An electroaffinity labelling platform for chemoproteomic-based target identification.

Target identification involves deconvoluting the protein target of a pharmacologically active, small-molecule ligand, a process that is critical for early drug discovery yet technically challenging. Photoaffinity labelling strategies have become the benchmark for small-molecule target deconvolution, but covalent protein capture requires the use of high-energy ultraviolet light, which can complicate downstream target identification. Thus, there is a strong demand for alternative technologies that allow for controlled activation of chemical probes to covalently label their protein target. Here we introduce an electroaffinity labelling platform that leverages the use of a small, redox-active diazetidinone functional group to enable chemoproteomic-based target identification of pharmacophores within live cell environments. The underlying discovery to enable this platform is that the diazetidinone can be electrochemically oxidized to reveal a reactive intermediate useful for covalent modification of proteins. This work demonstrates the electrochemical platform to be a functional tool for drug-target identification.

Targeted activation in localized protein environments via deep red photoredox catalysis.

State-of-the-art photoactivation strategies in chemical biology provide spatiotemporal control and visualization of biological processes. However, using high-energy light (λ < 500 nm) for substrate or photocatalyst sensitization can lead to background activation of photoactive small-molecule probes and reduce its efficacy in complex biological environments. Here we describe the development of targeted aryl azide activation via deep red-light (λ = 660 nm) photoredox catalysis and its use in photocatalysed proximity labelling. We demonstrate that aryl azides are converted to triplet nitrenes via a redox-centric mechanism and show that its spatially localized formation requires both red light and a photocatalyst-targeting modality. This technology was applied in different colon cancer cell systems for targeted protein environment labelling of epithelial cell adhesion molecule (EpCAM). We identified a small subset of proteins with previously known and unknown association to EpCAM, including CDH3, a clinically relevant protein that shares high tumour-selective expression with EpCAM.

SPATA2 and CYLD inhibit T cell infiltration into colorectal cancer via regulation of IFN-γ/STAT1 axis.

Introduction: Colorectal cancer (CRC) is largely refractory to currently available immunotherapies such as blockade of programmed cell death protein-1 (PD-1). Results: In this study, we identified SPATA2 and its protein partner CYLD as novel regulators of CXC-ligand 10 (CXCL10), a T-cell-attractant chemokine, in CRC. By specifically deleting SPATA2 and CYLD in human and mouse CRC cell lines, we showed that these two proteins inhibit STAT1 accumulation and activation and subsequently CXCL10 expression in tumor cells. At steady-state, STAT1 is highly ubiquitinated in a SPATA2/CYLD-dependent manner. Finally, we demonstrated that tumor-specific deletion of SPATA2 and CYLD enhances anti-PD-1 response in vivo. Discussion: Our data suggest that SPATA2 and CYLD represent two potential novel targets for treatment of immune-excluded, PD-1-resistant tumors.

Proteomic mapping of intercellular synaptic environments via flavin-dependent photoredox catalysis.

Receptor-ligand interactions play essential signaling roles within intercellular contact regions. This is particularly important within the context of the immune synapse where protein communication at the surface of physically interacting T cells and antigen-presenting cells regulate downstream immune signaling responses. To identify protein microenvironments within immunological synapses, we combined a flavin-dependent photocatalytic labeling strategy with quantitative mass spectrometry-based proteomics. Using α-PD-L1 or α-PD-1 single-domain antibody (VHH)-based photocatalyst targeting modalities, we profiled protein microenvironments within the intercellular region of an immune synapse-forming co-culture system. In addition to enrichment of both PD-L1 and PD-1 with either targeting modality, we also observed enrichment of both known immune synapse residing receptor-ligand pairs and surface proteins, as well as previously unknown synapse residing proteins.

Nanoscale Mapping of EGFR and c-MET Protein Environments on Lung Cancer Cell Surfaces via Therapeutic Antibody Photocatalyst Conjugates.

Receptor tyrosine kinases are involved in essential signaling roles that impact cell growth, differentiation, and proliferation. The overexpression or mutation of these proteins can lead to aberrant signaling that has been directly linked to a number of diseases including cancer cell formation and progression. This has led to intense clinical focus on modulating RTK activity through direct targeting of signaling activity or cell types harboring aberrant RTK behavior. In particular, epidermal growth factor receptor (EGFR) has attracted intense clinical attention due to the impact of inhibiting this RTK on tumor growth. However, mutations incurred through targeting EGFR have led to therapeutic resistance that involves not only direct mutations to the EGFR protein but also the involvement of other RTKs, such as c-MET, that can overcome therapeutic-based EGFR inhibition effects. This has, not surprisingly, led to co-targeting strategies of RTKs such as EGFR and c-MET to overcome resistance mechanisms. While the ability to co-target these proteins has led to success in the clinic, a more comprehensive understanding of their proximal environments, particularly in the context of therapeutic modalities, could further enhance both our understanding of their signaling biology and provide additional avenues for targeting these surface proteins. Thus, to investigate EGFR and c-MET protein microenvironments, we utilized our recently developed iridium photocatalyst-based microenvironment mapping technology to catalog EGFR and c-MET surface environments on non-small cell lung cancer cell lines. Through this approach, we enriched EGFR and c-MET from the cell surface and identified known EGFR and c-MET associators as well as previously unidentified proximal proteins.

Reverse Translating Molecular Determinants of Anti-Programmed Death 1 Immunotherapy Response in Mouse Syngeneic Tumor Models.

Targeting the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway with immunotherapy has revolutionized the treatment of many cancers. Somatic tumor mutational burden (TMB) and T-cell-inflamed gene expression profile (GEP) are clinically validated pan-tumor genomic biomarkers that can predict responsiveness to anti-PD-1/PD-L1 monotherapy in many tumor types. We analyzed the association between these biomarkers and the efficacy of PD-1 inhibitor in 11 commonly used preclinical syngeneic tumor mouse models using murinized rat anti-mouse PD-1 DX400 antibody muDX400, a surrogate for pembrolizumab. Response to muDX400 treatment was broadly classified into three categories: highly responsive, partially responsive, and intrinsically resistant to therapy. Molecular and cellular profiling validated differences in immune cell infiltration and activation in the tumor microenvironment of muDX400-responsive tumors. Baseline and on-treatment genomic analysis showed an association between TMB, murine T-cell-inflamed gene expression profile (murine-GEP), and response to muDX400 treatment. We extended our analysis to investigate a canonical set of cancer and immune biology-related gene signatures, including signatures of angiogenesis, myeloid-derived suppressor cells, and stromal/epithelial-to-mesenchymal transition/TGFß biology previously shown to be inversely associated with the clinical efficacy of immune checkpoint blockade. Finally, we evaluated the association between murine-GEP and preclinical efficacy with standard-of-care chemotherapy or antiangiogenic agents that previously demonstrated promising clinical activity, in combination with muDX400. Our profiling studies begin to elucidate the underlying biological mechanisms of response and resistance to PD-1/PD-L1 blockade represented by these models, thereby providing insight into which models are most appropriate for the evaluation of orthogonal combination strategies.

Toward developing a metastatic breast cancer treatment strategy that incorporates history of response to previous treatments.

BACKGROUND: Information regarding response to past treatments may provide clues concerning the classes of drugs most or least likely to work for a particular metastatic or neoadjuvant early stage breast cancer patient. However, currently there is no systematized knowledge base that would support clinical treatment decision-making that takes response history into account. METHODS: To model history-dependent response data we leveraged a published in vitro breast cancer viability dataset (84 cell lines, 90 therapeutic compounds) to calculate the odds ratios (log (OR)) of responding to each drug given knowledge of (intrinsic/prior) response to all other agents. This OR matrix assumes (1) response is based on intrinsic rather than acquired characteristics, and (2) intrinsic sensitivity remains unchanged at the time of the next decision point. Fisher's exact test is used to identify predictive pairs and groups of agents (BH p < 0.05). Recommendation systems are used to make further drug recommendations based on past 'history' of response. RESULTS: Of the 90 compounds, 57 have sensitivity profiles significantly associated with those of at least one other agent, mostly targeted drugs. Nearly all associations are positive, with (intrinsic/prior) sensitivity to one agent predicting sensitivity to others in the same or a related class (OR > 1). In vitro conditional response patterns clustered compounds into five predictive classes: (1) DNA damaging agents, (2) Aurora A kinase and cell cycle checkpoint inhibitors; (3) microtubule poisons; (4) HER2/EGFR inhibitors; and (5) PIK3C catalytic subunit inhibitors. The apriori algorithm implementation made further predictions including a directional association between resistance to HER2 inhibition and sensitivity to proteasome inhibitors. CONCLUSIONS: Investigating drug sensitivity conditioned on observed sensitivity or resistance to prior drugs may be pivotal in informing clinicians deciding on the next line of breast cancer treatments for patients who have progressed on their current treatment. This study supports a strategy of treating patients with different agents in the same class where an associated sensitivity was observed, likely after one or more intervening treatments.

LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody-Induced Tumor Cell Killing by Effector CD8+ T Cells.

Elicitation of tumor cell killing by CD8+ T cells is an effective therapeutic approach for cancer. In addition to using immune checkpoint blockade to reinvigorate existing but unresponsive tumor-specific T cells, alternative therapeutic approaches have been developed, including stimulation of polyclonal T cell cytolytic activity against tumors using bispecific T cell engager (BiTE) molecules that simultaneously engage the TCR complex and a tumor-associated Ag. BiTE molecules are efficacious against hematologic tumors and are currently being explored as an immunotherapy for solid tumors. To understand mechanisms regulating BiTE molecule--mediated CD8+ T cell activity against solid tumors, we sought to define human CD8+ T cell populations that efficiently respond to BiTE molecule stimulation and identify factors regulating their cytolytic activity. We find that human CD45RA+CCR7- CD8+ T cells are highly responsive to BiTE molecule stimulation, are enriched in genes associated with cytolytic effector function, and express multiple unique inhibitory receptors, including leukocyte Ig-like receptor B1 (LILRB1). LILRB1 and programmed cell death protein 1 (PD1) were found to be expressed by distinct CD8+ T cell populations, suggesting different roles in regulating the antitumor response. Engaging LILRB1 with its ligand HLA-G on tumor cells significantly inhibited BiTE molecule-induced CD8+ T cell activation. Blockades of LILRB1 and PD1 induced greater CD8+ T cell activation than either treatment alone. Together, our data suggest that LILRB1 functions as a negative regulator of human CD8+ effector T cells and that blocking LILRB1 represents a unique strategy to enhance BiTE molecule therapeutic activity against solid tumors.

Overcoming resistance to single-agent therapy for oncogenic BRAF gene fusions via combinatorial targeting of MAPK and PI3K/mTOR signaling pathways.

Pediatric low-grade gliomas (PLGGs) are frequently associated with activating BRAF gene fusions, such as KIAA1549-BRAF, that aberrantly drive the mitogen activated protein kinase (MAPK) pathway. Although RAF inhibitors (RAFi) have been proven effective in BRAF-V600E mutant tumors, we have previously shown how the KIAA1549-BRAF fusion can be paradoxically activated by RAFi. While newer classes of RAFi, such as PLX8394, have now been shown to inhibit MAPK activation by KIAA1549-BRAF, we sought to identify alternative MAPK pathway targeting strategies using clinically relevant MEK inhibitors (MEKi), along with potential escape mechanisms of acquired resistance to single-agent MAPK pathway therapies. We demonstrate effectiveness of multiple MEKi against diverse BRAF-fusions with novel N-terminal partners, with trametinib being the most potent. However, resistance to MEKi or PLX8394 develops via increased RTK expression causing activation of PI3K/mTOR pathway in BRAF-fusion expressing resistant clones. To circumvent acquired resistance, we show potency of combinatorial targeting with trametinib and everolimus, an mTOR inhibitor (mTORi) against multiple BRAF-fusions. While single-agent mTORi and MEKi PLGG clinical trials are underway, our study provides preclinical rationales for using MEKi and mTORi combinatorial therapy to stave off or prevent emergent drug-resistance in BRAF-fusion driven PLGGs.

Peripheral Blood Biomarkers Associated With Toxicity and Treatment Characteristics After 131I- Metaiodobenzylguanidine Therapy in Patients With Neuroblastoma.

PURPOSE: Few tools exist to predict clinical outcomes after radiopharmaceutical therapy. Our goal was to identify associations between blood-based biomarkers of radiation effect and clinical outcomes after 131I-metaiodobenzylguanidine (131I-MIBG) therapy in patients with neuroblastoma. METHODS AND MATERIALS: We conducted a prospective, single-center cohort study in children with advanced neuroblastoma treated with 131I-MIBG as monotherapy or in combination with systemic putative radiation sensitizers. We collected serial peripheral blood samples after 131I-MIBG infusions and quantified a panel of protein and messenger RNA markers. We plotted relative change from baseline to assess degree of modulation over time and then evaluated association of marker modulation with toxicity and response endpoints. RESULTS: The cohort included 40 patients (30 male/10 female; median age 7 years). We observed significant modulation of the majority of markers between baseline and hour 72 after 131I-MIBG. Greater fold increase of plasma FLT3 ligand was associated with subsequent grade 4 neutropenia (P=.039). Modulation of peripheral blood BCLXL and DDB2 was associated with grade 3+ nonhematologic toxicity (P=.043 and .048, respectively). No markers were associated with tumor response. Greater plasma FLT3 ligand, BCLXL, and BCL2 modulation was observed in patients receiving 131I-MIBG in combination with radiation sensitizers. Among 9 patients who received 2 courses, the degree of modulation in serum amylase was significantly lower after the second course (P=.012). CONCLUSIONS: Peripheral blood biomarkers relevant to radiation exposure are significantly modulated during the acute period after 131I-MIBG. The degree of modulation of a subset of these markers is associated with toxicity and receipt of concomitant radiation sensitizers.

BRAF Status in Personalizing Treatment Approaches for Pediatric Gliomas.

PURPOSE: Alteration of the BRAF/MEK/MAPK pathway is the hallmark of pediatric low-grade gliomas (PLGGs), and mTOR activation has been documented in the majority of these tumors. We investigated combinations of MEK1/2, BRAFV600E and mTOR inhibitors in gliomas carrying specific genetic alterations of the MAPK pathway. EXPERIMENTAL DESIGN: We used human glioma lines containing BRAFV600E (adult high-grade: AM-38, DBTRG, PLGG: BT40), or wild-type BRAF (pediatric high-grade: SF188, SF9427, SF8628) and isogenic systems of KIAA1549:BRAF-expressing NIH/3T3 cells and BRAFV600E-expressing murine brain cells. Signaling inhibitors included everolimus (mTOR), PLX4720 (BRAFV600E), and AZD6244 (MEK1/2). Proliferation was determined using ATP-based assays. In vivo inhibitor activities were assessed in the BT40 PLGG xenograft model. RESULTS: In BRAFV600E cells, the three possible doublet combinations of AZD6244, everolimus, and PLX4720 exhibited significantly greater effects on cell viability. In BRAFWT cells, everolimus + AZD6244 was superior compared with respective monotherapies. Similar results were found using isogenic murine cells. In KIAA1549:BRAF cells, MEK1/2 inhibition reduced cell viability and S-phase content, effects that were modestly augmented by mTOR inhibition. In vivo experiments in the BRAFV600E pediatric xenograft model BT40 showed the greatest survival advantage in mice treated with AZD6244 + PLX4720 (P < 0.01). CONCLUSIONS: In BRAFV600E tumors, combination of AZD6244 + PLX4720 is superior to monotherapy and to other combinatorial approaches. In BRAFWT pediatric gliomas, everolimus + AZD6244 is superior to either agent alone. KIAA1549:BRAF-expressing tumors display marked sensitivity to MEK1/2 inhibition. Application of these results to PLGG treatment must be exercised with caution because the dearth of PLGG models necessitated only a single patient-derived PLGG (BT40) in this study. Clin Cancer Res; 22(21); 5312-21. ©2016 AACR.

An Atlas of the Human Kinome Reveals the Mutational Landscape Underlying Dysregulated Phosphorylation Cascades in Cancer.

Kinase inhibitors are used widely to treat various cancers, but adaptive reprogramming of kinase cascades and activation of feedback loop mechanisms often contribute to therapeutic resistance. Determining comprehensive, accurate maps of kinase circuits may therefore help elucidate mechanisms of response and resistance to kinase inhibitor therapies. In this study, we identified and validated phosphorylatable target sites across human cell and tissue types to generate PhosphoAtlas, a map of 1,733 functionally interconnected proteins comprising the human phospho-reactome. A systematic curation approach was used to distill protein phosphorylation data cross-referenced from 38 public resources. We demonstrated how a catalog of 2,617 stringently verified heptameric peptide regions at the catalytic interface of kinases and substrates could expose mutations that recurrently perturb specific phospho-hubs. In silico mapping of 2,896 nonsynonymous tumor variants identified from thousands of tumor tissues also revealed that normal and aberrant catalytic interactions co-occur frequently, showing how tumors systematically hijack, as well as spare, particular subnetworks. Overall, our work provides an important new resource for interrogating the human tumor kinome to strategically identify therapeutically actionable kinase networks that drive tumorigenesis. Cancer Res; 76(7); 1733-45. ©2016 AACR.

Survival advantage combining a BRAF inhibitor and radiation in BRAF V600E-mutant glioma.

Radiation (RT) is critical to the treatment of high-grade gliomas (HGGs) but cures remain elusive. The BRAF mutation V600E is critical to the pathogenesis of 10-20% of pediatric gliomas, and a small proportion of adult HGGs. Here we aim to determine whether PLX4720, a specific BRAF V600E inhibitor, enhances the activity of RT in human HGGs in vitro and in vivo. Patient-derived HGG lines harboring wild-type BRAF or BRAF V600E were assessed in vitro to determine IC50 values, cell cycle arrest, apoptosis and senescence and elucidate mechanisms of combinatorial activity. A BRAF V600E HGG intracranial xenograft mouse model was used to evaluate in vivo combinatorial efficacy of PLX4720+RT. Tumors were harvested for immunohistochemistry to quantify cell cycle arrest and apoptosis. RT+PLX4720 exhibited greater anti-tumor effects than either monotherapy in BRAF V600E but not in BRAF WT lines. In vitro studies showed increased Annexin V and decreased S phase cells in BRAF V600E gliomas treated with PLX4720+RT, but no significant changes in ß-galactosidase levels. In vivo, concurrent and sequential PLX4720+RT each significantly prolonged survival compared to monotherapies, in the BRAF V600E HGG model. Immunohistochemistry of in vivo tumors demonstrated that PLX4720+RT decreased Ki-67 and phospho-MAPK, and increased γH2AX and p21 compared to control mice. BRAF V600E inhibition enhances radiation-induced cytotoxicity in BRAF V600E-mutated HGGs, in vitro and in vivo, effects likely mediated by apoptosis and cell cycle, but not senescence. These studies provide the pre-clinical rationale for clinical trials of concurrent radiotherapy and BRAF V600E inhibitors.

Intravital imaging reveals distinct responses of depleting dynamic tumor-associated macrophage and dendritic cell subpopulations.

Tumor-infiltrating inflammatory cells comprise a major part of the stromal microenvironment and support cancer progression by multiple mechanisms. High numbers of tumor myeloid cells correlate with poor prognosis in breast cancer and are coupled with the angiogenic switch and malignant progression. However, the specific roles and regulation of heterogeneous tumor myeloid populations are incompletely understood. CSF-1 is a major myeloid cell mitogen, and signaling through its receptor CSF-1R is also linked to poor outcomes. To characterize myeloid cell function in tumors, we combined confocal intravital microscopy with depletion of CSF-1R-dependent cells using a neutralizing CSF-1R antibody in the mouse mammary tumor virus long-terminal region-driven polyoma middle T antigen breast cancer model. The depleted cells shared markers of tumor-associated macrophages and dendritic cells (M-DCs), matching the phenotype of tumor dendritic cells that take up antigens and interact with T cells. We defined functional subgroups within the M-DC population by imaging endocytic and matrix metalloproteinase activity. Anti-CSF-1R treatment altered stromal dynamics and impaired both survival of M-DCs and accumulation of new M-DCs, but did not deplete Gr-1(+) neutrophils or block doxorubicin-induced myeloid cell recruitment, and had a minimal effect on lung myeloid cells. Nevertheless, prolonged treatment led to delayed tumor growth, reduced vascularity, and decreased lung metastasis. Because the myeloid infiltrate in metastatic lungs differed significantly from that in mammary tumors, the reduction in metastasis may result from the impact on primary tumors. The combination of functional analysis by intravital imaging with cellular characterization has refined our understanding of the effects of experimental targeted therapies on the tumor microenvironment.

Targeting Wee1 for the treatment of pediatric high-grade gliomas.

BACKGROUND: We investigated the efficacy of the Wee1 inhibitor MK-1775 in combination with radiation for the treatment of pediatric high-grade gliomas (HGGs), including diffuse intrinsic pontine gliomas (DIPGs). METHODS: Gene expression analysis was performed for 38 primary pediatric gliomas (3 grade I, 10 grade II, 11 grade III, 14 grade IV) and 8 normal brain samples using the Agilent 4 × 44 K array. Clonogenic survival assays were carried out in pediatric and adult HGG cell lines (n = 6) to assess radiosensitizing effects of MK-1775. DNA repair capacity was evaluated by measuring protein levels of γ-H2AX, a marker of double strand DNA breaks. In vivo activity of MK-1775 with radiation was assessed in 2 distinct orthotopic engraftment models of pediatric HGG, including 1 derived from a genetically engineered mouse carrying a BRAF(V600E) mutation, and 1 xenograft model in which tumor cells were derived from a patient's DIPG. RESULTS: Wee1 is overexpressed in pediatric HGGs, with increasing expression positively correlated with malignancy (P = .007 for grade III + IV vs I + II) and markedly high expression in DIPG. Combination treatment of MK-1775 and radiation reduced clonogenic survival and increased expression of γ-H2AX to a greater extent than achieved by radiation alone. Finally, combined MK-1775 and radiation conferred greater survival benefit to mice bearing engrafted, orthotopic HGG and DIPG tumors, compared with treatment with radiation alone (BRAF(V600E) model P = .0061 and DIPG brainstem model P = .0163). CONCLUSION: Our results highlight MK-1775 as a promising new therapeutic agent for use in combination with radiation for the treatment of pediatric HGGs, including DIPG.

Poly (ADP-Ribose) polymerase inhibitor MK-4827 together with radiation as a novel therapy for metastatic neuroblastoma.

BACKGROUND/AIM: To assess poly (ADP-ribose) polymerase (PARP) inhibitor MK-4827 together with radiation for the treatment of neuroblastoma. MATERIALS AND METHODS: Clonogenic survival assays were used to assess MK-4827, radiation and combination thereof in four neuroblastoma cell lines. In vivo efficacy was tested in a murine xenograft model of metastatic neuroblastoma. In vivo targeted inhibition and biological effects included measurement of cleaved caspase-3, γ-H2AX, and Ki 67 by immunohistochemistry (IHC) and poly-ADP-ribose by Enzyme-Linked Immunosorbent Assay. RESULTS: Treatment of neuroblastoma cell lines reduced clonogenicity and resulted in additive effects with radiation. In vivo treatment with MK-4827 and radiation prolonged survival (p<0.01) compared to single modalities. In vivo superiority of MK-4827 plus radiation was further documented by significant elevations of cleaved caspase-3 and γ-H2AX in tumors from the combination group compared to single modality cohorts. CONCLUSION: Combination of MK-4827 and radiation might provide effective therapy for children with high-risk neuroblastoma.

Genotype-dependent cooperation of ionizing radiation with BRAF inhibition in BRAF V600E-mutated carcinomas.

BACKGROUND: A substantial proportion of solid tumors carry the BRAF V600E mutation, which causes activation of the MEK/MAPK pathway and is a poor prognostic indicator. Patients with locally advanced human cancers are often treated with external beam radiation therapy. Given the association of Raf overactivation with radioresistance, we hypothesized that, in BRAF V600E-mutated carcinomas, there would be combinatorial activity between radiation and PLX4720, a specific BRAF V600E-inhibitor. METHODS: Two BRAF V600E-mutated cancer cell lines and one BRAF-V600E wildtype (WT) cancer cell line were obtained. We performed cell viability assays and clonogenic assays using combinations of radiation and PLX4720. We assessed MEK and MAPK phosphorylation at different PLX4720 concentrations with western blotting, and cell cycle progression was evaluated by flow cytometry. RESULTS: Our results show combinatorial, additive activity between radiation and PLX4720 in BRAF V600E-mutated cell lines, but not in the BRAF WT line. In BRAF V600E-mutated cells, there was a PLX4720 concentration-dependent decrease in MEK and MAPK phosphorylation. In cells with BRAF V600E mutations, PLX4720 caused cell cycle arrest at G1, and, when combined with radiation, caused a combined G1 and G2 cell cycle arrest; this pattern of cell cycle effects was not seen in the BRAF WT cell line. CONCLUSIONS: These data suggest additive, combinatorial activity between radiation and PLX4720 in cancers carrying BRAF V600E mutations. Our data has potential for translation into the multimodality treatment of BRAF V600E-mutated cancers.