KRAS Wild-type Cell Lines Research Articles

CX-4945 (Silmitasertib) Induces Cell Death by Impairing Lysosomal Utilization in KRAS Mutant Cholangiocarcinoma Cell Lines.

Macropinocytosis is a non-selective form of endocytosis that facilitates the uptake of extracellular substances, such as nutrients and macromolecules, into the cells. In KRAS-driven cancers, including pancreatic ductal adenocarcinoma, macropinocytosis and subsequent lysosomal utilization are known to be enhanced to overcome metabolic stress. In this study, we investigated the role of Casein Kinase 2 (CK2) inhibition in macropinocytosis and subsequent metabolic processes in KRAS mutant cholangiocarcinoma (CCA) cell lines. The bovine serum albumin (BSA) uptake indicating macropinocytosis was performed by flow cytometry using the HuCCT1 KRAS mutant CCA cell line. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and western blot. The CX-4945 (Silmitasertib), CK2 inhibitor, was used to investigate the role of CK2 in macropinocytosis and subsequent lysosomal metabolism. The TFK-1, a KRAS wild-type CCA cell line, showed only apoptotic morphological changes. However, the HuCCT1 cell line showed macropinocytosis. Although CX-4945 induced morphological changes accompanied by the accumulation of intracellular vacuoles and cell death, the level of macropinocytosis did not change. These intracellular vacuoles were identified as late macropinosomes, representing Rab7+ vesicles before fusion with lysosomes. In addition, CX-4945 suppressed LAMP2 expression following the inhibition of the Akt-mTOR signaling pathway, which interrupts mature macropinosome and lysosomal metabolic utilization. Macropinocytosis is used as an energy source in the KRAS mutant CCA cell line HuCCT1. The inhibition of CK2 by CX-4945 leads to cell death in HuCCT1 cells through alteration of the lysosome-dependent metabolism.

Effectiveness of 225Ac-Labeled Anti-EGFR Radioimmunoconjugate in EGFR-Positive Kirsten Rat Sarcoma Viral Oncogene and BRAF Mutant Colorectal Cancer Models.

Eighty percent of colorectal cancers (CRCs) overexpress epidermal growth factor receptor (EGFR). Kirsten rat sarcoma viral oncogene (KRAS) mutations are present in 40% of CRCs and drive de novo resistance to anti-EGFR drugs. BRAF oncogene is mutated in 7%-10% of CRCs, with even worse prognosis. We have evaluated the effectiveness of [225Ac]Ac-macropa-nimotuzumab in KRAS mutant and in KRAS wild-type and BRAFV600E mutant EGFR-positive CRC cells invitro and invivo. Anti-CD20 [225Ac]Ac-macropa-rituximab was developed and used as a nonspecific radioimmunoconjugate. Methods: Anti-EGFR antibody nimotuzumab was radiolabeled with 225Ac via an 18-membered macrocyclic chelator p-SCN-macropa. The immunoconjugate was characterized using flow cytometry, radioligand binding assay, and high-performance liquid chromatography, and internalization was studied using live-cell imaging. In vitro cytotoxicity was evaluated in 2-dimensional monolayer EGFR-positive KRAS mutant DLD-1, SW620, and SNU-C2B; in KRAS wild-type and BRAFV600E mutant HT-29 CRC cell lines; and in 3-dimensional spheroids. Dosimetry was studied in healthy mice. The invivo efficacy of [225Ac]Ac-macropa-nimotuzumab was evaluated in mice bearing DLD-1, SW620, and HT-29 xenografts after treatment with 3 doses of 13 kBq/dose administered 10 d apart. Results: In all cell lines, invitro studies showed enhanced cytotoxicity of [225Ac]Ac-macropa-nimotuzumab compared with nimotuzumab and controls. The inhibitory concentration of 50% in the DLD-1 cell line was 1.8 nM for [225Ac]Ac-macropa-nimotuzumab versus 84.1 nM for nimotuzumab. Similarly, the inhibitory concentration of 50% was up to 79-fold lower for [225Ac]Ac-macropa-nimotuzumab than for nimotuzumab in KRAS mutant SNU-C2B and SW620 and in KRAS wild-type and BRAFV600E mutant HT-29 CRC cell lines. A similar trend was observed for 3-dimensional spheroids. Internalization peaked 24-48 h after incubation and depended on EGFR expression. In the [225Ac]Ac-macropa-nimotuzumab group, 3 of 7 mice bearing DLD-1 tumors had complete remission. Median survival was 40 and 34 d for mice treated with phosphate-buffered saline and [225Ac]Ac-macropa-rituximab (control), respectively, whereas it was not reached for the [225Ac]Ac-macropa-nimotuzumab group (>90 d). Similarly, median survival of mice bearing HT-29 xenografts was 16 and 12.5 d for those treated with [225Ac]Ac-macropa-rituximab and phosphate-buffered saline, respectively, and was not reached for those treated with [225Ac]Ac-macropa-nimotuzumab (>90 d). One of 7 mice bearing HT-29 xenografts and treated using [225Ac]Ac-macropa-nimotuzumab had complete remission. Compared with untreated mice, [225Ac]Ac-macropa-nimotuzumab more than doubled (16 vs. 41 d) the median survival of mice bearing SW620 xenografts. Conclusion: [225Ac]Ac-macropa-nimotuzumab is effective against KRAS mutant and BRAFV600E mutant CRC models.

KRAS-mutant colorectal cancer cell lines cause a prothrombotic state through the upregulation of thrombin: experimental study

Background: The KRAS genotype status is strongly associated with a prothrombotic state in colorectal cancer, and hypercoagulability and cancer-related thrombosis are among the significant events leading to poor prognosis. However, this correlation has not been confirmed at the cellular level. This study aimed to assess the maximum platelet aggregation rate and thrombin expression induced by colorectal cancer cells under different KRAS genotypes. Materials and methods: Platelet aggregation rate assay and western blotting analysis were used to detect platelet aggregation and thrombin expression induced by four colorectal cancer cells with different KRAS genotypes, including RKO, HCT116, SW480, and SW620. FVIIa/tissue factor and thrombin inhibitors were added to explore changes in platelet aggregation rates induced by colorectal cancer cells and the association between KRAS genotype status and hypercoagulable state. Results: KRAS-mutant cells were more likely to increase maximal platelet aggregation, with RKO, HCT116, SW480, and SW620 inducing 34.7%, 55.4%, 44.4%, and 63.8% of platelet aggregation, respectively. The maximum platelet aggregation rate was higher in the metastatic rectal cancer tumour strain SW620 than in the primary rectal cancer strain SW480. RKO cells had lower thrombin expression than the other three cells. Furthermore, the addition of thrombin inhibitors caused a more significant decrease in the platelet aggregation rate in KRAS-mutant cell lines compared to KRAS wild-type cell lines. Conclusion: Compared to KRAS wild-type colorectal cancer cells, KRAS-mutant colorectal cancer cell lines were more likely to be hypercoagulable through the upregulation of thrombin expression, which was mainly achieved through the TF-thrombin pathway.

Cetuximab Enhances the Efficacy of MRTX1133, a Novel KRASG12D Inhibitor, in Colorectal Cancer Treatment.

Kirsten Rat Sarcoma viral oncogene homolog (KRAS) has remained undruggable for decades. KRAS has predominantly been used to evaluate the applicability of anti-Epidermal Growth Factor Receptor (EGFR) antibody drugs. However, various KRAS inhibitors have recently emerged. Unfortunately, KRAS inhibitors have not been effective against colorectal cancer. Therefore, this study aimed to determine the effects of MRTX1133, a novel KRASG12D inhibitor, in combination with an anti-EGFR antibody, cetuximab, on signal transduction and cell proliferation. The KRASG12D-mutated LS513 and KRAS wild-type CACO-2 human colon cancer cell lines were utilized. The KRASG12D mutation was stably transduced into the CACO-2 cells using a retrovirus. We evaluated the effects of the drugs using the CCK-8 assay and assessed the activity of proteins related to the MAPK pathway using western blotting. We demonstrated that the administration of MRTX1133, a novel KRASG12D inhibitor, to KRASG12D-mutated colorectal cancer cells led to feedback activation of the ERK pathway via EGFR activation, inducing drug resistance. Intriguingly, when MRTX1133 was used in combination with cetuximab, KRASG12D-mutant colorectal cancer growth was effectively inhibited, both in vitro and in vivo. The combination of MRTX1133 and cetuximab serves as a potential and promising therapeutic approach for colorectal cancer with KRASG12D mutation. KRASG12D is a frequent genetic mutation not only in colorectal cancer, but also in pancreatic and lung cancer, and the results of this study open new avenues for potential treatment of many cancer patients.

An integrated model for predicting KRAS dependency.

The clinical approvals of KRAS G12C inhibitors have been a revolutionary advance in precision oncology, but response rates are often modest. To improve patient selection, we developed an integrated model to predict KRAS dependency. By integrating molecular profiles of a large panel of cell lines from the DEMETER2 dataset, we built a binary classifier to predict a tumor's KRAS dependency. Monte Carlo cross validation via ElasticNet within the training set was used to compare model performance and to tune parameters α and λ. The final model was then applied to the validation set. We validated the model with genetic depletion assays and an external dataset of lung cancer cells treated with a G12C inhibitor. We then applied the model to several Cancer Genome Atlas (TCGA) datasets. The final "K20" model contains 20 features, including expression of 19 genes and KRAS mutation status. In the validation cohort, K20 had an AUC of 0.94 and accurately predicted KRAS dependency in both mutant and KRAS wild-type cell lines following genetic depletion. It was also highly predictive across an external dataset of lung cancer lines treated with KRAS G12C inhibition. When applied to TCGA datasets, specific subpopulations such as the invasive subtype in colorectal cancer and copy number high pancreatic adenocarcinoma were predicted to have higher KRAS dependency. The K20 model has simple yet robust predictive capabilities that may provide a useful tool to select patients with KRAS mutant tumors that are most likely to respond to direct KRAS inhibitors.

Abstract B014: Phenotypic CRISPR genome-wide screening for the discovery of genetic regulators of allele-specific mutant KRAS

Abstract Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most frequently mutated oncogene detected in 30% of all human cancers. Drugs that can impede the function of oncogenic KRAS offer exceptional therapeutic value. However, direct targeting KRAS is challenging due to its protein structure that lacks deep binding pockets for small-molecule inhibitors. Despite the recent success in targeting the G12C allele, targeted therapy for another hotspot mutant – G12V mutant KRAS has not been described. Here, we utilize CRISPR-Cas9 based genome-wide knockout screens and leverage a high-throughput microfluidic cell sorting platform to identify genes that regulate mutant KRAS harboring G12V mutation. The screens were conducted in two colorectal cancer cell lines HT29 and SW480, expressing wild-type KRAS and homozygous G12V KRAS, respectively. We have identified a list of genes whose loss-of-function reduces wild-type or mutant G12V KRAS protein expression. Our top gene identified from the mutant KRAS screen - denoted as selective-mutant KRAS modulator (SMKM) has shown promising results in selectively decreasing G12V KRAS protein expression. Through multiple assays including live cell imaging, western blot, flow cytometry, and proteomic analysis in SMKM knockout cells and SMKM inhibitor-treated cells. We found highly selective reduction in G12V KRAS protein expression. More importantly, genetically or pharmacologically inhibiting SMKM has demonstrated anti-tumor activity. We conducted cell viability assays including CellTiter-glo, MTT, and clonogenic assay. We treated a panel of G12V KRAS mutant and wild-type KRAS cell lines with SMKM inhibitor and found significantly reduced cell viability in G12V KRAS mutant cell lines across colorectal and lung cancer. To further demonstrate the downstream impact on mutant KRAS signaling, secondary assays including western blot and Lumit immunoassay were conducted. We observe a reduction in the aberrant phospho-ERK and phospho-AKT signaling in SMKM inhibitor treated cells. Our in vivo efficacy trial using heterozygous G12V KRAS lung cancer NCI-H441 xenograft model has shown SMKM inhibition significantly reduces tumor growth and increases the survival rate. Together, these data indicate the feasibility of selectively targeting G12V KRAS with SMKM inhibition. Lastly, our results provide a first-in-class small molecule oral inhibitor based selective protein clearance as an effective anti-G12V mutant KRAS therapeutic strategy. Citation Format: Xiyue Hu, Randy Singh, Shana Kelley. Phenotypic CRISPR genome-wide screening for the discovery of genetic regulators of allele-specific mutant KRAS [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B014.

Abstract 2627: The small molecule KRAS inhibitor, TEB-17231, blocks tumor progression and overcomes KRASG12C inhibitor mediated resistance

Abstract KRAS pathways are frequently upregulated in cancers bearing KRAS mutations or by other signaling modifications. Also, resistance to KRASG12C inhibitors and other KRAS/RAF/MEK pathway drugs is commonly observed in the treated cancer patients. A vital unmet need is the development of agents that potently and safely inhibit the altered KRAS signaling in these settings. A broadly acting KRAS mutation small molecule inhibitor lead series was developed using KRAS domain structure-guided scaffold modifications starting from a group of KRASG12C inhibitors. Optimized properties of these compounds were evaluated by tumor cell selectivity in vitro and pharmacophore elaboration. A novel lead series was identified with robust tumor cell growth inhibition, exemplified with the TEB-17112 lead candidate, having IC50 of inhibition against multiple KRAS-altered cell lines: NCI-H358 (KRAS-G12C, 33.6nM), AGS (KRAS-G12D, 23.5 nM), SW480 (KRAS-G12V, 22.5 nM) and A375 (KRAS-wildtype, 58.9 nM) respectively. TEB-17112 demonstrated inhibition of SW480 and H358 mouse xenografts at 25 or 50 mg/kg PO QD (21 days) without significant body weight loss. On further optimization of the lead series, TEB-17231 had on average a higher potency against KRAS mutant tumor cell lines, including H358 (12 nM), AGS (5.4 nM), SW480 (3.6 nM), and A427 (KRAS-G12D, 9.7 nM). With a candidate group of 7 compounds from this lead series, there was consistently a 5- to 6-fold selectivity of inhibition for KRAS mutant over KRAS wildtype cell lines. The lead candidate TEB-17231 was also observed to inhibit cell lines with other KRAS mutations: NCI-H1734 (KRAS-G13C, 6.5 nM), MDA-MB-231 (KRAS-G13D,11.2 nM), RPMI-8226 (KRAS-G12A, 11.3 nM), Calu-6 (KRAS-Q61K,19.8 nM), KP-2 (KRAS-G12R, 24.1 nM), LS123 (KRAS-G12S, 21 nM), as well as HRAS and NRAS mutant cell lines. TEB-17231 inhibits phospho-ERK downstream signaling in several tumor cell lines. Further, TEB-17231 exhibited tumor growth inhibition (IC50) in KRAS-G12C Ba/F3 (14 nM) and derivative cell lines that acquired resistance to G12C inhibitor MRTX849 by the additional KRAS domain mutations as follows: H95Q (32 nM), H95D (40 nM), Y96C (39 nM), Y96D (20 nM) and R68S (22 nM), compared with greater than 1μM for MRTX849. TEB-17231 induces late apoptosis of NCI-H358 cells in a manner that is time and concentration dependent. In addition to the improved selectivity and potency, TEB-17231 demonstrated satisfactory pharmacokinetics and oral bioavailability in mice (55%). Furthermore, with the SW480 (G12V) in vivo xenograft model, TEB-17231 showed clear tumor growth inhibition with QD dosing for 4-8 mg/kg PO without body weight loss. These properties highlight TEB-17231 as a potential clinically actionable inhibitor for the KRAS-modified tumor population and warrant investigation of TEB-17231 in combination with other agents in the future. Citation Format: Zusheng Xu, David T. Weaver, Robert Drakas, Yangtong Lou. The small molecule KRAS inhibitor, TEB-17231, blocks tumor progression and overcomes KRASG12C inhibitor mediated resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2627.

(-)-Epigallocatechin Gallate is a Noncompetitive Inhibitor of NAD Kinase.

Nicotinamide adenine dinucleotide kinase (NADK) controls the intracellular NADPH content and provides reducing power for the synthesis of macromolecules and anti-ROS. Moreover, NADK is considered to be a synthetic lethal gene for KRAS mutations. To discover NADK-targeted probes, a high-throughput screening assay was established and optimized with a Z factor of 0.71. The natural product (-)-epigallocatechin gallate (EGCG) was found to be a noncompetitive inhibitor of NADK with K i = 3.28 ± 0.32 μΜ. The direct binding of EGCG to NADK was determined by several biophysical methods, including NMR spectroscopy, surface plasmon resonance (SPR) assay, and hydrogen-deuterium exchange mass spectrometry (HDX-MS). The SPR assay showed a K d of 1.78 ± 1.15 μΜ. The HDX-MS experiment showed that EGCG was bound at the non-substrate-binding sites of NADK. Besides, binding mode prediction and derivative activity analysis revealed a potential structure-activity relationship between EGCG and NADK. Furthermore, EGCG can specifically inhibit the proliferation of KRAS-mutated lung cancer cell lines without affecting KRAS wild-type lung cancer cell lines.

Efficacy of Combined Use of Everolimus and Second-Generation Pan-EGRF Inhibitors in KRAS Mutant Non-Small Cell Lung Cancer Cell Lines.

Background: EGFR mutations are present in approximately 15–50% of non-small cell lung cancer (NSCLC), which are predictive of anti-EGFR therapies. At variance, NSCLC patients harboring KRAS mutations are resistant to those anti-EGFR approaches. Afatinib and allitinib are second-generation pan-EGFR drugs, yet no predictive biomarkers are known in the NSCLC context. In the present study, we evaluated the efficacy of pan-EGFR inhibitors in a panel of 15 lung cancer cell lines associated with the KRAS mutations phenotype. Methods: KRAS wild-type sensitive NCI-H292 cell line was further transfected with KRAS mutations (p.G12D and p.G12S). The pan-EGFR inhibitors’ activity and biologic effect of KRAS mutations were evaluated by cytotoxicity, MAPK phospho-protein array, colony formation, migration, invasion, and adhesion. In addition, in vivo chicken chorioallantoic membrane assay was performed in KRAS mutant cell lines. The gene expression profile was evaluated by NanoString. Lastly, everolimus and pan-EGFR combinations were performed to determine the combination index. Results: The GI50 score classified two cell lines treated with afatinib and seven treated with allitinib as high-sensitive phenotypes. All KRAS mutant cell lines demonstrated a resistant profile for both therapies (GI50 < 30%). The protein array of KRAS edited cells indicated a significant increase in AKT, CREB, HSP27, JNK, and, importantly, mTOR protein levels compared with KRAS wild-type cells. The colony formation, migration, invasion, adhesion, tumor perimeter, and mesenchymal phenotype were increased in the H292 KRAS mutated cells. Gene expression analysis showed 18 dysregulated genes associated with the focal adhesion-PI3K-Akt-mTOR-signaling correlated in KRAS mutant cell lines. Moreover, mTOR overexpression in KRAS mutant H292 cells was inhibited after everolimus exposure, and sensitivity to afatinib and allitinib was restored. Conclusions: Our results indicate that allitinib was more effective than afatinib in NSCLC cell lines. KRAS mutations increased aggressive behavior through upregulation of the focal adhesion-PI3K-Akt-mTOR-signaling in NSCLC cells. Significantly, everolimus restored sensibility and improved cytotoxicity of EGFR inhibitors in the KRAS mutant NSCLC cell lines.

Abstract 1057: Significant efficacy demonstrated with the combination of ulixertinib (ERK1/2 inhibitor) and CDK4/6 inhibitors in MAPK altered models

Abstract Ulixertinib (BVD-523) is a first-in-class small molecule inhibitor of ERK1/2 currently being investigated in several oncology clinical trials, both as a single agent and in combination with other anti-cancer therapeutics. Palbociclib and ribociclib are FDA approved orally active, potent, and highly selective reversible inhibitors of the CDK4 and CDK6 kinases. As it is well established that ERK activation increases cyclin D levels and entry into the cell cycle, we hypothesized that the combination of ERK1/2 and CDK4/6 inhibition would have synergistic antitumor activity and cause tumor regression in vivo. Initial in vitro work combined ulixertinib with CDK4/6 inhibitors across a small panel of lung cancer cell lines. Cell lines carrying a KRAS mutation were more sensitive to ulixertinib relative to KRAS wild type cell lines based on single agent IC50 values. The single agent IC50 values for the CDK4/6 inhibitors were dependent on whether a metabolic or non-metabolic readout for cell viability was used. The combination interactions across a dose matrix of concentrations were determined by the Loewe Additivity and Bliss Independence models. The results of combining ulixertinib and CDK4/6 inhibitors ranged from additive to potentially synergistic. The combination of ulixertinib and palbociclib was then assessed against four xenograft models representing colorectal, melanoma, and pancreatic tumor types, each harboring an alteration within a component of the MAPK pathway. Palbociclib monotherapy across all models showed limited tumor growth inhibition (TGI) while ulixertinib monotherapy demonstrated modest TGI across all models. The combination groups demonstrated significant responses ranging from 75% - 90% TGIs. All treatment regimens were well tolerated across all models. Downstream assays were completed including reverse phase protein arrays (RPPA). Using RPPA, treatment effects on protein signaling was evaluated in the MAPK family, cell cycle regulation, and other associated feedback and compensatory pathways. Notably, suppression of protein targets downstream of ERK1/2 were seen with both ulixertinib monotherapy and combination therapy. Similarly, the combination therapy group reduced protein levels involved in cell cycle progression, which was not seen in either monotherapy group alone. The efficacy demonstrated with this preclinical work has proven to be translatable to the clinic as the combination of ulixertinib and palbociclib recently achieved MTD in a Phase I trial in advanced solid tumors including pancreatic cancer (NCT 03454035). Citation Format: Caroline M. Emery, Brian Corgiat, Justin Davis, David Sorrell, Mitch Johnson, Brent Kreider, Deborah Knoerzer. Significant efficacy demonstrated with the combination of ulixertinib (ERK1/2 inhibitor) and CDK4/6 inhibitors in MAPK altered models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1057.

Abstract 319: Imipridone ONC212 and trametinib combination therapy demonstrates anti-neoplastic effects through immune-mediated mechanisms in pancreatic ductal adenocarcinoma cell lines

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by limited therapeutic options and an extremely high mortality-to-incidence ratio. Chemotherapy remains the primary treatment for metastatic disease and results in only modest improvements in median overall survival, typically with significant toxicity. We previously reported a novel treatment approach with the combination of the imipridone ONC212 and the MEK inhibitor trametinib. This combination demonstrated synergy in multiple KRAS-mutated and KRAS wild-type pancreatic cancer cell lines (BxPC3, PANC1, HPAF-II, AsPC-1). Using Western Blot, we assessed markers of autophagy, including Beclin-1 and LC3B, as well as key second messenger pathway activation/suppression with p-AKT/AKT and p-ERK/ERK. The mechanism of this synergy appears to be heterogeneous, working through autophagy inhibition, MAPK/PI3K pathway perturbation, activation of the integrated stress response and increased cell surface expression of death receptor 5. Further investigation has revealed that ONC212 also appears to synergize with various autophagy inhibitors including hydroxychloroquine and chloroquine. We hypothesized that combining trametinib and ONC212 may also induce cell death in-part through immune cell-mediated mechanisms. To explore this, we performed immune cell co-culture experiments using HPAF-II PDAC cells and natural killer (NK-92) cells at a 1:1 effector-to-target cell ratio with or without ONC212, trametinib, or the combination of the two at different concentrations. We assessed the levels of NK cell mediated-tumor cell death 4, 8, and 24-hours after simultaneous treatment and initiation of co-culture using fluorescent microscopy. Compared to trametinib, ONC212 only treated co-culture showed greater NK cell-mediated tumor cell death. At 24-hours, we also observed an increase in NK cell-mediated killing of PDAC cells with dual treatment as compared to single agent alone or vehicle controls. Importantly, this combination did not appear to have any effect on NK or tumor cell viability. Thus, this combination may represent a potential therapeutic modality for PDAC and may hold promise if combined with immunotherapy. Further in vitro experiments will be conducted to evaluate the effect of ONC212, trametinib, and other autophagy inhibitors on the PDAC tumor microenvironment using a T cell co-culture system. Similarly, in vivo murine studies will also be performed to assess the translational potential of this combination. Citation Format: Alexander G. Raufi, Arielle De La Cruz, Lindsey Carlsen, Kelsey Huntington, Lanlan Zhou, Varun Prabhu, Joshua Allen, Wafik S. El-Deiry. Imipridone ONC212 and trametinib combination therapy demonstrates anti-neoplastic effects through immune-mediated mechanisms in pancreatic ductal adenocarcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 319.

Abstract 1006: Combination therapy with MEK inhibitors and a novel anti-neoplastic drug, imipridone ONC212, demonstrates synergy in pancreatic ductal adenocarcinoma cell lines

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with limited therapeutic options. Although KRAS is mutated or amplified in nearly 95% of PDAC, no effective KRAS directed therapy has entered clinical practice. There is emerging data suggesting that inhibition of downstream targets of KRAS, such as MEK or ERK, increases autophagic flux and dependence in KRAS-mutated tumors. Autophagy, a catabolic process used by many tumors to maintain viability, can be targeted with various agents such has hydroxychloroquine and chloroquine, and recent preclinical data has shown that the combination of these agents with inhibitors of MEK/ERK can synergistically induce cell death. Using viability assays, we established sensitivity of multiple KRAS-mutated and KRAS wild-type pancreatic cancer cell lines, BxPC3, PANC1, HPAF-II, AsPC-1 to ONC212, trametinib, chloroquine and combinations of these agents. Results were analyzed after 72 hours of incubation using Compusyn and Combenefit. We also performed Western Blotting on cell lysates after treatment for 48 hours with these agents. We found that trametinib exhibited strong synergy with both chloroquine and ONC212 in all cell lines. The synergistic potential appeared to be most profound in those cell lines treated with ONC212 and trametinib. We used MRC5, a normal lung fibroblast cell line, as a control and found that these combinations did not significantly induce cell death. Using Western Blotting, we assessed markers of autophagy, including Beclin-1 and LC3B, as well as key second messenger pathway activation/suppression with p-AKT/AKT and p-ERK/ERK. At increasing doses, trametinib increased conversion of LC3-I to LC3-II, more notably in KRAS mutant cell lines, suggesting increased autophagic flux. ONC212, however, reduced conversion of LC3-I to LC3-II and increased degradation of Beclin-1 suggesting inhibition of autophagy. The combination, did not appear to have as striking of an effect on this pathway. In addition, p-ERK, as expected, was downregulated with MEK inhibition. ONC212 also appeared to reduce p-ERK levels and, interestingly, increased p-AKT. This suggests possible PI3K pathway upregulation, however, this could be influenced by timing and dose of treatments. Western blot analysis also revealed increased cPARP/PARP with combination therapy across cell lines. In conclusion, the combination of trametinib and ONC212 produces a synergistic cytotoxic effect on tumor cells at doses that are non-toxic to normal cells. In KRAS-mutated pancreatic cancer cell lines, we hypothesize that this may, in part, be due to autophagy suppression by ON212. Similar synergistic effects were noted in a KRAS wild-type pancreatic cancer cell lines, suggesting an autophagy-independent mechanism. Thus, this combination may represent a potential therapeutic modality for PDAC. Citation Format: Alexander G. Raufi, Casssandra Parker, Lanlan Zhou, Varun V. Prabhu, Josh Allen, Wafik S. El-Deiry. Combination therapy with MEK inhibitors and a novel anti-neoplastic drug, imipridone ONC212, demonstrates synergy in pancreatic ductal adenocarcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1006.

KHDRBS3 promotes multi-drug resistance and anchorage-independent growth in colorectal cancer.

5‐Fluorouracil (5‐FU) is one of the most frequently used pharmacological agents in the treatment of colorectal cancer (CRC). Resistance to chemotherapy is a major cause of treatment failure of CRC, and it is a well known fact that cancer stem cells play a significant role in the acquisition of drug resistance. In this study, we focused on the KHDRBS3 gene that encodes KH RNA Binding Domain Containing, Signal Transduction Associated 3. We first clarified the relationship between KHDRBS3 and 5‐FU resistance. We then observed higher expression levels of KHDRBS3 in KRAS‐mutant organoids and cell lines in comparison with KRAS wild‐type organoids and cell lines. Immunohistochemical analysis using CRC cases revealed that the prognosis of KHDRBS3‐positive patients was significantly worse compared with that of KHDRBS3‐negative patients. Univariate and multivariate Cox proportional hazards analyses showed that KHDRBS3 was an independent prognostic factor in patients with CRC. We determined that KHDRBS3 might play a crucial role in the acquisition of stem cell properties, such as drug resistance and spheroid/organoid formation, by regulating CD44 variant expression and the Wnt signaling pathway. In an immunodeficient mouse model, KHDRBS3‐positive cells showed efficient tumor formation and formed metastatic lesions in the lungs. These results indicated that KHDRBS3 plays a crucial role in drug resistance and anchorage‐independent growth by maintaining stem cell‐like features in CRC cells. KHDRBS3 could be a promising candidate marker for predicting chemotherapeutic effect and prognosis in CRC patients.

Abstract 4384: Quantitative assessment of drug combination effects in NSCLC cell lines through network-based analysis of functional protein activity

Abstract Non-small cell lung cancer (NSCLC) is the most prevalent type of lung cancer, which is the leading cause of cancer death worldwide. The KRAS oncogene is one of the most common driver mutations in NSCLC patients but, despite that, there are no approved targeted therapies for tumors harboring this mutation. The MEK inhibitor Selumetinib (SE) turned out to be an ineffective drug against KRAS mutated lung cancer. Since combination therapy has shown to be beneficial in many tumors, we wanted to investigate the combination of MEK inhibition with other targeted therapies. Thus, we measured the proteomic response of KRAS mutant (MUT) and wild-type (WT) NSCLC cell lines (CLs) to the combination of SE with Everolimus (EV), an mTOR inhibitor, and Tamoxifen (TA), an Estrogen inhibitor. The expression level of 183 proteins was measured through Reverse Phase Protein Array (RPPA) in eight NSCLC CLs treated with SE, SE plus EV and SE plus TA at 6 time points: 5 minutes (min), 30 min, 1, 2, 6 and 24 hours (h). As control, measures were taken in baseline CLs and in CLs with only dimethyl sulfoxide. To study the drug combination effects, we applied a computational workflow centered on two algorithms initially developed for gene expression data. We analyzed the CLs with the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) to reconstruct a context specific signaling network of 2635 interactions between 73 kinases and 6 phosphatases and their substrates. Then, we analyzed the obtained network with the Virtual Inference of Protein activity by Enriched Regulon analysis (VIPER) algorithm to assess the mechanism of action of the drug compounds and how they dysregulate the activity of kinases and phosphatases over time. With respect to SE alone and apart from the drug targets, VIPER predicted the following proteins as highly inhibited over time across all CLs : 1) Aurora and LKB1 in SE+EV, 2) AMPK, c-RAF and ATR in SE+TA, 3) mTOR, p90, p70S6K, c-MET and GSK-3 in both combinations. On the other hand, Akt, ETK, Axl and FAK are all activated in SE+EV as well as PP2A, SGK1 and IGF1R in SE+TA, after 1h from the treatment. By running Gene Set Enrichment Analysis on the VIPER output against the primary drug target pathways, we found that mTOR signaling pathway exhibits a quite different evolution between KRAS MUT and WT CLs. On the other hand, other pathways revealed mechanisms of response that are CL specific. For instance, the main MEK pathways in H1734 CL (KRAS MUT) treated with SE+TA are all insensitive to the drug compounds. In conclusion, this computational approach successfully predicted protein-protein interactions and elucidated both proteomic mechanisms of drug combinations and CL specific dependencies. It allows to develop effective predictive and quantitative models reproducing each CL behavior, in order to realize the fullest potential of a targeted therapeutic approach. Citation Format: Chiara Antonini, George Rosenberger, Fortunato Bianconi, Lorenzo Tomassoni, Vienna Ludovini, Sara Baglivo, Sara Calandrini, Elisa Baldelli, Mariaelena Pierobon, Emanuel F. Petricoin, Andrea Califano. Quantitative assessment of drug combination effects in NSCLC cell lines through network-based analysis of functional protein activity [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 4384.

Abstract 6329: Carfilzomib: A potential drug against cisplatin-resistant lung adenocarcinoma

Abstract Recent advances in the treatment of lung cancer, such as small molecule inhibitors and immunotherapy, have transformed care. However, platinum-based chemotherapy, such as cisplatin, is the recommended treatment option for those that do not express markers that respond to targeted therapy. In most cases, prolonged exposure to cisplatin eventually renders cancer cells resistant to therapy and patients progress on this line of care. Our study has shown that integrin beta 4 (ITGB4) and paxillin (PXN) in the focal adhesion complex are highly expressed in lung adenocarcinoma (LUAD) and their interaction plays an important role in mediating cisplatin resistance. Disrupting this axis with an FDA-approved compound can potentially alleviate cisplatin resistance in non-small cell lung cancer. Upon screening 10 LUAD cell lines, we determined one KRAS mutant (H2009) and one KRAS wild-type cell line (H1993) with highest resistance to cisplatin using a cell viability assay. Proximity ligation assay and co-immunoprecipitation results confirmed interaction between ITGB4 and PXN in H2009 cells. By virtually screening a library of 1440 FDA-approved compounds that bind to the N-terminal region of PXN, the top 40 hits were screened using a cell-based assay with H358 cells. These results determined the top 11 compounds with the lowest IC50 values (0.8 nM to 1.54 μM). Drug sensitivity to these 11 compounds was tested again using the cisplatin-resistant cell line H2009. Carfilzomib, a proteasome inhibitor, was identified to be the most effective (IC50 = 193.4 nM). This compound was further tested in 3D cell line-derived spheroids and patient cancer tissue-derived organoids that were allowed to form in multi-well ultra-low attachment plates. These cultures were monitored in real-time using the IncuCyte S3 Live-Cell Analysis System. In cisplatin-resistant cell lines H2009 and H1993, carfilzomib was the most effective (IC50 = 193.4 nM, 1.77 μM, respectively), irrespective of KRAS status. Wound healing was impeded by 20% in H2009 with a submicromolar dose (200 nM). In both H2009 and H1993 cells, a sublethal dose of carfilzomib decreased expression of ITGB4 and phospho-Rb (S807/811) and increased expression of p27, γH2AX, and cleaved PARP, indicating cell cycle inhibition and cell death. In H2009 spheroids, carfilzomib (600 nM) was highly effective in decreasing spheroid viability by 60% and inducing a 2.5-fold increase in caspase-3/7-mediated apoptosis. Also in patient-derived organoids, carfilzomib induced an increase in apoptosis by 5-fold in one case and 50-fold in a second case compared to cisplatin. Another proteasome inhibitor ixazomib was among the top 11 compounds tested in H2009 cells but had a higher IC50 (2.93 μM) compared to carfilzomib, indicating greater efficacy of carfilzomib against cisplatin-resistant LUAD. These results impart a potential therapeutic approach for overcoming chemoresistance in LUAD by targeting the focal adhesion complex through repurposing carfilzomib, an FDA-approved compound. Citation Format: Arin Nam, Atish Mohanty, Supriyo Bhattacharya, Saumya Srivastava, Rajendra Pangeni, Dan Raz, John Orban, Prakash Kulkarni, Ravi Salgia. Carfilzomib: A potential drug against cisplatin-resistant lung adenocarcinoma [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 6329.

Abstract PR07: Combinatorial knockout of Rap1GDS1 and RhoA leads to lethality in KRAS-driven non-small cell lung cancer

Abstract Synthetic lethal-based approaches to targeting KRAS driven cancers have recently been receiving increasing attention. However, as KRAS activates multiple effector pathways, targeting single genes may not be sufficient to fully inhibit KRAS-driven oncogenesis. In addition, genome-wide screens can be limited by high noise and low signal. Furthermore, it is increasingly clear that standard screens based on 2D proliferation can miss critical phenotypes relevant to cancer biology. Therefore, we developed a multistage screening strategy that includes a) identification of KRAS-proximal protein-protein interaction (PPI) networks, b) targeted CRISPR-based screening for pairs of genes that together are synthetic lethal with KRAS, and c) validation in a 3D sphere system using both human and mouse NSCLC cells. We used affinity purification/mass spectrometry (AP/MS) to construct a detailed map of protein-protein interactions centered on KRAS. Based on this network, we designed a CRISPR/Cas9 library targeting pairwise combinations of 119 KRAS-interacting genes. We screened two KRAS-driven non-small cell lung cancer (NSCLC) cell lines (A549 and H23). This screen yielded a large number of gene pairs that synergistically impaired cell growth. We selected the 20 most promising targets for further screening in a panel of 5 KRAS-mutant and 4 KRAS wild-type NSCLC cell lines. From these screens we identified a KRAS-specific synergistic interaction between two genes—Rap1GDS1 and RhoA. Validation in 3D cultures in a panel of cell lines showed that combinatorial Rap1GDS1 and RhoA knockout selectively impairs sphere growth of KRAS-mutant NSCLC cells but has minimal effects on KRAS wild-type NSCLC cell lines or nontransformed lung cells. We demonstrate that this effect is primarily due to the induction of apoptosis. Rap1GDS1 has previously been reported to encode two different isoforms with distinct cellular functions. We observe that only one of the isoforms is involved in the mediation of the KRAS-dependent lethal effect. Current efforts are directed at elucidating the mechanistic basis for the isoform-specific combinatorial lethality between Rap1GDS1 and RhoA. Furthermore, we are actively exploring how to exploit this knowledge to define potential novel therapies for KRAS-driven cancer. Overall, we demonstrate that an integrated strategy combining proteomics, paired CRISPR screens and innovative 3D validation systems can identify novel synthetic lethal combinations. This approach may help identify novel synthetic lethal drug combinations for KRAS and other to-date undruggable oncogenes. This abstract is also being presented as Poster A40. Citation Format: Kaja Kostyrko, Marcus Kelly, Kyuho Han, Edwin Jeng, David Morgens, Michael Bassik, Peter Jackson, Alejandro Sweet-Cordero. Combinatorial knockout of Rap1GDS1 and RhoA leads to lethality in KRAS-driven non-small cell lung 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 PR07.

Abstract 1790: Investigation of oncogenic G-protein coupled receptor signaling pathways in Kras dependent pancreatic cancer cell lines

Abstract Pancreatic Ductal Adenocarcinoma (PDA) is the most common form of pancreatic cancer driven primarily by mutations in V-Ki-Ras2 Kirsten Rat Sarcoma Viral Oncogene Homolog (Kras) gene. Its most common mutation, G12D, cause KRAS to remain constitutively active. KrasG12D expressing pancreatic acinar cells undergo transdifferentiation into ductal phenotype and form early tumorigenic lesions called Pancreatic Intraepithelial Neoplasm (PanIN). Coincidentally occurring inactivating mutations of tumor suppressor genes, such as Cyclin-dependent Kinase Inhibitor 2A (Cdkn2a), Tumor Protein p53 (Tp53), and Mothers Against Decapentaplegic Homolog 4 (Smad4) trigger progression of PanINs through stages from 1A to 3, eventually leading to carcinoma in situ and metastasis. G-protein Coupled Receptors (GPCR) are transmembrane receptors that transfer extracellular signals to intracellular heterotrimeric G-proteins with Gα and Gβγ subunits. Upon activation, dissociated Gα and Gβγ subunits separately induce intracellular second messengers for key cellular responses involved in various aspects of development, metabolism, and cell motility. GPCR signaling is negatively regulated by intracellular Regulator of G-protein Signaling (RGS) proteins that deactivate G-protein by mediating re-association of Gα and Gβγ subunits. Although GPCRs are the largest non-antibiotic drug targets, their specific role in pancreatic cancer is not much known. GPCR signaling can influence RTK pathways in cross-talk via various second messengers. The interplay between these two major classes of cellular pathways employing both heterotrimeric and monomeric G-proteins has therefore potential to shed new light on pancreatic cancer research. We have previously identified Rgs16 expression as a marker of pancreatic tumor formation in Rgs16::GFP o/o; Ptf1aCre/+; Lox-STOP-Lox-KrasG12D/+; Cdkn2af/f (Rgs16::GFP-KIC) mice from the onset of earliest PanINs at two weeks of age (Dis. Model Mech. 8, 1201). Using ImageJ software to quantitatively determine eGFP expression in micrographs, we have developed a rapid in vivo assay (RIVA) where we represent pancreatic tumor burden by one month of age and compare outcomes of known and novel therapeutics following two week long drug regimens. We found that just like the recently developed drug, BGB324, targeting an important RTK, called Anexelekto (Axl), the anticoagulant warfarin was more effective in reducing tumor burden than standard chemotherapy alone. Currently, my research focus is centered on understanding active GPCR pathways that stimulate cell growth, survival, and migration by enhancement of Kras downstream signaling. My aim is comparison of the effects of general G-protein signaling inhibition in Kras mutant and wild-type cell lines, such as AsPC-1 in contrast to BxPC-3, on cell behavior and making connections to active GPCR signaling components. Citation Format: Ozhan Ocal, Raymond MacDonald, Rolf A. Brekken, Thomas Wilkie. Investigation of oncogenic G-protein coupled receptor signaling pathways in Kras dependent pancreatic cancer cell lines [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 1790.

Abstract 2254: Dynamic network topologies analysis in proteomics data of NSCLC cell lines using a new pipeline based on machine learning tools

Abstract Background: Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer, characterizing ~80% of lung cancer diagnoses. Thus, the development of novel therapies for NSCLC is of primary importance in oncology. The aim of this study was to develop a novel cancer systems biology approach to understand the complex interactions in NSCLC and predict the response to target treatments. We combined proteomic experimental data with a machine learning tool to discover the diverse signaling networks of both KRAS mutant (MUT) and wild-type (WT) NSCLC cell lines (CL). Material and Methods: Eight NSCLC CL (5 KRAS MUT and 3 KRAS WT) were subjected to reverse phase protein microarray (RPPA) to explore the activation level of 183 proteins treated with selumetinib (SE) and its combination with everolimus (EV) or tamoxifen (TA). Measures were taken at 6 time points: 5 minutes (min), 1, 2 , 6 and 24 hours (h). RPPA was performed also on the baseline and on CL with only dimethyl sulfoxide. Recursive Feature Elimination with Support Vector Machine (RFE-SVM) was used to identify the subset of proteins/features that optimally separated samples in 2 groups. In order to build the interaction network from this subset and browse the main pathways involved, we used Reactome FI and the Pathway Enrichment tool, two Cytoscape plugins for network-based data analysis. Results: We applied RFE-SVM to discover the most divergent proteins between control and treated samples in the whole CL panel. For all the 3 drug combinations, the network in output contained not only the target of the corresponding drug, but also showed the activation of other parallel pathways. The analysis revealed also that all drugs were mainly effective in a short time, since the number of significant proteins was considerably higher at 5 min and 1 h. For instance, SE treatment activated NGFR, mTOR, PI3K-Akt and proteoglycans pathways in addition to the MAPK pathway. Network at 1 h contained 25 proteins linked by 76 interactions, while at 6 and 24 h the distinctive subsets of features included only 2 and 4 proteins. Then, we applied RFE-SVM to analyze KRAS MUT vs WT CLs. The algorithm showed that treated KRAS MUT CL had a more complex signaling network than the WT ones. Indeed, in SE WT samples MER and ERK were the main relevant proteins to distinguish from the control. In treated KRAS MUT samples, a complex network of 70 proteins and 278 interactions was active during the first hour. Conclusion: The proposed framework processes high-throughput data and identifies new possible molecular targets in cancer research. This approach is highly flexible for a multitude of data and purposes. We applied it to a RPPA dataset of NSCLC CL and it revealed that all drug combinations had fast dynamics and that KRAS MUT CL had a more complicated response due to a higher number of active pathways. Citation Format: Chiara Antonini, Lorenzo Tomassoni, Elisa Baldelli, Vienna Ludovini, Sara Baglivo, Mariaelena Pierobon, Emanuel F Petricoin, Lucio Crinò, Paolo Valigi, Fortunato Bianconi. Dynamic network topologies analysis in proteomics data of NSCLC cell lines using a new pipeline based on machine learning tools [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 2254.

Abstract 5863: RGX-202, a first-in-class small-molecule inhibitor of the creatine transporter SLC6a8, is a robust suppressor of cancer growth and metastatic progression

Abstract Background: Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide with more than 140,000 patients diagnosed and nearly 50,000 deaths annually in the U.S. alone. Roughly 60% of patients present with locally advanced or distant metastatic disease, with the liver being a primary site of metastatic colonization. Creatine metabolism has been implicated in colon cancer progression and metastatic colonization of the liver. Metastatic colon cancer cells upregulate and release creatine kinase-B (CKB) into the extracellular space, where it phosphorylates creatine to generate the high-energy metabolite phosphocreatine. Phosphocreatine is imported via the creatine transporter SLC6a8. Intracellular phosphocreatine can be converted to ATP to fuel the survival of metastatic cancer cells within the hypoxic hepatic microenvironment. Consistent with this finding, genetic depletion of SLC6a8 in colon and pancreatic cancer cell lines significantly reduced liver colonization in mouse xenograft models. Results: We herein demonstrate that the novel small molecule RGX-202 is a robust inhibitor of creatine uptake in cancer cells, both in vitro and in vivo. Oral administration of RGX-202 induced apoptosis of colon cancer cells in vivo, and significantly suppressed colon cancer liver metastatic colonization and primary tumor growth, both in KRAS wild-type and KRAS mutant colon cancer cell lines as well as in human PDX mouse models. Using genetic studies, these effects were found to be dependent on tumoral expression of SLC6a8. In addition, combination treatment of the CT26 syngeneic colon cancer mouse model with 5-FU resulted in synergistic antitumor activity, with complete tumor regressions observed in more than 40% of treated mice. Similarly, combination treatment of the KPC syngeneic mouse model with gemcitabine significantly reduced the growth of primary pancreatic tumors. Definitive 28-day GLP toxicology and pharmacokinetics studies of RGX-202 are currently ongoing. Preliminary observations suggest good tolerability in several animal species with a favorable pharmacokinetic profile, including bioavailability. Conclusion: These results strongly support clinical development of RGX-202 in patients with gastrointestinal cancers, including colorectal and pancreatic cancer, both as monotherapy and in combination with standard-of-care treatment. Citation Format: Isabel Kurth, Celia Andreu, Shugaku Takeda, Helen Tian, Foster Gonsalves, Katya Leites, Subhasree Sridhar, Jia Min Loo, Rob Busby, Sohail Tavazoie, Masoud Tavazoie. RGX-202, a first-in-class small-molecule inhibitor of the creatine transporter SLC6a8, is a robust suppressor of cancer growth and metastatic progression [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 5863.

Abstract B100: Mutant KRAS-induced macrophage migration inhibitory factor (MIF) secretion promotes resistance to cetuximab in colorectal cancer

Abstract The tumor microenvironment is recognized as developing crosstalk between different cells by secretion of growth factors and cytokines, thus providing oncogenic signals enhancing tumor progression and drug resistance. Here, we hypothesized that tumor cells carrying KRAS mutation-induced cytokines may have a critical role in intercellular communication between microenvironment and tumor cells. We first investigated biologic evidence of secreted cytokines in KRAS mutant type (KRAS MT) cell lines. We explored the roles of cytokine that cell morphology, colony-formation ability, and wound-healing ability were enhanced when KRAS wild-type (KRAS WT) cell lines were exposed to conditioned media (CM) from KRAS MT cells, and we found macrophage migration inhibitory factor (MIF) was highly expressed in KRAS MT cells by analysis of proteomics. We also observed secreted MIF level between KRAS WT and MT cell lines, and it was highly secreted in KRAS MT cell lines. In addition, compared with patients whose KRAS mutation was not harbored, MIF expression was higher in patients who have KRAS mutation. The predictive marker of KRAS mutation in colorectal cancer patients is associated with resistance to antiepidermal growth factor receptor (EGFR) antibody cetuximab. Following the hypothesis, we investigated that secreted cytokines have an effect on the cetuximab resistance to surrounding cells including KRAS WT cells. Treatment of CM from KRAS MT cells led to cetuximab resistance in KRAS WT cells and MIF blockade prevented cetuximab resistance. Also, CM from knockout in KRAS MT cells by using CRISPR/Cas9 system resulted in sensitizing to cetuximab resistance compared with CM from KRAS MT cells. In conclusion, we demonstrated for the first time that MIF promotes the cetuximab resistance of KRAS WT colorectal cancer cells, and this effect is mediated by paracrine and autocrine signaling-induced activation of the intracellular AKT signaling pathways and regulated by NF-kb transcription factor through oncogenic KRAS mutation and MIF overexpression, respectively. These findings suggest that MIF may be a promising predictive biomarker for the cetuximab resistance of KRAS wild-type colorectal cancer patients. Citation Format: Jee Eun Jang, Hwang-Phill Kim, Sae Won Han, Tae You Kim. Mutant KRAS-induced macrophage migration inhibitory factor (MIF) secretion promotes resistance to cetuximab in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B100.