Reactivation Of MAPK Signaling Research Articles

Abstract 1997: The small GTPase ARF6 augments BRAF and PI3K expression to promote melanoma survival

Abstract RAF and MEK inhibitors greatly benefit BRAF-mutant melanoma patients, but eventually, nearly 80% of patients relapse. Reactivation of MAPK signaling and activation of PI3K/AKT signaling play a major role in the development of targeted therapy resistance. Therefore, understanding how tumor cells regulate these signaling pathways may yield new insights into drug resistance. The small GTPase ARF6, a member of the RAS superfamily, influences oncogenic signaling by controlling cytoskeletal rearrangements and through endomembrane trafficking. We previously reported that ARF6 is aberrantly activated in metastatic melanoma and that activation of ARF6 is sufficient to promote invasion and accelerate spontaneous metastasis in vivo through WNT5A/beta-catenin and PI3K/AKT signaling. In contrast to ARF6 activation, herein we show that tumor-specific deletion of Arf6 significantly limits development and progression of BRAFV600E melanoma in vivo. Mechanistically, we discovered these ARF6-dependent phenotypes may be attributed to a previously unknown role for ARF6 in augmenting BRAFV600E and PI3K protein levels. Specifically, pharmacologic or genetic activation ARF6 (ARF6-GTP) is sufficient to increase BRAFV600E and PI3K protein levels in both murine and human melanoma. As a result, ARF6-GTP is also sufficient to enhance activation of MEK, ERK and AKT. Importantly, murine tumors expressing constitutively active ARF6 (ARF6Q67L) show increased expression of anti-apoptotic proteins compared to ARF6WT tumors. Together these data suggest that ARF6 promotes tumor survival through augmentation of the BRAF/MAPK and PI3K/AKT pathways. To test this, we pharmacologically activated ARF6 during serum withdrawal or vemurafenib treatment. In both conditions, ARF6 activation is sufficient to prevent apoptosis. Surprisingly, we found that vemurafenib treatment leads to ARF6 activation, suggesting that ARF6 may be an important component of the cellular machinery that reactivates MAPK signaling after RAF inhibition. Indeed, pharmacologic activation of ARF6 maintains MAPK signaling (pERK levels) in the presence of vemurafenib and enhances pERK and pAKT-mediated suppression of apoptotic signaling. In summary, our data suggest that in addition to well-established roles in tumor cell invasion, ARF6 is a crucial mediator of survival during tumor progression and in the context of BRAF targeted therapy. More work is needed to understand how ARF6 activation increases BRAF and PI3K expression and if ARF6 activation contributes to the development of persister cells in melanoma. Citation Format: Junhua Wang, Coulson Rich, Aaron Rogers, Sheri Holmen, Roger Wolff, Allie Grossmann. The small GTPase ARF6 augments BRAF and PI3K expression to promote melanoma survival [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 1997.

Abstract 1661: Preclinical characterization of HMPL-295, a potent and selective ERK 1/2 inhibitor

Abstract Background: Although therapeutic agents targeting alterations in the MAPK pathway have achieved great clinical success, the overall benefit is still suboptimal. Several studies have shown that reactivation of MAPK signaling is the main basis to compromise the efficacy. Thus, co-inhibition of ERK, the terminal master kinase of MAPK pathway, and the upstream targets may effectively shut down the MAPK signaling cascade and induce deeper and more durable anti-tumor activities. Herein, we present the preclinical characterization of HMPL-295, a potent and selective ERK 1/2 inhibitor, discovered and being currently developed in phase I clinical trial (NCT04908046) by HUTCHMED. Methods: The inhibition on ERK 1/2 kinase was determined using Z-LYTE™ kinase assay. The selectivity of HMPL-295 was evaluated against 394 kinases (KinaseProfilerTM) and 86 safety-related proteins (SafetyScreen87TM) by Eurofins. Cellular target inhibition on phosphorylation of RSK (p-RSK) was detected by ELISA. In vitro anti-proliferation activity of HMPL-295 as a single agent was measured by CCK-8 or CellTiter-Glo luminescent assay. Colony formation assay was used to evaluate the combination effect of HMPL-295 with targeted therapy. Multiple tumor models with RAS/RAF/MAPK pathway activation were applied in immune-deficient mice to investigate the anti-tumor activity of HMPL-295 as a single agent or in combination with either chemotherapy or targeted therapy. Results: HMPL-295 strongly inhibited ERK1 and ERK2 kinase activities with equal IC50 at 4 nM. In human colon cancer cell line COLO 205 harboring BRAFV600E mutation, HMPL-295 inhibited p-RSK, a direct substrate of ERK1/2, with an average IC50 of 180 nM from 3 independent experiments. With the inhibition of ERK and its downstream substrates, HMPL-295 treatment induced tumor cells arresting at G0/G1 phase via down-regulation of cyclin D1/p-RB signaling and consequently attenuated cell growth in a panel of MAPK pathway dysregulated tumor cell lines, but barely affected the growth of normal cells. Furthermore, HMPL-295 demonstrated favorable selectivity in the kinase and safety pharmacology profiling, indicating a low off-target risk. For in vivo studies, oral administration of HMPL-295 induced time- and dose-dependent inhibition of p-RSK and resulted in dose-dependent anti-tumor activity in several tumor models harboring KRAS or BRAF mutations. Moreover, HMPL-295 significantly improved the anti-tumor activity of standard-of-care chemotherapy as well as targeted agents in KRAS- or BRAF-mutant tumor models both in vitro and in vivo. Conclusion: HMPL-295 is a potent and selective ERK1/2 inhibitor which demonstrates strong anti-tumor activity in preclinical models, supporting further clinical evaluation. Citation Format: Jia Hu, Jun Ni, Yuanzhi Lv, Wenjun Li, Hui Zhang, Xin Li, Ying Yu, Zeyu Zhong, Jian Wang, Yang Sai, Weiguo Qing, Yongxin Ren, Michael Shi, Weiguo Su. Preclinical characterization of HMPL-295, a potent and selective ERK 1/2 inhibitor [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 1661.

DDDR-04. CA-4948 IN COMBINATION WITH BRAF/MEK INHIBITION IN MELANOMA BRAIN METASTASES

Abstract Melanoma brain metastases (MBM) remain a primary driver of melanoma morbidity and mortality. It is estimated that as many as 60% of all metastatic melanoma patients will develop MBM during the course of their disease. Once found, dual checkpoint inhibitor therapy or BRAF/MEK inhibition in BRAF mutated cancers, are the best options currently available. Even with these treatments, the response to therapy is less than what is seen with disease outside the. In BRAF mutated cancers, which constitute 50% of all patients, initial response to targeted therapy is rapid but resistance to therapy develops 3 months sooner than resistance outside of the brain. Novel strategies to prevent onset of resistance or to work in concert with BRAF/MEK inhibition are needed for this treatment population. The exact mechanism of resistance to targeted therapy in MBM is not known, but mutations in EphA2, PI3K, pAKT, and re-activation of MAPK signaling through secondary mechanisms have all been identified as drivers of resistance. One pathway of MAPK reactivation is through activation of phospho-ERK (eIF2alpha kinase) which also activates the JNK pathway. Our group has previously shown that MBM express high levels of the inflammatory transcription factor IRAK-4, the terminal chaperone of the inflammatory myddosome downstream of TLR and IL1R signally. This increase in IRAK-4 results in constitutive activation of NF-KB, JNK, and MAPK. We have additionally shown the novel oral IRAK-4 inhibitor CA-4948 can reach therapeutically relevant concentrations past the blood brain barrier in both tumor-bearing and naïve mice. We additionally show CA-4948 inhibits MAPK expression in melanoma and suppresses the escape pathways pERK and JNK signaling which can contribute to the prevention of resistance to targeted therapy. This study supports further investigation into the combination potential of CA-4948 with BRAF/MEK inhibitors as triplet therapy to delay intracranial resistance.

159 (PB039) - A combination vertical inhibition approach with inhibitors of SHP2 and ERK provides improved activity in KRAS-mutant pancreatic and colorectal cancer models

Background: Inhibitors of the MAPK pathway are approved therapeutic agents with a high initial response rate, however single agent use of MAPK inhibitors in the clinic is often limited by resistance. Resistance commonly occurs through mechanisms which result in reactivation of MAPK signaling, such as activation of receptor tyrosine kinases (RTKs). Vertical pathway combination strategies are therefore of interest for addressing resistance through pathway reactivation. Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) is a key regulator of the MAPK pathway downstream of RTKs and upstream of RAS, whilst ERK1/2 (ERK) is the final node of the kinase cascade.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 2361: The functional roles of miRNA-193a-3p in colorectal cancer

Abstract Background: Patients with BRAF-mutated colorectal cancer (CRC) have a poor prognosis despite recent therapeutic advances such as combination therapy with BRAF, MEK, and EGFR inhibitors. Aim: Our aim was to clarify the functions of the microRNAs (miRNAs) involved in oncogenesis and sensitivity to BRAF inhibitors and MEK inhibitors in BRAF-mutated CRC. Methods and Results: To identify miRNAs that sensitize a BRAF inhibitor dabrafenib (DAB) and a MEK inhibitor trametinib (TRA), we screened 240 miRNAs in BRAF-mutated CRC cell line RKO. Among the screened miRNAs, we tried to elucidate the novel functional roles of miR-193a-3p in CRC cell lines. We found that miR-193a-3p overexpression inhibited cell proliferation in CRC cell lines. The protein array analysis revealed that protein phosphorylation levels were altered by miR-193a-3p overexpression in various pathways such as cell-cycle-related pathways and PI3K-AKT pathways. The enhanced cytotoxicity by miR-193a-3p in addition to DAB and TRA was validated in multiple BRAF-mutated cell lines. Furthermore, the enhanced cytotoxicity by miR-193a-3p in addition to DAB and TRA was shown to be through inhibiting reactivation of MAPK signaling. This enhanced cytotoxicity by miR-193a-3p was even observed under treatment of DAB, TRA, and an anti-EGFR antibody. Conclusions: Our study shows that miR-193a-3p regulates multiple proteins involved in oncogenesis, and proposes that the combination therapy of miR-193a-3p and BRAF/MEK inhibitors could be a promising treatment strategy in BRAF-mutated CRC. Citation Format: Sakura Hiraide, Masanobu Takahashi, Yuya Yoshida, Keigo Komine, Chikashi Ishioka. The functional roles of miRNA-193a-3p in colorectal cancer [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 2361.

Abstract 926: Extrinsic and intrinsic activation of RAS/MAPK signaling enables resistance to FLT3 inhibitor, gilteritinib, in acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) remains a highly fatal disease due to the development of drug resistance. Mutational activation of kinases is a frequent event in leukemia, driving malignant cell growth. The most commonly mutated kinase in AML is the FMS-like tyrosine kinase 3 (FLT3) receptor. Internal tandem duplication (ITD) events in the juxtamembrane domain of FLT3 have been reported in ~ 20% of patients and lead to constitutive activation of FLT3. Previous work from our laboratory has shown that fibroblast growth factor 2 (FGF2) secreted by mesenchymal stromal cells protects FLT3-ITD AML cells from quizartinib, a FLT3 inhibitor (Cancer Research, 2016). FGF2 binds the FGFR1 receptor and activates MAPK signaling, enabling leukemia cells to become drug resistant. Over months of treatment, FGF2-protected leukemia cells acquired FLT3 resistance mutations. This two-step mechanism of resistance in vitro mirrors what happens in patients treated with quizartinib. Newer FLT3 inhibitors, such as gilteritinib, have been developed that have activity against both ITD and kinase domain mutations that impart resistance to quizartinib. We used a similar approach and treated the FLT3-ITD+ AML cell line, MOLM-14, with 100 nM of gilteritinib in media alone (n=4), or supplemented with 10 ng/ml FGF2 (n=4) or FLT3 ligand (FL, n=4). After 7 weeks, all cultures supplemented with FGF2 or FL eventually resumed growth. In contrast, no MOLM-14 cells cultured in gilteritinib alone resumed growth even after 16 weeks, suggesting the importance of extrinsic factors in facilitating early drug resistance. Selective pressure was then put on the ligand-dependent cultures by removal of FGF2 and FL. This transiently restored sensitivity to gilteritinib, however within a month the cultures resumed exponential growth. To further explore the mechanism of resistance, we performed immunoblots of kinase signaling pathways on MOLM-14 resistant cultures pre- and post-withdrawal of ligands. We saw robust activation of FLT3 receptor in resistant cultures with FL. FGF2 activated the MAPK pathway, circumventing inhibition by gilteritinib. After removal of extrinsic FGF2 and FL, there was re-activation of MAPK signaling but FLT3 itself remained inhibited. Whole Exome Sequencing of resistant cells identified NRAS mutations as the source of MAPK signaling after ligand withdrawal. Two of the four FGF2-supplemented cultures also developed novel FLT3 point mutations, although there was no clear re-activation of FLT3 signaling in these cultures. Our in vitro results suggest that extrinsic signals from the microenvironment can re-activate MAPK pathways enough to keep cells alive but continued selective pressure eventually leads to direct activation of MAPK through RAS mutations. As such, there is a need to consider rational inhibitor combinations early in disease therapy to prevent the emergence of resistance. Citation Format: Sunil K. Joshi, Stephen Christy, Renata Scopim Ribeiro, Shannon McWeeney, Jeffrey W. Tyner, Cristina E. Tognon, Brian J. Druker, Elie Traer. Extrinsic and intrinsic activation of RAS/MAPK signaling enables resistance to FLT3 inhibitor, gilteritinib, in acute myeloid leukemia [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 926.

Abstract 2492: BRAF inhibitors synergize with BH3 mimetics to induce apoptosis in BRAF mutant colorectal cancer cells

Abstract Activating mutations in BRAF (BRAFV600E) occur in ~10% of colorectal cancers (CRCs) and drive tumorigenesis through constitutive activation of MAPK signaling. In metastatic CRC, BRAF mutations are associated with poorer prognosis and resistance to conventional therapies, necessitating an urgent need to develop new treatments for these patients. BRAF inhibitors such as vemurafenib and dabrafenib have significant clinical activity in BRAF-mutant melanoma, however BRAF-mutant CRCs are largely refractory to these agents, due at least in part to feedback-relief mediated reactivation of MAPK signaling or alternate signaling pathway activation. Strategies to enhance the activity of BRAF inhibitors in BRAF-mutant CRC are therefore needed. Consistent with clinical observations, treatment of a panel of BRAF-mutant melanoma and CRC cell lines with vemurafenib resulted in significantly increased apoptosis in melanoma cell lines compared to CRC cell lines, where effects were largely cytostatic. To determine the mechanisms for this differential response we interrogated vemurafenib-induced gene expression changes in the two tumor types, focusing on altered expression of components of the intrinsic apoptotic pathway. Vemurafenib induced a more pronounced increase in expression of the pro-apoptotic genes BIM, BMF and PUMA and suppression of pro-survival gene MCL1 in melanoma cells compared to CRC cells. These findings suggested that the extent to which expression of pro and anti-apoptotic genes are altered by vemurafenib in CRC cells may be insufficient to reach the threshold required for apoptosis initiation. We therefore postulated that BH3-mimetics may synergize with vemurafenib to induce apoptosis in BRAF-mutant CRC cells. Analysis of quantitative proteomic data of BRAF-mutant CRC cell lines revealed significantly higher basal expression of the pro-survival proteins Bcl-xL and MCL1 compared to BCL2 and BCLW, suggesting CRC cells may be particularly dependent on Bcl-xL and MCL1 for survival. Indeed, combination treatment of BRAF-mutant CRC cells with the Bcl-xL inhibitor A-1331852 significantly enhanced apoptosis in the majority of BRAF-mutant CRC lines. Comparatively, combination treatment of vemurafenib with the MCL1 inhibitor S63845 induced a modest increase in apoptosis, while combination treatment with the BCL2 inhibitor ABT-199 had no effect on apoptosis, consistent with the low levels of BCL2 expression in these lines. Finally, we investigated the effect of combination treatment of vemurafenib with inhibitors of both Bcl-xL and MCL1. The triple combination further enhanced apoptosis in 3/5 cell lines, suggesting these cell lines are likely dependent on both Bcl-xL and MCL1 for survival. Collectively, these findings demonstrate that combining BRAF-inhibitors with Bcl-xL and/or MCL1 inhibitors may represent a novel strategy for treating BRAF-mutant CRC. Citation Format: Laura J. Jenkins, Fiona Chionh, Ian Y. Luk, Erinna F. Lee, Amardeep S. Dhillon, Niall Tebbutt, Walter D. Fairlie, John M. Mariadason. BRAF inhibitors synergize with BH3 mimetics to induce apoptosis in BRAF mutant colorectal cancer cells [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 2492.

Abstract 5467: Efficacy of the ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 in patient-derived RAS-mutant NSCLC models

Abstract Introduction: RAS mutations are present in 30% of non-small cell lung cancer (NSCLC) and, despite activated MAPK signaling, MEK inhibitors have disappointed in clinical trials. MAPK pathway reactivation and activation of anti-apoptotic signaling via the PI3K/mTOR axis are major resistance mechanisms, among others. ERK inhibitors are believed to overcome some of the restrictions of MEK inhibitors, and combinations with PI3K/mTOR inhibitors could potentially increase therapeutic efficacy. Past trials of MEK and PI3K inhibitor combinations, however, showed that toxicities and side effects are likely to be expected also for the ERK-PI3K/mTOR inhibitor combination. Therefore, more specific and potent drugs as well as alternating dosing schedules may overcome toxicities and improve treatment outcome. Methods: We so far established five patient-derived RAS-mutant NSCLC cell lines (1/5 NRASQ61K, 1/5 KRASQ61K, 2/5 KRASG12C, 1/5 KRASG12D) and xenograft models (PDX), characterized them by next-generation sequencing (DFCI Oncopanel) and tested the antiproliferative (CellTiter-Glo® assay) and apoptosis-inducing (Caspase3/7 activity, IncuCyte® technology) efficacy of LY3214996 (ERK inhibitor) and LY3023414 (PI3K/mTOR inhibitor) in vitro. Effects on signal transduction were investigated by Western blot analysis. Results: The ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 exhibit single-agent antiproliferative and apoptosis-inducing activity in patient-derived RAS-mutant NSCLC cell lines (for LY3214996: 1/5 sensitive (IC50<1μM), 2/5 intermediate sensitive (IC50<5μM), 2/5 resistant (IC50>10μM); for LY3023414: all IC50 <3μM, 3/5 <1μM). Despite reactivation of MAPK signaling (pMEK) upstream of ERK during LY3214996 treatment, ERK downstream targets (cMyc, DUSP4, SPRY2) were strongly suppressed. In sensitive cell lines, LY3214996 induced PARP cleavage and Bim accumulation in a dose- and time-dependent manner. Sensitivities to ERK and PI3K/mTOR inhibition varied between cell lines, indicating different pathway dependencies for proliferation and cancer cell survival. Conclusions and Outlook: Both LY3214996 and LY3023414 have single-agent in vitro efficacy in patient-derived NSCLC models with different RAS mutations and co-mutational landscapes. Single-agent in vivo studies and tolerability studies of drug combinations are ongoing, and treatment predictors and intrinsic/adaptive resistance mechanisms will be investigated via RNAseq/GSEA and phospho-receptor tyrosine kinase arrays. Citation Format: Jens Köhler, Prafulla C. Gokhale, Jiaqi Li, Margaret K. Wilkens, Hong Tiv, Shripad V. Bhagwat, Greg Donoho, Patrick Brueck, Ramon V. Tiu, Amanda J. Redig, Emily S. Chambers, Atsuko Ogino, Jihyun Choi, Man Xu, Paul Kirschmeier, Pasi A. Jänne. Efficacy of the ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 in patient-derived RAS-mutant NSCLC models [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 5467.

Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells

BRAF is the most frequently mutated gene in melanoma. Constitutive activation of mutant BRAFV600E leads to aberrant Ras-independent MAPK signaling and cell transformation. Inhibition of mutant BRAF is a current frontline therapy for such cases, with improved survival compared with chemotherapy. Unfortunately, reactivation of MAPK signaling by several mechanisms has been shown to cause drug resistance and disease recurrence. In this work, we describe the co-occurrence of an in-frame deletion within an amplified BRAFV600E locus and a missense point mutation of the transcriptional repressor BCORL1 in vemurafenib-resistant A375 melanoma cells. Functional data confirmed that truncated p47BRAFV600E and mutant BCORL1Q1076H both contribute to resistance. Interestingly, either endogenous BCORL1 silencing or ectopic BCORL1Q1076H expression mimicked the effects of a CRISPR/Cas9-edited BCORL1Q1076H locus, suggesting a complex mixture of loss- and gain-of-function effects caused by the mutation. Transcriptomic data confirmed this hypothesis. Finally, we show that the pan-RAF inhibitor sorafenib is not affected by expression of BRAF deletion variant and effectively synergizes with vemurafenib to block resistant cells, suggesting a possible intervention for this class of mutants.

Abstract IA15: Overcoming drug resistance and tumor heterogeneity in gastrointestinal cancers

Abstract Personalized cancer medicine approaches, inhibiting kinases in tumors driven by defined genomic alterations, have demonstrated striking efficacy in many cancer types. However, the development of drug resistance is a major limitation to the efficacy of targeted therapies in oncology. Identifying and understanding the molecular mechanisms driving resistance may foster opportunities to develop therapeutic strategies to overcome resistance. For example, in the ~10% of colorectal cancer patients whose tumors harbor BRAF V600 mutations, we have found that feedback reactivation of MAPK signaling, often mediated by EGFR, underlies the relative insensitivity of these cancers to RAF inhibitors. The development of targeted therapy combination strategies to block feedback reactivation of MAPK signaling has led to marked improvements in response rates for these patients from ~5% to >30% over the past few years. However, in patients who respond to these targeted combinations, the rapid development of acquired resistance still limits clinical benefit. Acquired resistance in BRAF mutant colorectal cancer and in other molecularly defined tumor types can be marked by the development of extensive molecular heterogeneity due to the selection of sub-clonal tumor cell populations, capable of growing under drug pressures, which poses significant diagnostic and therapeutic challenges. We will present data demonstrating how a single-lesion biopsy at disease progression to diagnose the mechanism of acquired resistance can vastly underrepresent the molecular heterogeneity of resistant tumor clones in an individual patient, and may fail to detect the existence of distinct but important resistance mechanisms that can drive mixed or lesion-specific responses and treatment failure to subsequent targeted therapy. By contrast, liquid biopsy approaches analyzing circulating tumor DNA have the potential to detect the presence of simultaneous resistance mechanisms residing in separate metastases in a single patient and to monitor the effects of subsequent therapies on specific sub-clonal tumor cell populations. These findings highlight the critical role of tumor heterogeneity in driving therapeutic resistance and how convergent targeted therapy strategies blocking common signaling nodes capable of overcoming multiple resistance mechanisms may be needed. Citation Format: Ryan B. Corcoran. Overcoming drug resistance and tumor heterogeneity in gastrointestinal cancers [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr IA15.

Abstract IA23: Overcoming drug resistance and tumor heterogeneity in gastrointestinal cancers

Abstract Personalized cancer medicine approaches, inhibiting kinases in tumors driven by defined genomic alterations, have demonstrated striking efficacy in many cancer types. However, the development of drug resistance is a major limitation to the efficacy of targeted therapies in oncology. Identifying and understanding the molecular mechanisms driving resistance may foster opportunities to develop therapeutic strategies to overcome resistance. For example, in the ~10% of colorectal cancer patients whose tumors harbor BRAF V600 mutations, we have found that feedback reactivation of MAPK signaling, often mediated by EGFR, underlies the relative insensitivity of these cancers to RAF inhibitors. The development of targeted therapy combination strategies to block feedback reactivation of MAPK signaling has led to marked improvements in response rates for these patients from ~5% to >30% over the past few years. However, in patients who respond to these targeted combinations, the rapid development of acquired resistance still limits clinical benefit. Acquired resistance in BRAF mutant colorectal cancer and in other molecularly-defined tumor types can be marked by the development of extensive molecular heterogeneity due to the selection of sub-clonal tumor cell populations, capable of growing under drug pressures, which poses significant diagnostic and therapeutic challenges. We will present data demonstrating how a single-lesion biopsy at disease progression to diagnose the mechanism of acquired resistance can vastly underrepresent the molecular heterogeneity of resistant tumor clones in an individual patient, and may fail to detect the existence of distinct but important resistance mechanisms that can drive mixed or lesion-specific responses and treatment failure to subsequent targeted therapy. By contrast, liquid biopsy approaches analyzing circulating tumor DNA have the potential to detect the presence of simultaneous resistance mechanisms residing in separate metastases in a single patient and to monitor the effects of subsequent therapies on specific sub-clonal tumor cell populations. These findings highlight the critical role of tumor heterogeneity in driving therapeutic resistance and how convergent targeted therapy strategies blocking common signaling nodes capable of overcoming multiple resistance mechanisms may be needed. Citation Format: Ryan B. Corcoran. Overcoming drug resistance and tumor heterogeneity in gastrointestinal cancers [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr IA23.

Abstract B175: A novel plant sesquiterpene lactone derivative DETD suppresses BRAFV600E mutant melanoma growth and overcomes acquired vemurafenib resistance in mice

Abstract Melanoma is the most lethal form of skin cancer and cases are increasing year by year. BRAFV600E mutation is found approximately in 50% of melanoma patients that drives MAPK (RAF/MEK/ERK) signaling for cancer progression. Vemurafenib, a small molecule that specifically targets BRAFV600E mutation, received FDA approval for treatment of late-stage melanoma in 2011. Unfortunately, patients develop acquired vemurafenib resistance via reactivation of MAPK signaling or bypass mechanisms. Recent combination therapy such as MEK inhibitor combined with vemurafenib shows improvement in major clinical end points but percentage of patients with adverse toxic events are higher compared to vemurafenib monotherapy and most patients relapse ultimately. It is therefore an urgent need to develop new anti-melanoma drug and/or adjuvant agent for vemurafenib therapy. In this study, we created a novel semi-organically modified derivative DETD from deoxyelephantopin (DET), a plant sesquiterpene lactone demonstrated as an anti-inflammatory and anti-mammary tumor agent in our laboratory. DETD suppressed both parental human BRAFV600E mutant melanoma (A375) and vemurafenib resistance melanoma (A375-R) cell proliferation in vitro. Furthermore, through compound drug combination assay, DETD and vemurafenib was found to show synergism in suppressing proliferation, conoly formation and inducing apoptosis of A375 melanoma cell. In A375 xenograft study, DETD suppressed tumor growth and reduced tumor mass by 70.5%, as effective as vemurafenib (71.9%), compared to tumor control group. Moreover, compound-drug combinational treatment with alternate administration of DETD and vemurafenib also showed similar tumor growth inhibition efficacy by 72.3%, suggesting in vivo synergistic or additive effect of DETD and vemurafenib. In A375-R xenograft study, vemurafenib treatment showed little or no anti-tumor activity with similar tumor growth rate and sizes relative to the tumor control group, whereas DETD could reduce A375-R tumor growth and mass with 46.9%. Notably, the combination of DETD and vemurafenib treatment exhibited the most significant effect, with a 65.3% reduction in tumor mass. Our mechanistic studies revealed the activation of non-receptor tyrosine kinase Src and its downstream signaling pathways, including MAPK, Akt and STAT3 pathways in the vemurafenib resistant melanoma A375-R, and DETD could de-regulate the activation of all these multi- kinase signaling pathways. Overall, our results offer strong evidence of DETD in inhibiting BRAFV600E melanoma growth and its acquired vemurafenib resistance in mice that suggest DETD might have great therapeutic or adjuvant potential in management of melanoma patients with BRAFV600E mutation. Citation Format: Jia-Hua Feng, Kyoko Nakagawa-Goto, Kuo-Hsiung Lee, Lie-Fen Shyur. A novel plant sesquiterpene lactone derivative DETD suppresses BRAFV600E mutant melanoma growth and overcomes acquired vemurafenib resistance in mice. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B175.

Abstract SY10-01: Strategies to overcome resistance to MAPK inhibition.

Abstract Activating mutations in BRAF and KRAS, key signaling components of the MAPK pathway, are among the most common oncogenic mutations in human cancer, occurring in ~7% and ~20% of all cancers, respectively. Consequently, inhibitors of MAPK signaling, including selective RAF and MEK inhibitors, have been under active clinical development. Yet, key resistance signals have limited the clinical effectiveness of these inhibitors. Although selective RAF inhibitors have proven remarkably effective in BRAF mutant melanoma (~60-80% response rates), efficacy has been disappointing in the ~10-15% of colorectal cancers (CRCs) that also harbor BRAF mutations (~5% response rate). Compared to BRAF mutant melanoma cells, BRAF mutant CRC cells were less sensitive to the RAF inhibitor vemurafenib (PLX4032), and phospho-ERK (P-ERK) suppression was not sustained in response to treatment. Although transient inhibition of P-ERK by vemurafenib was observed in BRAF mutant CRC, rapid ERK reactivation occurred through EGFR-mediated activation of RAS and CRAF. BRAF mutant CRCs expressed higher levels of phospho-EGFR than BRAF mutant melanomas, suggesting that BRAF mutant CRCs are specifically poised to exhibit EGFR-mediated resistance. Combined RAF and EGFR inhibition blocked reactivation of MAPK signaling in BRAF mutant CRC cells and markedly improved efficacy in vitro and in vivo, leading to tumor regressions in BRAF mutant CRC xenograft models. These findings support evaluation of combined RAF and EGFR inhibition in BRAF mutant CRC patients, and clinical trials evaluating this strategy are currently underway. Although KRAS is the most commonly mutated oncogene in human cancer, KRAS has proven difficult to target pharmacologically, and no effective therapies exist for KRAS mutant cancers. Recently, there has been evidence that targeted therapy combinations inhibiting multiple downstream effectors of KRAS may be a promising approach for KRAS mutant cancers. We developed a pooled shRNA-drug screen strategy to identify genes that, when inhibited, cooperate with MEK inhibitors to kill KRAS mutant cancer cells. The anti-apoptotic BH3 family gene BCL-XL emerged as a top hit through this approach. ABT-263 (navitoclax), a chemical inhibitor that blocks the ability of BCL-XL to bind and inhibit pro-apoptotic proteins, in combination with a MEK inhibitor led to dramatic apoptosis in the vast majority of KRAS mutant cell lines tested from different tissue types. Mechanistic studies revealed that MEK inhibition led to marked induction of the pro-apoptotic protein BIM in KRAS mutant cancer cells, but that BIM remained bound and inhibited by BCL-XL. Pharmacologic inhibition of BCL-XL with ABT-263 disrupted this inhibitory complex, allowing BIM to trigger apoptosis. Epithelial differentiation and E-cadherin expression correlated with increased sensitivity to this inhibitor combination across a panel of 30 KRAS mutant cell lines, while epithelial-to-mesenchymal transition (EMT) correlated with resistance. This combination also caused marked in vivo tumor regressions in three independent KRAS mutant xenografts and in established lung tumors in two genetically-engineered KRAS driven lung cancer mouse models. These data support combined BCL-XL/MEK inhibition as a promising therapeutic approach for evaluation in future clinical trials for patients with KRAS mutant cancers. Citation Format: Ryan B. Corcoran. Strategies to overcome resistance to MAPK inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY10-01. doi:10.1158/1538-7445.AM2013-SY10-01

Abstract PR8: Insensitivity to RAF inhibition by vemurafenib in BRAF mutant colorectal cancer by EGFR-mediated reactivation of MAPK signaling

Abstract BRAF mutations occur in 10–15% of colorectal cancers (CRCs) and confer adverse outcome in the metastatic setting. While RAF inhibitors such as vemurafenib (PLX4032) have proven effective in BRAF mutant melanoma (∼60–80% response rates), they are surprisingly ineffective in BRAF mutant CRCs (∼5% response rate), and the reason for this disparity remains unclear. Compared to BRAF mutant melanoma cells, BRAF mutant CRC cells were less sensitive to vemurafenib, and P-ERK suppression was not sustained in response to treatment. Although transient inhibition of phospho-ERK by vemurafenib was observed in CRC, rapid ERK re-activation occurred through EGFR-mediated activation of RAS and CRAF. BRAF mutant CRC cell lines and patient tumor specimens expressed significantly higher levels of phospho-EGFR than BRAF mutant melanoma cell lines and primary tumor specimens, suggesting that CRCs are specifically poised for EGFR-mediated resistance. Combined RAF and EGFR inhibition blocked reactivation of MAPK signaling in BRAF mutant CRC cells and markedly improved efficacy in vitro. In vivo, combined RAF and EGFR inhibition led to sustained suppression of MAPK signaling and to robust tumor regressions in BRAF mutant colorectal xenografts. These findings support evaluation of combined RAF and EGFR inhibition in clinical trials in patients with BRAF mutant CRC. This proffered talk is also presented as Poster B11.

Abstract LB-350: EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition by vemurafenib

Abstract Oncogenic BRAF mutations occur in 10-15% of colorectal cancers (CRCs) and confer adverse outcome. While RAF inhibitors such as vemurafenib (PLX4032) have produced dramatic response rates in BRAF mutant melanoma (∼60-80%), they are surprisingly ineffective in BRAF mutant CRCs (∼5% response rate), and the reason for this disparity remains unclear. Compared to BRAF mutant melanoma cells, BRAF mutant CRC cells were less sensitive to vemurafenib, and P-ERK suppression was not sustained in response to treatment. Although transient inhibition of phospho-ERK by vemurafenib was observed in CRC, rapid ERK re-activation occurred through EGFR-mediated activation of RAS and CRAF. BRAF mutant CRC cell lines and patient tumor specimens expressed higher levels of phospho-EGFR than BRAF mutant melanoma cell lines and primary tumor specimens, suggesting that CRCs are specifically poised for EGFR-mediated resistance. Combined RAF and EGFR inhibition blocked reactivation of MAPK signaling in BRAF mutant CRC cells, markedly improved efficacy in vitro, and led to robust tumor regressions in vivo in BRAF mutant xenografts models. These findings support combined RAF and EGFR inhibition as a novel and promising therapeutic strategy for evaluation in clinical trials in patients with BRAF mutant CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-350. doi:1538-7445.AM2012-LB-350