IκB kinase-α coordinates BRD4 and JAK/STAT signaling to subvert DNA damage-based anticancer therapy.

Activation of the IκB kinase (IKK) complex has recurrently been linked to colorectal cancer (CRC) initiation and progression. However, identification of downstream effectors other than NF-κB has remained elusive. Here, analysis of IKK-dependent substrates in CRC cells after UV treatment revealed that phosphorylation of BRD4 by IKK-α is required for its chromatin-binding at target genes upon DNA damage. Moreover, IKK-α induces the NF-κB-dependent transcription of the cytokine LIF, leading to STAT3 activation, association with BRD4 and recruitment to specific target genes. IKK-α abrogation results in defective BRD4 and STAT3 functions and consequently irreparable DNA damage and apoptotic cell death upon different stimuli. Simultaneous inhibition of BRAF-dependent IKK-α activity, BRD4, and the JAK/STAT pathway enhanced the therapeutic potential of 5-fluorouracil combined with irinotecan in CRC cells and is curative in a chemotherapy-resistant xenograft model. Finally, coordinated expression of LIF and IKK-α is a poor prognosis marker for CRC patients. Our data uncover a functional link between IKK-α, BRD4, and JAK/STAT signaling with clinical relevance.

IKKα Kinase Regulates the DNA Damage Response and Drives Chemo-resistance in Cancer.

Phosphorylated IKKα(p45) is a nuclear active form of the IKKα kinase that is induced by the MAP kinases BRAF and TAK1 and promotes tumor growth independent of canonical NF-κB signaling. Insights into the sources of IKKα(p45) activation and its downstream substrates in the nucleus remain to be defined. Here, we discover that IKKα(p45) is rapidly activated by DNA damage independent of ATM-ATR, but dependent on BRAF-TAK1-p38-MAPK, and is required for robust ATM activation and efficient DNA repair. Abolishing BRAF or IKKα activity attenuates ATM, Chk1, MDC1, Kap1, and 53BP1 phosphorylation, compromises 53BP1 and RIF1 co-recruitment to sites of DNA lesions, and inhibits 53BP1-dependent fusion of dysfunctional telomeres. Furthermore, IKKα or BRAF inhibition synergistically enhances the therapeutic potential of 5-FU and irinotecan to eradicate chemotherapy-resistant metastatic human tumors in vivo. Our results implicate BRAF and IKKα kinases in the DDR and reveal a combination strategy for cancer treatment.

Efficacy of CDK4/6 inhibitors in preclinical models of malignant pleural mesothelioma.

BACKGROUND: There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy. METHODS: We aimed to investigate the antitumor activity of CDK4/6 inhibitors using in vitro and in vivo preclinical models of MPM. RESULTS: Based on publicly available transcriptomic data of MPM, patients with CDK4 or CDK6 overexpression had shorter overall survival. Treatment with abemaciclib or palbociclib at 100 nM significantly decreased cell proliferation in all cell models evaluated. Both CDK4/6 inhibitors significantly induced G1 cell cycle arrest, thereby increasing cell senescence and increased the expression of interferon signalling pathway and tumour antigen presentation process in culture models of MPM. In vivo preclinical studies showed that palbociclib significantly reduced tumour growth and prolonged overall survival using distinct xenograft models of MPM implanted in athymic mice. CONCLUSIONS: Treatment of MPM with CDK4/6 inhibitors decreased cell proliferation, mainly by promoting cell cycle arrest at G1 and by induction of cell senescence. Our preclinical studies provide evidence for evaluating CDK4/6 inhibitors in the clinic for the treatment of MPM.

Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program.

Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease.

Epigenetic inactivation of the p53-induced long noncoding RNA TP53 target 1 in human cancer.

Long noncoding RNAs (lncRNAs) are important regulators of cellular homeostasis. However, their contribution to the cancer phenotype still needs to be established. Herein, we have identified a p53-induced lncRNA, TP53TG1, that undergoes cancer-specific promoter hypermethylation-associated silencing. In vitro and in vivo assays identify a tumor-suppressor activity for TP53TG1 and a role in the p53 response to DNA damage. Importantly, we show that TP53TG1 binds to the multifaceted DNA/RNA binding protein YBX1 to prevent its nuclear localization and thus the YBX1-mediated activation of oncogenes. TP53TG1 epigenetic inactivation in cancer cells releases the transcriptional repression of YBX1-targeted growth-promoting genes and creates a chemoresistant tumor. TP53TG1 hypermethylation in primary tumors is shown to be associated with poor outcome. The epigenetic loss of TP53TG1 therefore represents an altered event in an lncRNA that is linked to classical tumoral pathways, such as p53 signaling, but is also connected to regulatory networks of the cancer cell.

RAS-ON inhibition overcomes clinical resistance to KRAS G12C-OFF covalent blockade.

Selective KRASG12C inhibitors have been developed to covalently lock the oncogene in the inactive GDP-bound state. Two of these molecules, sotorasib and adagrasib, are approved for the treatment of adult patients with KRASG12C-mutated previously treated advanced non-small cell lung cancer. Drug treatment imposes selective pressures leading to the outgrowth of drug-resistant variants. Mass sequencing from patients' biopsies identified a number of acquired KRAS mutations -both in cis and in trans- in resistant tumors. We demonstrate here that disease progression in vivo can also occur due to adaptive mechanisms and increased KRAS-GTP loading. Using the preclinical tool tri-complex KRASG12C-selective covalent inhibitor, RMC-4998 (also known as RM-029), that targets the active GTP-bound (ON) state of the oncogene, we provide a proof-of-concept that the clinical stage KRASG12C(ON) inhibitor RMC-6291 alone or in combination with KRASG12C(OFF) drugs can be an alternative potential therapeutic strategy to circumvent resistance due to increased KRAS-GTP loading.

Stromal interaction molecule 2 (STIM2) is frequently overexpressed in colorectal tumors and confers a tumor cell growth suppressor phenotype.

Allelic imbalances at chromosome 4p have been largely documented in many different tumor types. In colorectal cancer, loss of heterozygosity (LOH) at 4p15 has been associated with tumor aggressiveness and poor patient outcome, however no target genes in the region have been identified to date. Since stromal interaction molecule 2 (STIM2) is located at 4p15.2 and has been proposed as a candidate gene for this region in glioblastoma multiforme, we aimed at investigating the role of STIM2 in colorectal cancer. We studied STIM2 transcript expression levels in a collection of xenografted primary colorectal tumors (n = 20) and a well-annotated tumor series of colorectal cancer (n = 140). We observed an overexpression of STIM2 in 63.5% of the cases that was associated with a less invasive phenotype. In vitro and in vivo functional studies with colon cancer cell lines revealed that overexpression of STIM2 reduced cell proliferation and tumor growth, respectively. Our work presents several lines of evidence indicating that STIM2 overexpression is a frequent trait in colorectal cancer that results in cell growth suppression, certifying that even in the absence of somatic genetic or epigenetic alterations, recurrent regions of LOH should still be considered a hallmark for the presence of relevant genes for tumorigenesis.

A High-Throughput Screening Platform Identifies Novel Combination Treatments for Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumors (MPNST) are soft-tissue sarcomas that are the leading cause of mortality in patients with Neurofibromatosis type 1 (NF1). Single chemotherapeutic agents have shown response rates ranging from 18% to 44% in clinical trials, so there is still a high medical need to identify chemotherapeutic combination treatments that improve clinical prognosis and outcome. We screened a collection of compounds from the NCATS Mechanism Interrogation PlatE (MIPE) library in three MPNST cell lines, using cell viability and apoptosis assays. We then tested whether compounds that were active as single agents were synergistic when screened as pairwise combinations. Synergistic combinations in vitro were further evaluated in patient-derived orthotopic xenograft/orthoxenograft (PDOX) athymic models engrafted with primary MPNST matching with their paired primary-derived cell line where synergism was observed. The high-throughput screening identified 21 synergistic combinations, from which four exhibited potent synergies in a broad panel of MPNST cell lines. One of the combinations, MK-1775 with Doxorubicin, significantly reduced tumor growth in a sporadic PDOX model (MPNST-SP-01; sevenfold) and in an NF1-PDOX model (MPNST-NF1-09; fourfold) and presented greater effects in TP53 mutated MPNST cell lines. The other three combinations, all involving Panobinostat (combined with NVP-BGT226, Torin 2, or Carfilzomib), did not reduce the tumor volume in vivo at noncytotoxic doses. Our results support the utility of our screening platform of in vitro and in vivo models to explore new therapeutic approaches for MPNSTs and identified that combination MK-1775 with Doxorubicin could be a good pharmacologic option for the treatment of these tumors.

Generation and Integrated Analysis of Advanced Patient-Derived Orthoxenograft Models (PDOX) for the Rational Assessment of Targeted Therapies in Endometrial Cancer.

Clinical management of endometrial cancer (EC) is handicapped by the limited availability of second line treatments and bona fide molecular biomarkers to predict recurrence. These limitations have hampered the treatment of these patients, whose survival rates have not improved over the last four decades. The advent of coordinated studies such as The Cancer Genome Atlas Uterine Corpus Endometrial Carcinoma (TCGA_UCEC) has partially solved this issue, but the lack of proper experimental systems still represents a bottleneck that precludes translational studies from successful clinical testing in EC patients. Within this context, the first study reporting the generation of a collection of endometrioid-EC-patient-derived orthoxenograft (PDOX) mouse models is presented that is believed to overcome these experimental constraints and pave the way toward state-of-the-art precision medicine in EC. The collection of primary tumors and derived PDOXs is characterized through an integrative approach based on transcriptomics, mutational profiles, and morphological analysis; and it is demonstrated that EC tumors engrafted in the mouse uterus retain the main molecular and morphological features from analogous tumor donors. Finally, the molecular properties of these tumors are harnessed to assess the therapeutic potential of trastuzumab, a human epidermal growth factor receptor 2 (HER2) inhibitor with growing interest in EC, using patient-derived organotypic multicellular tumor spheroids and in vivo experiments.

p53 wild-type colorectal cancer cells that express a fetal gene signature are associated with metastasis and poor prognosis.

Current therapy against colorectal cancer (CRC) is based on DNA-damaging agents that remain ineffective in a proportion of patients. Whether and how non-curative DNA damage-based treatment affects tumor cell behavior and patient outcome is primarily unstudied. Using CRC patient-derived organoids (PDO)s, we show that sublethal doses of chemotherapy (CT) does not select previously resistant tumor populations but induces a quiescent state specifically to TP53 wildtype (WT) cancer cells, which is linked to the acquisition of a YAP1-dependent fetal phenotype. Cells displaying this phenotype exhibit high tumor-initiating and metastatic activity. Nuclear YAP1 and fetal traits are present in a proportion of tumors at diagnosis and predict poor prognosis in patients carrying TP53 WT CRC tumors. We provide data indicating the higher efficacy of CT together with YAP1 inhibitors for eradication of therapy resistant TP53 WT cancer cells. Together these results identify fetal conversion as a useful biomarker for patient prognosis and therapy prescription.

Combination of chemotherapy with BRAF inhibitors results in effective eradication of malignant melanoma by preventing ATM-dependent DNA repair.

Invasive malignant melanoma (MM) is an aggressive tumor with no curative therapy in advanced stages. Chemotherapy has not demonstrated its efficacy in MM and current treatment for tumors carrying the most frequent BRAFV600E mutation consists of BRAF inhibitors alone or in combination with MAPK pathway inhibitors. We previously found that BRAF inhibition prevents activation of the DNA-damage repair (DDR) pathway in colorectal cancer thus potentiating the effect of chemotherapy. We now show that different chemotherapy agents inflict DNA damage in MM cells, which is efficiently repaired, associated with activation of the ATM-dependent DDR machinery. Pharmacologic inhibition of BRAF impairs ATM and DDR activation in these cells, leading to sustained DNA damage. Combination treatments involving DNA-damaging agents and BRAF inhibitors increase tumor cell death in vitro and in vivo, and impede MM regrowth after treatment cessation. We propose to reconsider the use of chemotherapy in combination with BRAF inhibitors for MM treatment.

Extramedullary multiple myeloma patient-derived orthotopic xenograft with a highly altered genome: combined molecular and therapeutic studies.

Extramedullary multiple myeloma (EMM) has an overall survival of 6 months and occurs in 20% of multiple myeloma (MM) patients. Genetic and epigenetic mechanisms involved in EMM and the therapeutic role of new agents for MM are not well established. Besides, well-characterized preclinical models for EMM are not available. Herein, a patient-derived orthotopic xenograft (PDOX) was generated from a patient with an aggressive EMM to study in-depth genetic and epigenetic events, and drug responses related to extramedullary disease. A fresh punch of an extramedullary cutaneous lesion was orthotopically implanted in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ(NSG) mouse. The PDOX mimicked histologic and phenotypic features of the tumor of the patient. Cytogenetic studies revealed a hyperploid genome with multiple genetic poor-prognosis alterations. Copy number alterations (CNAs) were detected in all chromosomes. The IGH translocation t(14;16)(q32;q23)IGH/MAF was already observed at the medullary stage and a new one, t(10;14)(p?11-12;q32), was observed only with extramedullary disease and could be eventually related to EMM progression in this case. Exome sequencing showed 24 high impact single nucleotide variants and 180 indels. From the genes involved, only TP53 was previously described as a driver in MM. A rather balanced proportion of hyper/hypomethylated sites different to previously reported widespread hypomethylation in MM was also observed. Treatment with lenalidomide, dexamethasone and carfilzomib showed a tumor weight reduction of 90% versus non-treated tumors, whereas treatment with the anti-CD38 antibody daratumumab showed a reduction of 46%. The generation of PDOX from a small EMM biopsy allowed us to investigate in depth the molecular events associated with extramedullary disease in combination with drug testing.

Use of patient derived orthotopic xenograft models for real-time therapy guidance in a pediatric sporadic malignant peripheral nerve sheath tumor.

BACKGROUND: The aim of this study was to test the feasibility and utility of developing patient-derived orthotopic xenograft (PDOX) models for patients with malignant peripheral nerve sheath tumors (MPNSTs) to aid therapeutic interventions in real time. PATIENT & METHODS: A sporadic relapsed MPNST developed in a 14-year-old boy was engrafted in mice, generating a PDOX model for use in co-clinical trials after informed consent. SNP-array and exome sequencing was performed on the relapsed tumor. Genomics, drug availability, and published literature guided PDOX treatments. RESULTS: A MPNST PDOX model was generated and expanded. Analysis of the patient's relapsed tumor revealed mutations in the MAPK1, EED, and CDK2NA/B genes. First, the PDOX model was treated with the same therapeutic regimen as received by the patient (everolimus and trametinib); after observing partial response, tumors were left to regrow. Regrown tumors were treated based on mutations (palbociclib and JQ1), drug availability, and published literature (nab-paclitaxel; bevacizumab; sorafenib plus doxorubicin; and gemcitabine plus docetaxel). The patient had a lung metastatic relapse and was treated according to PDOX results, first with nab-paclitaxel, second with sorafenib plus doxorubicin after progression, although a complete response was not achieved and multiple metastasectomies were performed. The patient is currently disease free 46 months after first relapse. CONCLUSION: Our results indicate the feasibility of generating MPNST-PDOX and genomic characterization to guide treatment in real time. Although the treatment responses observed in our model did not fully recapitulate the patient's response, this pilot study identify key aspects to improve our co-clinical testing approach in real time.

PRL-3 overexpression in epithelial cells is induced by surrounding stromal fibroblasts.

We isolate and culture carcinoma-associated fibroblasts (CAFs) from primary tumour (CAFpt), CAFs from corresponding synchronous liver metastasis (CAFlm) as well as normal colonic fibroblasts (NCF) from the same patient. From these cultures, conditioned media (CM) was obtained. Culture of a wide panel of colorectal and pancreatic cell lines in CM from CAFlm resulted in overexpression of mRNA PRL-3 and higher overexpression in CAFs than in non-activated fibroblasts. Moreover PRL-3 mRNA expression correlates with expression of alpha-SMA and deposition of collagen fibrils in the stroma. We demonstrate that products secreted by CAFs trigger PRL-3 overexpression in cancer cells. Identification of these factors may contribute to new stroma-targeted therapies for desmoplastic tumours.

The transcribed pseudogene RPSAP52 enhances the oncofetal HMGA2-IGF2BP2-RAS axis through LIN28B-dependent and independent let-7 inhibition.

One largely unknown question in cell biology is the discrimination between inconsequential and functional transcriptional events with relevant regulatory functions. Here, we find that the oncofetal HMGA2 gene is aberrantly reexpressed in many tumor types together with its antisense transcribed pseudogene RPSAP52. RPSAP52 is abundantly present in the cytoplasm, where it interacts with the RNA binding protein IGF2BP2/IMP2, facilitating its binding to mRNA targets, promoting their translation by mediating their recruitment on polysomes and enhancing proliferative and self-renewal pathways. Notably, downregulation of RPSAP52 impairs the balance between the oncogene LIN28B and the tumor suppressor let-7 family of miRNAs, inhibits cellular proliferation and migration in vitro and slows down tumor growth in vivo. In addition, high levels of RPSAP52 in patient samples associate with a worse prognosis in sarcomas. Overall, we reveal the roles of a transcribed pseudogene that may display properties of an oncofetal master regulator in human cancers.

Orthoxenografts of Testicular Germ Cell Tumors Demonstrate Genomic Changes Associated with Cisplatin Resistance and Identify PDMP as a Resensitizing Agent.

Purpose: To investigate the genetic basis of cisplatin resistance as efficacy of cisplatin-based chemotherapy in the treatment of distinct malignancies is often hampered by intrinsic or acquired drug resistance of tumor cells.Experimental Design: We produced 14 orthoxenograft transplanting human nonseminomatous testicular germ cell tumors (TGCT) in mice, keeping the primary tumor features in terms of genotype, phenotype, and sensitivity to cisplatin. Chromosomal and genetic alterations were evaluated in matched cisplatin-sensitive and their counterpart orthoxenografts that developed resistance to cisplatin in nude mice.Results: Comparative genomic hybridization analyses of four matched orthoxenografts identified recurrent chromosomal rearrangements across cisplatin-resistant tumors in three of them, showing gains at 9q32-q33.1 region. We found a clinical correlation between the presence of 9q32-q33.1 gains in cisplatin-refractory patients and poorer overall survival (OS) in metastatic germ cell tumors. We studied the expression profile of the 60 genes located at that genomic region. POLE3 and AKNA were the only two genes deregulated in resistant tumors harboring the 9q32-q33.1 gain. Moreover, other four genes (GCS, ZNF883, CTR1, and FLJ31713) were deregulated in all five resistant tumors independently of the 9q32-q33.1 amplification. RT-PCRs in tumors and functional analyses in Caenorhabditis elegans (C. elegans) indicate that the influence of 9q32-q33.1 genes in cisplatin resistance can be driven by either up- or downregulation. We focused on glucosylceramide synthase (GCS) to demonstrate that the GCS inhibitor DL-threo-PDMP resensitizes cisplatin-resistant germline-derived orthoxenografts to cisplatin.Conclusions: Orthoxenografts can be used preclinically not only to test the efficiency of drugs but also to identify prognosis markers and gene alterations acting as drivers of the acquired cisplatin resistance. Clin Cancer Res; 24(15); 3755-66. ©2018 AACR.

Tumor xenograft modeling identifies an association between TCF4 loss and breast cancer chemoresistance.

Understanding the mechanisms of cancer therapeutic resistance is fundamental to improving cancer care. There is clear benefit from chemotherapy in different breast cancer settings; however, knowledge of the mutations and genes that mediate resistance is incomplete. In this study, by modeling chemoresistance in patient-derived xenografts (PDXs), we show that adaptation to therapy is genetically complex and identify that loss of transcription factor 4 (TCF4; also known as ITF2) is associated with this process. A triple-negative BRCA1-mutated PDX was used to study the genetics of chemoresistance. The PDX was treated in parallel with four chemotherapies for five iterative cycles. Exome sequencing identified few genes with de novo or enriched mutations in common among the different therapies, whereas many common depleted mutations/genes were observed. Analysis of somatic mutations from The Cancer Genome Atlas (TCGA) supported the prognostic relevance of the identified genes. A mutation in TCF4 was found de novo in all treatments, and analysis of drug sensitivity profiles across cancer cell lines supported the link to chemoresistance. Loss of TCF4 conferred chemoresistance in breast cancer cell models, possibly by altering cell cycle regulation. Targeted sequencing in chemoresistant tumors identified an intronic variant of TCF4 that may represent an expression quantitative trait locus associated with relapse outcome in TCGA. Immunohistochemical studies suggest a common loss of nuclear TCF4 expression post-chemotherapy. Together, these results from tumor xenograft modeling depict a link between altered TCF4 expression and breast cancer chemoresistance.

Absence of MxA induction is related to a poor clinical response to interferon beta treatment in multiple sclerosis patients.

The aim of this study is to investigate whether induction of myxovirus resistance protein A (MxA) mRNA after 3 months of interferon-ß administration is related to the treatment response in multiple sclerosis (MS) patients. In this prospective study, MS patients were enrolled before starting treatment. Demographic, clinical and radiological variables were recorded. Blood samples were obtained before, and at 3 and 12 months after interferon-ß treatment. Real-time PCR was used to analyze MxA mRNA expression. Patients were classified as MxA-low or -high depending on MxA levels at baseline, and as MxA-induced or -non-induced according to whether an increase in MxA expression was detected at month 3. Time to the next relapse was investigated using Cox proportional hazards regression analysis. One hundred and four patients were selected and followed for a median of 2.2 years (IQR 1.6-3.5). On Cox regression analysis, a higher EDSS score before treatment (HR 1.57; 95 % CI 1.02-2.40; p = 0.039), MxA-high status at baseline (HR 2.71; 95 % CI 1.26-5.81; p = 0.010), and MxA-non-induced at month 3 (HR 2.49; 95 % CI 1.08-5.68; p = 0.031), were predictors of poor response to interferon-ß in naïve MS patients. Patients showing a lower capacity for MxA induction following 3 months of interferon-ß treatment are more likely to be non-responders to this therapy.

MxA mRNA expression as a biomarker of interferon beta response in multiple sclerosis patients.

Myxovirus resistance protein A (MxA) is a molecule induced after interferon-ß injection. The aim of this study was to investigate whether MxA determination one year after starting interferon-ß can predict treatment response in multiple sclerosis patients. MxA mRNA expression was evaluated in blood samples obtained at baseline and at month 12. Clinical variables were prospectively recorded. A threshold of 5 was defined to establish MxA induction. On survival analysis, time to the next relapse and to EDSS progression were significantly longer in patients showing MxA induction, suggesting that MxA induction after one year may be useful to identify interferon-ß responders.

BRAF-induced tumorigenesis is IKKα-dependent but NF-κB-independent.

KRAS mutations contribute to cell proliferation and survival in numerous cancers, including colorectal cancers (CRC). One pathway through which mutant KRAS acts is an inflammatory pathway that involves the kinase IKK and activates the transcription factor NF-κB. BRAF, a kinase that is downstream of KRAS, is mutated in a subset of CRC and is predictive of poor prognosis and therapeutic resistance. We found that, in contrast to mutant KRAS, mutant BRAF (BRAF(V600E)) did not trigger NF-κB activation but instead triggered the phosphorylation of a proteolytic fragment of IKKα (p45-IKKα) in CRC cells. BRAF(V600E) CRC cells had a high abundance of phosphorylated p45-IKKα, which was decreased by a RAF inhibitor. However, the abundance and DNA binding of NF-κB in these cells were unaffected by the RAF inhibitor, and expression of BRAF(V600E) in human embryonic kidney-293T cells did not activate an NF-κB reporter. Moreover, BRAF-induced transformation of NIH-3T3 cells and BRAF-dependent transcription required phosphorylation of p45-IKKα. The kinase TAK1, which was associated with the endosomal compartment, phosphorylated p45-IKKα. Inhibition of endosomal vacuolar adenosine triphosphatase (V-ATPase) with chloroquine or bafilomycin A1 blocked p45-IKKα phosphorylation and induced apoptosis in BRAF-mutant CRC cells independent of autophagy. Treating mice with V-ATPase inhibitors reduced the growth and metastasis of BRAF(V600E) xenograft tumors in the cecum of mice.