KRAS Mutant CRC Research Articles

Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation.

Background and Purpose: RAS mutations limit the effectiveness of anti-epidermal growth factor receptor (EGFR) monoclonal antibodies in combination with chemotherapy for metastatic colorectal cancer (mCRC) patients. Therefore, new cell death forms have focused on identifying indirect targets to inhibit Ras-induced oncogenesis. Recently, emerging evidence has shown the potential of triggering ferroptosis for cancer therapy, particularly for eradicating aggressive malignancies that are resistant to traditional therapies.Methods: KRAS mutant CRC cell HCT116 and Lovo were treated with cetuximab and β-elemene, a bioactive compound isolated from Chinese herb Curcumae Rhizoma. Ferroptosis and epithelial-mesenchymal transformation (EMT) were detected in vitro and in vivo. Orthotopic CRC animal model were established and the tumor growth was monitored by IVIS bioluminescence imaging. Tumor tissues were collected to determine ferroptosis effect and the expression of EMT markers after the treatment.Results: CCK-8 assay showed that synergetic effect was obtained when 125 µg/ml β-elemene was combined with 25 µg/ml cetuximab in KRAS mutant CRC cells. AV/PI staining suggested a non-apoptotic mode of cell death after the treatment with β-elemene and cetuximab. In vitro, β-elemene in combination with cetuximab was shown to induce iron-dependent reactive oxygen species (ROS) accumulation, glutathione (GSH) depletion, lipid peroxidation, upregulation of HO-1 and transferrin, and downregulation of negative regulatory proteins for ferroptosis (GPX4, SLC7A11, FTH1, glutaminase, and SLC40A1) in KRAS mutant CRC cells. Meanwhile, combinative treatment of β-elemene and cetuximab inhibited cell migration and decreased the expression of mesenchymal markers (Vimentin, N-cadherin, Slug, Snail and MMP-9), but promoted the expression of epithelial marker E-cadherin. Moreover, ferroptosis inhibitors but not other cell death suppressors abrogated the effect of β-elemene in combination with cetuximab on KRAS mutant CRC cells. In vivo, co-treatment with β-elemene and cetuximab inhibited KRAS mutant tumor growth and lymph nodes metastases.Conclusions: Our data for the first time suggest that the natural product β-elemene is a new ferroptosis inducer and combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant CRC cells by inducing ferroptosis and inhibiting EMT, which will hopefully provide a prospective strategy for CRC patients with RAS mutations.

Abstract 4294: C6 Ceramide potentiates paclitaxel and gemcitabine mediated antitumor effects against pancreatic cancer via inhibition of prosurvival PI3k/AKT/mTOR and ERK pathways

Abstract The 5% survival of pancreatic cancer emphasizes need for new treatment strategies. Our studies suggest potential value of C6Ceramide (C6Cer) as a chemotherapeutic adjunct. The current study demonstrates that combination chemotherapy of Paclitaxel with adjunct C6Cer significantly increased cell death (apoptosis) in pancreatic cancer cell lines (L3.6,PANC-1, MIA (PaCa-2) in vitro as well as in vivo - enhancing tumor regression in SCID mice bearing L3.6 transplants. A similar synergistic effect was observed with addition of C6Cer to cetuximab which is not effective in KRAS mutant Pancreatic or Colorectal cancer and thus is not used clinically. At the molecular level combining C6Cer with Gemcitabine or Paclitaxel resulted in significant decreased p-AKT and p-GSK-a/b(PI3K/AKT (survival)pathway in L3.6 cells. Conclusion: C6Cer significantly inhibited key cancer survival pathways resulting in significant synergism with chemotherapeutics in therapy of Pan Ca and KRAS mutant CRC. Citation Format: Harold J. Wanebo. C6 Ceramide potentiates paclitaxel and gemcitabine mediated antitumor effects against pancreatic cancer via inhibition of prosurvival PI3k/AKT/mTOR and ERK pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4294.

Warburg metabolic shift induced by vitamin C in KRAS mutant CRC

Warburg metabolic shift induced by vitamin C in KRAS mutant CRC

Abstract 911: Targeting the mutant KRAS/BRAF-induced enhancer as an approach to overcome resistance to 5-fluorouracil-based chemotherapy in colorectal cancer

Abstract Colorectal cancer (CRC) is the third most common cancer and is expected to cause approximately 50,000 deaths in the US in 2017. The antimetabolite 5-fluorouracil (5-FU) is the cornerstone of first-line adjuvant chemotherapy regimens for CRC. Resistance to 5-FU-based chemotherapies develops over time and is the greatest obstacle to effective management in this disease. However, the resistance mechanisms of 5-FU are largely unknown. Approximately 50% of the CRC cases have activating mutations in KRAS or BRAF. Based on recent clinical data, it is now appreciated that KRAS/BRAF mutations are associated with worse survival in 5-FU-based chemotherapies for CRC. Therefore, there is an urgent unmet need for improved therapeutic options to treat mutant KRAS/BRAF tumors. We analyzed the differential activation status of enhancers in KRAS/BRAF mutant and wildtype CRC cell lines. Enhancers that associated with genes regulating pyrimidine metabolism were significantly enriched in KRAS/BRAF mutant CRC cells. Among them, the differential activation of an enhancer located ~400kb upstream of the DPYD promoter was most significant. By using CRISPR-dCas9 system, specifically targeting of p300 to the enhancer significantly increased DPYD expression, and Ezh2 significantly decreased expression. Knockout (KO) of this enhancer resulted in reductions in DPD expression and increased resistance to 5-FU. In mouse xenograft studies, enhancer-KO cells responded to doses of 5-FU that were otherwise ineffective against control cells. Treating KRAS mutant CRC cells with CBP30, H3K27 acetyl-transferase inhibitor, significantly reduced DPYD expression. Synergistic interactions was identified between CBP30 and 5-FU. Finally, mouse xenograft studies strongly suggest that inhibition of H3K27ac may significantly improve 5-FU effectiveness against KRAS mutant tumors. In summary, the present studies advance our understanding of how mutant KRAS/BRAF epigenetically regulates DPYD expression and results in 5-FU resistance in CRC. We showed that clinical treatment of CRC might be significantly improved by the development of future pharmacologic approaches targeting enhancers that are activated by mutant KRAS/BRAF. Citation Format: Rentian Wu, Qian Nie, Phillip A. Kubica, Zhiquan Wang, Shikshya Shrestha, Colbren S. Trogstad-Isaacson, Calvin R. Jerde, Erin E. Tapper, Kelly J. Bouchonville, Steven M. Offer, Robert B. Diasio. Targeting the mutant KRAS/BRAF-induced enhancer as an approach to overcome resistance to 5-fluorouracil-based chemotherapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 911.

P3.02-066 Wild-Type KRAS Mediates Growth Inhibition and Resistance to MEK Inhibitors through Dimerization with Mutant KRAS in Lung Adenocarcinoma

Mutations in KRAS are the most frequent RAS alterations in human cancers and the prevalent driver event in lung adenocarcinoma (LUAD). There are no effective targeted therapies for KRAS-driven LUAD and chemotherapy remains the standard of care. Small-molecule inhibitors of the MAPK pathway, one of the prominent downstream KRAS mediators, show minimal clinical activity either as single agents or in combination with chemotherapy. Recently, wild-type KRAS (KRASWT) was shown to enhance tumor fitness in KRAS mutant AML and CRC cell lines while concomitantly increasing sensitivity to MEK inhibition. We hypothesized that dimerization between KRAS proteins could be a key regulator for lung adenocarcinoma biology and determinant of treatment response. To study the role of wild-type KRAS in the context of KRAS-driven cancer cells, we used genetically inducible models of KRAS loss of heterozygosity (LOH). We developed an isogenic KRASMUT inducible system that lacks endogenous HRas/NRas but harbors conditional CREERT2-controlled KRaslox alleles. Furthermore, we reconstituted KRASWT and dimerization-deficient KRASD154Q in KRAS-driven murine and human LUAD cell lines lacking the wild-type KRAS allele and evaluated the in vitro and in vivo impact on tumor progression and response to MEK inhibition. KRASWT decreased in vitro and in vivo fitness of human and murine KRAS mutant LUAD tumor cells. However, this phenotype was reverted upon MEK inhibition, with KRAS LOH cells being more sensitive than KRASWT expressing cells. Interestingly, both effects were dependent on wild-type/mutant KRAS dimerization and not observed with the dimerization-deficient KRASD154Q. We provide a mechanistic model of the ambivalent function of KRASWT, linking its tumor suppressor function with increased MEK inhibitor resistance through dimerization with mutant KRAS. KRASWT affects cellular fitness in KRAS-driven LUAD. KRASWT impairs response to MEK inhibitors in KRAS-driven LUAD. KRASWT inhibitory effect is dependent on dimerization with mutant KRAS. Impaired wild-type/mutant KRAS dimerization restores sensitivity to MEK inhibitors in vivo.

Phase II trial of capecitabine +/- erlotinib in advanced colorectal cancer, with retrospective KRAS and primary tumor site analysis.

781 Background: Palliative capecitabine (X) monotherapy for advanced colorectal colorectal cancer (aCRC) is generally well tolerated by elderly or frail pts. Epidermal growth factor receptor (EGFR) monoclonal antibodies improve efficacy when added to combination chemotherapy for mCRC. Erlotinib (E), an oral EGFR tyrosine kinase inhibitor (TKI) may add benefit when added to X. We conducted a randomized phase II trial to investigate the novel “all-oral” combination of X and E in aCRC. Methods: Pts with untreated aCRC who were either deemed unfit for, or chose against, combination chemotherapy were randomized to X (1000 mg/m2 PO BID x 14 days) alone or in combination with E (150 mg PO OD) on a 3-week schedule. Primary endpoint was time to disease progression (TTP); secondary endpoints included: objective response rate (ORR), overall survival (OS), and safety. Tumours were designated as left-sided if there were distal to the transverse colon. KRAS status was retrospectively analyzed for 72 of 82 pts. Results: From 2004 to 2008, 82 pts were randomized to X alone (40 pts) or XE (42 pts). TTP was not different between X and XE (7.9 m vs. 9.2 m; p = 0.890), however, KRAS subgroup analysis revealed pts with KRAS mutations did significantly worse when treated with XE compared to X alone (1.9m vs 7.4m; HR = 2.63; P = 0.038). Pts with KRAS wild-type (WT) treated with XE had an improved TTP compared to those treated with X (11.7m vs. 8.4m, HR = 0.73; Wilcoxon P = 0.061). KRAS-WT pts treated with XE with left-sided disease had an improved OS compared to pts with right-sided disease (16m vs. 12.1m, not significant). KRAS-WT pts with left-sided primaries treated with XE had a non-significant improvement in TTP compared to pts treated with X alone (11.7m vs 8.4m). XE was well tolerated but had significantly higher rates of diarrhea and acneform skin rash. Conclusions: The addition of E to X is generally well tolerated but associated with additional toxicities. E may benefit pts with KRAS-wild-type CRC, specifically those with left-sided primary tumours, and likely harms those with KRAS-mutated CRC. Further study of oral EGFR-TKIs in left-sided KRAS-wild-type aCRC is warranted.

C6-ceramide's effect on KRAS-defined colorectal cancer cells treated with oxaliplatin, 5-fluoruracil with and without cetuximab.

619 Background: Cetuximab (Cet) is beneficial for patients with metastatic KRAS Wild type (WT) colorectal cancer (mCRC) only. C6-Ceramide (C6-Cer) can act synergistically with chemotherapy to induce cancer cell death. The aim of this study was to compare growth inhibition percentage (GIP) of cytostatics 5-fluoruracil (5-FU), oxaliplatin (Ox) and Cet with or without C6-Cer in KRAS WT and KRAS mutant (KRAS Mut) CRC cell lines (SW48 and SW480, respectively). Methods: Both cell lines were incubated with IC50 concentrations of test drugs. Drug concentrations included 0.8µM for 5-FU, 0.04µM for Ox, 25 µg/mL for Cet, and C6-Cer concentrations ranged from 5 to 10 µM. Cell survival was assessed 72h after using 0.4% Trypan Blue. Results: With above mentioned concentrations, C6-Cer’s GIP was 78.3% for SW-480 (vs. 33.33% for SW-48). It was also noted that the addition of C6-Cer to a combination of steady concentrations of Ox, Cet and 5-FU increased GIP with an especially significant effect on SW-480. Addition of 5 and 7.5µM resulted in doubling of GIP (75% and 86.25%, respectively, vs 32.5% of 5-FU + Ox + Cet). The greatest effect was seen when 10µM of C6-Cer was added to the IC50 concentrations of chemotherapeutic agents (using the 25µg/mL concentration of Cet), where GIP increased from 32.5% to 92.5% in SW-480. Same concentration of drugs increased GIP for SW-48 to a similar 93.5%. Conclusions: C6-Cer appears to have direct inhibitory properties, especially on KRAS Mut cells. Additionally when added to Ox, 5-FU and Cet, C6-Cer reversed the apparent insensitivity of KRAS Mut to Cet. Also, the study showed C6-Cer can provide additional synergism to their cytostatic properties in KRAS WT CRC cell lines. The true mechanism of these observations is still undergoing analysis, however the effect of isolated C6-Cer on KRAS Mut raises possibility of a different pathway that could bypass EFGR pathway. We believe the results of this study provide a starting point for clinical studies with C6-Ceramide in patients with relapsing or metastatic KRAS Mut CRC in combination with standard chemotherapy plus molecular target agents hoping they will translate into clinical benefit for this difficult to treat patient population.

Abstract PR12: Discovery and preclinical evaluation of cEt-modified KRAS antisense oligonucleotide inhibitors

Abstract KRAS is one of the most frequently mutated genes in cancer and its activation is thought to underlie the pathogenesis of up to 30% of all human tumors. However, to date KRAS has proven difficult to target with traditional pharmacologic approaches. Antisense technology is particularly attractive for such difficult drug targets as antisense oligonucleotide (ASO) inhibitors can be designed based on a targets RNA sequence alone. cEt ASOs have been recently described and demonstrated to have significantly increased potency over previous generation ASO chemistries. Moreover, STAT3Rx/AZD9150, a cEt modified ASO targeting STAT3 mRNA was recently shown to produce robust STAT3 depletion in a broad range of xenograft models (AACR 2013) and importantly has also demonstrated promising single-agent antitumor activity in patients with advanced treatment-refractory cancers in phase I studies (ASCO 2013, EORTC 2014). Here, we describe the preclinical evaluation of potent and selective cEt ASOs targeted to human or mouse KRAS mRNA. A human-specific KRAS cEt ASO which potently and selectively down-regulated KRAS demonstrated anti-proliferative effects and the expected down-stream pathway inhibition in a panel of KRAS mutant NSCLC, CRC and PDAC cells. The KRAS ASO was differentiated from MAPK pathway inhibitors selectively inhibiting the proliferation of KRAS mutant not KRAS wild type cells and not causing feedback reactivation of the MAPK or PI3K pathways. Systemic delivery of the KRAS ASO to mice bearing KRAS mutant NSCLC or CRC xenografts resulted in significant inhibition of KRAS tumour expression and antitumor activity. Importantly, the KRAS ASO also showed significant target knockdown and antitumor effects in KRAS mutant NSCLC patient-derived xenografts (PDX). Finally, potent and selective murine-specific KRAS ASO produced robust target knockdown in a broad set of tissues without detectable tolerability signals associated with ASO-mediated systemic KRAS inhibition. Taken together these data suggest that KRAS ASOs are an attractive therapeutic approach to target KRAS for the treatment of human cancers. Citation Format: Alexey S. Revenko, Sarah J. Ross, Lyndsey L. Hanson, Rebecca Ellston, Chris May, Sanjay K. Pandey, Linda K. Buckett, Stephanie K. Klein, Mitchell Revill, Kevin Hudson, Brett P. Monia, David C. Blakey, Paul Lyne, Allan R. MacLeod. Discovery and preclinical evaluation of cEt-modified KRAS antisense oligonucleotide inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PR12.

Molecular profiling of 6,892 colorectal cancer samples suggests different possible treatment options specific to metastatic sites

Metastatic colorectal cancer (mCRC) carries a poor prognosis with an overall 5-year survival of 13.1%. Therapies guided by tumor profiling have suggested benefit in advanced cancer. We used a multiplatform molecular profiling (MP) approach to identify key molecular changes that may provide therapeutic options not typically considered in mCRC. We evaluated 6892 mCRC referred to Caris Life Sciences by MP including sequencing (Sanger/NGS), immunohistochemistry (IHC) and in-situ hybridization (ISH). mCRC metastases to liver, brain, ovary or lung (n = 1507) showed differential expression of markers including high protein expression of TOPO1 (52%) and/or low RRM1 (57%), TS (71%) and MGMT (39%), suggesting possible benefit from irinotecan, gemcitabine, 5FU/capecitabine and temozolomide, respectively. Lung metastases harbored a higher Her2 protein expression than the primary colon tumors (4% vs. 1.8%, p = 0.028). Brain and lung metastases had higher KRAS mutations than other sites (65% vs 59% vs 47%, respectively, p = 0.07, <0.01), suggesting poor response to anti-EGFR therapies. BRAF-mutated CRC (n = 455) showed coincident high protein expression of RRM1 (56%), TS (53%) and low PDGFR (22%) as compared with BRAF wild-type tumors. KRAS-mutated mCRC had higher protein expression of c-MET (47% vs. 36%) and lower MGMT (56% vs. 63%), suggesting consideration of c-MET inhibitors and temozolomide. KRAS-mutated CRC had high TUBB3 (42% vs. 33%) and low Her2 by IHC (0.5%) and HER2 by FISH (3%, p <0.05). CRC primaries had a lower incidence of PIK3CA and BRAF mutations in rectal cancer versus colon cancer (10% and 3.3%, respectively). MP of 6892 CRCs identified significant differences between primary and metastatic sites and among BRAF/KRAS sub-types. Our findings are hypothesis generating and need to be examined in prospective studies. Specific therapies may be considered for different actionable targets in mCRC as revealed by MP.

Antitumor activity of a potent MEK inhibitor, TAK-733, against colorectal cancer cell lines and patient derived xenografts.

CRC is a significant cause of cancer mortality, and new therapies are needed for patients with advanced disease. TAK-733 is a highly potent and selective investigational novel MEK allosteric site inhibitor. In a preclinical study of TAK-733, a panel of CRC cell lines were exposed to varying concentrations of the agent for 72 hours followed by a sulforhodamine B assay. Twenty patient-derived colorectal cancer xenografts were then treated with TAK-733 in vivo. Tumor growth inhibition index (TGII) was assessed to evaluate the sensitivity of the CRC explants to TAK-733 while linear regression was utilized to investigate the predictive effects of genotype on the TGII of explants. Fifty-four CRC cell lines were exposed to TAK-733, while 42 cell lines were deemed sensitive across a broad range of mutations. Eighty-two percent of the cell lines within the sensitive subset were BRAF or KRAS/NRAS mutant, whereas 80% of the cell lines within the sensitive subset were PIK3CA WT. Twenty patient-derived human tumor CRC explants were then treated with TAK-733. In total, 15 primary human tumor explants were found to be sensitive to TAK-733 (TGII ≤ 20%), including 9 primary human tumor explants that exhibited tumor regression (TGII > 100%). Explants with a BRAF/KRAS/NRAS mutant and PIK3CA wild-type genotype demonstrated increased sensitivity to TAK-733 with a median TGII of -6%. MEK-response gene signatures also correlated with responsiveness to TAK-733 in KRAS-mutant CRC. The MEK inhibitor TAK-733 demonstrated robust antitumor activity against CRC cell lines and patient-derived tumor explants. While the preclinical activity observed in this study was considerable, single-agent efficacy in the clinic has been limited in CRC, supporting the use of these models in an iterative manner to elucidate resistance mechanisms that can guide rational combination strategies.

Abstract 3575: Novel targets and monoclonal antibodies for antibody-drug conjugate therapy

Abstract The study focuses on two novel potential therapeutic targets identified by an immune-histochemistry (IHC) screening with a large collection of polyclonal antibodies (approximately 1600) raised against marginally characterized human proteins. Here we describe the molecular characterization of two surface-exposed proteins (EXN91 and EXN36) associated to different cancer types. EXN91 is an adhesion molecule and it acts as a signaling receptor, cell communication and motility. Prevalence studies showed that the protein is mainly detected in colon cancer (CRC)with high frequency (approximately 90%), both in early and advanced colon cancer stages, with a statistical association for early cancer stages. Moreover, the protein is clearly surface exposed in liver metastasis from colon. Experiments are ongoing to assess whether EXN91 is also expressed in precancerous conditions of colon and rectum that have the potential to develop into cancer. Interestingly, EXN91 is over-expressed in KRAS and BRAF mutant CRC with high frequency. Moreover, an expanded IHC analysis revealed that it is also over-expressed and surface exposed in a number of other cancers, including HCC, RCC, bladder and endometrium cancer. Preliminary data showed that EXN91 behaves as an auto-antigen being able to induce specific autoantibodies in CRC patients. EXN36 is mainly over-expressed in ovary and breast cancers (frequency of approximately 30-40%), including triple negative breast cancer. EXN36 prevalence studies in different cancer entities are ongoing. Moreover, EXN36 is involved in cell proliferation, migration and invasiveness. Murine monoclonal antibodies (mAb) able to recognize EXN91 and EXN36 on the surface of cancer cells show potential for specific therapeutic indications. In particular, a murine mAb recognizes EXN91 on the surface of colon cancer cells, including KRAS and BRAF mutant CRC. This antibody inhibits growth of colon cancer in xenograft mouse models. Concerning anti-EXN36 antibodies, they are able to bind the surface of different breast and ovary cancer cells. EXN36 and EXN91 mAbs show a high number of binding sites on the cell surface. The mAb specificity has been confirmed in different immunoassays by gene silencing and peptide competition assays. Moreover, they show limited IHC reactivity in normal tissues. The anti-EXN36 and antiEXN91 antibodies are efficiently internalized by cancer cells, suggesting that they can be exploited for the development of Antibody-Drug-Conjugate (ADC). Initial results from in vitro studies show that these antibodies, when tested in drug screening assays with Protein G conjugated to commercially available drugs show significant anti-tumor activity with a specific conjugation chemistry. Overall, the results indicate that EXN36, EXN91 and their mAbs could be developed for the targeted therapy of different cancer indications at high medical need. Citation Format: Matteo Parri, Susanna Campagnoli, Alberto Grandi, Elisa De Camilli, Paolo Sarmientos, Boquan Jin, Guido Grandi, Giuseppe Viale, Luigi Terracciano, Piero Pileri, Renata Maria Grifantini. Novel targets and monoclonal antibodies for antibody-drug conjugate therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3575. doi:10.1158/1538-7445.AM2015-3575

Abstract 2695: Using eicosapentaenoic acid to improve cetuximab sensitivity in KRAS mutants

Abstract It is known that epidermal growth factor receptor (EGFR) inhibitor, cetuximab, does not respond to cells with KRAS or BRAF mutations; therefore, a large proportion (more than 40%) of colorectal cancer patients are unable to receive this type of target therapy, and can only undergo surgical as a way of management. In this study, we aim to provide an alternative therapeutic strategy to KRAS or BRAF mutated neoplasms. Our previous studies have demonstrated that there may be better sensitivity to anti-EGFR antibody therapy in KRAS or BRAF mutants with increasing expression levels of miR-378. As known, miR-378 is embedded within PPARGC1b, which encodes PGC-1β, and could be stimulated by feeding lauric acid in cells. However, lauric acid is a type of saturated fatty acid, and therefore daily intake in humans is no recommended. Consequently, we tried to replace it with eicosapentaenoic acid (EPA or also icosapentaenoic acid), an omega-3 unsaturated fatty acid that is FDA-approved. Following this, we modulated the expression level of miR-378 in KRAS or BRAF mutants by feeding EPA in vitro, and eventually revealed the mechanism of cancer cells remission that currently remains unknown. The western blotting and ELISA assay were performed to analyze the MAPK/ERK pathway and caspase pathway in three cell lines (SW480 and HCT116 with contain KRAS mutants, and HT29 with contain BRAF mutants), including cell lines before and after feeding with 40 μM concentration of EPA. All experiments were then compared to the wild type cells (caco2), the control cell line. The results showed higher expression of miR-378 in all types of mutated cells, except the wild type CRC cells. In addition, lower cancer cell survival rate was observed in cells that were given 0.2 μM of cetuximab treatment, especially in KRAS mutated cells as well as control wild type cells (p = 0.010∼0.013). The total ERK1/2 protein in the KRAS mutant CRC cells and wild type CRC cell showed lower expression levels after receiving 40 μM EPAee for 24 hours (p = 0.022∼0.035). A higher phosphorylated proteins status of ERK1/2 was also seen (p = 0.006∼0.047). However, the opposite result was noted in BRAF mutant cells. In cells further treated with anti-EGFR antibody for 48 hours, cell viability was significantly lowered in KRAS mutant and control wild type cells (p = 0.006∼0.013), but there was no sensitivity of anti-EGFR antibody reaction to the EPA-fed BRAF mutant cell. The data demonstrated with EPA may indeed significantly induce the expression of miR-378, and further restored the sensitivity of anti-EGFR antibody in KRAS mutant cells. In conclusion, KRAS mutant cells may restore sensitivity to cetuximab after up-regulation of the miR-378 induced by EPA. This finding has offered a new alternative therapeutic solution for future patients suffering from KRAS mutated colorectal cancer. Citation Format: Chih-Wei Chen, Li-Wei Kuo, Yeu-Jye Pang, Wai-Hung Leung, Wen-Hui Weng. Using eicosapentaenoic acid to improve cetuximab sensitivity in KRAS mutants. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2695. doi:10.1158/1538-7445.AM2015-2695

A Phase II Study of Sorafenib Combined With Cetuximab in EGFR-Expressing, KRAS-Mutated Metastatic Colorectal Cancer

BackgroundMutations in the KRAS gene predict for resistance to anti-epidermal growth factor receptor (EGFR) therapies, including cetuximab. Upregulation of vascular endothelial growth factor (VEGF)-A has been implicated in resistance to anti-EGFR treatment. Abrogation of the VEGF and RAS/RAF/MEK/ERK pathways has the potential to restore cetuximab sensitivity. Patients and MethodsAdult patients with histologically documented, measurable, EGFR-expressing, KRAS-mutated metastatic colorectal cancer (mCRC) that had progressed after 5-fluorouracil–based regimens were treated with sorafenib 400 mg orally twice daily and intravenous cetuximab weekly in 28-day cycles. The primary endpoint was the response rate (complete response, partial response, and stable disease at 4 cycles). The secondary endpoints included plasma biomarker analysis of angiogenic cytokines and correlative imaging studies with dynamic contrast-enhanced magnetic resonance imaging and zirconium 89-panitumumab. ResultsOf the 30 patients enrolled, 26 were evaluable for response. Of the 26 patients evaluated, 4 had stable disease at 4 cycles and 1 had stable disease at 8 cycles. The median progression-free survival was 1.84 months. The common toxicities were rash, diarrhea, and liver enzyme elevations. Of the angiogenic cytokines evaluated, only the placental growth factor increased significantly with treatment (P < .0001). No pharmacodynamic parameters were associated with the treatment response. ConclusionWe report the results of a trial that combined cetuximab and sorafenib for the treatment of KRAS-mutated mCRC, with correlative imaging studies and pharmacodynamic angiogenic cytokine profiling as downstream markers of EGFR and VEGF receptor (VEGFR) signaling. No objective responses were observed. Additional development of biomarkers for patient selection is needed to evaluate combined EGFR and VEGFR blockade as a therapeutic option in KRAS-mutated CRC.

Abstract 4397: Therapeutic microRNA agaisnt KRAS mutant colorectal cancer

Abstract Background: The anti-epidermal growth factor receptor (EGFR) monoclonal antibodies are the molecular target drugs, dependent on v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations status in metastatic colorectal cancer (mCRC). Anti-EGFR monoclonal antibody based chemotherapy is the standard treatment for mCRC with KRAS wild-type. However, the issue of resistance to this therapy in CRC with KRAS mutations (approximately 50% of CRC) remains unresolved. The aim of this study is to identify microRNAs (miRNAs) that govern the resistance to anti-EGFR monoclonal antibody in CRC with KRAS mutations. Experimental Design: We first established the KRAS mutant cell model using HEK293 and MRC5 cells by introducing KRASG12V plasmid. The miRNA expression profiles were compared between these cells using miRNA microarray analysis. We selected the candidate a miRNA which down-regulates the downstream signal of KRAS using luciferase reporter assays. Assessment of target genes of the candidate a miRNA was performed in vitro. Next, anti-tumor effects by supplement of a miRNA were evaluated in vitro and in vivo. Results: We picked up miRNAs, of which expressions decreased in both HEK293 and MRC5 cells by introduction of KRASG12V plasmid in miRNA array analysis. Among them, administration of mature-miR-X suppressed SRE and AP1 activity in KRAS mutant CRC cell lines such as DLD1G13D, SW480 G12V, and HCT116 G13D using dual luciferase reporter assay. Furthermore, miR-X regulated not only Ras/Raf/MAPK pathway but also suppressed PI3K/Akt pathway in vitro. Administration of mature-miR-X suppressed proliferation and induced apoptosis of the CRC cell lines with KRAS mutations in vitro. Systemic mature-miR-X administration into tail veins of nude mice significantly inhibited the growth of pre-established DLD1G13D xenografts. miR-X level was significantly lower in tumor tissues compared to normal mucosa. Conclusions: miR-X may overcome anti-EGFR therapy resistance in CRC with KRAS mutations by regulating Ras/Raf/MAPK pathway and PI3K/Akt pathway. Our data suggests that miR-X could be suitable for a novel therapy for mCRC patients with KRAS mutations. Citation Format: Masayuki Hiraki, Junichi Nishimura, Mamoru Uemura, Taishi Hata, Ichiro Takemasa, Tsunekazu Mizushima, Hirofumi Yamamoto, Yuichiro Doki, Masaki Mori. Therapeutic microRNA agaisnt KRAS mutant colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4397. doi:10.1158/1538-7445.AM2014-4397

Effect of simvastatin plus cetuximab/irinotecan for KRAS mutant colorectal cancer and predictive value of the RAS signature for treatment response to cetuximab

Preclinical data has demonstrated the potential of simvastatin to overcome cetuximab resistance in KRAS mutant CRC patients. Therefore, we designed a study using simvastatin/cetuximab/irinotecan for KRAS mutant CRC patients who are refractory to irinotecan and oxaliplatin-based chemotherapy. In this phase II study, patients received 500 mg/m(2) cetuximab, 150-180 mg/m(2) (day 1), and 80 mg simvastatin (once daily, days 1-14, every 2 weeks). The primary endpoint was the objective response rate (ORR). Secondary endpoints were progression-free survival (PFS), overall survival (OS), the disease control rate (DCR), and safety. We also analyzed the relationship between the RAS gene expression signature score and treatment response to simvastatin/cetuximab/irinotecan. Fifty-two KRAS mutant CRC patients were enrolled. The ORR (complete response [CR], 0; partial response [PR], 1) was 1.9 % (95 % confidence interval [CI], -1.8-5.6). The DCR (CR, 0; PR, 1; stable disease, 33) was 65.4 % (95 % CI, 52.5-78.3). The median PFS and OS from the time of study drug administration were 7·6 months (95 % CI, 4.4-10.8) and 12.8 months (95 % CI, 9.5-16.2), respectively. The most common grade 3/4 adverse events were anemia (28.8 %), neutropenia (13.5 %), and diarrhea (7.7 %). The RAS signature score was significantly correlated with the maximal change in target lesions from baseline (r = 0.57, P = 0.014). The simvastatin/cetuximab/irinotecan regimen showed promising efficacy and safety in KRAS mutant CRC patients who failed irinotecan and oxaliplatin-based chemotherapy. The RAS signature may be a novel predictor of treatment response to cetuximab-combined chemotherapy in CRC patients.

A phase II study of combination epigenetic therapy in metastatic colorectal cancer (mCRC): A phase II consortium (P2C)/Stand Up 2 Cancer (SU2C) study.

3539 Background: Therapy with decitabine and entinostat (ENT: HDAC inhibitor) shows synergistic effect in re-expression of tumor-suppressor genes and growth inhibition in CRC cell lines and in vivo studies. Methods: We conducted a phase II, multi-institutional study of SQ 5-azacitidine (AZA) and oral ENT in mCRC pts. A 28 day cycle included: AZA at 40 mg/m2 d1-5 and 8-10 with ENT 7 mg d3 and 10. Initial eligibility criteria included: ECOG PS 0-1, good end organ function, and biopsiable disease for cohort A (CoA). An interim analysis indicated that toxicity which crossed pre-specified safety boundary was secondary to disease. A 2nd cohort B (CoB) with eligibility restrictions: &lt;2 prior regimens in KRAS-mutated CRC pts, &lt;3 prior regimens if KRAS wild-type, and liver disease limited to &lt;30% of volume was accrued. Serial tumor biopsies and research blood were collected to assess for methylation/expression changes in circulating tumor DNA and biopsies, respectively. The primary endpoint was response as measured by RECIST criteria using a 2-stage Simon design. Results: 47 pts were initially enrolled (24 CoA, 23 CoB). Pts received a median of 2 cycles on both cohorts (1-16 CoA, 2-6 CoB). Pts had a median of 4 prior therapies for CoA (range 2-9) and 3 for CoB (range 2-6). Gr 4 AEs attributable to treatment for CoA included hyperglycemia (1) and hypokalemia (1); other common Gr 3 AEs included anemia (3), decreased lymphocytes (7), fatigue (3), and nausea (3). CoB pts experienced grade 3 chest pain (1), neutropenia (2), leucopenia (2), urinary tract obstruction (1), and hypophosphatemia (1). No responses have been observed. Results of translational objectives will be presented at the meeting. Conclusions: SQ AZA and ENT therapy does not have clinical activity as defined by confirmed response in aCRC. Follow-up for survival, response to subsequent therapy, and correlative analysis are ongoing. Supported in part by N01-CM-2011-00099. Clinical trial information: NCT01105377. [Table: see text]

Immunomodulatory effects of a dipeptidyl peptidase inhibitor, ARI-4175, and NK cell activation in a colon cancer model.

3058 Background: A possible mechanism of the antitumor effect of therapeutic antibodies is antibody-dependent cellular cytotoxicity (ADCC). We reported that an inhibitor of dipeptidyl peptidase (DPP)4-like serine proteases, Ari-4175, significantly slowed the growth of KRAS-mutated HCT-116 xenografts in nude mice, either as a single agent or with cetuximab (Ctx). Ari-4175 is not cytotoxic in vitro, and since it was able to overcome the resistance to Ctx due to the KRAS mutation in the HCT-116 line, we hypothesized that the antitumor activity involves the activation of NK cells and enhancement of ADCC. Methods: Ari-4175 was administered orally to nude mice at 200 µg q.d. x5 days/week. Peripheral blood or spleens were assayed for immune parameters, ex vivo, at various time points. Expression of surface markers on myeloid and NK cells were monitored by flow. Natural cytotoxicity and ADCC were assayed using HCT116 cells and Ctx using a flow based assay. Cytokines were measured using Luminex assays. Results: Ari-4175 induced upregulation of CD69 on NK cells on day 2, followed by upregulation of CD16 on day 7. Coordinate with these changes in peripheral blood, splenocytes from treated mice exhibited significantly increased in vitro cytotoxicity toward the HCT116 cells. Increased serum levels of inflammatory cytokines, including IL-1b, IL-6, MCP-1, GCSF, and IL-2 were seen. A significant expansion of a distinct CD45+CD14+Gr-1+MHC-II-CDllb+CD11cvariable myeloid cell population in both peripheral blood and spleens of mice was also seen after one week of treatment. Conclusions: Ari-4175 showed dramatic anti-tumor effect in KRAS-mutant CRC xengrafts when given alone or in combination with Ctx. In vitro data suggest that the therapeutic effect of 4175 might partially be due to the augmentation of ADCC through elevated expression of CD16 on NK cells. In addition, Ari-4175 appears, in vivo, to expand a unique myeloid cell population, which may be responsible for an inflammatory cytokine response and subsequent activation of NK cells. Our study provides a mechanistic rationale for testing Ari-4175 in a clinical trial and possible biomarker endpoints for evaluation in peripheral blood samples.

A multicenter phase I study of intravenous administration of reolysin in combination with irinotecan/fluorouracil/leucovorin (FOLFIRI) in patients (pts) with oxaliplatin-refractory/intolerant KRAS-mutant metastatic colorectal cancer (mCRC).

450 Background: Reolysin (reovirus serotype 3) contains a naturally occurring, ubiquitous, non-enveloped human dearing strain reovirus. Reovirus replicates in KRAS-mutant cells resulting in cell lysis. In phase I evaluation, CRC pts received single agent Reolysin with tumor stabilization and CEA response without significant toxicity. Reolysin and irinotecan (IRI) are synergistic in KRAS-mutant preclinical CRC models, providing rationale for this phase I study. Methods: This was a phase I dose escalation study of FOLFIRI + Reolysin. Eligible pts were &gt;18 yrs with histologically confirmed KRAS-mutant mCRC, measurable disease, ECOG PS 0-1, &lt;3 metastatic regimens, and adequate organ function. Standard FOLFIRI was administered with escalating Reolysin doses (range 1x1010 TCID50 to 3x1010 TCID50) in cohorts of 3-6 pts. Reolysin was given IV over 1 hr days 1-5 every 28d (1 cycle). Primary objectives were dose-limiting toxicity (DLT) to determine MTD and pharmacokinetics. Secondary endpoints were antitumor activity, response rate, progression-free and overall survival (PFS and OS). Results: 21 pts enrolled; median age 62 (range 39-77); 5 M; 16 F; FOLFIRI-naïve: 9/21 pts. 2 pts had DLTs in cycle 1 at the highest dose of 180 mg/m2 of IRI. Common (&gt;10%) grade 3-4 toxicity include: neutropenia (n=11), anemia (n=4), and thrombocytopenia (n=3). One patient died of acute renal failure. The DLT is neutropenia. The recommended phase II dose is IRI 150 mg/m2 and Reolysin at 3x1010 TCID50 on days 1-5, q 28 days. 18 pts evaluable for response: PR (1pt; 5%), SD (9 pts; 50%), PD (8pts; 44%). 3 pts taken off study before evaluation. Median PFS: FOLFIRI-naïve pts = 7.4 mo. (95% CI = 1.9 mo., 12.9 mo.); Median PFS FOLFIRI non-naïve pts was not reached; overall median PFS = 7.4 mo. (95% CI = 0.6 mo., 14.1 mo.) Conclusions: The combination of Reolysin and FOLFIRI in pts with KRAS-mutant mCRC was safe, well tolerated and resulted in disease control in the majority of pts, including pts who previously progressed on IRI. We are encouraged by this activity and safety profile, and are planning additional studies. Clinical trial information: NCT01274624.

P1.28 Dusp4 Expression as A Marker of Heterogeneous Signaling in Colorectal Cancer Patients

ABSTRACT Introduction Previous analysis of the gene expression data of mutational subgroups in the PETACC3 trial, revealed a very homogenous gene expression pattern in BRAF mutant CRC patients; while KRAS mutant CRC contain at least three different patient groups in terms of gene expression. One of these KRAS mutant groups together with 10% of KRAS/BRAF WT (WT2) patients, resembled the gene expression found in the BRAF mutants. This BRAF-like subgroup is characterized by poor prognosis. In this article we investigate, if heterogeneity in CRC patients can be partly explained by diverse signaling downstream of KRAS, to which the DUSPs take part. Material and methods The expression of DUSP1, DUSP4 and DUSP6, DUSPs previously shown to be induced by mutations in genes of the MAPK pathway, was analyzed in 4 different CRC patient microarray datasets (PETACC3, cetuximab, TCGA and Agendia dataset). We investigated, if patients with low and high DUSP expression behave differently in terms of response, OS, PFS and differ in clinical characteristics both in PETACC3 and TCGA dataset. Findings Unlike DUSP1, DUSP4 and DUSP6 were differentially overexpressed between BRAF mutants and KRAS mutants on one side and between BRAF mutants and WT2 patients on the other side. When we subdivided the patients into low and high DUSP expression within KRAS mutant and WT2 patients, we found a significant difference in OS in both mutational subgroups with high DUSP4 expression in KRAS mutants associated with a better OS and in WT2 associated with a worse OS. Multivariate analysis in the BRAF WT patients in both PETACC3 and cetuximab dataset showed that high DUSP4 expression was still associated with a worse OS (HR = 1.23; p Conclusions DUSP4 is predictive for OS in CRC patients with KRAS mutations. Interestingly, within KRAS mutant and WT2 patients high DUSP4 expression correlates with clinical characteristics that are typical for BRAF mutant CRC patients. If DUSP4 may represent a novel biomarker for a subset of CRC patients needs further research. Download : Download full-size image Fig. 1 . Box plots with DUSP4 expression according to different mutational groups in PETACC3 dataset. Download : Download full-size image Fig. 2 . Kaplan-Meier curve for overall survival according to DUSP4 expression (low and high) in KRAS WT patients.

A phase I study of selumetinib (AZD6244/ARRY-142866) in combination with cetuximab (cet) in refractory solid tumors and KRAS mutant colorectal cancer (CRC).

3103 Background: KRAS mutations have been recognized as clinically important predictors of resistance to EGFR-directed therapies in CRC. Oncogenic activation of the RAS/RAF/MEK/ERK signaling cascade mediates proliferation independent of growth factor receptor signaling. We hypothesized that targeting MEK with selumetinib could overcome resistance to cet in KRAS mutant CRC. A phase I study (NCT01287130) was undertaken to determine the tolerability, and pharmacokinetic profiles of the combination of selumetinib and cet, with an expanded cohort in KRAS mutant CRC at the MTD dose to evaluate preliminary anti-tumor activity. Methods: In the dose escalation portion, patients (pts) with advanced solid tumors received fixed dose cet with escalating doses of selumetinib in cohorts of 3-6 pts. In the expansion cohort, 14 pts with KRAS mutant CRC were enrolled at the MTD level. Results: 15 pts (9 M, 6 F), average age of 60 (41-73) years were treated at 3 dose levels in the dose escalation cohort and 14 pts were treated in the expansion cohort. Pts had the following tumor types: CRC 73%, NSCLC 13%, and H&amp;N 13%, and had received a median of 4 (1-8) prior lines of therapy. 33% (only CRC) had prior EGFR-directed therapies. ECOG PS 0 (40%), 1 (53%), 2 (7%). 13 of 15 pts were evaluable for tolerability and response. One DLT for grade 4 hypomagnesemia occurred, and no other grade 4 toxicities were seen. Grade 3 (20%) toxicities included; rash, hyponatremia, and headache. The most common cycle 1 grade 1 and 2 adverse events included acneiform rash (100%), fatigue (54%), nausea/vomiting, (54%), diarrhea (54%), dry skin (46%), fever (23%), and hypomagnesemia (15%). Most pts (60%) required no dose modifications. The MTD was established at selumetinib 75 mg PO BID and cet 250 mg/m2 weekly following a 400 mg/m2load. Best response included 2 PR in pts with CRC and SD in 4 pts (1 SCC of the tonsil, 1 NSCLC, and 2 CRC). Conclusions: The combination of selumetinib and cet is well tolerated, and preliminary anti-tumor activity was observed in multiple pts. Results of the KRAS mutant CRC expansion cohort will be presented.