KRAS Mutant Colorectal Cancer Research Articles

Reliability of digital PCR in detecting KRAS mutation in colorectal cancer using plasma sample

Background:Test on the KRAS somatic mutation status is necessary before cetuximab and panitumumab treatments are given to colorectal cancer patients. Metastatic colorectal cancer patients sometimes lack tumor tissue samples, and the testing of KRAS mutation in plasma samples requires highly sensitive methods.Objectives:The aim of this study was to evaluate the accuracy of digital PCR in detecting KRAS mutation in plasma samples of colorectal cancer patients.Data sources:Literature research was conducted in Pubmed, Embase, and Cochrane Library.Study eligibility criteria, participants, and interventions:Database searching found 188 relevant studies. After removing duplicates, eligible studies were selected from 151 publications using the following exclusion criteria:1.did not discuss colorectal cancer;2.did not use digital PCR method;3.lacked plasma sample or tissue sample;4.did not measure KRAS status;5.un-interpretable data.Study appraisal and synthesis methods:Data were extracted from the eligible studies by 2 independent researchers. Pooled accuracy parameters were calculated from those extracted data using Meta-DiSc and STATA software.Results:Twelve eligible studies were selected for the systematic review and meta-analysis. After calculation, the pooled sensitivity and specificity were 0.83 (95% CI: 0.79–0.86) and 0.91 (95%CI: 0.88–0.93), respectively. Pooled positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio were 7.30 (95%CI: 4.78–11.17), 0.22 (95%CI: 0.15–0.32), and 41.00 (95%CI: 21.07–79.78), respectively. Area under curve of the summarized ROC curve was 0.9322.Limitations:Although no significant bias was identified, number of included studies was still quite small, especially in subgroup analysis.Conclusions and implication of key findings:Digital PCR showed high accuracy and could be a reliable detection method for KRAS mutation in plasma samples. Large-cohort prospective study is required to further confirm the usefulness of digital PCR in KRAS mutation detection.

Dual inhibition of VEGF and PARP suppresses KRAS-mutant colorectal cancer

The addition of bevacizumab to chemotherapy has prolonged overall and progression-free survival rates for metastatic colorectal cancer (mCRC). However, KRAS-mutant (KRAS-mut) CRC, lacking an ideal targeted agent, represents an inferior-response subgroup of patients. In the present study, we investigated a combination approach of bevacizumab + olaparib in KRAS-mut CRC in a preclinical setting. The combined therapy effectively prevented tumor growth in a KRAS-mut cancer cell-derived xenograft model, although this effect was not observed in vitro. Under bevacizumab treatment, we detected intratumor hypoxia and impaired homologous recombination repair (HRR), accompanied by vascular regression. We explored the underlying mechanism of this combined therapy by mimicking a hypoxic condition in vitro using cobalt chloride (CoCl2). The results showed that hypoxia impairs HRR and therefore sensitized KRAS-mut CRC cell lines HCT-116, SW620, and Lovo to olaparib. Furthermore, under this hypoxic condition, olaparib could arrest the cell cycle in the G2/M phase, increase DNA damage and dramatically induce cell apoptosis in KRAS-mut CRC cells. Taken together, these results indicated that the combination of bevacizumab + olaparib could be a potential therapeutic approach in a KRAS-mut CRC cohort.

Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe

To unravel vulnerabilities of KRAS-mutant CRC cells, a shRNA-based screen specifically inhibiting MAPK pathway components and targets was performed in CaCo2 cells harboring conditional oncogenic KRASG12V. The custom-designed shRNA library comprised 121 selected genes, which were previously identified to be strongly regulated in response to MEK inhibition. The screen showed that CaCo2 cells expressing KRASG12V were sensitive to the suppression of the DNA replication licensing factor minichromosome maintenance complex component 7 (MCM7), whereas KRASwt CaCo2 cells were largely resistant to MCM7 suppression. Similar results were obtained in an isogenic DLD-1 cell culture model. Knockdown of MCM7 in a KRAS-mutant background led to replication stress as indicated by increased nuclear RPA focalization. Further investigation showed a significant increase in mitotic cells after simultaneous MCM7 knockdown and KRASG12V expression. The increased percentage of mitotic cells coincided with strongly increased DNA damage in mitosis. Taken together, the accumulation of DNA damage in mitotic cells is due to replication stress that remained unresolved, which results in mitotic catastrophe and cell death. In summary, the data show a vulnerability of KRAS-mutant cells towards suppression of MCM7 and suggest that inhibiting DNA replication licensing might be a viable strategy to target KRAS-mutant cancers.

Discovery of Novel PDEδ Degraders for the Treatment of KRAS Mutant Colorectal Cancer.

KRAS-PDEδ protein-protein interaction represents an appealing target for cancer therapy. However, fast release of high-affinity inhibitors from PDEδ hampered drug binding affinity and antiproliferative activity. To overcome the limitations, the first proteolysis-targeting chimeric (PROTAC) small molecules targeting PDEδ were designed. By employment of PDEδ inhibitor deltazinone (2) and cereblon ligand pomalidomide (6), a series of potent PROTAC PDEδ degraders were obtained. The most promising compound 17f efficiently induced PDEδ degradation and demonstrated significantly improved antiproliferative potency in KRAS mutant SW480 cells. Compound 17f also achieved significant tumor growth inhibition in the SW480 colorectal cancer xenograft model. This proof-of-concept study provided a new strategy to validate the druggability of KRAS-PDEδ interaction and offered an effective lead compound for the treatment of KRAS mutant cancer.

Active Compound of Pharbitis Semen (Pharbitis nil Seeds) Suppressed KRAS-Driven Colorectal Cancer and Restored Muscle Cell Function during Cancer Progression.

Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven colorectal cancer (CRC) is notorious to target with drugs and has shown ineffective treatment response. The seeds of Pharbitis nil, also known as morning glory, have been used as traditional medicine in East Asia. We focused on whether Pharbitis nil seeds have a suppressive effect on mutated KRAS-driven CRC as well as reserving muscle cell functions during CRC progression. Seeds of Pharbitis nil (Pharbitis semen) were separated by chromatography and the active compound of Pharbitis semen (PN) was purified by HPLC. The compound PN efficiently suppressed the proliferation of mutated KRAS-driven CRC cells and their clonogenic potentials in a concentration-dependent manner. It also induced apoptosis of SW480 human colon cancer cells and cell cycle arrest at the G2/M phase. The CRC related pathways, including RAS/ERK and AKT/mTOR, were assessed and PN reduced the phosphorylation of AKT and mTOR. Furthermore, PN preserved muscle cell proliferation and myotube formation in cancer conditioned media. In summary, PN significantly suppressed mutated KRAS-driven cell growth and reserved muscle cell function. Based on the current study, PN could be considered as a promising starting point for the development of a nature-derived drug against KRAS-mutated CRC progression.

Identification of PIGU as the Hub Gene Associated with KRAS Mutation in Colorectal Cancer by Coexpression Analysis.

Colorectal cancer (CRC) patients with KRAS mutation are refractory and usually have poor prognosis. We aimed to identify the hub gene associated with KRAS mutant CRCs. Weighted gene coexpression network analysis (WGCNA) was used to calculate the key module and the hub genes in GSE39582. Combined with the protein-protein interaction (PPI) network and survival analysis, the real hub gene was identified and further validated. With the highest module significance value and correlation coefficient, the blue module was selected as the key module, 19 genes were identified as the hub gene candidates. The above genes were significantly downregulated in KRAS mutant CRCs compared with the wild type. Four genes (AAR2, PSMA7, NELFCD, and PIGU) were further screened as the potential hub genes by the PPI network. Low expression of PIGU for KRAS mutant patients had a poor prognosis. Therefore, PIGU was identified as the hub gene. PIGU expression was also downregulated in other two CRC datasets. "MAPK SIGNALING PATHWAY" was enriched in PIGU lowly expressed samples. PIGU was identified and validated to be closely related to KRAS mutation. It could be a potential prognosis biomarker and a novel treatment target for KRAS mutant CRC patients.

Blocking histone methyltransferase SETDB1 inhibits tumorigenesis and enhances cetuximab sensitivity in colorectal cancer.

The histone methyltransferase SETDB1 catalyzes the addition of methyl groups to histone H3 at lysine 9, and upregulation of SETDB1 is associated with poor prognosis in cancer patients. Here, we describe how overexpression of SETDB1 contributes to colorectal cancer (CRC) tumorigenesis and drug resistance. We show that SETDB1 is upregulated in CRC, and its level correlates with poor clinical outcome. SETDB1 attenuation inhibits CRC cell proliferation Mechanistically, SETDB1 promotes cell proliferation by upregulating Akt activation. Further, SETDB1 is essential for the tumorigenic activity of Akt. Functional characterization revealed that inhibition of SETDB1 reduces cell growth in CRC resistant to targeted treatments in vitro and in vivo, KRAS-mutated CRC included. Taken together, our results indicate that SETDB1 is a major driver of CRC and may serve as a potential target for the treatment of KRAS-mutated CRC.

MICA ∗012:01 Allele Facilitates the Metastasis of KRAS-Mutant Colorectal Cancer.

Major histocompatibility complex (HLA) class I chain-related protein A (MICA) regulates immune surveillance through activation of NKG2D (natural killer group 2D) receptor. However, the genetic association, potential function, and predictive ability of MICA alleles with colorectal cancer (CRC) prognosis remain undefined. In this study, we characterized MICA alleles in tissue samples from 104 patients with CRC and 536 healthy controls and carried out genetic association studies by molecular and clinical CRC phenotypes. Preliminary sequence analysis revealed that MICA ∗009:01 or ∗049 alleles were significantly decreased in patients with CRC (p = 0.0049), and further stratification analysis indicated that MICA ∗012:01 allele was associated with patients with CRC and carrying KRAS codon 12 mutation (p = 0.027). The functional consequences of MICA alleles were examined via transfected CRC cell lines which showed that overexpression of MICA ∗012:01 enhanced the proliferation, invasion, and metastatic phenotype of CRC. Preliminary analysis of disease-free survival time in patients with and without MICA ∗012:01 suggest this allele may be predictive for poor prognosis of patients with KRAS codon 12 mutated CRC, as no somatic mutation of MICA gene was detected in CRC tumors compared to paracancerous tissues. Our study indicates that MICA ∗012:01 allele is associated with KRAS-mutated CRC, facilitates CRC invasion and metastasis, and possibly reduces the survival of patients with KRAS-mutated CRC.

Pharmacodynamic effects in blood and tumor tissue of eftozanermin alfa, a tumor necrosis factor-related apoptosis-inducing ligand receptor agonist.

e15668 Background: Eftozanermin alfa (eftoza; formerly known as ABBV-621), a 2nd-generation tumor necrosis factor-related apoptosis-inducing ligand receptor agonist, is being evaluated in previously treated solid and hematologic malignancies (NCT03082209). In a dose-expansion cohort, patients (pts) with KRAS-mutant colorectal cancer (n = 24) and pancreatic cancer (n = 24) were evaluated at 3 dose levels with 12 mandatory paired biopsies per tumor type (pretreatment [Tx] and on-Tx collection). Following eftoza dosing, RNA and protein expression including posttranslational modifications were assessed in tumor biopsies to understand the target engagement and downstream pathway activation. Plasma was evaluated for changes in somatic mutant allele frequency and M30, M65 (circulating apoptotic markers). Methods: Biopsies were collected anytime during the screening period (pre-Tx) and 24±4 h following 2nd or 3rd infusion (on-Tx). Of the requested 4–6 fresh biopsy cores, 1–2 cores were collected as formalin fixed paraffin embedded (FFPE) and the rest were frozen tissue. FFPE tissue was analyzed by multiplex immunohistochemistry (IHC) and RNAseq; reverse phase protein array was used for frozen cores. Plasma was collected at cycle 1 predose and 2, 8, 24, 48, and 168 h postdose and analyzed for M30, M65 (by ELISA) and circulating tumor DNA (64-gene PlasmaSELECT assay). Results: Twenty-five pts consented to biopsies; paired biopsies were obtained from 16 pts at a 64% success rate: FFPE (n = 15) and frozen cores (n = 12). Tumor cells were detected in 11/15 (73%) FFPE and 4/12 (33%) frozen cores. Increase in M30, activated caspases, and cleaved PARP levels was observed in on-Tx biopsy samples compared with pre-Tx, thus serving as evidence for apoptosis induction in tumors following eftoza dosing. Changes in the tumor microenvironment were observed post-Tx by RNAseq and multiplex IHC (eg, CD68 level). Downregulation of prosurvival signaling pathways (eg, AKT/MEK) was also observed following eftoza dosing. Thirteen out of 16 pts showed transient increase in mutant allele fractions post eftoza Tx that correlated with increased plasma circulating tumor markers M30 and M65 at similar time points, suggesting activation of apoptosis pathway. Increase in M30, M65 levels also preceded increase in liver enzymes (ALT/AST) at 2, 48 hr post-Tx. Conclusions: Pharmacodynamic effect of eftoza was successfully demonstrated in blood and tumor tissue, including induction of apoptosis and modulation of PI3K and MEK signaling pathways. Clinical trial information: NCT03082209.

Phase I study of afatinib plus selumetinib in patients with KRAS mutation-positive colorectal, non-small cell lung and pancreatic cancer.

3613 Background: Mutations in the KRAS gene result in a constitutively activated RAS-RAF-MEK-ERK (MAPK) pathway. In KRAS mutant tumors, the anti-tumor activity of MEK inhibitors is limited due to intrinsic resistance caused by feedback activation of upstream epidermal growth factor receptors (HER). This upstream activation not only reactivates MAPK, but also the phosphoinositide 3-kinase (PI3K)-AKT pathway in preclinical research. Based on these data, a phase I clinical trial was initiated with the combination of the orally administered pan-HER inhibitor afatinib and the MEK inhibitor selumetinib in patients with KRAS mutant and PIK3CA wildtype colorectal cancer (CRC), non-small cell lung cancer (NSCLC), or pancreatic cancer to determine the recommended phase 2 regimen (RP2R). Methods: In this multicentre study, patients received escalating doses of afatinib and selumetinib according to a 3+3 design starting with 20 mg afatinib once daily (QD) continuously and 25 mg selumetinib twice daily (BID) in a 21 days on/7 days off schedule. Continuous and intermittent dosing were explored to assess optimal exposure and tolerability. The primary aim was determining the RP2R. Secondary objectives included assessment of anti-tumor activity and the analyses of pharmacokinetic and pharmacodynamic parameters for target inhibition. Clinicaltrials.gov identifier: NCT2450656. Results: In total, 26 mostly heavily pretreated patients with CRC (n=19), NSCLC (n=6) and pancreatic cancer (n=1) were enrolled among 5 dose-levels. Dose-limiting toxicities (DLTs) occurred in 6 patients and consisted of grade 3 diarrhea (n=3), decreased appetite (n=1), nausea/vomiting (n=1), dehydration (n=2) and mucositis (n=1). Clinical efficacy was limited with no responses according to RECIST v1.1 and stable disease for 221 days in a patient with NSCLC as best response. Conclusions: The RP2R was determined at 20 mg afatinib QD continuously and 25 mg selumetinib BID 21 days on/7 days off for continuous dosing. The 3 patients treated in the escalation cohort of the ongoing intermittent dose-level with 20 mg afatinib QD and 25 mg selumetinib BID 5 days on/2 days off, experienced no DLTs. Pending the latest safety results of the expansion cohort for this ongoing dose-level, the RP2R of intermittent dosing has not been established at the moment. Clinical trial information: NCT2450656 .

A phase Ib/II study of the polo-like kinase 1 (PLK1) inhibitor, onvansertib, in combination with FOLFIRI and bevacizumab for second-line treatment of patients with KRAS-mutated metastatic colorectal cancer (mCRC).

TPS4116 Background: Chemotherapy in combination with targeted agents are standard-of-care options for patients for mCRC with response rates >50% in first line. In the second line setting, efficacy of chemotherapy and targeted agents are much lower with response rates of 5% for FOLFIRI (5-fluorouracil, leucovorin, irinotecan) + bevacizumab (anti-VEGF). New treatment options are urgently needed in particular for the 50 % of patients harboring a KRAS mutation. PLK1 is a serine/threonine kinase, master regulator of the mitotic checkpoint and cell division. PLK1 is overexpressed in CRC and its overexpression is associated with poor prognostic. A genome wide RNAi screen identified PLK1 as a synthetic lethal target in KRAS mutant CRC cells, inducing cell cycle arrest and apoptosis upon inhibition. Onvansertib is an oral, highly selective PLK1 inhibitor that demonstrates single agent and synergistic activity with irinotecan in preclinical CRC models. Additionally, KRAS mutated vs wild-type cells showed higher sensitivity to onvansertib. PLK1 inhibition is a potential target in KRAS-mutated mCRC, and the combination of onvansertib + FOLFIRI + bevacizumab may provide a new second-line treatment option. Methods: The primary objective of this single-arm Phase 1b/2 study is to assess the safety and preliminary efficacy of onvansertib in combination with FOLFIRI and bevacizumab in the second line setting for KRAS-mutated mCRC patients. For the Phase 1b segment, a standard 3 + 3 dose-escalation design is used to determine the maximum tolerated dose or recommended phase 2 dose (RP2D) of onvansertib. As of January 24, 2020, enrollment in the second dose level is ongoing. Efficacy will be determined by objective response rate (ORR) according to RECIST v1.1 (primary endpoint), progression-free survival and reduction in KRAS allelic burden in liquid biopsies (secondary endpoints). In the phase 2, based on a one-sided one sample log-rank test with 10% Type I error, there will be at least 90% power to detect an improvement in ORR from 5% to 20% with 26 patients. Exploratory endpoints include genomic studies of circulating tumor cells and ctDNA to evaluate altered pathways that correlate with patient clinical response. Clinical trial information: NCT03829410 .

Phase I monotherapy dose escalation of RGX-202, a first-in-class oral inhibitor of the SLC6a8/CKB pathway, in patients with advanced gastrointestinal (GI) solid tumors.

3504 Background: About 65% of advanced colorectal cancer (CRC) patients (pts) have creatine kinase B (CKB) expressing tumors. CKB expressing (CKB+) GI cancer cells import creatine via the creatine transporter SLC6a8 and utilize it to generate intracellular ATP. RGX-202, a small molecule inhibitor of SLC6a8, reduces intracellular creatine and ATP levels, leading to apoptosis. RGX-202 treatment triggers complete tumor regressions in multiple CKB+ preclinical models, including KRAS mutant CRC. Methods: RGX-202-001 is a phase I escalation/expansion study of RGX-202 +/- FOLFIRI in pts with advanced GI tumors. The primary safety objective during dose escalation is to identify the maximum tolerated dose (MTD), or the maximum tested dose at which multiple dose-limiting toxicities (DLTs) are not observed. The primary efficacy objective is to estimate the antitumor activity of RGX-202 by RECIST. Results: As of January 31, 2020, 17 pts have been treated in 4 single agent dose escalation cohorts: 600 mg BID (3 pts), 1200 mg BID (4 pts), 2400mg BID (5 pts) and 3600mg BID (5pts) given continuously. No DLTs were observed and an MTD was not reached. Treatment-related adverse events (TRAEs) occurring in > 2 pts are shown in the Table. There were no Grade 4 TRAEs. At the highest dose, 2 of 3 CRC pts had prolonged disease control: a patient with a KRAS G13D mutant cancer had SD for 14 weeks; and a patient with KRAS G12V mutant (MSS) cancer had a confirmed PR ongoing at 30 weeks. Exposure to RGX-202 was greater than dose-proportional and the average AUC0-24 ranged from ~15,700 ng-hr/mL in cohort 1 to 241,097 ng-hr/mL in in Cohort 4. Serum and urine creatine levels, pharmacodynamic markers of SLC6a8 inhibition, correlated with systemic exposure to RGX-202. Conclusions: Among 17 patients treated with single agent therapy, no DLTs occurred; notably, exposures predicted to be sufficient to inhibit human tumor growth from preclinical models were achieved along with concomitant pharmacodynamic effects. These data, along with a durable PR observed in the highest dose cohort, support further development of RGX-202. Consequently, dose escalation in combination with FOLFIRI in patients with advanced GI cancers is underway with plans for expansion in CKB+ CRC pts. Clinical trial information: NCT03597581 . [Table: see text]

Novel Polymethoxylated Chalcones as Potential Compounds Against KRAS-Mutant Colorectal Cancers.

KRAS-mutant colorectal cancers (CRC) are tumors that are associated with poor prognosis. However, no effective treatments are available to target them. Therefore, we designed and synthesized novel chalcone analogs, small organic molecules, to investigate their effects on KRAS-mutant CRC cells. Fourteen new chalcone analogs were synthesized, optimized, characterized, and tested against two KRAS-mutant CRC cell lines (HCT-116 and LoVo), one p-53 and BRAF mutant CRC cell line (HT-29) and one normal immortalized colon cells (NCE-1 E6/E7). Effects on cell viability, apoptosis, cell cycle, migration, colony formation, EMT, and angiogenesis were investigated. Compounds 3 and 14 were the most effective. Compound 3 showed potent activity against HCT-116 and LoVo cell lines (GI50 of 6.10 μM and 7.00 μM, respectively). While compound 14 showed GI50 of 8.60 μM and 8.80 μM on HCT-116 and LoVo cell lines, respectively. Both compounds were approximately 2-3 times more selective toward cancer cells rather than normal colon cells. Compound 3 was effective in inducing apoptosis in HCT-116 cells via Bax upregulation and Bcl-2 downregulation. Invasion and metastasis of KRAS-mutant cells were modulated by compounds 3 and 14 through significant inhibition of cell migration and the prevention of colony formation. In addition, they reversed EMT by downregulation of EMT markers (vimentin, fascin, and β- catenin) and upregulation of cell-cell adhesion marker, E-cadherin. Furthermore, compounds 3 and 14 had significantly inhibited angiogenesis in ovo. Compounds 3 and 14 represent potent and selective leads for KRAS-mutant CRC cells, thus, further in vitro and in vivo studies are necessary to confirm their effect on KRAS-mutant CRCs.

KRAS G12C Metastatic Colorectal Cancer: Specific Features of a New Emerging Target Population

BackgroundKirsten rat sarcoma viral oncogene (KRAS) G12C mutation occurs in about 4% of colorectal cancers (CRCs). Recently, KRAS G12C was identified to be a potential drug target and predictor of response to the novel on AMG510 target treatment. We described the clinicopathologic features and prognosis of KRAS G12C-mutated metastatic CRCs compared to other KRAS mutation. Patients and MethodsClinicopathologic features and outcome data of KRAS-mutated metastatic CRC (mCRC) patients referred to 3 Italian oncology units from January 2010 to December 2018 were collected. A cohort of KRAS-mutant mCRC patients referred to the Department of Medical Oncology at Fondazione IRCCS Istituto Nazionale dei Tumori, Milan (Italy) within the same time frame was included as external validation. ResultsA total of 839 KRAS-mutated mCRC cases were included in the main patient population. A total of 145 patients (17%) had KRAS G12C mutation. Our analyses showed that patients harboring KRAS G12C mutation were more likely to be men and to present lung and liver metastases, and were less likely to have peritoneal spread. KRAS G12C mutation was associated with shorter overall survival compared to other KRAS mutations (hazard ratio, 1.32; 95% confidence interval, 1.07-1.63; P = .009). Such results were confirmed in the external validation cohort. ConclusionThe knowledge of the distinctive traits of KRAS G12C-mutated CRC patients is crucial to future translational research studies, clinical trial design, and proper interpretation of results.

Abstract B50: ERK MAPK inhibition enhances the immunogenicity of KRAS-mutant colorectal cancer

Abstract The three RAS genes are the most commonly mutated oncogenes in cancer and are refractory to conventional therapies. Oncogenic mutation of KRAS in 44% of colorectal cancers (CRC) leads to aberrant activation of downstream effector pathways, including the ERK MAPK signaling cascade, which can lead to an immunosuppressive tumor microenvironment. Single-agent therapies targeting the ERK MAPK cascade or immune checkpoint (PD-1/PD-L1) have been mostly unsuccessful in KRAS-mutant CRC, and efforts are under way to identify effective combination strategies. We employed an unbiased RNA-seq approach to identify gene signatures significantly upregulated upon ERK MAPK pathway inhibition in a panel of KRAS-mutant CRC cell lines. We found that treatment with either the MEK inhibitor trametinib or ERK inhibitor Vx-11e induced upregulation of interferon gene signatures and JAK/STAT pathway signaling components. Inhibition of MEK or ERK also transcriptionally increased levels of MHC Class I and antigen presentation machinery. Upregulation of MHC Class I was reversed with either pharmacologic inhibition of JAK/STAT signaling or genetic knockdown of the transcription factor IRF1. We propose that combining MAPK inhibition with checkpoint immunotherapy may provide an effective treatment in KRAS-mutant CRC tumors in vivo and that potential synergy may be due to enhancing the immunogenicity of tumors through priming of interferon response pathways and antigen presentation machinery. Citation Format: Meagan B. Ryan, Ferran Fece de la Cruz, Leanne G. Ahronian, Sarah Phat, David T. Myers, Heather A. Shahzade, Catriona Hong, Ryan B. Corcoran. ERK MAPK inhibition enhances the immunogenicity of KRAS-mutant 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 B50.

PCC0208023, a potent SHP2 allosteric inhibitor, imparts an antitumor effect against KRAS mutant colorectal cancer

The non-receptor tyrosine phosphatase SHP2, encoded by PTPN11, plays an indispensable role in tumors driven by oncogenic KRAS mutations, which frequently occur in colorectal cancer. Here, PCC0208023, a potent SHP2 allosteric inhibitor, was synthesized to evaluate its inhibitory effects against the SHP2 enzyme, and the KRAS mutant colorectal cancer in vitro and in vivo, and its impart on the RAS/MAPK pathway. Consistent with an allosteric mode of inhibition, PCC0208023 can non-competitively inhibit the activity of full-length SHP2 enzyme, but lacks activity against the free catalytic domain of SHP2. Furthermore, PCC0208023 inhibited the proliferation of KRAS mutation-driven human colorectal cancer cells by inhibiting the RAS/MAPK signaling pathway in vitro. Importantly, PCC0208023 displayed good anti-tumor efficacy against KRAS-driven LS180 and HCT116 xenograft models in nude mice with the decreased Ki67 and p-ERK level, and increased cleaved caspase-3 expression in tumors. Interestingly, PCC0208023 maintained high levels in LS180 tumors within 24 h after administration and was mainly distributed in both intestines and lungs. Molecular docking studies revealed a higher affinity of PCC0208023 with key residues in the SHP2 allosteric pocket than RMC-4550. PCC0208023 deserves further optimization to identify additional low-toxic and potent SHP2 allosteric inhibitors with novel scaffolds for the treatment of patients with KRAS mutation-positive colorectal cancer.

Gene editing particle system as a therapeutic approach for drug-resistant colorectal cancer

The epidermal growth factor receptor (EGFR) pathway plays an important role in the progression of colorectal cancer (CRC). Anti-EGFR drugs based on antibodies have been widely used for treating CRC through inducing the cell death pathway. However, the majority of CRC patients will inevitably develop drug-resistance during anti-EGFR drug treatment, which is mainly caused by a point mutation in the KRAS oncogene. We developed a nanoliposomal (NL) particle containing the Cas9 protein and a single-guide RNA (sgRNA) complex (Cas9-RNP), for genomic editing of the KRAS mutation. The NL particle is composed of bio-compatible lipid compounds that can effectively encapsulate Cas9-RNP. By modifying the NL particle to include the appropriate antibody, it can specifically recognize EGFR expressing CRC and effectively deliver the gene-editing complexes. The conditions of NL treatment were optimized using a KRAS mutated CRC in vivo mouse model. Mice with KRAS-mutated CRC showed drug resistance against cetuximab, a therapeutic antibody drug. After treating the mice with the KRAS gene-editing NL particles, the implanted tumors showed a dramatic decrease in size. Our results demonstrated that this genomic editing complex has great potential as a therapeutic carrier system for the treatment of drug-resistant cancer caused by a point mutation.

The proprotein convertase furin is a pro-oncogenic driver in KRAS and BRAF driven colorectal cancer.

Mutations in KRAS and/or BRAF that activate the ERK kinase are frequently found in colorectal cancer (CRC) and drive resistance to targeted therapies. Therefore, the identification of therapeutic targets that affect multiple signaling pathways simultaneously is crucial for improving the treatment of patients with KRAS or BRAF mutations. The proprotein convertase furin activates several oncogenic protein precursors involved in the ERK-MAPK pathway by endoproteolytic cleavage. Here we show that genetic inactivation of furin suppresses tumorigenic growth, proliferation, and migration in KRAS or BRAF mutant CRC cell lines but not in wild-type KRAS and BRAF cells. In a mouse xenograft model, these KRAS or BRAF mutant cells lacking furin displayed reduced growth and angiogenesis, and increased apoptosis. Mechanistically, furin inactivation prevents the processing of various protein pecursors including proIGF1R, proIR, proc-MET, proTGF-β1 and NOTCH1 leading to potent and durable ERK-MAPK pathway suppression in KRAS or BRAF mutant cells. Furthermore, we identified genes involved in activating the ERK-MAPK pathway, such as PTGS2, which are downregulated in the KRAS or BRAF mutant cells after furin inactivation but upregulated in wild-type KRAS and BRAF cells. Analysis of human colorectal tumor samples reveals a positive correlation between enhanced furin expression and KRAS or BRAF expression. These results indicate that furin plays an important role in KRAS or BRAF-associated ERK-MAPK pathway activation and tumorigenesis, providing a potential target for personalized treatment.

Synthesis and in-vitro anti-cancer evaluations of multi-methoxylated asymmetrical diarylpentanoids as intrinsic apoptosis inducer against colorectal cancer

In the present study, a series of nine stable 3,4,5-methoxylphenyl-containing asymmetrical diarylpentanoids, derivatives of curcuminoids, have been synthesized, characterized and evaluated for their in-vitro anti-cancer potential against a panel of BRAF- and KRAS-mutated colorectal cancer cell lines including T84, LoVo and SW620, HT29, RKO and NCI-H508, respectively. Structure-activity relationship study on cytotoxicity of tested compounds suggested that the presence of meta-hydroxyl and adjacent dimethoxyl groups are crucial for enhanced cytotoxicity of diarylpentanoids. Among the evaluated analogs, 8 has been identified as the lead compound due to its highest chemotherapeutic index of 9.9 and nano molar scale cytotoxicity against SW620 and RKO. Colonies formation and cell cycle analyses on 8-treated RKO cells showed that 8 exhibits strong anti-proliferative activity by inducing G2/M-phase cell arrest. Subsequent flow cytometry based annexin-V and DCFHDA studies suggested that 8 could induce apoptosis through intracellular ROS-dependent pathway. Further Western blot studies confirmed that 8 has induced intrinsic apoptosis in RKO cells through the up-regulations of Bad and Bax pro-apoptotic proteins and down-regulations of Bcl-2 and Bcl-xL pro-survival proteins. In all, the present results suggest that 8 could be a potent lead which deserves further modification and investigation in the development of small molecule-based anti-colorectal cancer agents.

Methylglyoxal Scavengers Resensitize KRAS-Mutated Colorectal Tumors to Cetuximab

The use of cetuximab anti-epidermal growth factor receptor (anti-EGFR) antibodies has opened the era of targeted and personalized therapy in colorectal cancer (CRC). Poor response rates have been unequivocally shown in mutant KRAS and are even observed in a majority of wild-type KRAS tumors. Therefore, patient selection based on mutational profiling remains problematic. We previously identified methylglyoxal (MGO), a by-product of glycolysis, as a metabolite promoting tumor growth and metastasis. Mutant KRAS cells under MGO stress show AKT-dependent survival when compared with wild-type KRAS isogenic CRC cells. MGO induces AKT activation through phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin 2 (mTORC2) and Hsp27 regulation. Importantly, the sole induction of MGO stress in sensitive wild-type KRAS cells renders them resistant to cetuximab. MGO scavengers inhibit AKT and resensitize KRAS-mutated CRC cells to cetuximab invivo. This study establishes a link between MGO and AKT activation and pinpoints this oncometabolite as a potential target to tackle EGFR-targeted therapy resistance in CRC.