3-Aminoisoquinolines inhibit selectively phosphodiesterase 4B in KRAS-mutated colorectal cancer cell lines in-vitro and in-vivo.

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.

Introduction and Aims Efficacy of targeted anticancer drugs is limited by de novo resistances related to cross talk within and between complex signal transduction networks. Rational combinatorial targeted therapies are one of the main solutions to increase activity or overcome resistance. We aimed to investigate the effects of inhibiting different nodes within the MAPK pathway (RAF and MEK) and PI3K-AKT pathway (PI3K, AKT and m-TORC1/2) either alone or in different combinations, on cell growth in a panel of KRAS mutant and KRAS wt NSCLC cell lines. Material & Methods We used dabrafenib, trametinib, GDC-0941, MK2206 and AZD2014 to inhibit RAF, MEK, PI3K, AKT and m-TORC1/2 respectively. Inhibition of signalling output in MEK, PI3K, AKT and m-TORC1/2 was assessed by maximal reduction of p-ERK, p-AKT (Thr308), p-AKT (Ser473) and p-S6 by trametinib, GDC-0941, MK2206 and AZD2014 respectively. In keeping with its mechanism of action, maximal induction of p-MEK was considered as inhibition of RAF by dabrafenib in the cell line panel (none had mutations in BRAF). Quantification of phosphoproteins was done using MesoScale Discovery ELISA. Using concentrations of drugs shown to inhibiting signalling though these nodes, the effects of inhibiting the nodes alone or in combination on cell growth was studied using WST-1 assays. The NSCLC cell line panel included KRAS wild-type (H522; H1838; H1651) or KRAS mutant (A-549; Calu-6; H23) cells. Results The concentration range of dabrafenib, trametinib, GDC-0941, MK2206 and AZD2014 to inhibit RAF, MEK, PI3K, AKT and m-TORC1/2 related to the different cell lines was [40-1000 nM], [40-200 nM], [1000 - 10 000 nM], >50 000 nM, and [500-1500 nM] respectively. The degree of growth inhibition following inhibition of an individual node varied between 5% and 75%. Highest inhibitions of cell proliferation by drugs alone were obtained with PI3K-AKT pathways compared to the MAPK pathway inhibitors for 6/6 cell lines. Drug combinations improved inhibition of cell proliferation in all cell lines. In 3/3 KRAS mutant cells, best combinations included MEK inhibitor and any other of the PI3K-AKT pathway as compared to inhibition of different nodes within the PI3K-AKT or MAPK pathways. In 3/3 KRAS wt cell lines, vertical combinations within the PI3K-AKT pathway induced highest inhibitions of cell proliferation compared to vertical combinations within the MAPK pathway or horizontal combinations between the PI3K-AKT and MAPK pathways. Discussion/Conclusions In the cell line panel tested, KRAS mutant cell lines were more sensitive to horizontal combinations than vertical combinations with the MAPK and PI3K-AKT pathways. KRAS wt cells were more sensitive to vertical combinations within the PI3K-AKT pathway. This preclinical study has clinical implications while planning combinations of targeted agents to treat NSCLC. Citation Format: Sophie Broutin, Adam Stewart, Parames Thavasu, Angelo Paci, Jean-Michel Bidart, Udai Banerji. The effect of horizontal and vertical inhibition of nodes within the MAPK and PI3K-AKT pathways in NSCLC models. [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 5343. doi:10.1158/1538-7445.AM2015-5343

Despite the availability of several active combination regimens for advanced colorectal cancer (CRC), the 5-year survival rate remains poor at less than 10%, supporting the development of novel therapeutic approaches. In this study, we focused on the preclinical assessment of a rationally based combination against KRAS-mutated CRC by testing the combination of the MEK inhibitor, selumetinib, and vorinostat, a histone deacetylase (HDAC) inhibitor. Transcriptional profiling and gene set enrichment analysis (baseline and posttreatment) of CRC cell lines provided the rationale for the combination. The activity of selumetinib and vorinostat against the KRAS-mutant SW620 and SW480 CRC cell lines was studied in vitro and in vivo. The effects of this combination on tumor phenotype were assessed using monolayer and 3-dimensional cultures, flow cytometry, apoptosis, and cell migration. In vivo, tumor growth inhibition, (18)F-fluoro-deoxy-glucose positron emission tomography (FDG-PET), and proton nuclear magnetic resonance were carried out to evaluate the growth inhibitory and metabolic responses, respectively, in CRC xenografts. In vitro, treatment with selumetinib and vorinostat resulted in a synergistic inhibition of proliferation and spheroid formation in both CRC cell lines. This inhibition was associated with an increase in apoptosis, cell-cycle arrest in G(1), and reduced cellular migration and VEGF-A secretion. In vivo, the combination resulted in additive tumor growth inhibition. The metabolic response to selumetinib and vorinostat consisted of significant inhibition of membrane phospholipids; no significant changes in glucose uptake or metabolism were observed in any of the treatment groups. These data indicate that the rationally based combination of the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, selumetinib, with the HDAC inhibitor vorinostat results in synergistic antiproliferative activity against KRAS-mutant CRC cell lines in vitro. In vivo, the combination showed additive effects that were associated with metabolic changes in phospholipid turnover, but not on FDG-PET, indicating that the former is a more sensitive endpoint of the combination effects.

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.

Activation of the mitogen-activated protein kinase (MAPK) pathway by oncogenic mutations in RAS and RAF are frequent drivers of human cancer. Targeting specific components of the MAPK pathway is a precise and rational route to deliver benefits to cancer patients. RAS and RAF proteins are core pathway components that interact directly to mediate MAPK signaling. Direct and indirect inhibition of this key MAPK pathway intersection is critical in driving responses in patients with RAS and RAF mutations. JZP815 (flezurafenib) is an oral pan-RAF inhibitor under development by Jazz Pharmaceuticals. We examined in vitro and in vivo effects of JZP815 pan-RAF inhibition as a single agent and in combination, in multiple RAS and RAF mutant cell and tumor model systems, and those arising from KRAS G12C inhibitor therapy. Lastly, we established a pharmacodynamic (PD) profile indicating JZP815 MAPK pathway engagement. Specifically, in vitro studies in a broad KRAS and NRAS mutant tumor cell line dataset revealed a complex and extensive potency profile of JZP815. Mutant NRAS lines demonstrated significant anti-proliferative response, whereas KRAS mutant lines showed a more diverse response profile. JZP815 inhibited cell proliferation in KRAS mutant cell lines with acquired resistance mutations known to arise in patients after KRAS inhibitor therapy, including acquired KRAS and BRAF mutations. JZP815, in combination with a MEK inhibitor, generated synergistic antiproliferative activity in mutant NRAS melanoma cells lines. In vivo CDX and PDX models showed JZP815 activity against KRAS, NRAS, and BRAF mutant (class 1, 2, 3) xenografts. In BRAF class 1 melanoma PDX models, JZP815 monotherapy generated a more robust antitumor effect compared to 2nd-generation RAF inhibitors. JZP815 demonstrated activity alone and in combination with SOC agents in CRC PDX models, derived from patients who had progressed on KRAS G12C inhibitors. Using bioinformatic analysis, PD profiles were generated that indicated direct impact of JZP815 in MAPK signaling on xenograft tumors bearing KRAS mutations. Whole transcriptome profiling of a KRAS G13D mutant tumor model showed JZP815 suppressed a clinically relevant transcriptional biomarker of MAPK pathway inhibition (MPAS). In addition, the suppression of MPAS gene signature was associated with JZP815 exposure. These PD data support dose- and time-dependent MAPK pathway and target engagement by JZP815 in preclinical models. In summary, JZP815, a potent selective pan-RAF inhibitor, is active against a variety of KRAS, NRAS, and BRAF mutants, and KRAS inhibitor generated mutants, as a single agent and in combination. JZP815 elicits a clinically verified PD profile that could be used to confirm target engagement in patients. JZP815 is currently in phase 1 testing (NCT05557045). Citation Format: Robert Hauptschein, Ankur Karmokar, Emanuele Loro, Yang Yuan, Graham Brock, Robin C. Humphreys, Alexey Sorokin, Preeti Kanikarla, Scott Kopetz. JZP815, a potent and selective pan-RAF inhibitor, is effective against a spectrum of mutant KRAS and NRAS solid tumor cancers, as monotherapy and in combination, generating a gene expression profile of MAPK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6830.

KRAS mutation is a predictive biomarker for resistance to cetuximab (Erbitux®) in metastatic colorectal cancer (mCRC). This study sought to determine if KRAS mutant CRC lines could be sensitized to cetuximab using dasatinib (BMS-354825, sprycel®) a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src Family Kinases. We analyzed 16 CRC lines for: 1) KRAS mutation status, 2) dependence on mutant KRAS signaling, 3) expression level of EGFR and SFKs. From these analyses, we selected three KRAS mutant (LS180, LoVo, and HCT116) cell lines, and two KRAS wild type cell lines (SW48 and CaCo2). In vitro, using Poly-D-Lysine/laminin plates, KRAS mutant cell lines were resistant to cetuximab whereas parental controls showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater anti-proliferative effect on KRAS mutant line as compared to either agent alone both in vitro and in vivo. To investigate potential mechanisms for this anti-proliferative response in the combinatorial therapy we performed Human Phospho-kinase Antibody Array analysis measuring the relative phosphorylation levels of phosphorylation of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in LS180, LoVo and HCT116 cells. The results of this experiment showed a decrease in a broad spectrum of kinases centered on the β-catenin pathway, the classical MAPK pathway, AKT/mTOR pathway and the family of STAT transcription factors when compared to the untreated control or monotherapy treatments. Next we analyzed tumor growth with cetuximab, dasatinib or the combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an anti-tumor response when treated with cetuximab. KRAS mutant tumors exhibited minimal response to dasatinib monotherapy. However, as in vitro, KRAS mutant lines exhibited a response to the combination of cetuximab and dasatinib. Combinatorial treatment of KRAS mutant xenografts resulted in decreased cell proliferation as measured by Ki67 and higher rates of apoptosis as measured by TUNEL. The data presented herein indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key-signaling pathways. Further, these results suggest that signaling via the EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. This data strengthen the rationale for clinical trials in this genetic setting combining cetuximab and dasatinib.

Background: Treatment with KRASG12C inhibitors results in an objective response in only small fraction of patients with KRASG12C mutant colorectal cancer (CRC) (7.1% and 22% objective response rates (ORR) in two prospective trials), in contrast to lung cancer where the ORR exceeds 35%. We hypothesize that co-mutation of other oncogenes and/or transcriptomic factors may contribute to the resistance of KRASG12C CRC to KRAS inhibitors. Methods: Mutation profiles of CRC patients were obtained from the AACR Project-GENIE v14 CRC cohort (N = 9441). The impact of KRAS knockout in 59 CRC cell lines was queried from the DepMap database. KRASG12C mutant CRC cell lines SW837 and SW1463 were treated with Sotorasib (KRASG12C inhibitor), Vismodegib and Taladegib (Hedgehog inhibitors), and cell viability was measured up to 96 hours after treatment. Results: In the AACR Project-GENIE CRC (N = 9441) cohort, KRASG12C tumors (2.6%, 245/9441) were significantly co-mutated with PIK3CAmut (19.2%, 47/245; Odds ratio = 1.7 (95% CI: 1.2 - 2.4), Chi-Square test, p-value = 0.002). Dependence on KRAS in CRISPR knockout viability assays was higher in KRASmut vs. wildtype CRC cell lines (median CERES score -1.3 vs -0.5, p-value < 0.001). Interestingly, KRASmut cell lines with PIK3CA co-mutation (n=10) were more resistant to KRAS knockout than PIK3CAwt cells (n=22) (median -1.0 vs -1.4, p-value = 0.03). The SW837 and SW1463 cell lines initially displayed high sensitivity to Sotorasib (IC50: 0.92 µM and 0.90 µM). However, overexpression of PIK3CAE545K mutant induced resistance to the same treatment (IC50: 5.18 µM and 6.45 µM). Furthermore, gene set enrichment analysis (GSEA) showed that Hedgehog signaling was significantly enriched in KRAS knockout resistant KRASmut CRC cell lines. SW837 cell line treated with Sotorasib (S) and a fixed dosage of 25 µM of Vismodegib (V) or Taladegib (T) showed higher sensitivities in combination treatment assays (IC50 decreased: ~11 folds in S+V vs S; and ~15 folds in S+T vs S) as compared to Sotorasib (S) treatment alone. Similar results were seen for the SW1463 cell line (IC50 decreased: ~18 folds in S+V vs S; and ~15 folds in S+T vs S). Conclusion: Resistance in CRC to KRASG12C inhibitors may be attributed to activated PIK3CA mutations and the Hedgehog signaling pathway. The future study will focus on detailed investigations involving comprehensive transcriptome and exome profiles in tumors and PDX models treated with anti-KRAS drugs in monotherapy and combination therapy. Citation Format: Saikat Chowdhury, Jibran Ahmed, Valsala Haridas, David S. Hong, Scott Kopetz, John Paul Shen. Activating PIK3CA mutations and hedgehog signaling may confer resistance to KRAS inhibition in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1250.

BackgroundPrevious studies showed that the combination of an anti-Epidermal growth factor (EGFR) and a MEK-inhibitor is able to prevent the onset of resistance to anti-EGFR monoclonal antibodies in KRAS-wild type colorectal cancer (CRC), while the same combination reverts anti-EGFR primary resistance in KRAS mutated CRC cell lines. However, rapid onset of resistance is a limit to combination therapies in KRAS mutated CRC.MethodsWe generated four different KRAS mutated CRC cell lines resistant to a combination of cetuximab (an anti-EGFR antibody) and refametinib (a selective MEK-inhibitor) after continuous exposure to increasing concentration of the drugs. We characterized these resistant cell lines by evaluating the expression and activation status of a panel of receptor tyrosine kinases (RTKs) and intracellular transducers by immunoblot and qRT-PCR. Oncomine comprehensive assay and microarray analysis were carried out to investigate new acquired mutations or transcriptomic adaptation, respectively, in the resistant cell lines. Immunofluorescence assay was used to show the localization of RTKs in resistant and parental clones.ResultsWe found that PI3K-AKT pathway activation acts as an escape mechanism in cell lines with acquired resistance to combined inhibition of EGFR and MEK. AKT pathway activation is coupled to the activation of multiple RTKs such as HER2, HER3 and IGF1R, though its pharmacological inhibition is not sufficient to revert the resistant phenotype. PI3K pathway activation is mediated by autocrine loops and by heterodimerization of multiple receptors.ConclusionsPI3K activation plays a central role in the acquired resistance to the combination of anti-EGFR and MEK-inhibitor in KRAS mutated colorectal cancer cell lines. PI3K activation is cooperatively achieved through the activation of multiple RTKs such as HER2, HER3 and IGF1R.

BACKGROUND. Proteosome inhibition is associated with induction of the proapoptotic BH3 protein NOXA. Therefore, we hypothesize that the combination of the irreversible proteosome inhibitor, carfilzomib, will cooperatively enhance apoptosis induction by the BH3 mimetic drug, ABT-263. Given that nononcogenic addiction to proteosome activity has been reported in KRAS mutant cells, we utilized isogenic colorectal cancer (CRC) cell lines in which the endogenous wild-type (wt) or mutant alleles had been inactivated through targeted homologous recombination. Since proteasome blockade results in proteotoxic stress, we determined whether an autophagy inhibitor can enhance chemosensitivity. EXPERIMENTAL METHODS. Isogenic KRAS mutant (MUT) CRC cells (HCT116, DLD1; obtained from B. Vogelstein, JHU) were incubated with carfilzomib (25,50nM) alone or combined with ABT-263 (1 µM) and apoptosis was measured by caspase cleavage and annexin V+ labeling. Synergy was measured by calculation of the combination index. Effect of carfilzomib on Bcl-2 family proteins was determined by immunoblotting. Interaction of NOXA with MCL-1 was examined by immunoprecipitation. Knockdown of NOXA was achieved by lentiviral shRNA. Cells were incubated with drugs alone or combined with chloroquine, and autophagy (LC3I/II conversion) and apoptosis were analyzed. RESULTS. Carfilzomib was shown to potently and synergistically enhance ABT-263-induced apoptosis (2-fold) in both KRAS MUT and wild type (WT) cell lines, with the effect being modestly higher in wild type cells, as shown by annexin V+ staining and caspase cleavage. Carfilzomib induced NOXA expression in both KRAS MUT and WT cells, and reduced MCL-1 expression. Induced NOXA was shown to bind and sequester MCL-1 proteins. Knockdown of NOXA attenuated drug-induced apoptosis. Autophagy inhibition by chloroquine accumulated LC3II, but only modestly increased carfilzomib-induced cell death. CONCLUSION. Irreversible proteasome inhibition by carfilzomib synergistically enhanced ABT-263-induced cell death that is mediated by NOXA upregulation and its sequestration/neutralization of MCL-1. The finding of similar efficacy in KRAS MUT vs WT cells suggests that this combination may represent a novel therapeutic strategy in this tumor subset. Citation Format: Shengbing Huang, Koichi Okamoto, Frank A. Sinicrope. Carfilzomib synergistically enhances ABT-263-induced apoptosis due to NOXA induction in KRAS wild type and mutant colorectal cancer cell lines. [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 337. doi:10.1158/1538-7445.AM2014-337

Control of the PI3K/AKT Pathway by Phosphodiesterase 4B Modulates Glucocorticoid Response in B-Cell Lymphomas.

422 Background: The MAPK pathway is a crucial regulator of cell proliferation, survival, and resistance to apoptosis. Hyperactivation of this pathway due to mutations in KRAS have been reported in up to 50% of CRC cases. Clinical trials have shown that KRAS patients do not benefit from therapies targeting EGFR, highlighting the need for new therapeutic options. Utilizing differential gene array analyses, we have identified the hedgehog (HH) signaling pathway as a potential mediator of resistance to AZD6244. Based on these results, we tested the rational combination of selumetinib and the HH inhibitor, cyclopamine against human CRC cell lines. Methods: CRC cell lines were exposed to varying concentrations of selumetinib and cyclopamine. For AZD6244, cell lines with IC50≤ 0.1 μM were considered extremely sensitive (ES) and those with IC50≥ 1μM were deemed extremely resistant (ER). Four KRAS mutant cell lines (2ES, 2ER) were selected for combination studies. The antiproliferative effects were assessed using the sulforhodamine B (SRB) cell viability assay, and potential synergy was evaluated using the Chou and Talalay method. Apoptosis was analyzed using bioluminescent caspase 3/7 detection. Results: In all four cell lines tested, synergistic antiproliferative effects of selumetinib and cyclopamine were observed, including resistant lines to selumetinib. We observed significant induction of apoptosis when cell lines were exposed to the combination treatment, independent of their responsiveness to selumetinib in the SRB assay. Conclusions: Treatment of KRAS mutant CRC cell lines with selumetinib and cyclopamine resulted in synergistic inhibition of proliferation, regardless of sensitivity to selumetinib. Interestingly, a significant increase in apoptosis was observed in response to the combination, which may explain the synergy observed by the combination index (CI). In vivo analyses of this combination in cell lines and human CRC explants are ongoing to further validate these results. These preclinical data may suggest a rational combination strategy for patients with KRAS mutant CRC. No significant financial relationships to disclose.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most difficult human malignancies to treat due to its innate and acquired therapeutic resistance. Our hypothesis is that cyclin-dependent kinase 4 (CDK4) mediates therapeutic resistance to targeting of the KRAS-MAPK pathway in PDAC. Experimental Procedure: We characterized the expression of total and phosphorylated Retinoblastoma (Rb) and MEK protein levels in KRAS wild-type (BxPC3) and KRAS mutant (PANC1, MiaPaca2) human PDAC cell lines at baseline and with MEK and CDK4 inhibition alone or in combination. We then assessed the effects of combined therapy on cell-cycle progression and tumorigenicity (in vitro and in vivo). Finally, using the Ptf1a cre/+;LSL-Kras G12D/+;Tgfbr2 flox/flox (PKT) mouse model of PDAC, we assessed the in vivo overall tumor growth and survival after combined treatment with CDK4/6 and MEK inhibitors. Results: Rb functions as a tumor suppressor, and it is inactivated when phosphorylated by CDK4-Cyclin-D1. The CDK4/6 inhibitor (LEE011) effectively inhibits phosphorylation of Rb in cell lines regardless of KRAS mutational status. Combined inhibition of CDK4/6 and MEK (MEK162) decreased phosphorylation of RB and MAPK expression synergistically in the KRAS mutant cell lines, but not the KRAS wild-type BxPC3 cell line. Cell cycle progression was delayed effectively with MEK inhibition alone in the KRAS wild-type cell line, yet only combined CDK4/6 and MEK inhibition effectively delayed cell cycle progression in the KRAS mutant cell lines. Colony formation and invasion were also significantly decreased when Kras mutant cells were treated with combined CDK4/6 and MEK inhibition compared to all monotherapy and control groups. Finally, treatment of PKT mice resulted in a modest increase in OS with MEK inhibition alone, but mice receiving combined CDK4/6 and MEK inhibition exhibited a four-fold increase in OS. Conclusions: Combined inhibition of CDK4/6 and MEK results in significantly enhanced therapeutic efficacy and prolonged survival in the aggressive PKT mouse model of PDAC. This study suggests that concurrent inhibition of CDK4/6 and MEK may be an effective treatment for PDAC. Citation Format: Jason A. Castellanos, Nagaraj Nagathihalli, Michael N. Van Saun, Cameron Kasmai, Yanhua Xiong, Nipun Merchant. CDK4/6 inhibition synergizes with KRAS-MAPK pathway targeting in pancreatic cancer. [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 2537. doi:10.1158/1538-7445.AM2015-2537

Recent reports have documented the paradoxical activation of the MAPK pathway in response to pharmacological RAF inhibition in cells bearing either wild-type (wt) or mutant RAS, as evidenced by hyperphosphorylation of ERK in human tumor cells exposed to BRAF inhibitors in vitro. ARQ 680 is a pan-RAF kinase inhibitor (IC50 values of 2.6, 2.7, & 7.3 nM respectively for BRAF, mutant BRAF(V600E), and CRAF) which is the active moiety of the highly soluble pro-drug ARQ 736, whose biological properties and anti-tumor activities have been described previously. Here we compared the activity of ARQ 680 and PLX 4032, the latter a structurally distinct pan-RAF inhibitor currently in advanced clinical trials, in various cell lines with various RAS/RAF genotypes. ARQ 680 and PLX 4032 show comparable degrees of selectivity across the human kinome, with ARQ 680 inhibiting 11 other kinases within 100-fold of its activity against BRAF. Interestingly, there is no discernable overlap between the off-target kinases of ARQ 680 and PLX 4032. In human melanoma cell lines (A375, SK-MEL-28) bearing mutant BRAF(V600E), both compounds exhibited comparable concentration-dependent inhibition of phospho-ERK as shown by quantitative western blotting (EC50 ∼ 10 – 30 nM). In 3 colon cancer cell lines harboring a mutant kras gene (HCT-116, SW480, & DLD-1), both ARQ 680 and PLX 4032 induced robust phosphorylation of ERK (1.8 to 5-fold increases) at varying concentrations, with ARQ 680 exhibiting the phenomenon at lower concentrations than PLX 4032. Importantly, ARQ 680 induced a concentration-dependent decrease in, and ultimately a nearly complete inhibition of, ERK phosphorylation at concentrations of 3 μM and above in all KRAS-mutant cell lines. In contrast, PLX 4032 exerted no inhibition of ERK phosphorylation below untreated controls at concentrations up to 100 μM, with the exception of the SW480 cell line, where the elevated p-ERK levels decreased to control levels at 10 to 100 μM. In an NRAS-mutant melanoma cell line (SK-MEL-2), ARQ 680 exerted a concentration-dependent inhibition of ERK phosphorylation (EC50 ∼ 400 nM) with no paradoxical activation of p-ERK at any concentration, while PLX 4032 induced an increase in p-ERK at concentrations up to 10 μM. Finally, in a wt RAS/RAF colon epithelial cell line (NCM-460), both ARQ 680 and PLX 4032 doubled p-ERK levels at sub-micromolar concentrations, with ARQ 680 exhibiting a concentration-dependent abrogation of this effect at concentrations between 1 and 10 μM while PLX 4032 sustained a 2-fold increase in p-ERK at concentrations as high as 100 μM. In summary, we have delineated the pharmacodynamic profile for a novel RAF inhibitor, ARQ 680. The molecular mechanisms by which ARQ 680 inhibits pERK in KRAS and NRAS mutant cell lines are under investigation and may be due in part to the suite of kinases inhibited by ARQ 680 in cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3565. doi:10.1158/1538-7445.AM2011-3565

Combined HASPIN and mTOR inhibition is synergistic against KRAS-driven carcinomas

Immune checkpoint inhibitors (ICIs), specifically those which target the T-cell surface receptor Programmed Cell Death-1 (PD-1) and its ligand, Programmed cell death ligand-1 (PD-L1), have demonstrated substantial clinical benefit in patients with non-small cell lung cancer (NSCLC). Tumoral PD-L1 expression may be important for response to therapy, but the precise mechanism of regulation has yet to be fully elucidated. It is known that PD-L1 expression is elevated in cancers harboring mutations in the RAS family of genes, specifically KRAS. Mutations in the KRAS gene are found in up to 30% of NSCLC tumors, and there are no targeted treatment options available for this subset of patients. The tyrosine phosphatase, SHP-2, has been shown to be a critical regulator of the KRAS signaling cascade. Therefore, genes regulated by the RAS/MAPK signaling cascade, including PD-L1, may be modulated by SHP-2 inhibition. Thus, this study aims to investigate the impact of SHP-2 activity on PD-L1 expression in NSCLC. The allosteric inhibitor of SHP-2, SHP099, was used to ablate SHP-2 activity in KRAS-mutant NSCLC cell lines, H460, A549, H2122, and EGFR-mutant NSCLC cell line PC9. PD-L1 levels were quantified by western blot and flow cytometry analyses. PD-L1 mRNA was measured by quantitative real time PCR (qRT-PCR) analysis. SHP099 activity was confirmed by SHP-2, siRNA transfection in KRAS active cell lines. Inhibition of SHP-2 led to increased levels of PD-L1 expression in the KRAS-mutant cell lines, but not in the EGFR-mutant cells. Analysis of PD-L1 surface expression by flow cytometry confirmed that surface PD-L1 levels increased following exposure to SHP099. To determine whether SHP-2 controlled expression at the transcriptional level, mRNA quantification by qRT-PCR displayed increased levels of PD-L1 mRNA following SHP099 treatment. Together, these results suggest that SHP-2 regulates PD-L1 expression in KRAS-active NSCLC at the level of PD-L1 gene expression. It is presently unclear whether SHP-2 also has a role in controlling protein turnover. Using SHP-2 mutant constructs that mimic a constitutively active phosphatase, we hope to gain further insight into the roles of both SHP-2 catalytic and scaffolding functionality on PD-L1 expression. Citation Format: Keller Toral. The tyrosine phosphatase, SHP-2, is involved in regulating PD-L1 expression in non-small cell lung cancer [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 1773.