Molecular Characterization ofKRASWild-type Tumors in Patients with Pancreatic Adenocarcinoma
Philip A. PhilipIbrahim AzarJoanne XiuMichael J. HallAndrew HendifarEmil LouJimmy J. HwangJun GongRebecca FeldmanMichelle EllisPhil StaffordDavid SpetzlerMoh’d KhushmanDavendra SohalA. Craig LockhartBenjamin A. WeinbergWafik S. El‐DeiryJohn L. MarshallAnthony F. ShieldsW. Michael Korn
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KRAS mutation (MT) is a major oncogenic driver in pancreatic ductal adenocarcinoma (PDAC). A small subset of PDACs harbor KRAS wild-type (WT). We aim to characterize the molecular profiles of KRAS WT PDAC to uncover new pathogenic drivers and offer targeted treatments.Abstract Allelic imbalance is reported to be a frequent event in cancers and a common feature associated with oncogenes such as KRAS and BRAF. However the functional and therapeutic consequences of such imbalances are poorly understood. Mutations in the KRAS oncogene are one of the most prevalent events in human cancers and heterozygous KRAS mutations are well-described functionally and are thus viewed as sufficient for tumorigenesis. Recent evidence shows high incidence of KRAS gene dosage changes in human cancers, including loss of the normal wild-type allele of KRAS. However, there is still much debate over the function of wild-type KRAS in tumour initiation, progression and therapy response. Using advanced genetically engineered mouse models of colorectal cancer (VillinCRE Apcfl/fl; LSL-KrasG12D/+ (AK), LSL-KrasG12D/+; Trp53fl/fl; Rosa26N1iCD/+ (KPN)) we investigated the functional impact of wild-type Kras in oncogenic Kras driven tumour initiation (AK), progression and metastasis (KPN) in vivo. Mechanistically, wild-type Kras restrains oncogenic Kras signalling and significantly affects the efficiency of the oncogenic Kras induced transformation and response to therapy. We demonstrate a suppressive role for wild-type Kras during tumorigenesis and highlight the critical impact of wild-type Kras upon therapeutic response and tumour progression in Kras mutant CRC. Wild-type KRAS-deficient colorectal tumours are characterized by increased MAPK signalling and transcriptional activation of MAPK regulators. Importantly, loss of wild-type Kras in oncogenic KRAS-driven aggressive KPN tumours significantly alter tumour progression and liver metastasis, showing increased immune infiltration and WNT signalling. In addition, loss of wild-type Kras modulates response to therapeutic intervention and sensitizes wild-type Kras deficient tumours to MEK1/2 inhibition. This study demonstrates a suppressive role for wild-type Kras during tumour initiation and highlights the critical impact of wild-type Kras upon therapeutic response to MAPK and tumour progression in KRAS mutant CRC. Citation Format: Arafath K. Najumudeen, Sigrid K. Fey, Andrew D. Campbell, Owen J. Sansom. KRAS allelic imbalance drives an epithelial MAPK-dependent tumor initiation program that is inefficient in provoking metastasis in colorectal cancer in vivo [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A004.
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e19324 Background: KRAS G12C mutations are present in 15% of non-small cell lung cancer (NSCLC) and have recently been shown to confer sensitivity to KRAS(G12C) inhibitors. This study aims to assess the clinical features and outcomes with KRAS G12C mutant NSCLC in a real-world setting. Methods: Patients enrolled in an Australian prospective cohort study, Thoracic Malignancies Cohort (TMC), between July 2012 to October 2019 with metastatic or recurrent non-squamous NSCLC, with available KRAS test results, and without EGFR, ALK, or ROS1 gene aberrations, were selected. Data was extracted from TMC and patient records. Clinicopathologic features, treatment and overall survival was compared for KRAS wildtype ( KRAS WT ) and KRAS mutated ( KRAS mut ) patients, and between KRAS G12C ( KRAS G12C ) and other ( KRAS other ) mutations. Results: Of 1386 patients with non squamous NSCLC, 1040 were excluded for: non metastatic or recurrent (526); KRAS not tested (356); ALK, EGFR or ROS1 positive (154); duplicate (4). Of 346 patients analysed, 202 (58%) were KRAS WT and 144 (42%) were KRAS mut , of whom 65 (45%) were KRAS G12C . 100% of pts with KRAS G12C were smokers, compared to 92% of KRAS other and 83% of KRAS WT . The prevalence of brain metastases over entire follow-up period was similar between KRAS mut and KRAS WT (33% vs 40%, p = 0.17), and KRAS G12C and KRAS other (40% vs 41%, p = 0.74). Likewise, there was no difference in the proportion of patients receiving one or multiple lines of systemic therapy. Overall survival (OS) was also similar between KRAS mut and KRAS WT (p = 0.54), and KRAS G12C and KRAS other (p = 0.39). Conclusions: In this real-world prospective cohort, patients had comparable clinical features regardless of having a KRAS mut , KRAS G12C or KRAS other mutation, or being KRAS WT . Treatment and survival were also similar between groups. While not prognostic, KRAS G12C may be an important predictive biomarker as promising KRAS G12C covalent inhibitors continue to be developed.
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74 Background: KRAS mutation is rare ( < 5%) in gastroesophageal cancer (GEC). However, the incidence of KRAS gene amplification (amp+), consequent protein levels, and prognostic and/or therapeutic implications are unknown. Methods: 410 GEC samples and 30 cell lines were assessed for KRAS gene copy number (GCN) by fluorescence in situ hybridization (FISH) (n = 90), Kras expression by selected reaction monitoring mass spectrometry (Kras-SRM-MS) (n = 393), and Kras-SRM level evaluated for correlation with KRAS amp+ status (n = 73). Survival analysis was performed comparing KRAS amp+ versus non-amp+ patients. When possible, concurrent 315 gene next-generation sequencing was also performed. Four KRAS-amplified xenograft lines (CAT-2,12,14,15) were established from malignant effusions. Tumorigenic activity of KRAS amp+ lines (CAT lines, MKN-1) were assessed using MTT and soft agar assays in vitro and subcutaneous xenograft models, compared to non-amp+ lines. Inhibitory assays were performed using KRAS siRNA and CRIPSR, and commercial inhibitors targeting downstream effectors MEK and/or PIK3CA. Results: KRAS FISH revealed clustered gene amp+ in 28.9% (26/90); these patients had worse prognosis than non-amp+ patients. GCN significantly correlated with Kras expression. All KRAS amp+ cell lines significantly overexpressed Kras protein and were tumorigenic in xenograft subcutaneous models. KRAS siRNA and KRAS CRISPR of KRAS amp+ cell lines demonstrated inhibition in MTT viability and soft agar assays, compared to appropriate controls, and demonstrated significant and durable xenograft growth reduction. Conversely, inhibition using MEK and/or PI3K inhibitors demonstrated only transient growth reduction in vivo. Conclusions: KRAS gene amp+ was observed in a large subset (26%) of GEC patients, which correlated with extreme expression by mass spectrometry. Established xenograft lines serve as models to investigate therapeutic strategies for KRAS amp+ patients. Inhibition using MEK/PIK3CA inhibitors provided transient benefit for KRAS amp+ tumors while durable inhibition was observed with Kras protein knockdown, suggesting potential benefit from novel siRNA therapeutics currently in development.
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Abstract Background : KRAS is the most frequently mutated oncogene in cancer, however efforts to develop targeted therapies have been largely unsuccessful. Recently, two small-molecule inhibitors, AMG 510 and MRTX849, have shown promising activity in KRAS G12C-mutant solid tumors. The current study aims to assess the molecular profile of KRAS G12C in colorectal (CRC) and non-small-cell lung cancer (NSCLC) tested in a clinical certified laboratory. Methods : CRC and NSCLC samples submitted for KRAS testing between 2017 and 2019 were reviewed. CRC samples were tested for KRAS and NRAS by pyrosequencing, while NSCLC samples were submitted to next generation sequencing of KRAS, NRAS, EGFR, and BRAF. Results : The dataset comprised 4,897 CRC and 4,686 NSCLC samples. Among CRC samples, KRAS was mutated in 2,354 (48.1%). Most frequent codon 12 mutations were G12D in 731 samples (15.2%) and G12V in 462 (9.6%), followed by G12C in 167 (3.4%). KRAS mutations were more frequent in females than males (p=0.003), however this difference was exclusive of non-G12C mutants (p<0.001). KRAS mutation frequency was lower in the South and North regions (p=0.003), but again KRAS G12C did not differ significantly (p=0.80). In NSCLC, KRAS mutations were found in 1,004 samples (21.4%). As opposed to CRC samples, G12C was the most common mutation in KRAS, in 346 cases (7.4%). The frequency of KRAS G12C was higher in the South and Southeast regions (p=0.012), and lower in patients younger than 50 years (p<0.001). KRAS G12C mutations were largely mutually exclusive with other driver mutations; only 11 NSCLC (3.2%) and 3 CRC (1.8%) cases had relevant co-mutations. Conclusions : KRAS G12C presents in frequencies higher than several other driver mutations, represent a large volume of patients in absolute numbers. KRAS testing should be considered in all CRC and NSCLC patients, independently of clinical or demographic characteristics.
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Abstract Pancreatic cancer is characterized by the prevalence of oncogenic mutations in KRAS. Previous studies have reported that altered KRAS gene dosage drives progression and metastasis in pancreatic cancer. While the role of oncogenic KRAS mutations is well characterized, the relevance of the partnering wild-type KRAS allele in pancreatic cancer is less well understood and controversial. Using in vivo mouse modelling of pancreatic cancer, we demonstrated that wild-type KRAS restrains the oncogenic impact of mutant KRAS and dramatically impacts both KRAS-mediated tumorigenesis and therapeutic response. Mechanistically, deletion of wild-type Kras increased oncogenic KRAS signaling through the downstream MAPK effector pathway, driving pancreatic intraepithelial neoplasia (PanIN) initiation. In addition, in the KPC mouse model, a more aggressive model of pancreatic cancer, lack of wild-type KRAS led to accelerated initiation but delayed tumor progression. These tumors had altered stroma and an enrichment of immunogenic gene signatures. Importantly, loss of wild-type Kras sensitized Kras mutant tumors to MEK1/2 inhibition though tumors eventually became resistant and then rapidly progressed. This study demonstrates the repressive role of wild-type KRAS during pancreatic tumorigenesis and highlights the critical impact of the presence of wild-type KRAS in both tumor progression and therapeutic response in pancreatic cancer.
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KRAS遺伝子変異は非小細胞肺癌を含むヒトの癌で頻度の高いがん遺伝子変異の一つである．発見から30年以上のKRAS変異陽性癌の治療法開発にもかかわらず，臨床的有用性を示す薬物は得られず，創薬不能な標的とされてきた．理由として，KRASとGTPの親和性は高く結合阻害は困難，KRASの下流シグナルや膜結合に必要な翻訳後修飾はいくつも平行しており，単一の経路や修飾反応の阻害では他の活性化が起こる，KRAS変異陽性癌は必ずしもKRASに生死が依存していないことなどが考えられる．2013年にGDP結合KRASに低分子化合物がはまるポケットが見出され，G12C変異KRASに限定的ながら，KRASを不活性なGDP結合型に非可逆的に固定する化合物が報告された．この発見に基づき，ソトラシブやアダグラシブなどのG12C特異的阻害剤が開発され，前者は2021年に米国で，2次治療以降のKRASG12C変異陽性非小細胞肺癌に対し迅速承認された．今後，G12C以外の直接阻害剤，G12C阻害剤との併用療法，耐性獲得後の対策，有効な患者選択のためのバイオマーカーなどについて，さらなる研究開発が待たれる．
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Abstract Background KRAS is the most frequently mutated oncogene in cancer, however efforts to develop targeted therapies have been largely unsuccessful. Recently, two small-molecule inhibitors, AMG 510 and MRTX849, have shown promising activity in KRAS G12C-mutant solid tumors. The current study aims to assess the molecular profile of KRAS G12C in colorectal (CRC) and non-small-cell lung cancer (NSCLC) tested in a clinical certified laboratory. Methods CRC and NSCLC samples submitted for KRAS testing between 2017 and 2019 were reviewed. CRC samples were tested for KRAS and NRAS by pyrosequencing, while NSCLC samples were submitted to next generation sequencing of KRAS , NRAS , EGFR , and BRAF . Results The dataset comprised 4897 CRC and 4686 NSCLC samples. Among CRC samples, KRAS was mutated in 2354 (48.1%). Most frequent codon 12 mutations were G12D in 731 samples (14.9%) and G12V in 522 (10.7%), followed by G12C in 167 (3.4%). KRAS mutations were more frequent in females than males ( p = 0.003), however this difference was exclusive of non-G12C mutants ( p < 0.001). KRAS mutation frequency was lower in the South and North regions ( p = 0.003), but again KRAS G12C did not differ significantly ( p = 0.80). In NSCLC, KRAS mutations were found in 1004 samples (21.4%). As opposed to CRC samples, G12C was the most common mutation in KRAS , in 346 cases (7.4%). The frequency of KRAS G12C was higher in the South and Southeast regions ( p = 0.012), and lower in patients younger than 50 years ( p < 0.001). KRAS G12C mutations were largely mutually exclusive with other driver mutations; only 11 NSCLC (3.2%) and 1 CRC (0.6%) cases had relevant co-mutations. Conclusions KRAS G12C presents in frequencies higher than several other driver mutations, and may represent a large volume of patients in absolute numbers. KRAS testing should be considered in all CRC and NSCLC patients, independently of clinical or demographic characteristics.
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// Annalisa Conti 1 , Maria Teresa Majorini 1 , Richard Elliott 2 , Alan Ashworth 2,8 , Christopher J. Lord 2 , Carlotta Cancelliere 3,4,5 , Alberto Bardelli 3,4,5 , Pierfausto Seneci 6 , Henning Walczak 7 , Domenico Delia 1 and Daniele Lecis 1 1 Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 2 The Breakthrough Breast Cancer Research Centre and CRUK Gene Function Laboratory, The Institute of Cancer Research, London, UK 3 Department of Oncology, University of Torino, Candiolo, Torino, Italy 4 Candiolo Cancer Institute - FPO, IRCCS, Candiolo, Torino, Italy 5 FIRC Institute of Molecular Oncology (IFOM), Milano, Italy 6 Università Degli Studi di Milano, Dipartimento di Chimica, Milan, Italy 7 Centre for Cell Death, Cancer, and Inflammation, University College London, London, UK 8 Current Address: UCSF Helen Diller Family Comprehensive Cancer Centre, San Francisco, California, USA Correspondence: Daniele Lecis, email: // Keywords : KRAS, Smac mimetics, colorectal cancer, camptothecin Received : January 23, 2015 Accepted : February 21, 2015 Published : March 12, 2015 Abstract KRAS is mutated in about 20-25% of all human cancers and especially in pancreatic, lung and colorectal tumors. Oncogenic KRAS stimulates several pro-survival pathways, but it also triggers the trans-activation of pro-apoptotic genes. In our work, we show that G13D mutations of KRAS activate the MAPK pathway, and ERK2, but not ERK1, up-regulates Noxa basal levels. Accordingly, premalignant epithelial cells are sensitized to various cytotoxic compounds in a Noxa-dependent manner. In contrast to these findings, colorectal cancer cell sensitivity to treatment is independent of KRAS status and Noxa levels are not up-regulated in the presence of mutated KRAS despite the fact that ERK2 still promotes Noxa expression. We therefore speculated that other survival pathways are counteracting the pro-apoptotic effect of mutated KRAS and found that the inhibition of AKT restores sensitivity to treatment, especially in presence of oncogenic KRAS. In conclusion, our work suggests that the pharmacological inhibition of the pathways triggered by mutated KRAS could also switch off its oncogene-activated pro-apoptotic stimulation. On the contrary, the combination of chemotherapy to inhibitors of specific pro-survival pathways, such as the one controlled by AKT, could enhance treatment efficacy by exploiting the pro-death stimulation derived by oncogene activation.
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4065 Background: KRAS mutation is rare (< 5%) in gastroesophageal cancer (GEC). However, the incidence of KRAS gene amplification (amp+), consequent protein levels, and prognostic and/or therapeutic implications are unknown. Methods: 410 GEC samples and 30 cell lines were assessed for KRAS gene copy number (GCN) by fluorescence in situ hybridization (FISH) (n = 90), Kras expression by selected reaction monitoring mass spectrometry (Kras-SRM-MS) (n = 393), and Kras-SRM level evaluated for correlation with KRAS amp+ status (n = 73). Survival analysis was performed comparing KRAS amp+ versus non-amp+ patients. When possible, concurrent 315 gene next-generation sequencing was also performed. Four KRAS-amplified xenograft lines (CAT-2,12,14,15) were established from malignant effusions. Tumorigenic activity of KRAS amp+ lines (CAT lines, MKN-1) were assessed using MTT and soft agar assays in vitro and subcutaneous xenograft models, compared to non-amp+ lines. Inhibitory assays were performed using KRAS siRNA and CRIPSR, and commercial inhibitors targeting downstream effectors MEK and/or PIK3CA. Results: KRAS FISH revealed clustered gene amp+ in 28.9% (26/90); these patients had worse prognosis than non-amp+ patients. GCN significantly correlated with Kras expression. All KRAS amp+ cell lines significantly overexpressed Kras protein and were tumorigenic in xenograft subcutaneous models. KRAS siRNA and KRAS CRISPR of KRAS amp+ cell lines demonstrated inhibition in MTT viability and soft agar assays, compared to appropriate controls, and demonstrated significant and durable xenograft growth reduction. Conversely, inhibition using MEK and/or PI3K inhibitors demonstrated only transient growth reduction in vivo. Conclusions: KRAS gene amp+ was observed in a large subset (26%) of GEC patients, which correlated with extreme expression by mass spectrometry. Established xenograft lines serve as models to investigate therapeutic strategies for KRAS amp+ patients. Inhibition using MEK/PIK3CA inhibitors provided transient benefit for KRAS amp+ tumors while durable inhibition was observed with Kras protein knockdown, suggesting potential benefit from novel siRNA therapeutics currently in development.
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