPC-1 cell lines with the IC50s of 0.05, 0.83 and 0.92 nM, respectively. The selectivity over KRAS WT PC9 cells, KRAS G12C and G12V mutant cell lines was more than 500-fold. Additionally, SHR1127 displayed excellent human hepatocyte stability (T1/2 > 1000 min) and desirable physicochemical properties. Moreover, SHR1127 exhibited favorable pharmacokinetic profiles with dose-dependent exposure and acceptable oral bioavailability. In the GP2d xenograft model, SHR1127 showed a robust anti-tumor activity in a dose-dependent manner, leading to tumor regression (TGI = 125%) as low as 5 mg/kg (p.o. dosage, bid), without body-weight loss during 21 days of treatment. Furthermore, SHR1127 demonstrated no off-target activities at 1000 nM in 97 kinase panel and 78 safety panel screening. SHR1127 showed good safety window in the 14-day toxicology study in rat and dog. In summary, SHR1127 has been successfully developed as an orally bioavailable KRAS G12D inhibitor, exhibiting excellent cellular activities with good selectivity over KRAS WT. More importantly, SHR1127 showed favorable pharmacokinetic profiles across species, and robust tumor regression in a GP2d xenograft model at the dose of 5 mg/kg. In addition, SHR1127 demonstrated no off-target toxicity and good safety window. An IND submission for SHR1127 is planned in the first half of 2024. Citation Format: Xin Li, Feng Shen, Limin Zhang, Wei Wang, Luyao Kong, Yong Bao, Yuchang Mao, Zaiyong Wang, Sophie Lin, Zhe Zhang, Jun Feng, Min Hu, Feng He. Discovery of SHR1127, an orally bioavailable, highly potent and selective KRAS G12D inhibitor [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 7279.

Abstract 3318: GFH375 (VS-7375): An oral, selective KRAS G12D (ON/OFF) inhibitor with potent anti-tumor efficacy

Abstract Background: KRAS is the best-known oncogene but had long been considered ‘undruggable’ until the approval of the first KRAS G12C inhibitor sotorasib in 2021. However, most current clinical-stage KRAS inhibitors target the KRAS G12C mutation. Inhibitors of other KRAS mutants such as KRAS G12D, the most frequent KRAS mutation in human cancer, are needed for patients. We have developed GFH375 (VS-7375), an oral, selective KRAS G12D inhibitor targeting both “ON” (GTP-bound) and “OFF” (GDP-bound) states of KRAS proteins. The in vitro potency, selectivity, and in vivo efficacy of monotherapy and combination therapy with avutometinib, a unique RAF/MEK clamp, were evaluated in preclinical studies. Methods: Biochemical and cellular assays were used to investigate inhibition to KRAS cycling and signaling. CellTiter-Glo assay was performed to determine the effects on proliferation of tumor cell lines. Several KRAS G12D CDX tumor models were employed to study the in vivo pharmacodynamic and anti-tumor effects. Results: GFH375 inhibited both nucleotide exchange on GDP-bound KRAS G12D and interaction between GMPPNP-bound KRAS G12D with RAF1 with single-digit nanomolar IC50 values. GFH375 suppressed phosho-ERK1/2 (p-ERK) level with sub-nanomolar IC50 values and potently inhibited cell proliferation across a panel of KRAS G12D tumor cell lines. GFH375 showed high selectivity for KRAS G12D relative to non-G12D KRAS variants, KRAS wild type cells, and NRAS, HRAS, or BRAF mutated cells. Following a single oral dose, GFH375 produced deep and durable inhibition of p-ERK in KRAS G12D CDX tumors. GFH375 demonstrated dose-dependent anti-tumor activity with tumor regressions at 10 or 30 mg/kg given orally twice daily in multiple KRAS G12D PDAC and CRC CDX tumor models. GFH375 also showed strong anti-tumor efficacy in an intracranial KRAS G12D tumor model starting as low as 10 mg/kg orally twice daily. Strong synergy between GFH375 and avutometinib was observed in vitro and was validated in vivo as anti-tumor activity of GFH375 was further enhanced by avutometinib. Conclusions: GFH375 is a highly potent and selective orally active inhibitor of KRAS G12D (ON/OFF) and demonstrated promising anti-tumor activity in multiple KRAS G12D tumor models. GFH375 was also effective in an intracranial tumor model. GFH375 showed strong synergy with avutometinib in vitro and in vivo. GFH375 is currently in IND-enabling development in preparation for clinical studies of monotherapy and potentially in combination with other agents for patients with KRAS G12D mutant tumors. Citation Format: Feng Yan, Tao Jiang, Tao Liang, Lijian Cai, Leitao Zhang, Xiaoming Xu, Yanhui Zhao, Xiaoling Lan, Xiaohui Zhang, Meng Liu, Qiang Liu, Jinting Gao, Fubo Xie, Xueyan Gao, Li Wang, Jingyang Zhang, Hongcan Ren, Dong Liu, Siyuan Le, Lili Tang, Silvia Coma, Yaofeng Cheng, Nathan Sanburn, Jonathan A. Pachter, Fusheng Zhou, Jiong Lan, Qiang Lu. GFH375 (VS-7375): An oral, selective KRAS G12D (ON/OFF) inhibitor with potent anti-tumor efficacy [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 3318.

Abstract 1943: Discovery and characterization of potential drivers of resistance to the KRAS G12D inhibitor, MRTX1133, in cancer cell line models

Abstract Introduction: KRASG12D is the most common KRAS mutation and is present in approximately 34% of pancreatic cancer, 10-12% of colorectal cancer, 4% of lung adenocarcinoma and in a number of other cancer types. We previously identified MRTX1133, a potent, selective, and non-covalent KRASG12D inhibitor. The advancement of MRTX1133 to clinical trials warrants continued study of the role of the KRASG12D mutation in cancer pathogenesis and progression. Methods: Previous pharmacodynamic and pharmacogenomic profiling in sensitive and partially resistant models identified mechanisms implicated in limiting the anti-tumor response including signaling pathways that induce feedback reactivation and/or bypass KRAS dependence. Thus, cell lines (GP2D and SNU-1033, colorectal cancer; HPAC and AsPC-1, pancreatic cancer) were utilized to study acquired resistance to MRTX1133 following continuous treatment at increasing concentrations over time. These cell lines were confirmed to be stably resistant as determined by evaluation of response to MRTX1133 in vitro and in vivo. These cell lines were extensively profiled to determine underlying mechanisms of resistance utilizing molecular profiling strategies as well as drug combination screens to interrogate pathways that could circumvent resistance. Results: In one of the four generated cell lines with acquired resistance, evidence of secondary KRAS mutations Y96N and H95Q were confirmed by RNAseq and are likely drivers of resistance in this cell line and are consistent with known mechanisms of KRASG12C inhibitor resistance. The three other MRTX1133 resistant cell lines did not show evidence of secondary KRAS mutations and were further evaluated for mechanisms that bypass KRAS dependence. Combinations of MRTX1133 with therapies that target pathways including upstream RTK signaling, mTOR, or AKT broadly enhanced the anti-tumor activity of MRTX1133 in both in vitro cell viability and in vivo efficacy studies in the resistant cell lines. Conclusion: Collectively, these data provide early insight onto potential mechanisms of resistance to selective KRASG12D inhibitors and demonstrate the importance of developing rational combination strategies designed to address drug resistance. In addition, these studies provide a catalogue of potentially druggable vulnerabilities that complement KRAS blockade. Citation Format: Xousaen M. Helu, Ellie Griffiths, Andrew Calinisan, Allan Hebbert, Larry Yan, Natalie Hoffman, David Trinh, Laura Hover, Julio Fernandez-Banet, David M. Briere, Ella Lifset, Peter Olson, James G. Christensen, Jill Hallin. Discovery and characterization of potential drivers of resistance to the KRAS G12D inhibitor, MRTX1133, in cancer cell line models [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 1943.

Abstract 4312: KRAS oncogenic activation in Gramd2 + AT1 cells form lung adenocarcinoma with distinct transcriptomic and tumor microenvironment signatures

Abstract Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer and presents with a high degree of biological heterogeneity and distinct clinical outcomes. The current paradigm of LUAD etiology recognizes alveolar epithelial type II (AT2) cells as the primary cell of origin, while the role of AT1 cells in LUAD oncogenesis remains relatively unknown. Here, we examined transformation via activation of the oncogenic KRASG12D mutation in a Gram-domain containing 2 (Gramd2)+ AT1 cell lineage tracing model system, which resulted in multifocal LUAD, primarily of papillary histology. Spatial transcriptomic sequencing revealed significant differences of transcriptomic signatures and signaling activities between Gramd2+ AT1 cell-derived and Sftpc+ AT2 cell-derived LUAD. Single-cell RNA sequencing of macrodissected LUAD lesions demonstrated heterogeneous cell composition of the tumor microenvironment (TME), with Gramd2+ AT1 cell-derived LUAD fostering a proinflammatory, immunoreactive TME and Sftpc+ AT2-derived LUAD presenting with an immunosuppressive TME. Collectively, our study suggests that cell of origin for LUAD is associated with histologic subtype, transcriptomic aberrations, and immune cell composition within LUAD that may influence response to therapy. Citation Format: Minxiao Yang, Jonathan Castillo, Edgar Gonzalez, Chunli Yan, Evanthia Roussos Torres, Zea Borok, Crystal Marconett. KRAS oncogenic activation in Gramd2 + AT1 cells form lung adenocarcinoma with distinct transcriptomic and tumor microenvironment signatures [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 4312.

Abstract 6512: BPI-501836: A highly potent small molecule inhibitor targeting KRASG12D mutation

Abstract As one of the most notorious un-druggable oncogenes, mutant KRAS impairs the transition of RAS from its active GTP-binding form to the inactive GDP-binding form, leading to sustained activation of downstream MAPK pathway and ultimately tumorigenesis. The recent approved KRASG12C inhibitors have demonstrated clinical benefits in KARSG12C mutant patients. However, the other prevalent KRAS mutations, such as KRASG12D, remain a huge unmet medical challenge. There’s no approved KRASG12D specific inhibitors yet. Here we present BPI-501836, a highly potent and selective small molecular inhibitor for KRASG12D. In vitro, BPI-501836 exhibited very high potency by disrupting the KRASG12D and cRAF interaction in a cell-free assay. In cell-based assays, BPI-501836 inhibited proliferation of KRASG12D mutant cell lines with sub-nanomolar IC50 values, without affecting wide type or non-G12D KRAS mutant cell lines. Moreover, it also attenuated ERK phosphorylation in KRASG12D mutant cells. In vivo, tumor regression was observed following the administration of 6 mg/kg (iv, QW) BPI-501836 in KRASG12D human pancreatic Panc04.03 xenograft mouse models, or in the KRASG12D human pancreatic PK-59 xenograft mouse models with an intraperitoneal injection of 20 mg/kg per week. Noteworthy was the prolonged half-life of BPI-501836 observed across various species, coupled with its high distribution in tumor tissues. Moreover, sustained suppression of ERK phosphorylation in tumor tissues persisted for up to 144 hours after the last dose of BPI-501836 in both xenograft mouse models. This extended pharmacodynamic effect supports the feasibility of a once-weekly dosing schedule in clinical applications. Safety assessments conducted through a 4-week repeated infusion (intravenous, twice weekly) of BPI-501836 in beagle dog demonstrated favorable safety profiles. In conclusion, BPI-501836 emerges as a highly potent and selective KRASG12D inhibitor, exhibiting promising pharmacokinetics, pharmacodynamics, and safety profiles in pre-clinicalstudies. BPI-501836 is currently in IND-enabling stage and holds potential for further development as a therapeutic agent. Citation Format: Xiang Yang, Yuan Lu, Renqi Xu, Xiaoqiang Kong, Yongqing Sun, Xingjie Zhu, Xiaoyun Liu, Zhengyao Zou, Haibo Chen, Yanju Liu, Jialing Sun, Han Han, Cheng Yang, Jing Guo, Hong Lan, Quan Zhou, Lieming Ding, Hao Wu, Jiabing Wang. BPI-501836: A highly potent small molecule inhibitor targeting KRASG12D mutation [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 6512.

Abstract 3938: A novel mouse model reveals potential therapeutic strategies for RIT1-driven lung adenocarcinomas

Abstract Gain-of-function oncogenic mutations in RIT1 have been identified in lung adenocarcinoma and are mutually exclusive with other reported driver mutations, highlighting the role of RIT1 as a cancer driver. RIT1 is a poorly-studied non-classical RAS GTPase and, to date, there are no reported genetically-engineered mouse models of RIT1-driven lung adenocarcinoma. Here, we have generated a conditional knock-in allele within the endogenous mouse Rit1 locus, introducing the most frequent mutation observed in lung adenocarcinomas (RIT1M90I). Upon intranasal administration of adenovirus encoding the Cre recombinase to induce somatic recombination in the mouse lungs, we observed the formation of discrete adenomas. Although Rit1M90I mutation can promote lung tumor initiation in mice, we found that this phenotype is less penetrant than the common KrasG12D mutation. Immunohistochemistry analysis of Rit1M90I and KrasG12D tumors showed expression of typical histological markers associated with lung adenocarcinoma (i.e., TTF1 and SPC), as well as increased levels of Ki67 and pERK expression. Next, to develop potential pharmacological strategies for RIT1-driven lung tumors, we first explored drugs that inhibit the RAS/MAPK pathway and are either FDA-approved or under clinical development. In vitro experiments with cell lines carrying the RIT1M90I mutation demonstrated high sensitivity to SHP2, MEK, RAF, and RAS inhibitors. In vivo, SHP2 and RAS, but not MEK, inhibitors slowed down tumor progression in both cell line and patient-derived xenografts. Our results indicate that, although RAS signaling might be needed for RIT1-dependent growth, these tumors are not sensitive to MAPK inhibition as a monotherapy and, hence, other pathways could contribute to tumor growth. Therefore, we decided to explore whether targeting RIT1 directly, could be a more beneficial strategy for these tumors. In order to inhibit oncogenic RIT1 directly, we have undertaken two complementary approaches. First, because RIT1 mutations were shown to impair its proteolysis, we explored whether restoring RIT1 proteasomal degradation could be a promising strategy. Recently, the development of Proteolysis Targeting Chimeras (PROTAC) has demonstrated that targeted degradation can be used as a pharmacological approach for the treatment of cancer. Therefore, we have tested successfully several proof-of-concept PROTAC approaches that caused degradation of oncogenic RIT1 in different cellular models. Second, we used a chemical biology approach to assess whether covalent inhibitors directed against the closely-related GTPase KRAS could cross-react and bind to oncogenic RIT1. We have identified a molecule that binds RIT1 and we are currently characterizing it in our preclinical models. Overall, our work provides comprehensive insights into the oncogenic role of RIT1 in lung cancer emphasizing its potential as a therapeutic target. Citation Format: Alessandro Mauro Mozzarelli, Antonio Cuevas-Navarro, Anatoly Urisman, Pau Castel. A novel mouse model reveals potential therapeutic strategies for RIT1-driven lung adenocarcinomas [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 3938.

Abstract 2667: Mucin 5AC inhibition via intratracheal RNAi delivery as an effective alternative strategy for prevention and treatment of Kras mutant lung cancer

Abstract Lung cancer, including lung adenocarcinoma (LUAD), is the leading cause of cancer-related deaths in the United States. KRAS mutations are the most prevalent oncogenic driver alteration in human LUAD. We have previously shown that high expression of Mucin 5 AC (MUC5AC), a main airway secretory mucin, is significantly associated with poor prognosis. Mucins are essential for mucociliary clearance and the maintenance of airway homeostasis, however, their overproduction could negatively affect the lung immune microenvironment and has been shown to promote airway diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer. Previously, we have shown that genetic deletion of MUC5AC in a murine model of K-ras mutant lung adenocarcinoma (KM-LUAD) impedes tumor development and reduces pro-tumor inflammatory and immune phenotypes. These findings indicate that MUC5AC is a potential druggable target against KM-LUAD. In this study, we sought a translational approach and inhibited MUC5AC in our K-rasG12D mutant mouse model of LUAD via RNA interference (RNAi). Six-week-old mice were given RNAi (5mg/kg) or vehicle (saline) via biweekly intratracheal administration for 8 weeks. This approach led to a significant decrease in tumor burden (40%) and a trending increase in total white blood cell counts, predominantly macrophages in the lung tumor microenvironment which was associated with phenotypic changes in macrophages characterized by reduced expression of Fizz1, and M2 pro-tumor macrophage marker. Additionally, RNAi-treated mice had reduced expression of Siglec-E, a mucin receptor and known inhibitor of the TLR4 endocytosis and promoter of NF-ĸB signaling, as well as a significant decrease in IL-6, a product of the NF-ĸB pathway and promoter of STAT3 signaling which we have previously shown to have an essential role in promotion of KM-LUAD. In summary, these findings elucidate a potential mechanism by which MUC5AC protects tumor cells from apoptosis and immune-mediated cytotoxic activity but also highlights how MUC5AC RNAi is an effective indirect preventive and therapeutic strategy in high-risk individuals (smokers with COPD) and patients with early stage KM-LUAD, respectively. Citation Format: Melvin Zarghooni, Maria T. Grimaldo, Avantika Krishna, Michael J. Clowers, Linda Phan, Yasmina H. Rezai, Arnav Gaitonde, Carlos Rodriguez Reyna, Walter Velasco Torrez, Erik Bush, Seyed Javad Moghaddam. Mucin 5AC inhibition via intratracheal RNAi delivery as an effective alternative strategy for prevention and treatment of Kras mutant lung cancer [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 2667.

Abstract 5306: PTPN22 targeting partners with 41BB/OX40 costimulation to overcome anti-PD1 resistance in pancreatic ductal adenocarcinoma

Abstract Background: The pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) is notable for a scarcity of cytotoxic T cells and an overabundance of immunosuppressive myeloid and regulatory T cells. Checkpoint immunotherapies targeting PD/L1 or CTLA4 have shown efficacy in immunogenic cancers but remain ineffective in PDAC, highlighting a need for novel approaches. Abrogating protein tyrosine phosphatase non-receptor type 22 (PTPN22), a phosphatase in the TCR signaling cascade, has been shown to modulate multiple mechanisms of immune resistance. Furthermore, 41BB costimulatory pathway has recently been investigated as a potential strategy to enhance immunotherapy against PDAC. Using immune-resistant murine models of PDAC, we investigated PTPN22 as an immunotherapeutic target in combination with 41BB co-stimulation and PD1 inhibition against PDAC. Methods: We employed a well-characterized murine PDAC cell line expressing mutant KrasG12D and Trp53R172H under the Pdx1-cre promoter (KPCY 6419c5). This tumor line is resistant to combination immuno- and chemotherapies. To determine treatment efficacy, PTPN22 deficient mice (KO) were inoculated with KPCY6419c5 cells either subcutaneously or orthotopically into the pancreas. To extend the translatability of PTPN22 targeting, we also injected PTPN22 competent mice with the same tumor cells and treated them with our novel PTPN22 inhibitor L1. Harvested tumors were processed for FFPE and mass cytometry (CyTOF) analysis to track changes in the immune compartment. Results: When PTPN22 was abrogated, treatment with anti-PD1 and 41BB agonism led to a 10-day delay in substantial tumor formation and roughly 75% reduction in tumor volume by 21-days post treatment. These tumors showed elevated levels of OX40 and OX40L primarily in the setting of PTPN22 KO/PD1/41BB therapy. In a subsequent in vivo experiment using an OX40L neutralizing antibody, we found that OX40 signaling is essential for the efficacy of PTPN22 KO/PD1/41BB therapy. Furthermore, combining OX40 agonist with anti-PD1 in PTPN22 KO sufficiently mimicked the efficacy seen with the anti-PD-1 and 41BB combination. CyTOF analysis indicated that OX40 expression is enriched in Tregs and OX40L is enriched in myeloid derived suppressor cells (MDSCs). Moreover, combination therapy efficacy was reproduced using the PTPN22 inhibitor L1. Conclusions: PTPN22 abrogation enhances the success of PD-1 inhibition given with either OX-40 or 41BB agonist therapies. In addition, PTPN22 abrogation together with PD1 inhibition and 41BB costimulation reduces MDSC and Treg suppression of effector T cells via the OX40L-OX40 axis. Finally, PTPN22 can be blocked by a small molecule inhibitor strengthening its candidacy as a therapeutic target. Citation Format: Soren Charmsaz, Nicole E. Gross, Jae W. Lee, Jianping Lin, Alexei G. Hernandez, Erin M. Coyne, Sarah M. Shin, Courtney Cannon, Xuan Yuan, Zhong-Yin Zhang, Elizabeth M. Jaffee, Won Jin Ho. PTPN22 targeting partners with 41BB/OX40 costimulation to overcome anti-PD1 resistance in pancreatic ductal adenocarcinoma [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 5306.

Abstract 6610: Metabolic patterns of pancreatic cancer cachexia: Cross-tissue lipid networks predict cachexia progression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal of all cancer types. A key — yet poorly understood — facet in the disease state is cachexia, a multi-organ pathological state characterized by physical wasting and tissue catabolism. It occurs in 80% of PDAC patients, and to date there are no preventative or early detection methods. Cachexia leads to limited tolerance to anti-cancer therapy and contributes to disease lethality. Here, we present the first-of-its-kind systemic metabolomic analysis across cachectic stages to better understand disease progression. We used the well-established mutant KRASG12D(LSL/+) mutant p53 inducible mouse model of PDAC. Model physiology faithfully recaptures human cachexia: we observe progressive overall weight loss as well as loss of skeletal muscle and adipose tissue. As with human disease, weight loss occurs prior to loss of appetite in the animals, suggesting physiological changes independent of nutrition. Importantly, our model captures the period prior to weight loss (pre-CAC), as well as early-CAC (<10% weight loss) and late-CAC (>10% weight loss). We profiled the metabolomes of the pancreas, interstitial fluid, plasma, liver, 3 different adipose tissues, and 3 skeletal muscles, from male and female mice, control and tumor-bearing, across all 3 stages of cachexia. Each tissue has a unique metabolic trajectory across cachexia stages; pathway and correlation analyses showed a particular emphasis on lipid alterations in peripheral tissues. Strikingly, we find systemic metabolic changes prior to tissue wasting, including rewiring in the liver before the presence of metastases. This indicates systemic alterations as early as pre-CAC. We therefore used computational modeling to identify metabolites that may be participating in cross-tissue networks. A key finding, confirmed by 13C tracing, was the circulation of lactate and glucose and their uptake by the liver and pancreas. Network maps also suggest lipid rewiring in distal tissues, changes in abundances of these same lipid species in plasma, and their consequent alterations in the tumor and liver. A fundamental question remains: can we predict cachexia development before the manifestation of symptoms? We used feature selection algorithms based on statistical learning, training the models only on a subset of data. Specific sphingolipids and triglycerides were the key lipid species that distinguished control and pre-CAC. We find that the model — trained only on pre-CAC — is able to determine early-CAC with an 85% accuracy and late-CAC with a 90% accuracy. Thus, a limited set of metabolites that are altered in pre-CAC could have predictive value for future cachexia development. Overall, our work provides a resource for the field and advances our understanding of systemic metabolism in PDAC cachexia. We hope this will lay the foundation for cachexia prevention and treatment. Citation Format: Deepti Mathur, Blanca Majem, Courtney Beaulieu, Lucas Dailey, Sarah Jeanfavre, Joao Xavier, Clary Clish, Nada Kalaany. Metabolic patterns of pancreatic cancer cachexia: Cross-tissue lipid networks predict cachexia progression [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 6610.

Abstract 5633: Unravelling the pleiotropic role of HNF4α in invasive mucinous adenocarcinoma of the lung (IMA)

Abstract IMA is a distinct subtype of lung adenocarcinoma (LUAD), marked by a cellular identity shift from a pulmonary to a gastric lineage with the loss of the pulmonary lineage specifier NKX2-1. This distinctive transformation underscores the complexity of lung cancer heterogeneity and highlights the critical role of lineage-specifying transcription factors in dictating tumor behavior. Notably, both human and murine models of IMA overexpress HNF4α, a known lineage specifier for gastric differentiation in the adult mouse mucosa. Also, concomitant deletion of Hnf4a and Nkx2-1 at tumor initiation impairs tumor growth and the extent of HNF4α in regulating IMA tumor growth, cellular identity and drug response remains unknown at large. Here, we utilized genetically engineered mouse models (GEMM) that relies on a sequential recombination strategy to delete Hnf4a in established tumors. We induce tumor formation through intratracheal delivery of Flp-O virus, activating the expression of mutant KRASG12D and Cre in lung epithelial cells. Tamoxifen administration recombines either Nkx2-1 alone (KN) or in combination with Hnf4a (KNH). We have extensively studied the phenotypic role of HNF4α in GEMM and organoids and identified gene sets regulated by HNF4α in IMA at the bulk level. To complement our findings, we profiled both the tumor and stromal compartments via scRNAseq analysis on our GEMM to gain insights into the cellular diversity within the tumor microenvironment (TME) and the role HNF4α plays. Additionally, we performed ChIPSeq in organoids to elucidate the genome occupancy of HNF4α and determine the mechanisms of gene regulation in IMA. We find that deletion of Hnf4a impairs tumor growth in GEMM and this is phenocopied in organoids. We identified numerous differentially expressed genes (DEGs) in KNH compared to KN and validated several DEGs in vivo. GSEA analysis reveals a significant involvement in regulating cellular identity and suggests a potential impact on anti-tumor immune responses. To this, our scRNAseq data revealed an enrichment of N2-TAN (tumor associated neutrophils) associated genes in KNH compared to KN, suggesting a nuanced interplay between HNF4α and the TME. Additionally, HNF4α exhibited dual functionality, acting as an activator and indirectly as a repressor by modulating the activity of other transcription factors like FOXA1. FOXA1 was found to bind de novo target genes following the loss of HNF4α. These findings so far collectively illuminate the multifaceted role of HNF4α in IMA, primarily driving tumorigenesis in a cell autonomous manner and secondarily skewing the TME towards a protumor via N2-TAN following loss of HNF4α in tumors. Citation Format: Headtlove Essel Dadzie, Gabriela Fort, Katherine Gillis, Pengshu Fang, Walter Orellana, Eric L. Snyder. Unravelling the pleiotropic role of HNF4α in invasive mucinous adenocarcinoma of the lung (IMA) [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 5633.

Antineoplastic effect of compounds C14 and P8 on TNBC and radioresistant TNBC cells by stabilizing the K-Ras4BG13D/PDE6δ complex.

Breast cancer (BC) is the leading cause of cancer-related deaths among women, with triple-negative breast cancer (TNBC) representing one of the most aggressive and treatment-resistant subtypes. In this study, we aimed to evaluate the antitumor potential of C14 and P8 molecules in both TNBC and radioresistant TNBC cells. These compounds were chosen for their ability to stabilize the complex formed by the overactivated form of K-Ras4BG13D and its membrane transporter (PDE6δ). The antitumor potential of C14 and P8 was assessed using TNBC cell lines, MDA-MB-231, and the radioresistant derivative MDA-MB-231RR, both carrying the K-Ras4B> G13D mutation. We investigated the compounds' effects on K-Ras signaling pathways, cell viability, and tumor growth in vivo. Western blotting analysis determined the negative impact of C14 and P8 on the activation of mutant K-Ras signaling pathways in MDA-MB-231 and MDA-MB-231RR cells. Proliferation assays demonstrated their efficacy as cytotoxic agents against K-RasG13D mutant cancer cells and in inducing apoptosis. Clonogenic assays proven their ability to inhibit TNBC and radioresistant TNBC cell clonogenicity. In In vivo studies, C14 and P8 inhibited tumor growth and reduced proliferation, angiogenesis, and cell cycle progression markers. These findings suggest that C14 and P8 could serve as promising adjuvant treatments for TNBC, particularly for non-responders to standard therapies. By targeting overactivated K-Ras and its membrane transporter, these compounds offer potential therapeutic benefits against TNBC, including its radioresistant form. Further research and clinical trials are warranted to validate their efficacy and safety as novel TNBC treatments.

Albumin promoter-driven FlpO expression induces efficient genetic recombination in mouse liver.

Tissue-specific gene manipulations are widely used in genetically engineered mouse models. A single recombinase system, such as the one using Alb-Cre, has been commonly used for liver-specific genetic manipulations. However, most diseases are complex, involving multiple genetic changes and various cell types. A dual recombinase system is required for conditionally modifying different genes sequentially in the same cell or inducing genetic changes in different cell types within the same organism. A FlpO cDNA was inserted between the last exon and 3'-UTR of the mouse albumin gene in a bacterial artificial chromosome (BAC-Alb-FlpO). The founders were crossed with various reporter mice to examine the efficiency of recombination. Liver cancer tumorigenesis was investigated by crossing the FlpO mice with FSF-KrasG12D mice and p53frt mice (KPF mice). BAC-Alb-FlpO mice exhibited highly efficient recombination capability in both hepatocytes and intrahepatic cholangiocytes. No recombination was observed in the duodenum and pancreatic cells. BAC-Alb-FlpO-mediated liver-specific expression of mutant KrasG12D and conditional deletion of p53 gene caused the development of liver cancer. Remarkably, liver cancer in these KPF mice manifested a distinctive mixed hepatocellular carcinoma and cholangiocarcinoma phenotype. A highly efficient and liver-specific BAC-Alb-FlpO mouse model was developed. In combination with other Cre lines, different genes can be manipulated sequentially in the same cell, or distinct genetic changes can be induced in different cell types of the same organism.NEW & NOTEWORTHY A liver-specific Alb-FlpO mouse line was generated. By coupling it with other existing CreERT or Cre lines, the dual recombinase approach can enable sequential gene modifications within the same cell or across various cell types in an organism for liver research through temporal and spatial gene manipulations.

KRAS Mutation Detection with (2S,4R)-4-[18F]FGln for Noninvasive PDAC Diagnosis.

Pancreatic ductal adenocarcinoma (PDAC), which has a poor prognosis and nonspecific symptoms and progresses rapidly, is the most common pancreatic cancer type. Inhibitors targeting KRAS G12D and G12C mutations have been pivotal in PDAC treatment. Cancer cells with different KRAS mutations exhibit various degrees of glutamine dependency; in particular, cells with KRAS G12D mutations exhibit increased glutamine uptake. (2S,4R)-4-[18F]FGln has recently been developed for clinical cancer diagnosis and tumor cell metabolism analysis. Thus, we verified the heterogeneity of glutamine dependency in PDAC models with different KRAS mutations by a visual and noninvasive method with (2S,4R)-4-[18F]FGln. Two tumor-bearing mouse models (bearing the KRAS G12D or G12C mutation) were injected with (2S,4R)-4-[18F]FGln, and positron emission tomography (PET) imaging features and biodistribution were observed and analyzed. The SUVmax in the regions of interest (ROI) was significantly higher in PANC-1 (G12D) tumors than in MIA PaCa-2 (G12C) tumors. Biodistribution analysis revealed higher tumor accumulation of (2S,4R)-4-[18F]FGln and other metrics, such as T/M and T/B, in the PANC-1 mouse models compared to those in the MIAPaCa-2 mouse models. In conclusion, PDAC cells with the KRAS G12D and G12C mutations exhibit various degrees of (2S,4R)-4-[18F]FGln uptake, indicating that (2S,4R)-4-[18F]FGln might be applied to detect KRAS G12C and G12D mutations and provide treatment guidance.

Clinicopathologic features, concurrent genomic alterations, and clinical outcomes of patients with KRAS G12D mutations in resected lung adenocarcinoma

BackgroundIn light of the ongoing clinical development of KRAS G12D-specific inhibitors, we sought to investigate the clinicopathologic, co-occurring genomic features and outcomes of patients with KRAS G12D-mutant lung adenocarcinoma. Methods3828 patients with completely resected primary lung adenocarcinomas were examined for KRAS mutations between 2008 and 2020. The association between KRAS G12D and clinicopathologic features, molecular profiles, and outcomes was investigated. Results65 patients (1.7%) with KRAS G12D-mutant lung adenocarcinoma were identified. KRAS G12D mutation was more frequent in males, former/current smokers, radiologic solid tumors, and invasive mucinous adenocarcinoma. TP53 and STK11 were the two most frequent concomitant mutations in the KRAS G12D group. KRAS G12D mutation did not appear to be a prognostic factor in resected stage I-III lung adenocarcinomas, while KRAS non-G12D mutation was related to worse survival, especially in stage I tumors. KRAS G12D mutations were associated with positive but low (1–49%) PD-L1 expression compared to negative (<1%), while KRAS non-G12D mutation was associated with high PD-L1 expression (≥50%). TP53 co-mutation indicated higher PD-L1 expression, while STK11 co-mutation had a negligible impact on PD-L1 expression. Furthermore, data mining of MSK datasets from cBioPortal revealed that KRAS G12D and SKT11 co-mutation were associated with a diminished response to immunotherapy. ConclusionsKRAS G12D-mutant lung adenocarcinoma harbored unique clinicopathologic and genomic characteristics. Despite not being prognostic in resected lung adenocarcinoma, KRAS G12D might be a valuable biomarker in combination with certain co-mutations for identifying relevant subgroups of patients that could eventually influence treatment regimens