RAF Inhibitors Research Articles

Mutation-oriented profiling of autoinhibitory kinase conformations predicts RAF inhibitor efficacies

Kinase-targeted therapies have the potential to improve the survival of patients with cancer. However, the cancer-specific spectrum of kinase alterations exhibits distinct functional properties and requires mutation-oriented drug treatments. Besides post-translational modifications and diverse intermolecular interactions of kinases, it is the distinct disease mutation which reshapes full-length kinase conformations, affecting their activity. Oncokinase mutation profiles differ between cancer types, as it was shown for BRAF in melanoma and non-small-cell lung cancers. Here, we present the target-oriented application of a kinase conformation (KinCon) reporter platform for live-cell measurements of autoinhibitory kinase activity states. The bioluminescence-based KinCon biosensor allows the tracking of conformation dynamics of full-length kinases in intact cells and real time. We show that the most frequent BRAF cancer mutations affect kinase conformations and thus the engagement and efficacy of V600E-specific BRAF inhibitors (BRAFi). We illustrate that the patient mutation harboring KinCon reporters display differences in the effectiveness of the three clinically approved BRAFi vemurafenib, encorafenib, and dabrafenib and the preclinical paradox breaker PLX8394. We confirmed KinCon-based drug efficacy predictions for BRAF mutations other than V600E in proliferation assays using patient-derived lung cancer cell lines and by analyzing downstream kinase signaling. The systematic implementation of such conformation reporters will allow to accelerate the decision process for the mutation-oriented RAF-kinase cancer therapy. Moreover, we illustrate that the presented kinase reporter concept can be extended to other kinases which harbor patient mutations. Overall, KinCon profiling provides additional mechanistic insights into full-length kinase functions by reporting protein-protein interaction (PPI)-dependent, mutation-specific, and drug-driven changes of kinase activity conformations.

Antiglioma Potential of Coumarins Combined with Sorafenib.

Coumarins, which occur naturally in the plant kingdom, are diverse class of secondary metabolites. With their antiproliferative, chemopreventive and antiangiogenetic properties, they can be used in the treatment of cancer. Their therapeutic potential depends on the type and location of the attachment of substituents to the ring. Therefore, the aim of our study was to investigate the effect of simple coumarins (osthole, umbelliferone, esculin, and 4-hydroxycoumarin) combined with sorafenib (specific inhibitor of Raf (Rapidly Accelerated Fibrosarcoma) kinase) in programmed death induction in human glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cells lines. Osthole and umbelliferone were isolated from fruits: Mutellina purpurea L. and Heracleum leskowii L., respectively, while esculin and 4-hydroxycoumarin were purchased from Sigma Aldrich (St. Louis, MO, USA). Apoptosis, autophagy and necrosis were identified microscopically after straining with specific fluorochromes. The level of caspase 3, Beclin 1, PI3K (Phosphoinositide 3-kinase), and Raf kinases were estimated by immunoblotting. Transfection with specific siRNA (small interfering RNA) was used to block Bcl-2 (B-cell lymphoma 2), Raf, and PI3K expression. Cell migration was tested with the wound healing assay. The present study has shown that all the coumarins eliminated the MOGGCCM and T98G tumor cells mainly via apoptosis and, to a lesser extent, via autophagy. Osthole, which has an isoprenyl moiety, was shown to be the most effective compound. Sorafenib did not change the proapoptotic activity of this coumarin; however, it reduced the level of autophagy. At the molecular level, the induction of apoptosis was associated with a decrease in the expression of PI3K and Raf kinases, whereas an increase in the level of Beclin 1 was observed in the case of autophagy. Inhibition of the expression of this protein by specific siRNA eliminated autophagy. Moreover, the blocking of the expression of Bcl-2 and PI3K significantly increased the level of apoptosis. Osthole and sorafenib successfully inhibited the migration of the MOGGCCM and T98G cells.

Anti-tumor activities of the new oral pan-RAF inhibitor, TAK-580, used as monotherapy or in combination with novel agents in multiple myeloma.

Many RAS pathway inhibitors, including pan-RAF inhibitors, have shown significant anti-tumor activities in both solid and hematological tumors. The pan-RAF inhibitor, TAK-580, is a representative of the novel RAF inhibitors that act by disrupting RAF homo- or heterodimerization. In this study, we examined the anti-tumor effects of TAK-580 used as monotherapy or in combination with bortezomib, lenalidomide, or other novel agents in multiple myeloma (MM) cells in vitro. TAK-580 monotherapy potently targeted proteins in the RAS-RAF-MEK-ERK signaling pathway and induced potent cytotoxicity and apoptosis in MM cell lines and myeloma cells from patients with newly diagnosed and relapsed and/or refractory MM, compared with a representative RAF inhibitor, dabrafenib. Normal donor peripheral blood B lymphocytes and cord blood CD34-positive cells were not affected. Importantly, TAK-580 significantly inhibited phospho-FOXO3 and induced upregulation of BimL and BimS in a dose-dependent manner, finally leading to apoptosis in MM cells. Moreover, TAK-580 enhanced bortezomib-induced cytotoxicity and apoptosis in MM cells via the FOXO3-Bim axis and the terminal unfolded protein response. Importantly, TAK-580 also enhanced lenalidomide-induced cytotoxicity and apoptosis in MM cells. Taken together, our results provide the rationale for TAK-580 monotherapy and/or treatment in combination with novel agents to improve outcomes in patients with MM.

Receptor-Driven ERK Pulses Reconfigure MAPK Signaling and Enable Persistence of Drug-Adapted BRAF-Mutant Melanoma Cells.

Targeted inhibition of oncogenic pathways can be highly effective in halting the rapid growth of tumors but often leads to the emergence of slowly dividing persister cells, which constitute a reservoir for the selection of drug-resistant clones. In BRAFV600E melanomas, RAF and MEK inhibitors efficiently block oncogenic signaling, but persister cells emerge. Here, we show that persister cells escape drug-induced cell-cycle arrest via brief, sporadic ERK pulses generated by transmembrane receptors and growth factors operating in an autocrine/paracrine manner. Quantitative proteomics and computational modeling show that ERK pulsing is enabled by rewiring of mitogen-activated protein kinase (MAPK) signaling: from an oncogenic BRAFV600E monomer-driven configuration that is drug sensitive to a receptor-driven configuration that involves Ras-GTP and RAF dimers and is highly resistant to RAF and MEK inhibitors. Altogether, this work shows that pulsatile MAPK activation by factors in the microenvironment generates a persistent population of melanoma cells that rewires MAPK signaling to sustain non-genetic drug resistance.

Role of serine 365 in BRAF V600E sensitivity to RAF inhibition.

The serine-threonine kinase, BRAF, is an upstream regulator of the MEK-ERK1/2 pathway and is commonly mutated in cancer. 14-3-3 proteins bind to two sites in BRAF, N-terminal S365, and C-terminal S729. 14-3-3 binding modulates the activity and dimerization of both wild-type and non-V600 mutant forms of BRAF. In BRAF V600E mutants, the C-terminal S729 site affects dimerization of truncated splice variants. The N-terminal, S365, is removed in BRAF V600E splice variants but its importance in full-length BRAF V600 mutants remains uncertain. We tested the role of S365 in dimerization and RAF inhibitor resistance in full-length BRAF V600E. Mutating BRAF S365 site to an alanine (S365A) reduced 14-3-3 association and increased BRAF V600E homodimerization. BRAF V600E S365A displayed reduced sensitivity to RAF inhibitor at the level of MEK-ERK1/2 signaling, cell growth, and cell viability. These data suggest that alteration or removal of the S365 14-3-3 binding site may contribute to RAF inhibitor resistance.

A Systematic Analysis of Signaling Reactivation and Drug Resistance

Increasing evidence suggests that reactivation of initially inhibited signaling pathways causes drug resistance. Here, we reveal network topology features and molecular mechanisms that are necessary and sufficient for complete reactivation or overshoot of steady state signaling after drug treatment. Network-dependent drug resistance is commonly attributed to negative and positive feedback loops mediated by posttranslational modification or transcription and translation. We show that feedback loops, by themselves, cannot completely reactivate steady state signaling. De novo synthesized negative feedback regulators can lead to a transient overshoot but still cannot fully restore output signaling. At least two, activating and inhibitory, connection routes from an inhibited upstream protein to a downstream output must exist for complete reactivation of signaling. Irrespective of the network topology, drug-induced overexpression of a primary target or increase in its dimerization can restore or even paradoxically increase the pathway activity. Inhibitor-induced kinase dimerization cooperates with inhibitor-mediated alleviation of negative feedback. These findings will inform the development of new drugs that consider network context and guide the design of optimal combinations of existing drugs. As an example, we demonstrate that in RAS-mutant cancers the optimal combination to counterbalance ERK pathway reactivation and concomitant drug resistance is a combination of Type I½ and Type II RAF inhibitors.

CIC Is a Mediator of the ERK1/2-DUSP6 Negative Feedback Loop.

SummaryDUSP6 functions as an important negative feedback component of the MAPK/ERK signaling pathway. Although DUSP6 expression is tightly regulated by ERK1/2 signaling, the molecular mechanism of this regulation remains partially understood. In this work, we show that the transcriptional repressor CIC functions downstream of the ERK1/2 signaling to negatively regulate DUSP6 expression. CIC directly represses DUSP6 transcription by binding to three cis-regulatory elements (CREs) in DUSP6 promoter. p90RSK, a downstream target of ERK1/2, phosphorylates CIC at S173 and S301 sites, which creates a 14-3-3 recognition motif, resulting in 14-3-3-mediated nuclear export of CIC and derepression of DUSP6. Finally, we demonstrate that the oncogenic CIC-DUX4 fusion protein acts as a transcriptional activator of DUSP6 and its nuclear/cytoplasmic distribution remains regulated by ERK1/2 signaling. These results complete an ERK1/2/p90RSK/CIC/DUSP6 negative feedback circuit and elucidate the molecular mechanism of how RTK/MAPK signaling harnesses the transcriptional repressor activity of CIC in mammalian cells.

A validated LC-MS/MS method for the determination of RAF inhibitor LXH254: Application to pharmacokinetic study in rat.

In this study, a simple and sensitive UHPLC-ESI-MS/MS method was established for the determination of LXH254 in rat plasma. The developed method was validated according to the Food and Drug administration guidelines. After extraction using ethyl acetate, the sample was separated on an ACQUITY BEH C18 column. The mobile phase consisted of 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. The flow rate was 0.3 mL/min. A TSQ triple quadrupole mass spectrometer operated in positive-ion mode was used for mass detection, with multiple reaction monitoring transitions of m/z 503.3 > 459.1 and m/z 435.3 > 367.1 for LXH254 and olaparib (internal standard), respectively. An excellent linearity was achieved in the concentration range of 0.1-1000 ng/mL, with correlation coefficient >0.998. The mean recovery was more than 78.55%. Inter- and intra-day precision (percentage of relative standard deviation) did not exceed 12.87%, and accuracy was in the range of -2.50 to 13.50%. LXH254 was demonstrated to be stable under the tested storage conditions. The validated UHPLC-MS/MS method was further applied to the pharmacokinetic study of LXH254 in rat plasma after oral (2, 5, and 15 mg/kg) and intravenous (2 mg/kg) administrations. The pharmacokinetic study revealed that LXH254 showed low clearance, moderate bioavailability (~30%), and linear pharmacokinetic profile over the oral dose range of 2-15 mg/kg. To the best of our knowledge, this is the first report on the method development and validation of the determination of LXH254 and its application to pharmacokinetic study.

KRASQ61H Preferentially Signals through MAPK in a RAF Dimer-Dependent Manner in Non–Small Cell Lung Cancer

Assembly of RAS molecules into complexes at the cell membrane is critical for RAS signaling. We previously showed that oncogenic KRAS codon 61 mutations increase its affinity for RAF, raising the possibility that KRASQ61H, the most common KRAS mutation at codon 61, upregulates RAS signaling through mechanisms at the level of RAS assemblies. We show here that KRASQ61H exhibits preferential binding to RAF relative to PI3K in cells, leading to enhanced MAPK signaling in in vitro models and human NSCLC tumors. X-ray crystallography of KRASQ61H:GTP revealed that a hyperdynamic switch 2 allows for a more stable interaction with switch 1, suggesting that enhanced RAF activity arises from a combination of absent intrinsic GTP hydrolysis activity and increased affinity for RAF. Disruption of KRASQ61H assemblies by the RAS oligomer-disrupting D154Q mutation impaired RAF dimerization and altered MAPK signaling but had little effect on PI3K signaling. However, KRASQ61H oligomers but not KRASG12D oligomers were disrupted by RAF mutations that disrupt RAF-RAF interactions. KRASQ61H cells show enhanced sensitivity to RAF and MEK inhibitors individually, whereas combined treatment elicited synergistic growth inhibition. Furthermore, KRASQ61H tumors in mice exhibited high vulnerability to MEK inhibitor, consistent with cooperativity between KRASQ61H and RAF oligomerization and dependence on MAPK signaling. These findings support the notion that KRASQ61H and functionally similar mutations may serve as predictive biomarkers for targeted therapies against the MAPK pathway. SIGNIFICANCE: These findings show that oncogenic KRASQ61H forms a cooperative RAS-RAF ternary complex, which renders RAS-driven tumors vulnerable to MEKi and RAFi, thus establishing a framework for evaluating RAS biomarker-driven targeted therapies.

Inhibitors of BRAF dimers using an allosteric site

BRAF kinase, a critical effector of the ERK signaling pathway, is hyperactivated in many cancers. Oncogenic BRAFV600E signals as an active monomer in the absence of active RAS, however, in many tumors BRAF dimers mediate ERK signaling. FDA-approved RAF inhibitors poorly inhibit BRAF dimers, which leads to tumor resistance. We found that Ponatinib, an FDA-approved drug, is an effective inhibitor of BRAF monomers and dimers. Ponatinib binds the BRAF dimer and stabilizes a distinct αC-helix conformation through interaction with a previously unrevealed allosteric site. Using these structural insights, we developed PHI1, a BRAF inhibitor that fully uncovers the allosteric site. PHI1 exhibits discrete cellular selectivity for BRAF dimers, with enhanced inhibition of the second protomer when the first protomer is occupied, comprising a novel class of dimer selective inhibitors. This work shows that Ponatinib and BRAF dimer selective inhibitors will be useful in treating BRAF-dependent tumors.

Abstract 632: Novel BRAF gene fusions in pediatric histiocytic neoplasms respond differentially to RAF targeted therapies based on dimerization profiles

Abstract Introduction: Pediatric histiocytic neoplasms are clonal hematopoietic disorders driven by mutations activating the mitogen-activated protein kinase (MAPK) pathway, such as BRAF- V600E. In non-BRAFV600E cases, we investigated alternative MAPK mutations and found two novel BRAF gene fusions. We have previously shown effective targeting of BRAF fusions (KIAA1549-BRAF) in pediatric low-grade gliomas (PLGGs) with paradox breaker RAF inhibitor (RAFi), PLX8394 and MEK inhibitors (MEKi). Here, we investigate the distinct responsiveness of novel BRAF fusions to RAFi therapies and explore the mechanistic basis of such differential responses compared to other BRAF fusions. Method: Two histiocytic patient tumors were analyzed using the CHOP Comprehensive Next- Gen Sequencing Solid Tumor Panel and a targeted RNA-seq panel for 106 fusion partner genes. Novel BRAF fusions identified were sub-cloned to create heterologous cell models. Immunoblotting of serum starved cells measured MAPK and PI3K pathway activation and soft agar assay tested for oncogenic phenotypes driven by BRAF fusions, along with response to PLX8394, LY3009120 (pan-RAF dimer inhibitor), and MEKi. Co-immunoprecipitation assays using Myc- and Flag-tagged BRAF fusion constructs assessed dimerization profiles, with or without inhibitors. Result: In the two M- and L-type histiocytic neoplasms assessed, we found novel and rare BRAF gene fusions, MTAP-BRAF and MS4A6A-BRAF, respectively. Both BRAF fusions activated the MAPK/ PI3K pathways and showed homo- and hetero-dimerization with BRAF and the respective N-terminal fusion partner. Compared to common BRAF fusions, MTAP-BRAF and MS4A6A- BRAF did not respond to PLX8394 due to no disruption of active fusion homo- and hetero-dimers, which was in turn was due to the untargeted, stable dimerization mediated by the N-terminal fusion partners. Conversely, we observed robust suppression with LY3009120 that bound fusion dimers and kept them in an inactivate confirmation. MEKi selumetinib and trametinib also suppressed fusion driven signaling and oncogenic phenotypes. Conclusion: We show that novel MTAP-BRAF and MS4A6A-BRAF fusions, found in histiocytic tumors, do not respond to RAFi PLX8394 but are targeted by LY3009120 or MEKi. Our finding that PLX8394 does not disrupt MTAP-BRAF or MS4A6A-BRAF dimerization due to contribution of N-terminal partners defines a novel paradigm for the distinct mechanisms sought by BRAF fusions in response to RAFi therapy. We show potent suppression of these mechanistically distinct BRAF fusions with MEKi or LY3009120 which function independent of vulnerability to fusion dimerization. Overall, this study highlights the unique and differential biology hijacked by BRAF fusions in response to RAFi and further warrants detailed mechanistic classification of BRAF fusions based on their responsiveness to targeted agents. Citation Format: Payal Jain, Lea F. Surrey, Joshua Straka, Pierre Russo, Richard Womer, Marilyn Li, Phillip B. Storm, Angela J. Waanders, Michael Hogarty, Jennifer Picarsic, Adam C. Resnick. Novel BRAF gene fusions in pediatric histiocytic neoplasms respond differentially to RAF targeted therapies based on dimerization profiles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 632.

Abstract 5509: Breaking the paradox breakers - RAF inhibitor mechanisms

Abstract A number of ATP competitive inhibitors of RAF onco-protein have been successful in tumors with RAF V600 mutations but have failed for more common cases with upstream mutations. Instead, a paradoxical activation (PA) of downstream signaling is observed in response to these inhibitors when RAF is not the driver mutation. A detailed understanding of processes that potentiate PA would help in minimizing side effects, choosing appropriate mono- or combination treatments and guide future drug development. Several mechanisms of PA have been proposed in the past, such as negative co-operativity to additional drug binding and RAF dimer stabilization. However, the evidence for these mechanisms is indirect and does not explain PA for all the different types of drugs. In our work, we created a mathematical model of RAF activation solely based on canonical RAF signal regulatory processes to show how autoinhibition provides a general mechanism of PA. We also used this model to show that 14-3-3 proteins could further potentiate PA by stabilizing autoinhibited RAF. Based on the computational work, we predicted that transfecting 14-3-3 proteins would potentiate PA even with drugs which are otherwise expected to provide minimal or no PA. Our experiments with these drugs in SKMEL2 melanoma cells and SW48 colon cancer cells validated our predictions thereby providing support for our general model of PA. Citation Format: Gaurav Mendiratta, Thomas McFall, Edward Stites. Breaking the paradox breakers - RAF inhibitor mechanisms [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5509.

Baseline cardiovascular risk assessment in cancer patients scheduled to receive cardiotoxic cancer therapies: a position statement and new risk assessment tools from the Cardio-Oncology Study Group of the Heart Failure Association of the European Society of Cardiology in collaboration with the International Cardio-Oncology Society.

This position statement from the Heart Failure Association of the European Society of Cardiology Cardio-Oncology Study Group in collaboration with the International Cardio-Oncology Society presents practical, easy-to-use and evidence-based risk stratification tools for oncologists, haemato-oncologists and cardiologists to use in their clinical practice to risk stratify oncology patients prior to receiving cancer therapies known to cause heart failure or other serious cardiovascular toxicities. Baseline risk stratification proformas are presented for oncology patients prior to receiving the following cancer therapies: anthracycline chemotherapy, HER2-targeted therapies such as trastuzumab, vascular endothelial growth factor inhibitors, second and third generation multi-targeted kinase inhibitors for chronic myeloid leukaemia targeting BCR-ABL, multiple myeloma therapies (proteasome inhibitors and immunomodulatory drugs), RAF and MEK inhibitors or androgen deprivation therapies. Applying these risk stratification proformas will allow clinicians to stratify cancer patients into low, medium, high and very high risk of cardiovascular complications prior to starting treatment, with the aim of improving personalised approaches to minimise the risk of cardiovascular toxicity from cancer therapies.

Activating KRAS, NRAS, and BRAF mutants enhance proteasome capacity and reduce endoplasmic reticulum stress in multiple myeloma

KRAS, NRAS, and BRAF mutations which activate p44/42 mitogen-activated protein kinase (MAPK) signaling are found in half of myeloma patients and contribute to proteasome inhibitor (PI) resistance, but the underlying mechanisms are not fully understood. We established myeloma cell lines expressing wild-type (WT), constitutively active (CA) (G12V/G13D/Q61H), or dominant-negative (DN) (S17N)-KRAS and -NRAS, or BRAF-V600E. Cells expressing CA mutants showed increased proteasome maturation protein (POMP) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) expression. This correlated with an increase in catalytically active proteasome subunit β (PSMB)-8, PSMB9, and PSMB10, which occurred in an ETS transcription factor-dependent manner. Proteasome chymotrypsin-like, trypsin-like, and caspase-like activities were increased, and this enhanced capacity reduced PI sensitivity, while DN-KRAS and DN-NRAS did the opposite. Pharmacologic RAF or MAPK kinase (MEK) inhibitors decreased proteasome activity, and sensitized myeloma cells to PIs. CA-KRAS, CA-NRAS, and CA-BRAF down-regulated expression of endoplasmic reticulum (ER) stress proteins, and reduced unfolded protein response activation, while DN mutations increased both. Finally, a bortezomib (BTZ)/MEK inhibitor combination showed enhanced activity in vivo specifically in CA-NRAS models. Taken together, the data support the hypothesis that activating MAPK pathway mutations enhance PI resistance by increasing proteasome capacity, and provide a rationale for targeting such patients with PI/RAF or PI/MEK inhibitor combinations. Moreover, they argue these mutations promote myeloma survival by reducing cellular stress, thereby distancing plasma cells from the apoptotic threshold, potentially explaining their high frequency in myeloma.

Abstract B36: Therapy-induced YAP hyperactivation is a mechanism driving the evolution of residual disease and resistance to targeted cancer therapy

Abstract Initial profound responses of precision medicines against cancer are limited in duration in part due to residual disease states that are therapy refractory and serve as a prelude to acquired drug resistance and tumor relapse. A prominent clinical example is in the collection of BRAF mutant cancers, including lung cancer and melanoma, which often respond initially, but incompletely and temporarily, to targeted RAF and MEK inhibition. The Hippo pathway transcriptional coactivator, Yes-associated protein (YAP), is a chief mediator of RAF and MEK inhibitor response in BRAF mutant cancers. However, the underlying mechanism for YAP-driven resistance is not fully understood. Here, we modeled the kinetics of patient-derived and isogenic BRAF mutant (V600E) cell lines in response to BRAF inhibitor and MEK inhibitor treatment. Our analysis indicated that in response to drug treatment the cells evolve through a “persister,” residual disease state that is distinct from therapy-naive cells or cells with acquired resistance. We observed that drug treatment induced early nuclear translocalization of YAP and this response peaked at the “persister” cell state before returning towards baseline upon acquired resistance. Additionally, our functional analysis of the proteomic and transcriptional landscape of the “persister” cells demonstrated drug-dependent reversible and concomitant activation of AKT, YAP target gene expression, and an epithelial mesenchymal transition (EMT). These programs underlay acquired vulnerabilities, as pharmacologic perturbation of AKT and genetic ablation of YAP was lethal specifically in the “persister” cells. Remarkably, AKT was required and sufficient for drug-induced nuclear localization of YAP, indicating that therapy-mediated hyperactivation of AKT is required for YAP induction and YAP-mediated residual disease. Current studies are focused on characterizing the molecular events causing AKT-mediated hyperactivation of YAP in response to MAPK pathway inhibition. Overall, this work aims to deconvolute a novel molecular mechanism of YAP activation in cancer cells and establish the functional implications of YAP signaling on “persister” cell biology and the evolution of targeted cancer therapy resistance. Citation Format: Aubhishek Zaman, Trever Bivona. Therapy-induced YAP hyperactivation is a mechanism driving the evolution of residual disease and resistance to targeted cancer therapy [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B36.

A Cell-Based MAPK Reporter Assay Reveals Synergistic MAPK Pathway Activity Suppression by MAPK Inhibitor Combination in BRAF-Driven Pediatric Low-Grade Glioma Cells.

Pilocytic astrocytomas as well as other pediatric low-grade gliomas (pLGG) exhibit genetic events leading to aberrant activation of the MAPK pathway. The most common alterations are KIAA1549:BRAF fusions and BRAFV600E and NF1 mutations. Novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for the treatment of these single-pathway diseases. We aimed to develop an assay suitable for preclinical testing of MAPKi in pLGGs with the goal to identify novel MAPK pathway-suppressing synergistic drug combinations. A reporter plasmid (pDIPZ) with a MAPK-responsive ELK-1-binding element driving the expression of destabilized firefly luciferase was generated and packaged using a lentiviral vector system. Pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAFV600E mutation (BT-40) background, respectively, were stably transfected. Modulation of the MAPK pathway activity by MAPKi was measured using the luciferase reporter and validated by detection of phosphorylated protein levels. A screening of a MAPKi library was performed, and synergy of selected combinations was calculated. Screening of a MAPKi library revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC50s, followed by ERK and next-generation RAF inhibitors. Combination treatments with different MAPKi classes showed synergistic effects in BRAF fusion as well as BRAFV600E mutation backgrounds. Here, we report a novel reporter assay for medium- to high-throughput preclinical drug testing in pLGG cell lines. The assay confirmed MEK, ERK, and next-generation RAF inhibitors as potential treatment approaches for KIAA1549:BRAF and BRAFV600E-mutated pLGGs. In addition, the assay revealed that combination treatments synergistically suppressed MAPK pathway activity.

Clinical Relevance of BRAF V600E Mutation Status in Brain Tumors with a Focus on a Novel Management Algorithm.

The possible application of BRAF-targeted therapy in brain tumors is growing continuously. We have analyzed clinical strategies that address BRAF activation in primary brain tumors and verified current recommendations regarding screening for BRAF mutations. There is preliminary evidence for a range of positive responses in certain brain tumor types harboring the BRAF V600E mutation. National Comprehensive Cancer Network Guidelines for central nervous system cancers recommend screening for the BRAF V600E mutation in pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and ganglioglioma. We suggest additional testing in glioblastomas WHO grade IV below the age of 30 years, especially those with epithelioid features, papillary craniopharyngiomas, and pediatric low-grade astrocytomas. BRAF-targeted therapy should be limited to the setting of a clinical trial. If the patient harboring a V600E mutation does not qualify for a trial, multimodality treatment is recommended. Dual inhibition of both RAF and MEK is expected to provide more potent and durable effects than anti-BRAF monotherapy. First-generation RAF inhibitors should be avoided. Gain-of-function mutations of EGFR and KIAA fusions may compromise BRAF-targeted therapy. BRAF alterations that result in MAPK pathway activation are common events in several types of brain tumors. BRAF V600E mutation emerges as a promising molecular target. The proposed algorithm was designed to help oncologists to provide the best therapeutic options for brain tumor patients.

Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers

SUMMARYWe address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1, and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC.

Phase I dose-finding study of oral ERK1/2 inhibitor LTT462 in patients (pts) with advanced solid tumors harboring MAPK pathway alterations.

3640 Background: LTT462 is an investigational small molecule inhibitor of ERK1/2, which has demonstrated preclinical activity in multiple MAPK activated cancer cells and xenograft models. This first-in-human study was designed to evaluate the safety and tolerability of LTT462 in advanced solid tumors harboring MAPK pathway alterations (NCT02711345). Methods: The dose-escalation part of this Phase I, open-label study, enrolled adult and adolescent pts with advanced solid tumors harboring ≥1 documented MAPK pathway alteration with progressive disease (PD) despite standard therapy, or for whom there is no effective standard treatment. Oral LTT462 was given once daily (QD) at 45–600 mg or twice daily (BID) at 150 mg or 200 mg. Objectives were to determine the maximum tolerated dose (MTD) using a Bayesian hierarchical logistic regression model guided by escalation with overdose control, and characterize safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of LTT462. Results: Sixty-five pts (median age 60 years) including 1 pt aged 15 were enrolled in the dose-escalation; most pts (22%) had 3 prior therapies. Most common primary sites for cancer were in the colon (n = 21; 32%), ovary (n = 9; 14%), and pancreas (n = 7; 11%). All pts discontinued, the majority due to PD (n = 44; 68%). Eleven pts experienced DLTs; 6 pts experienced Grade 3 eye disorder DLTs (4 pts retinopathy, 2 pts chorioretinopathy). Treatment-related adverse events (TRAEs) were reported for 89% of pts, most commonly ( > 30%) diarrhea (n = 25; 38%) and nausea (n = 22; 34%). Grade 3/4 TRAEs were reported in 29% of pts; most common was retinopathy (n = 4; 6%). MTD of LTT462 was 400 mg QD and 150 mg BID. Overall, 8 pts (12%) had stable disease (SD) and 35 pts (54%) had PD. An unconfirmed partial response was reported in a pt with cholangiocarcinoma with BRAF mutation; best change in sum of target lesions per RECIST 1.1 was -33.9%. LTT462 increased plasma peak drug concentration and drug exposure at increasing doses between 45–450 mg QD. Exposure at LTT462 600 mg QD was lower than anticipated, indicating potential saturation of absorption at this dose. LTT462 inhibited ERK1/2 and reduced DUSP6 expression relative to baseline in most pts evaluated. Conclusions: LTT462 is well tolerated. Limited clinical activity was reported with single agent LTT462; best overall response was SD. An ongoing study is investigating LTT462 in combination with the RAF inhibitor, LXH254, in NSCLC and melanoma. Clinical trial information: NCT02711345 .

Abstract IA32: Interplay between RAF inhibitors and RAS

Abstract Ever since RAF was shown to be a key downstream effector of RAS in the early 90s, RAF inhibition has been a goal within the drug discovery community. Sorafenib, an early entrant to the RAF armamentarium, was discovered through biochemical RAF inhibition assays with the goal of disrupting RAS signaling. Anticancer efficacy that was demonstrated through clinical trials was likely through “off-target” inhibition of VEGF receptors, and RAF is not effectively inhibited in RAS-mutant tumor cells. Furthermore, BRAF-mutant tumors were also poorly targeted by sorafenib, most likely because the “type 2” binding mode is ineffective against the activated BRAF kinase. Subsequently, compounds such as vemurafenib, dabrafenib, and encorafenib were developed as “type 1” inhibitors that target the active form of BRAF kinase: these inhibitors show efficacy in many BRAF-mutant tumors. Curiously, these RAF inhibitors cause certain side effects that have been attributed to the “RAF inhibitor paradox”: the ERK pathway is paradoxically stimulated by RAF inhibitors in cells with an activated RAS pathway. To overcome this liability, the next generation of RAF inhibitors dubbed “paradox breakers” were developed. As a BRAF-specific dimer disruptor, PLX8394 selectively inhibits ERK signaling in tumors bearing both monomer- and dimer-dependent BRAF mutants, including BRAF fusions and splice variants, while sparing RAF function in normal cells and avoiding paradoxical ERK activation in RAS-mutant cells. A converse effort seeks to take advantage of the paradox to identify RAS cell liabilities. By hyperstimulating the ERK pathway, “paradox activators” could tip the balance in favor of cellular disharmony. Future efforts exploiting this mechanism will enhance the context for thinking about RAF and near-neighbor targets in RAS tumors and guide the judicious selection of combination therapies involving RAF-targeting agents and other therapeutics. Citation Format: Ying Zhang, Gideon Bollag. Interplay between RAF inhibitors and RAS [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA32.