BRAF-mutant Cell Lines Research Articles

Cetuximab promotes SN38 sensitivity via suppression of heat shock protein 27 in colorectal cancer cells with wild-type RAS.

Combination treatment with cetuximab and CPT-11 produces beneficial and synergistic effects in wild-type RAS metastatic colorectal cancer (mCRC) patients. However, the mechanism underlying this synergism is not yet understood. We examined whether cetuximab had a synergistic effect with CPT-11 and its active metabolite, SN38, and examined the molecular mechanism of the synergism between cetuximab and SN38 in CRC cells with various mutational status. We hypothesized that cetuximab promotes sensitivity to SN38 via suppression of heat shock protein27 (HSP27), a protein involved in multidrug resistance through blocking the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, which is associated with chemosensitivity. Four human CRC cell lines with different RAS and BRAF mutational status were used. Expression levels of HSP27 protein correlated with SN38 sensitivity in these cell lines (R=0.841, p=0.159). Exposure to cetuximab and various concentration of AG490, an inhibitor of JAK2, STAT3 and HSP27 protein levels, except in the KRAS G12V mutant line, SW620. A synergistic effect of cetuximab in combination with SN38 was observed in RAS and BRAF wild-type cells (here, Caco2), but not in the three other RAS- or BRAF-mutated cell lines. These results indicate that cetuximab may promote sensitivity to SN38 via suppression of HSP27 through blocking the JAK/STAT pathway in Caco2 cells. The mutational status of numerous downstream effectors, such as RAS and BRAF, is important in mono- or combination therapy with cetuximab. In conclusion, cetuximab may promote SN38 sensitivity via suppression of HSP27, through blocking the JAK/STAT signaling pathway, and shows synergistic effects when combined with SN38 in wild-type RAS CRC cells.

The Mitogen-Activated Protein Kinase Pathway Facilitates Resistance to the Src Inhibitor Dasatinib in Thyroid Cancer.

Advanced stages of papillary and anaplastic thyroid cancer represent a highly aggressive subset, in which there are currently few effective therapies. We and others have recently demonstrated that c-SRC is a key mediator of growth, invasion, and metastasis, and therefore represents a promising therapeutic target in thyroid cancer. However, clinically, Src inhibitor efficacy has been limited, and therefore further insights are needed to define resistance mechanisms and determine rational combination therapies. We have generated four thyroid cancer cell lines with a greater than 30-fold increase in acquired resistance to the Src inhibitor dasatinib. Upon acquisition of dasatinib resistance, the two RAS-mutant cell lines acquired the c-SRC gatekeeper mutation (T341M), whereas the two BRAF-mutant cell lines did not. Accordingly, Src signaling was refractory to dasatinib treatment in the RAS-mutant dasatinib-resistant cell lines. Interestingly, activation of the MAPK pathway was increased in all four of the dasatinib-resistant cell lines, likely due to B-Raf and c-Raf dimerization. Furthermore, MAP2K1/MAP2K2 (MEK1/2) inhibition restored sensitivity in all four of the dasatinib-resistant cell lines, and overcame acquired resistance to dasatinib in the RAS-mutant Cal62 cell line, in vivo Together, these studies demonstrate that acquisition of the c-SRC gatekeeper mutation and MAPK pathway signaling play important roles in promoting resistance to the Src inhibitor dasatinib. We further demonstrate that up-front combined inhibition with dasatinib and MEK1/2 or ERK1/2 inhibitors drives synergistic inhibition of growth and induction of apoptosis, indicating that combined inhibition may overcome mechanisms of survival in response to single-agent inhibition. Mol Cancer Ther; 15(8); 1952-63. ©2016 AACR.

Molecular Landscape of Acquired Resistance to Targeted Therapy Combinations in BRAF-Mutant Colorectal Cancer.

Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1 These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504-15. ©2016 AACR.

Abstract 4658: Activation mechanisms of Integrated Stress Response by inhibition of oncogenic BRAF in melanoma cell lines

Abstract The eIF2α-ATF4 signaling pathway termed “Integrated stress response (ISR)” plays an important role in protecting cells against various cellular stresses such as deprivation of glucose and amino acids, virus infection, and deficiency of heme. Under such stress conditions, eIF2α kinases, including PERK, GCN2, PKR and HRI, phosphorylate eIF2α, resulting in reduction of general protein synthesis while facilitating selective translation of ATF4, a central transcription factor that upregulates expression of stress-induced genes for cellular adaptation. Recently, it was reported that BRAF inhibitor vemurafenib triggers activation of the eIF2α-ATF4 signaling in BRAF-mutant melanoma cell lines; however, activation mechanisms of vemurafenib-induced ISR signaling not fully understood. In this study, we first compared vemurafenib-induced ISR activation in BRAF-mutant and -wild type melanoma cell lines. Consistent with previous studies, vemurafenib activated ISR signaling preferentially in BRAF-mutant cell lines, as determined by phosphorylation of eIF2α and ATF4 expression. We also found that the ISR activation by vemurafenib was impaired by knockdown of BRAF in BRAF-mutant cell lines. These results indicated that existence of mutated BRAF is important for vemurafenib to trigger activation of ISR signaling pathway. We next examined which eIF2α kinase is involved in the ISR signaling activation by vemurafenib. We found that knockdown of GCN2, an eIF2α kinase sensing depletion of amino acids, impaired phosphorylation of eIF2α and ATF4 induction by vemurafenib. In addition, we confirmed that vemurafenib induced phosphorylation of GCN2 on Thr899 that is known to occur in response to amino acids starvation. In agreement with these results, gene expression profiling with ontology analysis revealed that vemurafenib upregulated genes that are involved in amino acids biosynthesis via ISR signaling. Collectively, our results suggested that vemurafenib activated ISR signaling pathway possibly through mimicking amino acid deprivation in BRAF-mutant melanoma cell lines. Citation Format: Ikuko Nagasawa, Satomi Tsukahara, Kazuhiro Kunimasa, Akihiro Tomida. Activation mechanisms of Integrated Stress Response by inhibition of oncogenic BRAF in melanoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4658.

Abstract 187: ASN0007, a potent ERK1/2 inhibitor with strong antitumor activity in multiple RAS mutant models

Abstract The extracellular-signal-regulated kinases, ERK1 and ERK2 (ERK1/2), play a critical role in the RAS/RAF/MEK signaling pathway which is quite frequently activated by mutations in upstream targets such as BRAF, RAS and receptor tyrosine kinases. Most of the resistance mechanisms to BRAF and MEK inhibitors ultimately lead to increase in phosphorylation of ERK1/2 suggesting the importance of this node in the RAS/RAF/MEK pathway even in the resistance setting. Therefore, inhibition of ERK1/2 offers a promising strategy to address both innate and acquired resistance to BRAF and MEK inhibitors in various solid tumors. In order to inhibit this critical node of the RAS/RAF/MEK pathway, we discovered and characterized ASN007, a novel compound that shows potent inhibition of ERK1/2 kinases in a biochemical assay with low single-digit IC50 values. In mechanistic cell-based assays, ASN007 inhibited the phosphorylation of ERK1/2 substrates such as pRSK1, FRA1 and Elk1 in various cell lines. As expected, the compound did not have any effect on the phosphorylation of ERK1/2. ASN007 showed strong anti-proliferative activity in both BRAF and RAS mutant cell lines. Importantly, when compared to other ERK1/2 inhibitors such as BVD-523 and GDC-0994, ASN007 showed potent antiproliferative activity in a much broader panel of KRAS, NRAS and HRAS mutant cell lines representing various histological tumor types. The compound also caused cell cycle arrest at the G0-G1 phase and induced apoptosis in both BRAF and RAS mutant cell lines. In a daily oral dosing regimen, ASN007 demonstrated strong inhibition of tumor growth in multiple BRAF and KRAS mutant xenograft models in mice and was well tolerated at efficacious doses. ASN007 has a good pharmacokinetic profile in pre-clinical species and does not exhibit significant inhibition of CYP enzymes or hERG channel. Based on its profile in preclinical studies, ASN007 is expected to show strong efficacy in BRAF and RAS mutant cancers. ASN007 is currently in preclinical development. Citation Format: Sanjeeva P. Reddy, Dhanalakshmi Sivanandhan, Raghava Reddy Kethiri, Sreekala Nair, Purushottam Dewang, Mohd Zainuddin, Krishnakumar Vaithilingam, Roger A. Smith, Sandeep Gupta, Scott K. Thompson. ASN0007, a potent ERK1/2 inhibitor with strong antitumor activity in multiple RAS mutant models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 187.

Abstract 1247: ERK1/2 inhibitors with distinct modes of inhibition confer different response profiles in KRAS mutant cancers

Abstract The clinical benefit of targeting the MAPK pathway is demonstrated by the success of RAF and MEK inhibitors in the treatment of melanoma. However, response to such agents is short-lived due to the onset of resistance mechanisms that result in reactivation of ERK signalling. Additionally, many BRAF- and KRAS-mutant cancers exhibit de novo resistance. As the direct targeting of ERK may overcome the limitations of RAF or MEK inhibitors, there are several ERK1/2 inhibitors in development. SCH772984 (SCH, Merck-Schering) is an example of an ERK1/2 inhibitor that in addition to inhibiting ERK catalytic activity also inhibits the phosphorylation of residues within the ERK activation loop. Several ERK1/2 inhibitors have been described that inhibit ERK catalytic activity without modulating ERK phosphorylation e.g GDC-0994 (GDC, Genentech). Here we aim to characterise the biological relevance of the different modes of ERK inhibition. We investigated the biochemical mechanism by which SCH inhibits ERK phosphorylation and demonstrated that it prevents the phosphorylation of ERK by MEK (T202/Y204), but does not affect MEK activity. We characterised the effects of the distinct modes of ERK inhibition on ERK substrates, cellular pERK levels, pathway feedback activation and sensitivities of cell lines with different genetic backgrounds. In BRAFV600E-mutant cells e.g A375 (melanoma) and Colo205 (colorectal), 16 hr treatment with SCH and GDC significantly inhibited the phosphorylation of RSK (ERK substrate). SCH significantly inhibited ERK phosphorylation, whereas GDC did not modulate pERK levels. Neither compound induced pathway feedback activation. In proliferation assays A375 and Colo205 cells had remarkably similar sensitivities to SCH (EC50 40 nM and 30 nM, respectively) and GDC (EC50 = 140 nM in both). In contrast to BRAF-mutant cells, SCH and GDC treatment conferred pMEK induction in the KRAS-mutant cell line HCT116 (while still significantly inhibiting RSK phosphorylation). This is likely to be attributable to the feedback-driven C-RAF activation commonly associated with MAPK pathway inhibition in KRAS-mutant cells. In addition to pMEK, GDC also conferred an induction of pERK. However, SCH overcame pathway feedback and inhibited the ERK phosphorylation. The inhibition of pathway feedback conferred by SCH was associated with increased sensitivity of HCT116 cells to SCH (EC50 = 30 nM) compared to GDC (EC50 = 1.1 μM). In a cell panel screen comprising 25 BRAF-mutant and 63 KRAS-mutant cell lines, SCH and GDC had similar activity in BRAF-mutant cell lines. However, although KRAS-mutant cell lines were sensitive to SCH, they were largely resistant to GDC. These data suggest that due to their ability to overcome MAPK inhibitor-induced pathway feedback, ERK inhibitors that also inhibit the phosphorylation of ERK would be more effective in KRAS-mutant cancer than those that inhibit ERK activity alone. Citation Format: Joanne M. Munck, Brent Graham, Juan Castro, Aurelie Courtin, Sandra Muench, Sharna Rich, Harpreet K. Saini, Jessica Brothwood, John Lyons, Neil T. Thompson, Nicola G. Wallis, Chris Murray. ERK1/2 inhibitors with distinct modes of inhibition confer different response profiles in KRAS mutant cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1247.

Abstract 375: Combination of MEK and PI3K inhibition in BRAF wild-type and mutant melanoma

Abstract Introduction: Targeted therapy against BRAF-mutated melanoma with BRAF and MEK inhibitors has shown clinical efficacy. However, resistance to therapy occurred and lead to therapy failure. We evaluated the effect of combining downstream inhibition of RAS/RAF pathways with MEK (AZD6244) and PI3K (XL765) inhibition on BRAF wild-type and mutant melanoma with variable expression of NRAS as possible therapeutic option. Methods: Fifteen melanoma cell lines were screened for BRAF mutation status and NRAS expression. BRAF wildtype (Mel628, Mel1098) and BRAF-mutated (Mel1861, Mel1890) cell lines were selected. Drug concentration study with AZD6244 (A, 2.5-40 uM) and XL765 (X, 2.5-40 uM) were performed on Mel628 cells. Subsequently, cells were treated with A (5 uM), X (5 uM), or combination. Cell proliferation assay was performed using Cell Titer Blue assay. Western blotting was performed to for expression of PARP, caspase-9, LC3A/B, Beclin1, pMEK/MEK, pERK/ERK, p-P70S6/P70S6, and p4EBP1/4EBP1. GAPDH was used as internal control. Apoptosis was analyzed using FITC Annexin V Apoptosis Detection Kit (BD Biosciences). Cell migration assay was performed by artificial wounding of melanoma cell monolayer and observing migration of cells into the wound up to 72 hours. Data were presented as means ± SD for the three separate experiments. For comparison between groups, the student's t test was used and p< 0.05 was considered to be statically significant. Results: There was dose-dependent inhibition of melanoma cell proliferation with both A and X. Combination A and X had synergistic effect on the anti-melanoma proliferative effect of BRAF mutated cells regardless of expression level of NRAS. Significantly increased apoptosis by Annnexin V assay was detected in BRAF mutated cells (Mel 1860 and Mel1890). Significantly increased expression (p<0.05) of apoptosis markers (PARP and caspases-9) were observed in cells treated with A+X compared with A or X alone. Enhanced phosphorylation of ERK and P70S6 were observed in BRAF mutated cells. No significant difference in autophagy markers (LC3A/B and Beclin1) were observed between A, X, or combination. Variable to minimal additive effect of the combination treatment was observed in BRAF wild-type cells in terms of proliferation, apoptosis, and autophagy. The combination of A and X significantly inhibited melanoma migration compared with either drug alone regardless of BRAF mutation status in the wounding assay model. Conclusion: We have observed a synergistic effect on suppressing BRAF-mutated melanoma proliferation with combined inhibition of MEK and PI3K. The effect is not dependent on NRAS expression levels of melanoma. Citation Format: Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. Combination of MEK and PI3K inhibition in BRAF wild-type and mutant melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 375.

Abstract 2671: BRAF inhibitor resistance reprograms metabolic and survival pathways to sensitize melanoma cells to arginine deprivation

Abstract The combination of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) has been FDA approved to treat melanomas harboring (V600E) mutation. Although most BRAF mutant melanomas are highly responsive to these treatments, they all relapse ultimately. In this study, we found a unique mechanism which can be used to treat BRAFi resistant patients. Five BRAFi resistant (BR) cells were established from known BRAF mutant cell lines A375, MEL-1220, A2058, UACC-62, and SK-MEL28 by long-term exposure to BRAFi. These BR cells are hypersensitive to ADI-PEG20, an enzyme which degrades arginine to citrulline. ADI-PEG20 treatment resulted in a 2-3-fold decrease in BR cell viability (MTT assay) and 10-30% increase in apoptosis (Annexin V/PI) in BR cells compared to their paternal cells. The mechanisms involved are as follows: All BR cells express very low levels or do not express argininosuccinate synthetase (ASS, a key enzyme to generate arginine from citrulline). Thus, these BR cells rely primarily on exogenous arginine. Furthermore, these BR cells also have attenuation of glucose uptake which makes them rely mainly on amino acids. Importantly, their ability to undergo autophagy upon nutritional stress is also impaired. The underlying mechanism for low levels of ASS is due to diminished c-Myc expression which is a positive regulator for ASS. Additionally, AMPK-α1, which possesses phosphorylation site at Thr172 governing autophagy and glucose uptake, was attenuated in BR cells. Overexpression of AMPK-α1 using plasmid transfection in A2058BR and MEL-1220BR cells rescued 8-28% ADI-PEG20-induced apoptosis and enhanced autophagosome formation (detected by LysoTacker). Conversely, knockdown of AMPK-α1 using siRNA significantly enhanced ADI-PEG20-reduced cell viability (compared to non-targeting siRNA) in A2058 and MEL-1220 cells through attenuation of autophagy and glucose uptake. This is evidenced by decreased GLUT1 and LC3-II expression, and low activity of 2-NBDG uptake, which also can be seen in most BR cells. The xenograft model also showed that ADI-PEG20 aborted tumor growth of A2058BR and A375BR cells but retarded growth of A2058 and A375 cells. Immunohistochemical staining further confirmed lower levels of ASS and AMPK-α1 in A2058BR xenograft and in tumor tissues from 10 BR patients relative to their parental counterparts (average H-scores of ASS and AMPK in parental tissues vs. BR tissues are 58.2 vs. 7.8, and 146 vs. 78.3, respectively, p <0.03). Importantly, these findings also apply to cell lines and tumor samples from patients who failed both BRAF and MEK inhibitor. In summary, our data suggest that attenuated AMPK-α1 mediated autophagy and glucose uptake and decreased c-Myc mediated ASS re-expression sensitize BR cells to arginine deprivation. Thus, ADI-PEG20 is a potential salvage therapy for BR patients (Supported by R01CA152197). Citation Format: Ying-Ying Li, Chunjing Wu, Sumedh Shah, Shu-Mei Chen, Medhi Wangpaichitr, Lynn Feun, Macus Kuo, Miguel Suarez, Niramol Savaraj. BRAF inhibitor resistance reprograms metabolic and survival pathways to sensitize melanoma cells to arginine deprivation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2671.

Combination of a Selective HSP90α/β Inhibitor and a RAS-RAF-MEK-ERK Signaling Pathway Inhibitor Triggers Synergistic Cytotoxicity in Multiple Myeloma Cells.

Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/β inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/β inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM.

Predicting Anticancer Drug Responses Using a Dual-Layer Integrated Cell Line-Drug Network Model.

The ability to predict the response of a cancer patient to a therapeutic agent is a major goal in modern oncology that should ultimately lead to personalized treatment. Existing approaches to predicting drug sensitivity rely primarily on profiling of cancer cell line panels that have been treated with different drugs and selecting genomic or functional genomic features to regress or classify the drug response. Here, we propose a dual-layer integrated cell line-drug network model, which uses both cell line similarity network (CSN) data and drug similarity network (DSN) data to predict the drug response of a given cell line using a weighted model. Using the Cancer Cell Line Encyclopedia (CCLE) and Cancer Genome Project (CGP) studies as benchmark datasets, our single-layer model with CSN or DSN and only a single parameter achieved a prediction performance comparable to the previously generated elastic net model. When using the dual-layer model integrating both CSN and DSN, our predicted response reached a 0.6 Pearson correlation coefficient with observed responses for most drugs, which is significantly better than the previous results using the elastic net model. We have also applied the dual-layer cell line-drug integrated network model to fill in the missing drug response values in the CGP dataset. Even though the dual-layer integrated cell line-drug network model does not specifically model mutation information, it correctly predicted that BRAF mutant cell lines would be more sensitive than BRAF wild-type cell lines to three MEK1/2 inhibitors tested.

Abstract 3522: BET and BRAF inhibitors act synergistically against BRAF mutant melanoma

Abstract Purpose: Despite major advances in the treatment of metastatic melanoma, treatment failure is still inevitable in most cases. Manipulation of key epigenetic regulators, including inhibition of Bromodomain and extra-terminal domain (BET) family members impairs cell proliferation in vitro and tumor growth in vivo in different cancers, including melanoma. BET inhibition is thought to impair downstream transcriptional activity of the MAPK (MAPK) pathway and other key oncogenic signaling pathways, suggesting complementarity with MAPK inhibition. Here, we investigated the effect of combining the BET inhibitor JQ1 with the BRAF inhibitor Vemurafenib in in vitro and in vivo models of BRAF-mutant melanoma. We additionally screened potential synergism of JQ-1 with 27 additional compounds including inhibitors of the proteasome, histone deacetylases (HDACi) and MEK. Methods: We performed cytotoxicity and apoptosis assays, and a xenograft mouse model to determine the in vitro and in vivo efficacy of JQ1 in combination with Vemurafenib against BRAF-mutant melanoma cell lines. Further, to investigate the molecular mechanisms underlying the effects of this combined treatment, we conducted antibody arrays of in vitro drug-treated cell lines and RNA sequencing of drug-treated xenograft tumors. Synergism between JQ1 and other targeted compounds was examined by in vitro cytotoxicity assays. Results: The combination of JQ1 and Vemurafenib acted synergistically in BRAF-mutant cell lines. Additional combinations that were found synergistic included JQ-1 plus proteasome inhibitors (carfilzomib and marizomib, HDAC inhibitors (romidepsin and panobinostat), or anti-Bcl2 small molecules (Obatoclax and TW-37). The combination of JQ-1 and Vemurafenib resulted in marked apoptosis in vitro, with up-regulation of several pro-apoptotic proteins involved in both the intrinsic and extrinsic pathways of apoptosis. In vivo, this combination treatment suppressed tumor growth and significantly improved survival compared to either drug alone. RNA sequencing of tumor tissues revealed almost four thousand genes that were uniquely modulated by the combination. Furthermore, several anti-apoptotic genes such as MCL-1, BCL-XL and BCL-2 were significantly down-regulated. Conclusions: Collectively, our data provide a rationale for combinations of BET inhibitors with additional compounds including BRAF inhibitors, as a novel strategy for the treatment of melanoma. Citation Format: Luca Paoluzzi, Douglas Hanniford, Elena Sokolova, Iman Osman, Farbod Darvishian, Jinhua Wang, James E. Bradner, Eva Hernando. BET and BRAF inhibitors act synergistically against BRAF mutant melanoma. [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 3522. doi:10.1158/1538-7445.AM2015-3522

Abstract 1668: Effective targeting of colorectal cancer with a recombinant antibody mixture against EGFR, HER3 and IGF1R

Abstract The epidermal growth factor receptor (EGFR) plays an important role in the development and maintenance of the malignant phenotype of several human cancers. Therefore, it is a frequently pursued therapeutic target and several antibodies targeting EGFR have been clinically approved. However, the clinical responses to current anti-EGFR agents are often modest, indicating a need for improved therapeutics. Resistance mechanisms to anti-EGFR antibodies include ligand up-regulation, receptor tyrosine kinase plasticity, and mutations in downstream signaling molecules such as KRAS, BRAF, and PIK3CA. Here, we present data showing that a mixture of antibodies targeting HER3, EGFR, and IGF1R (HEI mixture) with high affinity ligand-blocking antibodies induced growth inhibition in a large panel of cancer cell lines. Synergies of simultaneous inhibition of the three targets were frequently seen in cell lines and xenograft tumor models. In particular, colorectal cancer cell lines were sensitive to the HEI mixture and superiority to cetuximab was seen in several cell lines. Surprisingly, the HEI mixture was effective at inhibiting the growth of KRAS and BRAF mutated cell lines, suggesting that the three receptor tyrosine kinases may still contribute to the malignant phenotype of tumors with these genomic alterations. Colorectal cancer is thought to be a ligand driven disease and hence the inhibitory activity of the HEI mixture was tested in the presence of EGFR, HER3 and IGF1R ligands and compared with the activity of cetuximab. As expected, the HEI mixture was able to block growth stimulation induced by all ligands, whereas heregulin and IGF1/IGF2 caused resistance to cetuximab. A clinically relevant subpopulation of colorectal cancer harbors amplification of IGF2, a major ligand for IGF1R, and hence should display sensitivity to IGF1R targeting. The HEI mixture was tested in three metastatic colorectal PDX models with IGF2 overexpression and induced tumor regression in all three models. In conclusion, our data indicate that simultaneous targeting of EGFR, HER3 and IGFR1 provides a broader efficacy than targeting a single receptor, and thereby provides a rationale for simultaneous targeting of the three receptors with a recombinant antibody mixture. Citation Format: Christina Egebjerg, Camilla Fröhlich, Karsten Wessel Eriksen, Thomas Tuxen Poulsen, Klaus Kofoed, Thomas Bouquin, Andrea Bretotti, Livio Trusolino, Ivan David Horak, Michael Kragh, Mikkel Wandahl Pedersen. Effective targeting of colorectal cancer with a recombinant antibody mixture against EGFR, HER3 and IGF1R. [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 1668. doi:10.1158/1538-7445.AM2015-1668

Abstract 2843: Resistance to ERK inhibitors as a result of an acquired novel P-loop mutation of ERK2

Abstract Genomic aberrations frequently target and activate the MAPK and PI3K pathways in human cancers and combined inhibition of these pathways is being explored widely in the clinic. In a panel of 45 human colorectal cancer cell lines we found the presence of a KRAS mutation resulted in reduced sensitivity to PI3K and MEK inhibitors when used as single treatments. In addition, prolonged exposure to the individual PI3K (GDC-0941) or MEK (GDC-0973) inhibitors resulted in resistance that was associated with acquisition of mutations of the respective pathway being targeted. Resistance to individual targeted agents is challenge in clinic and can be overcome by combining treatments and in our colorectal cancer panel combined MEK/PI3K inhibition exhibited potent synergy in the KRAS mutant lines. However, as with the single treatments prolonged exposure to the combination regime also resulted in resistance through acquisition of a mutation of the allosteric drug-binding site of MEK2 that abrogates binding of the inhibitor. The clinical development of small-molecule ERK inhibitors is of considerable interest as ERK signaling represents a key downstream effector of RAS mutations and ERK inhibitors may retain activity in cells resistant to RAF and MEK inhibitors. Consistent with the latter, the combination resistant line retained sensitivity to an ERK inhibitor (VTX-11e) that also rescued the synergistic interaction when combined with PI3K inhibition. However, prolonged exposure of this MEK/PI3K resistant line to the combination of VTX-11e/GDC0941 resulted in the acquisition of additional resistance to the ERK inhibitor component of the combination. Resistance to VTX-11e was a result of an ERK2 mutation, novel to protein kinases, in a conserved residue of the kinase domain P-loop. Recombinant wild-type and P-loop mutant ERK2 had similar Vmax and ATP Km values, but exhibited a 20-fold increase in IC50 for VTX-11e. Stable expression of the P-loop mutant ERK2 in parent cells resulted in resistance to VTX-11e. Development of an additional VTX-11e resistant line, in this case by prolonged exposure to a single treatment resulted in the acquisition of the same ERK2 mutation. The repeated development of resistance to the MEK/ERK inhibitor arm of the combination is intriguing as the parent cells were not particularly sensitive to treatment with MEK or ERK inhibitors alone, with a GI50 30-fold higher than BRAF mutant cell lines that are sensitive to these inhibitors. Our data show that dual combination of agents, at least for MEK and PI3K pathway inhibition, is insufficient to block the acquisition of resistance, an observation similar to that of clinical trials of BRAF and MEK inhibitors in melanoma. Finally we recommend sequencing of P-loop of the ERKs in clinical studies of ERK inhibitors to determine whether these mutations will occur in the clinic. Citation Format: Paul A. Clarke, Toby Roe, Kate Swabey, Craig McAndrew, Kathy Boxall, Isaac Westwood, Robert van Montfort, Bissan Al-Lazikhani, Paul Workman. Resistance to ERK inhibitors as a result of an acquired novel P-loop mutation of ERK2. [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 2843. doi:10.1158/1538-7445.AM2015-2843

Abstract 2900: Biochemical alterations in BRAF inhibitor (BRAFi) resistant melanoma cells increase their vulnerability to arginine deprivation

Abstract The BRAFi, has been applied to treat melanomas harboring (V600E) mutation. While the response rate is high, resistance is inevitable. Despite combination with MEK inhibitor which enhances the antitumor efficacy as well as increases duration of response, patients ultimately relapse. Therefore, there is an urgent need for second line treatment for melanoma patients who are resistant to BRAFi treatment. In this study, we found that BRAFi-resistant (BR) melanoma cells are susceptible to arginine deprivation due to their inability to turn on argininosuccinate synthetase (ASS), a key enzyme for arginine synthesis as well as their inability to undergo autophagy. Thus, arginine deprivation using pegylated arginine deiminase (ADI-PEG20, supplied by Polaris Inc.) which degrades arginine to citrulline results in cell death. We established 6 BR cell lines from BRAF mutant (V600E) cell lines including ASS-negative cell lines such as A375 and MEL-1220 (derived from a patient) and ASS-positive and/or inducible cell lines include UACC-62, SK-MEL28, A2058, and MEL-GP (derived from a patient) by continuous exposure to vemurafenib at IC50 over 30 weeks. MTT, caspase activity, and Annexin V/PI analyses showed that treatment with ADI-PEG20 (100ng/ml) for 72 hr induced 2-3 fold increase in growth inhibition and 10-30% increase in apoptosis in BR cells compared to their parental cells counterpart. The xenograft mouse model also confirmed that ADI-PEG20 abrogated tumor growth of A2058BR cells but only retarded growth seen in A2058 cells (tumor growth inhibition (T/C ratio) in A2058 and A2058BR is 38.7% vs. 8.3%; T/C<42% indicates significant inhibition). The results observed by cyto-ID staining combined with fluorescent microscope and electron microscope also exhibited 40-80% decrease in autophagosome formation in BR cells compared to parental cells after treatment with ADI-PEG20. We further found that BRAFi-resistance attenuated autophagy and ASS re-expression due to loss of Atg5 and c-Myc. Atg5-overexpressed plasmid rescued 12-35% apoptosis in MEL-1220BR and A2058BR cells through autophagic process as evidenced by more autophagosome formation following treatment with ADI-PEG20. The other mechanism leading to attenuated ASS re-expression is decreased c-Myc (positive regulator of ASS transcription). It is known that c-Myc stability is governed by ubiquitination-dependent proteasomal degradation. Using immunoprecipitation assay, our results showed that the levels of deubiquitinating enzyme, USP28, were too low to prevent c-Myc from ubiquitination. Overall, our data suggest that attenuated Atg5 which halted autophagy and decreased c-Myc which prevented re-expression of ASS made the BR cells vulnerable to cell death upon arginine deprivation. Thus, ADI-PEG20 is a potential candidate for salvage therapy in BR patients (Supported by R01CA152197). Citation Format: Ying-Ying Li, Niramol Savaraj, Chunjing Wu, Shumei Chen, Medhi Wangpaichitr, Macus T. Kuo, Lynn G. Feun. Biochemical alterations in BRAF inhibitor (BRAFi) resistant melanoma cells increase their vulnerability to arginine deprivation. [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 2900. doi:10.1158/1538-7445.AM2015-2900

Abstract 3502: Selective targeting of CTNBB1-, KRAS- or MYC-driven cell growth by combinations of existing drugs

Abstract Purpose: Combination therapy plays an important role in achieving durable responses in anticancer therapy. Ideally, compound combinations work synergistically in cancer cells and not in non-malignant cells. Here we present a rational approach for identifying pairs of selective inhibitors that show a synergistic interaction only in specific genetic backgrounds, i.e. mutated in the CTNNB1 (β-catenin) or KRAS genes, or expressing amplified MYC. Experimental Procedures: We have established a panel of sixty six genetically well-characterized cell lines that represents the most frequently occurring oncogenic drivers in cancer (1). A collection of more than 100 inhibitors targeting all important oncogenic signaling pathways, including many kinase and epigenetic regulators, was profiled in a subset of forty four cell lines, generating highly reproducible and high-quality cell proliferation data. The inhibitor response was correlated to the genetic background of the cell lines by Anova analysis (1). Compounds that targeted similar subsets of cell lines, were tested in combination for synergistic interaction, using in vitro proliferation assays with equipotent mixtures, followed by curve shift analysis, isobolograms and combination index scoring. The approach was validated by showing that the MEK inhibitor trametinib and the BRAF inhibitor dabrafenib, which are targeted to BRAF-mutant cell lines, show synergistic interaction in this subpopulation, and not in other cell line types. Results: For all inhibitors, the drug response in cell line panel (Oncolines™) was analyzed. Notably, the Wnt-pathway inhibitor ICG-001 and the MEK inhibitor trametinib were shown to preferentially inhibit CTNNB1-mutated cancer cell lines. Neratinib, a spectrum selective EGFR inhibitor, and GSK-1070916, an Aurora kinase inhibitor, were shown to target MYC-amplified cell lines. The ERBB2 inhibitor TAK-165 and trametinib targeted KRAS mutant cell lines. These compounds thus show a pharmacological synthetic lethal effect. Subsequent combination studies showed synergy on cell proliferation in a high number of cases. ICG-001 works synergistically with trametinib in a CTNNB1-mutant colon cell line, and not in CTNNB1-wild type colon cell lines. Neratinib and GSK-1070916 work synergistically in MYC-amplified cell lines and not in non-MYC-amplified lines. Conclusions: Pharmacogenomic analysis of single agent responses identified compounds that preferentially inhibit the growth of cell lines harbouring mutant CTNNB1 or KRAS, or amplified MYC. Synergy studies in the pertinent genetic background identified new combinations of existing drugs with enhanced targeting of CTNNB1-mutated or MYC-amplified cancer cell lines. Our approach can efficiently discover novel drug combinations that target cancer genes more selectively and more potently.

Kinase inhibitor screening identifies CDK4 as a potential therapeutic target for melanoma.

Despite recent advances in targeted therapies and immunotherapies metastatic melanoma remains only rarely curable. The objective of the present study was to identify novel therapeutic targets for metastatic melanoma. A library of 160 well-characterised and potent protein kinase inhibitors was screened in the BRAF mutant cell line Sk-Mel-28, and the NRAS mutant Sk-Mel-2, using proliferation assays. Of the 160 inhibitors tested, 20 achieved >50% growth inhibition in both cell lines. Six of the 20 were cyclin dependent kinase (CDK) inhibitors, including two CDK4 inhibitors. Fascaplysin, a synthetic CDK4 inhibitor, was further tested in 8 melanoma cell lines. The concentration of fascaplysin required to inhibit growth by 50% (IC50 value) ranged from 0.03 to 0.22 μM. Fascaplysin also inhibited clonogenic growth and induced apoptosis. Sensitivity to PD0332991, a therapeutic CDK4/6 inhibitor was also evaluated in the melanoma cell lines. PD0332991 IC50 values ranged from 0.13 to 2.29 μM. Similar to fascaplysin, PD0332991 inhibited clonogenic growth of melanoma cells and induced apoptosis. Higher levels of CDK4 protein correlated with lower sensitivity to PD0332991 in the cell lines. Combined treatment with PD0332991 and the BRAF inhibitor PLX4032, showed additive anti-proliferative effects in the BRAF mutant cell line Malme-3M. In summary, targeting CDK4 inhibits growth and induces apoptosis in melanoma cells in vitro, suggesting that CDK4 may be a rational therapeutic target for metastatic melanoma.

Low-concentration vemurafenib induces the proliferation and invasion of human HaCaT keratinocytes through mitogen-activated protein kinase pathway activation.

Cutaneous squamous cell carcinomas and keratoacanthomas commonly occur in patients treated with BRAF inhibitors. We investigated the effect of the BRAF inhibitor vemurafenib on normal immortalized human HaCaT keratinocytes to explore the mechanism of hyperproliferative cutaneous neoplasia associated with the use of BRAF inhibitors. Vemurafenib induced an increase in viable cell number in BRAF wild-type cell lines (SK-MEL-2 and HaCaT) but not in BRAF mutant cell lines (SK-MEL-24 and G361). In HaCaT keratinocytes, a low concentration (2μmol/L) of vemurafenib increased cell proliferation and activated mitogen-activated protein kinase kinase/extracellular signal-regulated kinase in a CRAF-dependent manner. Invasiveness of HaCaT cells in a Matrigel assay significantly increased upon cultivation of cells with 2μmol/L vemurafenib for 24h. Gelatin zymography, reverse transcription polymerase chain reaction and western blot results revealed that 2μmol/L vemurafenib treatment increased matrix metalloproteinase (MMP)-2 and MMP-9 expressions and activities in HaCaT cells. These results offer additional insight into the complex mechanism of paradoxical mitogen-activated protein kinase signaling involved in hyperproliferative cutaneous neoplasias that arise after BRAF inhibition and suggest a possible role for MMP in tumor progression and invasion.

Aloe-emodin exerts a potent anticancer and immunomodulatory activity on BRAF-mutated human melanoma cells

Aim of this study was to extend the knowledge on the antineoplastic effect of aloe-emodin (AE), a natural hydroxyanthraquinone compound, both in metastatic human melanoma cell lines and in primary stem-like cells (melanospheres). Treatment with AE caused reduction of cell proliferation and induction of SK-MEL-28 and A375 cells differentiation, characterized by a marked increase of transamidating activity of transglutaminase whose expression remained unmodified. In vitro antimetastatic property of AE was evaluated by adhesion and Boyden chamber invasion assays. The effect of AE on melanoma cytokines/chemokines production was determined by a multiplex assay: interestingly AE showed an immunomodulatory activity through GM-CSF and IFN-γ production. We report also that AE significantly reduced the proliferation, stemness and invasive potential of melanospheres. Moreover, AE treatment significantly enhanced dabrafenib (a BRAF inhibitor) antiproliferative activity in BRAF mutant cell lines. Our results confirm that AE possesses remarkable antineoplastic properties against melanoma cells, indicating this anthraquinone as a promising agent for differentiation therapy of cancer, or as adjuvant in chemotherapy and targeted therapy. Further, its mechanisms of action support a potential efficacy of AE treatment to counteract resistance of BRAF-mutated melanoma cells to target therapy.

Phase I trial of the CDK 4/6 inhibitor, P1446A-05 (voruciclib) in combination with the BRAF inhibitor (BRAFi), vemurafenib in advanced, BRAF-mutant melanoma.

9076 Background: P1446A-05 is a potent, selective CDK 4/6 inhibitor with activity in multiple BRAF-mutant and wild type cell lines. The BRAFi have been transformative in the therapy of BRAF-mutant ...

BRAF- and MEK-Targeted Small Molecule Inhibitors Exert Enhanced Antimelanoma Effects in Combination With Oncolytic Reovirus Through ER Stress

Reovirus type 3 (Dearing) (RT3D) infection is selective for cells harboring a mutated/activated RAS pathway. Therefore, in a panel of melanoma cell lines (including RAS mutant, BRAF mutant and RAS/BRAF wild-type), we assessed therapeutic combinations that enhance/suppress ERK1/2 signaling through use of BRAF/MEK inhibitors. In RAS mutant cells, the combination of RT3D with the BRAF inhibitor PLX4720 (paradoxically increasing ERK1/2 signaling in this context) did not enhance reoviral cytotoxicity. Instead, and somewhat surprisingly, RT3D and BRAF inhibition led to enhanced cell kill in BRAF mutated cell lines. Likewise, ERK1/2 inhibition, using the MEK inhibitor PD184352, in combination with RT3D resulted in enhanced cell kill in the entire panel. Interestingly, TCID50 assays showed that BRAF and MEK inhibitors did not affect viral replication. Instead, enhanced efficacy was mediated through ER stress-induced apoptosis, induced by the combination of ERK1/2 inhibition and reovirus infection. In vivo, combined treatments of RT3D and PLX4720 showed significantly increased activity in BRAF mutant tumors in both immune-deficient and immune-competent models. These data provide a strong rationale for clinical translation of strategies in which RT3D is combined with BRAF inhibitors (in BRAF mutant melanoma) and/or MEK inhibitors (in BRAF and RAS mutant melanoma).