G12V Mutation Research Articles

Active and Inactive Cdc42 Differ in Their Insert Region Conformational Dynamics

Cell division control protein 42 homolog (Cdc42) protein, a Ras superfamily GTPase, regulates cellular activities, including cancer progression. Using all-atom molecular dynamics (MD) simulations and essential dynamic analysis, we investigated the structure and dynamics of the catalytic domains of GDP-bound (inactive) and GTP-bound (active) Cdc42 in solution. We discovered substantial differences in the dynamics of the inactive and active forms, particularly in the “insert region” (residues 122–135), which plays a role in Cdc42 activation and binding to effectors. The insert region has larger conformational flexibility in the GDP-bound Cdc42 than in the GTP-bound Cdc42. The G2 loop and switch I at the effector lobe of the catalytic domain exhibit large conformational changes in both the GDP- and the GTP-bound systems, but in the GTP-bound Cdc42, the switch I interactions with GTP are retained. Oncogenic mutations were identified in the Ras superfamily. In Cdc42, the G12V and Q61L mutations decrease the GTPase activity. We simulated these mutations in both GDP- and GTP-bound Cdc42. Although the overall structural organization is quite similar between the wild type and the mutants, there are small differences in the conformational dynamics, especially in the two switch regions. Taken together, the G12V and Q61L mutations may play a role similar to their K-Ras counterparts in nucleotide binding and activation. The conformational differences, which are mainly in the insert region and, to a lesser extent, in the switch regions flanking the nucleotide binding site, can shed light on binding and activation. We propose that the differences are due to a network of hydrogen bonds that gets disrupted when Cdc42 is bound to GDP, a disruption that does not exist in other Rho GTPases. The differences in the dynamics between the two Cdc42 states suggest that the inactive conformation has reduced ability to bind to effectors.

Targeted Sequencing of Pancreatic Adenocarcinomas from Patients with Metachronous Pulmonary Metastases.

Mutation spectra of 250 cancer driver, druggable, and actionable genes were analyzed in surgically resected pancreatic ductal adenocarcinoma (PDAC) patients who developed metachronous pulmonary metastases. Targeted sequencing was performed in DNA from blood and archival samples of 15 primary tumors and three paired metastases. Results were complemented with the determination of G12V mutation in KRAS by droplet digital PCR. The median number of protein-changing mutations was 52 per patient. KRAS and TP53 were significantly enriched in fractions of mutations in hotspots. Individual gene mutation frequencies or mutational loads accounting separately for drivers, druggable, or clinically actionable genes, did not significantly associate with patients’ survival. LRP1B was markedly mutated in primaries of patients who generalized (71%) compared to those developing solitary pulmonary metastases (0%). FLG2 was mutated exclusively in primary tumors compared to paired metastases. In conclusion, signatures of prognostically differing subgroups of PDAC patients were generated for further utilization in precision medicine.

Abstract 1826: CRISPR mediated KRAS mutant gene editing in pancreatic and colorectal cancer therapy

Abstract Background: KRASG12V and KRASG12D mutations are among the most commonly observed mutations in pancreatic (30% and 51%) and colorectal (27% and 45%) adenocarcinomas and have been associated with poor prognosis. Activating mutations in KRAS play potent roles in cancer initiation, propagation, and maintenance. Although they represent important therapeutic targets, no effective treatment for direct inhibition of KRASG12V and KRASG12D mutants have been reported. We have developed a new potent strategy combing the potency of CRISPR for gene editing with a tumor selective nanocarrier, to impair cancer cell proliferation by directly targeting G12V and G12D mutations of the KRAS oncogene. Methods: ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles with CRISPR/Cas9. ADGN nanoparticles containing Cas9mRNA and gRNA targeting either KRASG12D or KRASG12V were evaluated on pancreas (PANC1, PK-45H) and colorectal (LS513, SW480, SW403) cancer cells harboring KRASG12D or KRASG12V mutations. KRAS mutant indel frequency was evaluated by T7E1 method and compared to KRASWT (HT29, HS-69) cells. Cell proliferation was determined by flow cytometry. In-vivo efficacy of IV-administered ADGN/mRNACas9/gRNA complexes was evaluated in PANC1-LUC (KRASG12D) and SW403 (KRASG12V) mouse xenografts. The phosphorylation of ERK and AKT was measured by western blot, KRAS mutation level in the tumor and variation of housekeeping genes were estimated by deep sequencing. Results: We have developed highly specific gRNAs targeting KRASG12V and KRASG12D mutants complexed with ADGN-nanoparticles. These complexes efficiently and selectively silenced KRASG12V and KRASG12D in colorectal and pancreatic cancer cells in vitro and in vivo. Silencing KRAS mutations reduced the cell proliferation by 70% and the phosphorylation of the downstream effector pathways ERK and AKT. ADGN-nanoparticles containing CRISPR/gRNA targeting KRASG12D abolished PANC1 tumor growth and induced KRASG12D gene editing in the tumor with an indel frequency of 76%. ADGN-nanoparticles containing CRISPR/gRNA targeting KRASG12V abolished SW403 tumor growth and induced KRASG12V gene editing in the tumor with an indel frequency of 82%. In contrast, no effect on tumor growth were observed with ADGN-nanoparticles containing non-specific gRNA or gRNA targeting other KRAS mutations. ADGN/mRNACas9/gRNA targeting KRASG12D or KRASG12V abolished pancreatic and colorectal tumor growth respectively in vivo, without inducing any significant toxicity, emergence of off target effects or other KRAS mutations. Conclusions: ADGN-nanoparticle containing mRNACas9/gRNA targeting KRASG12D or KRASG12V were effective in selectively targeting mutated KRAS both in vitro and in vivo. Our study provides a proof-of-concept that ADGN/CRISPR can be applied to target driver mutations of cancers in vivo and permanently disrupt the oncogenic alleles, leading to inhibition of tumor growth. We proposed a new strategy for targeting KRAS oncogenes that can be useful in treatment of pancreatic and colorectal cancers. Citation Format: Gilles Divita, Elodie Czuba, Melanie Guidetti, Audrey Gunenberger, Leslie Tempremant, Veronique Josserand, Neil Desai. CRISPR mediated KRAS mutant gene editing in pancreatic and colorectal cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1826.

Abstract 1453: Specific detection of mutant Ras oncoproteins in cell and tissue lysates by multiplex immunoassay

Abstract Ras proteins (HRAS, KRAS, and NRAS) are small GTPases that function as molecular switches by alternating between inactive GDP-bound and active GTP-bound states. Ras-GTP activates downstream pathways, including MAPK, by binding to Raf kinase and phosphatidylinositol 3-kinase (PI3K) to promote cellular proliferation, survival, growth and differentiation. Oncogenic mutations in Ras are found in approximately 25% of human cancers. Furthermore, 90% of pancreatic cancers harbor KRAS mutations with 80% of KRAS mutations occurring at codon 12 with G12V and G12D being the most prevalent. The Ras-Raf-MEK pathway can also be activated downstream of Ras. BRAF is a well-established oncogene, with the BRAF V600E mutation being prevalent in melanoma. While targeting this BRAF oncoprotein has had therapeutic efficacy, treating cancers driven by Ras oncoproteins remains an urgent unmet clinical need. We developed a novel multiplex immunoassay capable of simultaneously detecting total Ras, RasG12V, RasG12D, phospho-MEK1 (S217/S221), phospho-BRAF (S446) and phospho-CRAF (S338) in a single well. This multiplex assay detects the H-Ras, N-Ras, and K-Ras isoforms for total Ras, RasG12D, and RasG12V. To confirm specific recognition of mutant Ras oncoproteins, this kit was able to detect the known KRAS G12V mutation found in the COR-L23 cell line and the NRAS G12D mutation found in THP-1 cell line, respectively. In addition, the response to BRAF inhibitors vemurafenib and dabrafenib was compared between MDA-MB-435S cells, which harbor the BRAF V600E mutation, and insensitive cell lines, using multiplex immunoassays. While COR-L23 and THP-1 cells were not responsive to vemurafenib and dabrafenib, MDA-MB-435S cells showed a dramatic reduction in pMEK1 and pERK, consistent with inhibition of mutant BRAF protein. We then analyzed commercially-sourced tissue lysates, using tumor and adjacent tissue from a patient with colon cancer harboring a KRAS G12D mutation. While multiplex protein analysis revealed a similar phosphorylation pattern for Raf and MEK proteins, the RasG12D oncoprotein was specifically observed in lysate from the tumor but not the adjacent tissue. Finally, exosomes were enriched from pancreatic cancer serum and tested using the Ras-Raf Oncoprotein panel. These exosomal preparations yielded detectable signal for Total Ras and phospho-BRAF. Our data demonstrate the utility of studying the effects of Ras oncoproteins by multiplex immunoassay in multiple sample types, including cell, tissue, and exosomal lysates. Citation Format: Joseph B. Hwang, Anthony Saporita, Qiang Xiao. Specific detection of mutant Ras oncoproteins in cell and tissue lysates by multiplex immunoassay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1453.

Abstract LB-302: Mutant KRAS causes diverse metabolic reprogramming and subsequent autophagy early in the pancreatic carcinogenesis

Abstract Background: Pancreatic cancer is a lethal disease and is profoundly resistant to all forms of therapy. In recent years, understanding metabolic reprogramming in cancer has attracted interests as novel therapeutic targets. In addition to the well-established role of the altered glucose metabolism in cancer, recent studies have shown the altered amino acid metabolism, especially glutamine, in malignant cells. Because the initiation of most pancreatic carcinoma is triggered by KRAS gene mutation, we examined the state of metabolic reprogramming in KRAS gene mutation in human pancreatic ductal epithelial cells to clarify the metabolic reprogramming at the early stages during the pancreatic carcinogenesis. Methods: To investigate the specific pathway by which mutant KRAS exhibits metabolic alterations in human pancreatic epithelial cells, we introduced KRAS G12V mutation using CRISPR/Cas9 system in hTERT-HPNE cells, an immortalized human pancreatic non-cancerous epithelial cells. Comprehensive metabolome analyses were performed and further studies were done based on the results. Results: We confirmed the precise single nucleotide substitution by droplet digital PCR and by western blotting using mutant-specific antibodies. Mutant KRAS promoted cellular proliferation in a glucose- and glutamine-dependent manner. To clarify the metabolic alterations, CE mass spectrometry (CE/MS) metabolomic analyses were performed to characterize intracellular metabolites comprehensively. These analyses revealed that mutant KRAS caused the decreases in almost all amino acids, especially asparagine. Evaluation of the degree of protein translation by incorporation of puromycin, a tyrosyl-tRNA mimetic, showed that mutant KRAS caused enhanced protein synthesis. By quantitative RT-PCR, we also confirmed that various amino acid transporters including SLC1A5, SLC7A11/SLC3A2 was significantly upregulated by mutant KRAS, suggesting that KRAS mutant cells take in amino acids from extracellular space and actively consume them. Consistent to the previous reports, autophagy was activated, which was considered as a compensatory mechanism for the decreased amino acids levels. The lysosomal inhibitor, chloroquine, significantly inhibited the proliferation of the cells with mutant KRAS, suggesting that autophagy is required to promote oncogenic KRAS-driven tumorigenesis and to maintain tis metabolic reprogramming state. Conclusions: These results suggest that metabolic reprogramming and the subsequent autophagic activity are the key biological changes at the early stages of carcinogenetic changes in the cells harboring KRAS gene mutation and could be molecular targets to develop preventive methods against pancreatic carcinogenesis. Citation Format: Tatsunori Suzuki, Motoyuki Otsuka, Kazuhiko Koike. Mutant KRAS causes diverse metabolic reprogramming and subsequent autophagy early in the pancreatic carcinogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-302.

K-ras peptide mimotope induces a humoral immune response against G12V K-ras antigen in BALB/c mice

Background. KRAS mutations are highly prevalent in pancreatic, lung, and colorectal carcinomas with G12V point substitution being one of the most prevalent mutations. While developments of peptide vaccines for KRAS(+) cancers are usually associated with poor immunogenicity, coupling mutant K-ras vaccines with universal CD4+ carrier molecules may enhance its outcome. Additionally, recent immunotherapeutic advances also suggest the possibility of inducing mucosal immunity against cancers using Lactococcus lactis as a live gastrointestinal delivery vehicle. Methods. A region of wild-type K-ras peptide was previously modified with a V7D substitution flanking the G12V mutation, generating a K-ras peptide (termed 68-V) with improved predicted antigenicity. This peptide was fused with a diphtheria toxoid sequence, and cloned into pNZ8048 vector within Lactococcus lactis NZ9000. BALB/c mice were then immunized orally, and then subjected to T/B cells immunophenotyping, as well as IgG and IgA detection. Results. Modified 68-V K-ras peptide and controls were successfully cloned and detection of His-tagged proteins expressed following induction by nisin was observed. Populations of CD3-CD19+ immune cells increased following immunization of 68-V, while K-ras specific-IgG and -IgA sera titers were elevated compared to wild-type and G12V K-ras controls. Conclusion. 68-V K-ras mimotope was shown to induce humoral-mediated immunity, highlighting the ability of an additional mutation flanking the G12V KRAS mutation to induce B cell activation and production of K-ras specific antibodies, while diphtheria toxoid was unable to stimulate an enhanced response when fused to 68-V. Nevertheless, these findings showed that further assessments are required to understand the role of K-ras specific antibodies within a KRAS(+) environment.

Abstract A28: SPACEWALK: Plasma NGS for remote evaluation of ALK drug resistance in advanced NSCLC

Abstract Introduction: Precision therapy for cancer drug resistance requires detection of resistance mutations and treatment with appropriate targeted therapies. This paradigm is well established in EGFR-mutant NSCLC, yet our understanding of drug resistance in ALK-positive NSCLC is limited. Next-generation sequencing (NGS) of plasma cell-free DNA (cfDNA) now permits noninvasive interrogation of drug resistance. To facilitate improved understanding of ALK drug resistance and the effectiveness of treatment strategies, we launched this remote participation study. Methods: The SPACEWALK study offers plasma NGS to patients with advanced NSCLC with systemic progression on a next-generation ALK TKI to assess for mechanisms of resistance. Patients consent, enroll, and participate remotely, permitting a larger cohort of ALK positive patients to be studied. Remote phlebotomists can be dispatched to the patient’s home when necessary to ease the burden of study participation and to ensure study compliance. Blood is sent to Resolution Biosciences for ctDNA extraction and hybrid capture NGS, with results returned to the study team and the patient’s physician. Patients are followed for 2 years and blood is collected again after starting a new treatment or at progression. This study is supported by the Addario Lung Cancer Medical Institute (ALCMI). Results: Of 143 patients contacting us via the study website, 61 were offered consent and 53 signed consent. 46 enrolled from 23 different US states and 38 different medical centers. Plasma NGS results were returned a median of 13 days after blood draw (range 8-21). Of the 46 patients, the known ALK fusion was detected in 20 (43%) with a median ALK fusion AF of 3.5% (range 0.3%-37%). 10 patients also underwent plasma NGS with Guardant360, with a median time between the two plasma NGS draws of 1.5 weeks (range 0-22.6). In 4 cases, Resolution Bioscience identified an ALK fusion (AF 0.2-37%) not detected on Guardant360. Among the 20 patients with ALK fusions detected, 12 (60%) were found to carry ALK resistance mutations, 5 with G1202R; 2 patients had 2 or more ALK mutations detected. In addition, 6 participants (30%) had a MET amplification detected with a median of 8 copies (range 3-22) and 1 had a KRAS G12V mutation detected (10% AF). One patient with high MET amplification received combination therapy with alectinib and crizotinib and had dramatic radiographic response. Conclusions: Plasma NGS permits the detection of targetable resistance mechanisms in patients with ALK-positive NSCLC and drug resistance. Sensitivity of different plasma NGS assays for ALK fusions varies. This assay may help guide oncologists across the country to select best treatment options after resistance. Such remote-participation studies may offer a new mechanism for characterizing resistance to emerging targeted therapies in rare cancer populations. Citation Format: Marissa N. Lawrence, Rubii M. Tamen, Alicia Sable-Hunt, Seyed Ali Hosseini, George R. Simon, Jonathan W. Riess, Geoffrey R. Oxnard. SPACEWALK: Plasma NGS for remote evaluation of ALK drug resistance in advanced NSCLC [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A28.

Abstract A34: Defining KRAS mutation-specific kinome signatures and vulnerabilities in colorectal cancer

Abstract Activating mutations in KRAS occur in about half of colorectal cancers (CRC) and are associated with drug resistance and poor survival. A number of distinct oncogenic mutations in KRAS have been identified in CRC, including mutations in exon 2-producing (G12D, G12V, and G13D) and in exon 4 (A146T and A146V). Importantly, emerging clinical and basic science data indicate that these KRAS mutations are not all equal in their oncogenic properties and sensitivity to targeted therapies. To access and exploit the distinct kinase programs engaged by the different KRAS mutants, we employed an innovative mass spectrometry-based technique that enables the majority of the human kinome to be analyzed simultaneously, providing the means to explore the KRAS-induced kinome and its response to targeted therapy in an unbiased fashion. Using multiplexed inhibitor beads and mass spectrometry (MIB/MS), we measured differences in protein kinase signaling between the distinct KRAS mutants (G12D, G12V, G13D and A146T) at both baseline and in response to targeted MEK inhibitors in isogenic KRAS mutant xenografts. Kinome profiling of the tumors demonstrated that each KRAS mutant exhibits a unique basal activation signature, with G12D and A146T mutations showing the most divergence from wild-type (WT) KRAS kinome(s). Tumors harboring G12D or A146T knockin mutations displayed intrinsic resistance to MEKi trametinib, while G13D remained sensitive to MEK inhibition comparable to KRAS WT tumors. Notably, tumors expressing G12V KRAS mutations showed enhanced tumor regression relative to KRAS WT, suggesting G12V mutations may sensitize CRC cells to MEK inhibition. Analysis of kinome adaptations to trametinib using MIB/MS revealed that each mutant reprogrammed their kinome(s) uniquely, supporting the design of individualized MEK inhibitor combination therapies tailored to specific KRAS mutations. Our kinome profiling studies further support the notion that not all KRAS mutations are created equal in CRC and demonstrate mutation-specific kinome activation signatures that can be therapeutically exploited in kinase inhibitor combination therapies. Citation Format: Nishi Srivastava, Alison Kurimchak, Carlos Montavez, Jonathan Chernoff, James Duncan. Defining KRAS mutation-specific kinome signatures and vulnerabilities in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A34.

The effect of MEK1/2 inhibitors on cisplatin-induced acute kidney injury (AKI) and cancer growth in mice

In a clinically-relevant model of 4 week, low-dose cisplatin-induced AKI, mice were injected subcutaneously with non small cell lung cancer (NSCLC) cells that harbor an activating Kirsten rat sarcoma viral oncogene homolog (KRAS)G12V mutation. Phospho extracellular signal-regulated kinase1/2 (pERK1/2) expression in kidney and tumors was decreased by the MEK1/2 inhibitors, U0126 and trametinib, that potently inhibit pERK1/2. U0126 resulted in a significant improvement in kidney function, acute tubular necrosis (ATN) and tubular cell apoptosis in mice with AKI. Genes that were significantly decreased by U0126 were heat shock protein 1, cyclin-dependent kinase 4 (CDK4) and stratifin (14–3-3σ). U0126 resulted in a significant decrease in tumor weight and volume and significantly increased the chemotherapeutic effect of cisplatin. Trametinib, a MEK1/2 inhibitor that is FDA-approved for the treatment of cancer, did not result in functional protection against AKI or worse AKI, but dramatically decreased tumor growth more than cisplatin. Smaller tumors in cisplatin or MEK1/2 inhibitor-treated mice were not related to changes in microtubule-associated proteins 1A/1B light chain 3B (LC3-II), p62, cleaved caspase-3, granzyme B, or programmed death-ligand 1 (PD-L1). In summary, despite ERK inhibition by both U0126 and trametinib, only U0126 protected against AKI suggesting that the protection against AKI by U0126 was due to an off-target effect independent of ERK inhibition. The effect of U0126 to decrease AKI may be mediated by inhibition of heat shock protein 1, CDK4 or stratifin (14–3-3σ). Trametinib was more effective than cisplatin in decreasing tumor growth, but unlike cisplatin, trametinib did not cause AKI.

TCR-mimic antibody-drug conjugates targeting intracellular tumor-specific mutant antigen KRAS G12V mutation

Limited clinical application of antibody-drug conjugates (ADCs) targeting tumor associated antigens (TAAs) is usually caused by on-target off-tumor side effect. Tumor-specific mutant antigens (TSMAs) only expressed in tumor cells which are ideal targets for ADCs. In addition, intracellular somatic mutant proteins can be presented on the cell surface by human leukocyte antigen class I (HLA I)molecules forming tumor-specific peptide/HLA I complexes. KRAS G12V mutation frequently occurred in varied cancer and was verified as a promising target for cancer therapy. In this study, we generated two TCR-mimic antibody-drug conjugates (TCRm-ADCs), 2E8-MMAE and 2A5-MMAE, targeting KRAS G12V/HLA-A*0201 complex, which mediated specific antitumor activity in vitro and in vivo without obvious toxicity. Our findings are the first time validate the strategy of TCRm-ADCs targeting intracellular TSMAs, which improves the safety of antibody-based drugs and provides novel strategy for precision medicine in cancer therapy.

CDK4/6 Inhibitors Impair Recovery from Cytotoxic Chemotherapy in Pancreatic Adenocarcinoma

Inhibition of the cell-cycle kinases CDK4 and CDK6 is now part of the standard treatment in advanced breast cancer. CDK4/6 inhibitors, however, are not expected to cooperate with DNA-damaging or antimitotic chemotherapies as the former prevent cell-cycle entry, thus interfering with S-phase- or mitosis-targeting agents. Here, we report that sequential administration of CDK4/6 inhibitors after taxanes cooperates to prevent cellular proliferation in pancreatic ductal adenocarcinoma (PDAC) cells, patient-derived xenografts, and genetically engineered mice with Kras G12V and Cdkn2a-null mutations frequently observed in PDAC. This effect correlates with the repressive activity of CDK4/6 inhibitors on homologous recombination proteins required for the recovery from chromosomal damage. CDK4/6 inhibitors also prevent recovery from multiple DNA-damaging agents, suggesting broad applicability for their sequential administration after available chemotherapeutic agents.

Cell-free DNA next-generation sequencing liquid biopsy as a new revolutionary approach for arteriovenous malformation

ObjectiveSomatic mosaicism of KRAS gene is currently recognized as the only established molecular basis of arteriovenous malformations (AVM). However, given the limitations of the current technologies, KRAS somatic mutations are detected only in a limited proportion of AVMs and tissue biopsy remains an invasive high risky, sometimes life-threatening, diagnostic procedure. Next-generation sequencing liquid biopsy using cell-free DNA (cfDNA) has emerged as an innovative noninvasive approach for early detection and monitoring of cancer. This approach overcomes the space-time profile constraint of tissue biopsies opens a new scenario for vascular malformations owing to somatic mosaicism. Here, we propose a new approach as a fast noninvasive reliable tool in order to investigate the cfDNA coming from the AVMs. MethodsA group of five patients suffering from AVM were selected. Blood samples from peripheral vein and efferent vein from vascular malformation were collected and cfDNA was extracted. The cfDNA libraries were performed using Oncomine Pan-Cancer Cell-Free Assay. We used Ion Proton for sequencing and Ion Reporter Software for analysis (Life Technologies, Carlsbad, Calif). ResultsIn all cases, either G12D or G12V mutations in KRAS were identified. The mutational load was higher in the efferent vein than in peripheral blood, confirming the causative role of the identified mutation at a somatic level. ConclusionsWe demonstrate that cfDNA next-generation sequencing liquid biopsy is able to identify the KRAS mutation detected in affected tissues. Moreover, we have shown that blood sample withdrawal at the lesion site increases variant allele frequency with an order of magnitude above the limit of detection (usually 0.05%), decreasing the risk of a false negative. Finally, the noninvasiveness of the method avoids any risk of bleeding, being easily performed also in children. We propose this technique as the method of choice to better investigate AVMs and consequently to identify the therapy tailored to the genetic defect. Clinical RelevanceThis article highlights the importance of using liquid biopsy as a new method to investigate the molecular profile of AVMs. In view of the frequent inaccessibility of vascular tissues owing to the invasiveness of solid biopsy and the relative high incidence of biopsies with low diagnostic power, here we evaluated the efficacy of detecting cfDNA fragments released into the bloodstream from the affected tissue cells. Through a simple blood draw from the efferent vein at the vascular malformation site, the liquid biopsy allowed us to identify KRAS pathogenic mutations piloting a personalized therapeutic approach and opening a new scenario for new therapeutic strategies.

A CASE SERIES OF ADENOMATOID ODONTOGENIC TUMOR: CLINICOPATHOLOGIC AND MOLECULAR CHARACTERIZATION

We report a series of 22 adenomatoid odontogenic tumors (AOTs). The patients' age, tumor location, association with impacted teeth, and clinical size were obtained. Capsule thickness was evaluated. The patients’ mean age was 18.8 years (range 6-57 y). Considering the cases with known information, 16 occurred in women and 5 in men, 11 in the maxilla, and 10 in the mandible, 13 in the anterior regions, and 7 in the posterior regions, 8 in association with impacted teeth, and 9 not associated with impaction. We evaluated 3 AOT samples for mutations in a panel of 50 oncogenes and tumor suppressor genes by next-generation sequencing. The KRAS G12V mutation was detected in 3 samples. We screened the other 19 samples for KRAS codon 12 mutations. KRAS codon 12 mutations occurred in 15 out of 22 (68%), KRAS G12V in 14 out of 22 cases, and KRAS G12R in 1 out of 22. Codon 12 mutations were not associated with any of the clinicopathologic parameters tested (P > .05). Supported by FAPEMIG/CAPES/CNPq/Brazil. We report a series of 22 adenomatoid odontogenic tumors (AOTs). The patients' age, tumor location, association with impacted teeth, and clinical size were obtained. Capsule thickness was evaluated. The patients’ mean age was 18.8 years (range 6-57 y). Considering the cases with known information, 16 occurred in women and 5 in men, 11 in the maxilla, and 10 in the mandible, 13 in the anterior regions, and 7 in the posterior regions, 8 in association with impacted teeth, and 9 not associated with impaction. We evaluated 3 AOT samples for mutations in a panel of 50 oncogenes and tumor suppressor genes by next-generation sequencing. The KRAS G12V mutation was detected in 3 samples. We screened the other 19 samples for KRAS codon 12 mutations. KRAS codon 12 mutations occurred in 15 out of 22 (68%), KRAS G12V in 14 out of 22 cases, and KRAS G12R in 1 out of 22. Codon 12 mutations were not associated with any of the clinicopathologic parameters tested (P > .05). Supported by FAPEMIG/CAPES/CNPq/Brazil.

Abstract A129: Vertical inhibition overcomes adaptive resistance to KRASG12C inhibition

Abstract The three RAS genes are the most commonly mutated oncogenes in cancer and have long been considered an “undruggable” target. Alterations in KRAS occur at the highest frequency, with G12D, G12V, and G12C mutations being the most common. The recent development of novel covalent inhibitors selective for the KRASG12C mutation offers the unprecedented opportunity to target KRAS directly. However, prior efforts to target the RAS-MAPK pathway in have been hampered by adaptive feedback, which drives pathway reactivation and resistance. We find evidence of rapid adaptive RAS-MAPK pathway feedback reactivation following KRASG12C inhibition in the majority of KRASG12C models, including lung, colon, and pancreatic lines. Reactivation of the RAS-MAPK pathway correlates with activation of multiple upstream RTKs and subsequent increase of wild-type RAS activity. We find that co-inhibition of SHP2, a common node downstream of RTKs, or co-inhibition of individual RTKs such as EGFR or FGFR enhances the efficacy of KRASG12C inhibition both in vitro and in vivo by overcoming adaptive reactivation of the RAS-MAPK pathway. Notably, only the combination of KRASG12C and SHP2 inhibitor treatment displays tumor regressions in vivo in multiple models when compared to either inhibitor alone. We also see evidence that combining KRASG12C inhibitors with MEK or ERK inhibitors as an additional potential vertical inhibition strategy in KRASG12C models. We propose a vertical inhibition strategy anchored with KRASG12C inhibitors with either RTK or SHP2 inhibitors as a potential strategy for the treatment of KRASG12C mutant tumors. Citation Format: Meagan B Ryan, Ferran Fece de la Cruz, Sarah Phat, David T Myers, Heather A Shahzade, Catriona B Hong, Ryan B Corcoran. Vertical inhibition overcomes adaptive resistance to KRASG12C inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A129. doi:10.1158/1535-7163.TARG-19-A129

K-Ras-PI3K regulates H3K56ac through PCAF to elevate the occurrence and growth of liver cancer.

H3 modification is related to a wide range of tumors, including liver cancer. The Ras passageway is actuated in human diseases. Thus, we investigated the roles of Ras in liver cancer cells via acetylation of H3K56. Ras-carrying G12V and Y40C site mutation was transfected into liver cancer cell lines SNU-475 and SK-Hep-1. Acetylation of H3K56 and phosphatidylinositol 3-kinase (PI3K), P300/CBP-associated factor (PCAF) and Mouse double minute 2 homolog (MDM2) was tested via western blot. Cell activity, colonies, and migration were tested via Cell Counting Kit-8, soft-agar colony formation, and Transwell experiment, respectively. Sirtuin 6 (SIRT6) and PCAF were tested via quantitative reverse transcription polymerase chain reaction (qRT-PCR). Chromatin immunoprecipitation was employed to test the relationship between Ras and downstream elements. Flow cytometry was employed to test cell cycle series. We found that RasG12V/Y40C transfection reduced the acetylation of H3K56 and activated phosphorylation of protein kinase B. H3K56Q (H3K56ac overexpression) suppressed cell activity, colonies, and migration. H3K56ac changed Ras downstream factors expression. RasG12V/Y40C bound to Ras-PI3K downstream elements' promoters. SIRT6 silencing raised H3K56ac and suppressed cell activity, migration and S phage cell percentage. SIRT6 silence transformed expression of downstream elements. PCAF and H3K56ac demonstrated the close current while MDM2 was conversed. In summary, the Ras-PI3K passageway promoted cell growth and metastasis via decreasing H3K56ac, in which MDM2-mediated PCAF was involved.

P2.01-67 Are KRAS Mutations Predictive of Response to Immunotherapy in Non Small-Cell Lung Cancer? A Single Center Experience

Lung cancer is the leading cause of cancer-related mortality. In the last decade, the treatment landscape has evolved for non-small-cell lung cancer (NSCLC), in large part thanks to two therapeutic advances: targeted therapy for oncogene driven tumours and immune checkpoint-inhibitors (ICI). The latter can lead to very long survival, but only in a limited number of patients. Post-hoc analyses and preclinical data suggest KRAS mutated NSCLC may respond better to ICI. We retrospectively analysed all stage IV NSCLC patients treated in our center with immunotherapy from January 2016 to December 2017 and compared KRAS mutated to wildtype patients. The clinical outcomes analysed were disease control rate (DCR), partial response (PR), progression free survival (PFS) and overall survival (OS). We performed an exploratory analysis on the impact of KRAS mutation type and concurrent mutations. 45 patients treated with ICI. 7 were excluded due to insufficient data. 38 were included. 27 patients (71%) with nivolumab, 9 patients (24%) with pembrolizumab and 2 patients (5%) with atezolizumab. 22 patients (58%) were male and 16 (42%) female. The median age was 63. 21 patients (55%) presented KRAS mutations, of which 4 (19%) had concurrent p53 mutations and 1 (5%) an EGFR mutation. 17 patients (45%) were KRAS wild-type, of which 4 (24%) had EGFR mutations and 1 (6%) had a BRAF V600E mutation. In the KRAS mutated subgroup 59% were male and 41% were female, the median age was 61 years and all patients had PS 0-1. All patients received second line immunotherapy except for 2, one in third line, one fifth line. In the intention to treat population (ITT) the DCR was 71% with 55% PR, PFS was 11.3 months, and OS 17.7 month. In the KRAS wild-type subgroup DCR was 59% with 49% PR, the PFS was 8.4 months and OS 16.8 months. In the KRAS mutated subgroup, DCR was 81%, with 62% PR, PFS was 13.6 months and OS 18.5 months. An exploratory analysis based on KRAS mutation types or co-mutations was performed. The average PFS for G12C, G13C, G12V, G61H and other mutations was 19.1, 7.8, 9.4, 2.2, 13.9 months respectively. PFS for p53 co-mutated KRAS NSCLC was 23.5 months. Further analyses for STK11 co-mutations are ongoing, as these confer resistance to ICI. At the time of analysis 7 patients were still receiving immunotherapy. In our retrospective study, KRAS mutations in NSCLC were predictive of numerically superior response to ICI compared to wildtype patients. Currently, the data is conflicting and larger clinical trials are needed to clarify this hypothesis and ascertain whether and how KRAS should be part of the treatment algorithm for the selection of ICI patients. Furthermore, more research is needed to confirm the potential role of mutation types and clinical impact of co-mutations in KRAS.

Proteomic and Interactome Approaches Reveal PAK4, PHB-2, and 14-3-3η as Targets of Overactivated Cdc42 in Cellular Responses to Genomic Instability.

Cdc42, a member of the Rho GTPase family, is an intracellular signaling protein known for its roles in cytoskeleton rearrangements and, more recently, in apoptosis/senescence triggered by genotoxic stress. In some tumor cells, the overactivation of Cdc42 through the expression of constitutively active mutants (G12V or Q61L), GEF activation, or GAP downregulation functions as an antiproliferative or pro-aging mechanism. In this study, human cell lines with different P53 protein profiles were exposed to UV radiation, and the interactions between Cdc42 and proteins that are putatively involved in the DNA damage response and repair mechanisms were screened. The affinity-purified proteins obtained through pull-down experiments of the cell lysates using the recombinant protein baits GST, GST-Cdc42-WT, or GST-Cdc42-G12V were identified by mass spectrometry. The resulting data were filtered and used for the construction of protein-protein interaction networks. Among several promising proteins, three targets, namely, PAK4, PHB-2, and 14-3-3η, which are involved in the cell cycle, apoptosis, DNA repair, and chromatin remodeling processes, were identified. Biochemical validation experiments showed physical and proximal interactions between Cdc42 and the three targets in the cells, particularly after exposure to UV. The results suggest that the molecular mechanisms coordinated by overactivated Cdc42 (with the G12V mutation) to increase the cellular sensitivity to UV radiation and the susceptibility to cell death are collectively mediated by these three proteins. Therefore, the Cdc42 GTPase can potentially be considered another player involved in maintenance of the genomic stability of human cells during exposure to genotoxic stress.

G12V and G12C mutations in the gene KRAS are associated with a poorer prognosis in primary colorectal cancer.

The increased incidence of colorectal cancer (CRC) has necessitated the development of novel prognostic and predictive factors from which new diagnostic tests could evolve. Evidence suggests the KRAS gene represents such a factor; its mutations are considered to be early indicators of CRC progression. This study assessed the prognostic impact of specific known KRAS codon 12/13 mutations on survival in patients with CRC. Formalin-fixed paraffin-embedded tissue blocks or sections from primary were obtained from patients registered between 2014 and 2016 for genomic DNA extraction. KRAS gene was analyzed by direct sequencing or Luminex assay. The primary endpoint was the frequency of KRAS gene mutations and the secondary endpoints were differences in KRAS mutation rates by various stratification factors. Univariate and multivariate analyses were performed to investigate relationships between KRAS mutation rates and patient background factors. Sequencing of 200 CRC primary tumor samples demonstrated 74 (37.5%) with KRAS mutations in codons 12 (77%; 57/74) and 13 (23%; 17/74), all of which were TNM stages I-III. Tumors with KRAS mutations were more frequently located in the right side of the colon. Multivariate analysis indicated that G12V or G12C mutations were associated with poor prognosis [hazard ratio (HR) = 3.77, 95% confidence interval (CI), 1.54-8.39 and HR = 6.57; 95% CI, 1.90-17.7, respectively] in terms of recurrence-free survival. KRAS codon 12G-to-V or G-to-C mutations are independent prognostic factors in patients with stage I-III CRC.

ASSOCIATION OF MAPK/ERK PATHWAY ACTIVATION WITH KRAS MUTATIONS IN ADENOMATOID ODONTOGENIC TUMORS

Objectives Adenomatoid odontogenic tumors (AOT) are benign tumors derived from odontogenic epithelium, and they account for 2-7% of all odontogenic tumors. Intraosseous AOTs are thought to be associated with unerupted permanent teeth, although their pathogenesis is still unclear.KRAS mutation, which is involved in the pathogenesis of some malignant tumors as driver mutation, was recently detected in AOT suggesting its association with tumorigenesis.The aim of this study was to assess the frequency of KRAS mutation and his association with the presence of the MAPK / ERK signaling pathway proteins. Finding Paraffin-embedded tissue samples from 9 AOT patients (3-47 years old, mean 24.7 years) were obtained for this study. Genomic DNA was extracted from each sample, and in one case, genetic mutations in 50 cancer-associated genes were examined by next-generation sequencing. A KRAS G12D missense mutation was detected in the DNA sequence of the tumor cells, but it was not detected in that of the stroma tissue. Based on this result, hotspot mutations in the RAS family were analyzed by PCR-rSSO using the remaining 8 cases. KRAS G12V and KRAS G12R mutations were detected in 2 and 4 cases, respectively. Subsequently, in the paraffin blocks, immunohistochemistry was performed to visualize the presence of the proteins involved in the MAPK / ERK signaling pathway. All the cases were EGFR, KRAS,CRAF, BRAF positive, one case was ERK negative, and one case was MEK and ERK negative, all the other remaining cases were MEK and ERK positive. Conclusions In conclusion, KRAS mutation was frequently detected in AOT, suggesting its association with tumorigenesis of AOT. However, since EGFR was positive, how the mutation affects the tumor development is still unclear.

Molecular characterization and management of secondary resistance to serial TRK inhibitors.

e22547 Background: TRK inhibitor drugs such as the highly selective larotrectinib (Laro), have proven highly effective in malignancies harboring fusions of NTRK1, 2, or 3. Resistance in patients (pts) with progressive disease (PD) after response to initial TRK inhibitor therapy has been attributed to secondary mutations in the solvent front or gatekeeper domains of the NTRK fusion gene. LOXO-195 (L195) is a 2nd generation TRK inhibitor that overcomes these mutations. Mechanisms of resistance to L195 have not yet been well characterized. Methods: We analyzed molecular mechanisms of resistance in one adult pt with undifferentiated pleomorphic sarcoma (UPS) who had serial responses and PD on Laro and L195. Targeted DNA sequencing, RNA sequencing and multicolor cyclic immunofluorescence (CyCIF) were performed on pre- and post-PD specimens on both drugs. Results: The patient was enrolled on the phase 2 clinical trial of Laro (NCT02576431) with TPM3-NTRK1 fusion UPS. Multifocal PD and resistance to Laro developed after major objective response RECIST -74.9%, of 10 months (mo); the resistant tumor harbored both the initial TPM3-NTRK1 fusion but also evolved a new solvent front mutation in NTRK1 [c.1783G > A (p.G595R)]. A single pt protocol (NCT03206931) was designed to treat with L195. After an initial response to L195, PD limited to 2 sites developed; both sites were resected at 5 and 10 mo. The pt continues on L195 with systemic disease control 20 mo after initiation (in total 30 mo since Laro initiation). Analysis of tumor samples pre- and post-PD on L195 identified the emergence of a KRAS G12V mutation, with associated activation of the KRAS signaling pathway and a significant infiltration by inflammatory cells. A cell line and pt-derived xenografts (PDX), all harboring the initial TPM3-NTRK1 fusion, were generated from this pt. Conclusions: Resection of oligoclonal PD and continuation of L195 post-PD can be an effective treatment strategy. Oncogenic activation of the KRAS pathway is a possible mechanism of resistance to L195. Our studies indicate that the tumor microenvironment of TRK-fusion sarcomas resistant to TRK inhibitors may increase inflammatory cell infiltrates, which may provide clues for future combination therapy.