Rapid activation of ARF6 after RAF inhibition augments BRAFV600E and promotes therapy resistance

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Sorensen, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7133814/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Apr, 2026 Read the published version in Oncogene → Version 1 posted You are reading this latest preprint version Abstract The intrinsic ability of cancer cells to evade death underpins tumorigenesis, progression, metastasis and the survival of drug-tolerant persister (DTP) cells. Herein, we discovered that when activated, the small GTPase ARF6 plays a central role in tumor survival by facilitating expression of the BRAF V600E oncoprotein. Tumor-specific Arf6 deletion caused a significant reduction in BRAF V600E protein and MAPK signaling and prevented rapid tumor progression. In the context of targeted therapy, BRAF inhibition induced swift activation of ARF6, driving a positive feedback loop that restored MAPK-driven anti-apoptotic signaling, facilitated DTP cell survival during the early phases of treatment and contributed to drug-tolerant growth. In patient-derived melanoma cells with innate or clinically acquired resistance to MAPK inhibitors, ARF6 inhibition enhanced sensitivity to combined BRAF + MEK inhibition. Collectively, these findings elucidate an ARF6-dependent mechanism of BRAF oncoprotein synthesis that may be exploited in BRAF V600E driven cancers as a therapeutic vulnerability. Health sciences/Oncology/Cancer/Cancer therapy/Cancer therapeutic resistance Biological sciences/Cancer/Cancer models Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Avoiding cell death is fundamental to the progressive acquisition of hallmark cancer behaviors 1 , and to the survival of drug tolerant persister (DTP) cells that give rise to therapy resistance 2 . Proposed origins of DTP cells include clonal selection of pre-existing drug-resistant cells, and drug-induction of a reversible DTP state that enables the outgrowth of fixed resistance 3 , 4 . The molecular mechanisms of DTP cell emergence are complex and incompletely understood. Mitogen activated protein kinase (MAPK) signaling is hyperactivated in many cancer types and directly opposes cell death by inactivating pro-apoptotic proteins of the intrinsic pathway 5 . In cutaneous melanoma, where most cases harbor somatic mutations in BRAF, NRAS or NF1 that cause aberrant MAPK signaling 6 , resistance to MAPK targeted therapy remains a significant clinical challenge 7 . We detected abnormally elevated levels of ARF6-GTP, the active form of the small GTPase ARF6, in melanoma 8 . ARF6 activation in melanoma can occur through extracellular signals such as HGF 9 , 10 , WNT5a 11 and Interferon-g 12 ; or by altered expression of guanine exchange factors (GEFs) or GTPase activating proteins (GAPs) 8 , 12 , 13 . HGF and WNT5a signaling also mediate resistance to MAPK targeted therapy in melanoma 14 – 17 . We showed that ARF6-GTP promotes tumor development, progression, and acceleration of metastasis in murine models of BRAF V600E melanoma 8 , 11 , 12 . Functionally, ARF6 activation enhances tumor cell invasion and adaptive immune suppression 8 , 9 , 11 , 12 . Hence, ARF6 mediates the acquisition of at least two hallmark malignant behaviors 1 . Whether ARF6 is involved in DTP cell biology is unknown. ARF6 is a ubiquitously expressed protein critical for endomembrane trafficking and actin cytoskeleton remodeling 18 and has diverse physiologic roles across multiple organ systems 19 – 30 . To expand our understanding of ARF6 function in cancer, we interrogated proteomic alterations induced by ARF6 activation and discovered that ARF6 dynamically regulates expression of the BRAF oncoprotein in melanoma and other cancer types, impacting tumor cell survival, including during MAPK targeted therapy. RESULTS ARF6 augments a dynamic pool of oncogenic BRAF protein In early passage murine melanoma cell lines with homozygous BRAF V600E mutation, derived from our genetically engineered murine melanoma models 8 , 12 , proteomic analysis showed higher levels of BRAF V600E protein, and increased phosphorylated MEK1, ERK, RSK and Jun, in cells expressing constitutively active ARF6-GTP (ARF6 Q67L ) compared to ARF6 WT ( Fig. 1a ). In contrast, p38 MAPK-JNK signaling was unaltered ( Fig. 1a ). ARF6-GTP-induced BRAF V600E expression was confirmed by Western blot ( Fig. 1b ). These findings align with our previously published genomic data from this tumor model showing upregulation of genes in the MAPK cascade in bulk tumor transcriptomes 8 . Based on these findings, we hypothesized that ARF6 controls MAPK signaling by regulating oncogenic BRAF expression. In pursuit of this, we interrogated human melanoma cells and found that doxycycline-induced, ectopically expressed ARF6-GTP, in the form of ARF6 Q67L ( Fig. S1 a ), or adenoviral delivered ARF6 Q67L , augmented endogenous BRAF V600E expression in human melanoma cells ( Fig. 1c-d ). Consistent with genetic activation of ARF6, pharmacological activation of ARF6 with QS11 ( Fig. S1 b ), an inhibitor of ARF GTPase Activating Protein 1 31 , increased BRAF V600E protein expression in human melanoma, colorectal carcinoma and glioma cell lines ( Fig. 1e, S1c ). BRAF V600E protein levels rose quickly after treatment with the ARF6 agonist QS11, as early as two hours, and continued to accumulate over 48 hours ( Fig. 1f ). These data demonstrate that sustained ARF6 activation is sufficient to acutely increase endogenous BRAF V600E protein. The BRAF V600E oncoprotein is stabilized by the chaperone protein HSP90 32, 33 , limiting proteasome-mediated degradation. Unlike HSP90, ARF6 prevents lysosome mediated degradation of proteins through endosomal recycling 12 , 34 , 35 . Thus, we asked if BRAF V600E might be degraded by the lysosome. Blocking lysosomal degradation by Bafilomycin A1 failed to increase BRAF V600E protein ( Fig. S1 d ). Thus, it is unlikely that ARF6 regulates oncogenic BRAF expression through endolysosomal trafficking. ARF6-GTP did not alter HSP90 protein expression ( Fig. S1 e ). Nevertheless, the ARF6-regulated pool of BRAF V600E was HSP90 dependent because inhibition of HSP90 with 17-AAG prevented accumulation of BRAF V600E protein after QS11 treatment ( Fig. 1g ). Surprisingly, ARF6 mediated BRAF V600E expression was dependent on protein translation. Specifically, inhibition of protein translation with cycloheximide prevented the accumulation of BRAF V600E protein upon ARF6 activation (Fig. 1h). Activation of ARF6 failed to alter BRAF mRNA levels in A375 melanoma cells, which harbor a homozygous BRAF V600E mutation (Fig. 1i) , demonstrating that ARF6-mediated upregulation of BRAF V600E occurred without altering BRAF oncogene expression. These data confirm that ARF6 activation is sufficient to increase BRAF V600E protein levels and suggest a previously unknown role for ARF6 in regulating protein translation. ARF6 is necessary for maintenance of the BRAF protein In contrast to ARF6 activation, deletion of Arf6 in BRAF V600E murine melanoma tumors 12 reduced total BRAF V600E levels and downstream phosphorylated MEK (p-MEK) and ERK (p-ERK), detected by immunofluorescence in situ ( Fig. 2a, S1f ). Consistently, silencing of Arf6 downregulated BRAF V600E and p-MEK detection in murine melanoma cells ( Fig. 2b ). To test whether inactivation of ARF6 (ARF6-GDP) could produce the same effect, we treated human melanoma cells with SecinH3, an ARF6 guanine exchange factor inhibitor that reduces ARF6-GTP levels 11 , 36 ( Fig. S1 g ) and reduces spontaneous metastasis of human BRAF V600E melanoma xenograft tumors 11 . In human melanoma cells, SecinH3 significantly reduced BRAF V600E protein within 48 hours of treatment ( Fig. 2c ). NAV-2729, a direct inhibitor of ARF6 GTPase function 13 ( Fig. S1 h ), also reduced BRAF V600E protein after 48 hours ( Fig. 2d ). Finally, ectopic expression of inactive ARF6 (ARF6-GDP), in the form of ARF6 T27N , reduced BRAF V600E protein ( Fig. 2e ), suggesting that ARF6 activation is necessary to maintain expression of endogenous BRAF V600E . Together these data demonstrate that ARF6 may be necessary to maintain steady state levels of the BRAF V600E oncoprotein and suggest that targeted inhibition of ARF6 might be an alternative approach to reducing BRAF V600E oncoprotein expression. ARF6-GTP promotes tumor survival by protecting against apoptosis Because MAPK signaling opposes the intrinsic apoptotic signaling pathway 5 , we reasoned that ARF6-mediated fluctuations in BRAF V600E protein might be linked to survival. Proteomic clues to ARF6-mediated survival were evident in murine melanoma cell lines cultured in full serum ( Fig. 3a-b ). Compared to ARF6 WT , cells expressing ARF6 Q67L showed significantly increased levels of the anti-apoptotic protein MCL-1 and phosphorylation (inactivation) of BAD at residue S112 (pS112) 5 , as well as decreased levels of pro-apoptotic proteins BAX and FOXO3 ( Fig. 3a ). ARF6 dependent expression of MCL-1 and FOXO3 were confirmed by Western blot ( Fig. 3b ). ERK signaling has been reported to increase MCL-1 37 and decrease FOXO3 38 protein levels 5 . Thus, our data suggest that ARF6 activation might promote tumor cell survival through ERK-mediated anti-apoptotic signaling. To test whether ARF6 activation could protect against apoptosis, we deployed a doxycycline-inducible system to express either ectopic ARF6 Q67L or ARF6 WT in human melanoma cells ( Fig. S1 a, S2a ). Doxycycline alone did not alter viability of A375 parental cells, ( Fig. S2b ), while doxycycline-induced ARF6 Q67L significantly reduced apoptosis caused by serum withdrawal ( Fig. 3c ). In contrast, doxycycline-induced ectopic expression of ARF6 WT did not alter apoptosis caused by serum withdrawal ( Fig. 3c ), suggesting that the active form of ARF6 is required for the survival benefit. Consistent with ARF6 Q67L , pharmacological activation of ARF6 with QS11 protected against apoptosis caused by serum starvation ( Fig. 3d ). QS11 alone failed to alter cell viability during steady-state conditions, when cells were cultured in full serum ( Fig. S2c ), indicating that the compound does not stimulate proliferation. Overall, these data demonstrate that ARF6 activation can protect against apoptosis during growth signal deprivation. Given that ARF6 can regulate both PI3K-AKT 8 and BRAF V600E -MAPK signaling ( Fig. 1 ) and apoptosis upon serum withdrawal ( Fig. 3c-d ), we asked if ARF6 supports the viability of BRAF-mutant human cancer cells grown in full serum. Consistent with this, ARF6 silencing led to significantly reduced viability in multiple human melanoma cell lines ( Fig. 3e ). Similarly, treatment with NAV-2729, a direct inhibitor of ARF6 GTPase function 13 , reduced ARF6-GTP levels ( Fig. S1 h ) and decreased cell viability in most of the human melanoma cells tested ( Fig. S2d ), although not as effectively as ARF6 silencing ( Fig. 3e ). These data demonstrate that ARF6 can optimize survival during normal growth conditions. ARF6 is required for accelerated tumor progression caused by PTEN loss In parallel with the MAPK pathway, survival signaling can also originate from the PI3K-AKT pathway 39 and we previously reported that activation of ARF6 enhanced PI3K expression and PI3K-AKT signaling 8 . PTEN loss of function mutations activate the PI3K-AKT pathway, are frequently detected in cutaneous melanoma 6 , cooperate with mutant BRAF or NRAS to drive melanomagenesis 40 , 41 , and accelerate primary tumor growth in genetically engineered Dct::TVA, Braf V600E ; Cdkn2a flox/flox murine melanoma models induced in epidermal melanocytes of the ear pinnae 42 . Like pinnae tumors, deletion of Pten dramatically accelerated the growth of BRAF V600E melanoma induced in the flank ( Fig. 3f ). To test the necessity of ARF6 in this highly aggressive model, we crossed Arf6 flox/flox ( Arf6 f/f ) mice with the Dct::TVA, Braf V600E ; Cdkn2a f/f ; Pten f/f mice. In this model, tumor-specific loss of Arf6 significantly reduced tumor growth to a level equivalent to Pten WT tumors (measured from the time of tumor formation, Fig. 3g ), and prolonged overall survival despite the absence of PTEN ( Fig. 3h ). Unlike Pten WT mice 12 , loss of ARF6 did not reduce overall tumor incidence in Pten f/f mice ( Fig. S2e ), demonstrating that loss of PTEN is sufficient to overcome the weakened tumor initiation phenotype we previously observed with Arf6 knockout. Nevertheless, loss of ARF6 significantly delayed tumor onset in Pten f/f mice ( Fig. S2e ). Consistent with the Pten WT tumor cell lines ( Fig. 3b ), tumors from Pten f/f ; Arf6 f/f mice showed increased levels of pro-apoptotic proteins BAK and BIM ( Fig. 3i ), suggesting enhanced apoptosis signaling in the absence of ARF6. Given that Arf6 deletion prevented primary tumor acceleration caused by PTEN loss ( Fig. 3f-g ), there is a component of ARF6-dependent survival that is necessary for, and/or functions independently of the PI3K pathway. Indeed, ARF6-dependent survival may also originate from rheostatic control of BRAF V600E expression ( Figs. 1–2 ) and downstream, MAPK-mediated anti-apoptotic signaling. ARF6 is activated by RAF inhibition, protects against MAPK inhibitor-induced apoptosis, and potentiates resistance to MAPK inhibition Because ARF6 can regulate BRAF V600E protein expression ( Figs. 1–2 ), we asked if BRAF inhibition alters ARF6 activation. Remarkably, class I BRAF inhibitors, vemurafenib or dabrafenib, increased ARF6-GTP levels ( Fig. 4a ). This occurred both in the presence and absence of serum and is reproducible in independent BRAF V600E cell lines ( Fig. 4a and S3a ). Notably, the pan-mutant BRAF inhibitor PF-07799933, which inhibits BRAF mutant monomers and dimers and has antitumor activity in treatment refractory patients 43 , also increased ARF6-GTP levels in human melanoma ( Fig. 4a ). Importantly, ARF6 activation occurred rapidly after BRAF inhibition, as early as one hour ( Fig. 4a-b ), suggesting that ARF6 activation functions in an acute adaptive response pathway to BRAF-targeted therapy. Because ARF6 was rapidly activated upon RAF inhibition and ARF6-GTP promoted survival upon serum withdrawal ( Figs. 4a-b, 3c-d ), we asked whether ARF6 activation can facilitate survival during MAPK inhibitor (MAPKi) treatment. Indeed, genetic activation of ARF6 dramatically reduced apoptosis after 48 hours of vemurafenib ( Fig. 4c ), whereas silencing of Arf6 significantly increased apoptosis induced by vemurafenib ( Fig S3b ), consistent with a role for ARF6 in early tumor cell survival during targeted therapy. Overexpression of wildtype ARF6 also decreased vemurafenib-induced apoptosis, but to a lesser extent than ARF6 Q67L ( Fig. 4c ). Similar to ARF Q67L , pharmacological activation of ARF6 with QS11 almost completely abrogated vemurafenib induced apoptosis ( Fig. 4d ). Combination RAF + MEK inhibition is the preferred choice of MAPKi therapy in BRAF V600E melanoma patients, due to superior clinical outcomes compared to single agent RAF inhibition 44 . Thus, we interrogated ARF6 in this context. A375 melanoma cells are highly sensitive to both single-agent RAF inhibition and combination RAF + MEK inhibition in short-term cultures ( Fig S3c-d ). In contrast, A2058 melanoma cells are resistant to vemurafenib ( Fig S3c ), possibly due to a MAP2K1 P124S mutation 45 , but remain sensitive to the combination of dabrafenib + trametinib (Dab + Tram) ( Fig S3d ). Importantly, genetic or pharmacologic activation of ARF6 reduced Dab + Tram sensitivity in these cell lines by significantly reducing apoptosis ( Fig. 4e-f ). These combined data suggest that the consequence of ARF6 activation upon BRAF inhibition ( Fig. 4a-b ) might be the emergence of resistance. Because ARF6 activation can fortify BRAF V600E protein ( Fig. 1 ), we reasoned that ARF6 might facilitate recovery of MAPK signaling after RAF inhibition. Indeed, ARF6 activation by QS11 resulted in a markedly faster recovery of phosphorylated ERK (pERK) after vemurafenib treatment ( Fig. 4g and Fig. S3e ). Additional evidence that ARF6-GTP boosted MAPK recovery manifested in ERK-mediated inhibition of the apoptotic proteins BAD and BIM 5 . Unlike the control, QS11 significantly recovered ERK-mediated phosphorylation (inhibition) of BAD 24–48 hours after vemurafenib ( Fig. 4g and S3e ). Furthermore, downregulation of BIM was more pronounced with QS11 ( Fig. 4g and S3e ). These findings demonstrate that ARF6 activation can potentiate MAPK reactivation and anti-apoptotic signaling after BRAF inhibition. To test if ARF6-GTP promotes the emergence of DTP cells, leading to therapy resistance, we quantified colony formation during vemurafenib ( Fig. 4h ) or Dab + Tram treatment ( Fig. 4i ). Activation of ARF6 with QS11 significantly increased drug-resistant colony formation in both conditions ( Fig. 4h-i, S3f-g ). Hence, our overall data supports that ARF6 is activated in the early phases of adaptive resistance, acutely responding to diminished MAPK signaling, and facilitating the survival of drug-tolerant persister cells in melanoma. ARF6 inhibition sensitizes patient-derived, MAPK inhibitor-resistant melanoma cells Because ARF6 activation significantly reduced tumor cell death after MAPKi ( Fig. 4c-f ), we asked whether inhibition of ARF6 could sensitize melanoma to clinically acquired or innate MAPKi resistance. For this, we pivoted to early-passage, patient-derived xenograft (PDX) melanoma cell lines ( Table S1 , Fig. 5a ). We recently reported that the MET gene is amplified in MTG013/CM013 PDX cells 46 , which may explain the patient’s history of disease progression through vemurafenib treatment because HGF-MET signaling is a common mechanism of reactivation of MAPK signaling after RAFi 14 . Similar to the patient’s clinical outcome (progression through vemurafenib), MTG013 PDXs are resistant to high dose Dab + Tram 47 . We transduced these PDX cells with a doxycycline-inducible shRNA construct to conditionally knockdown ARF6 expression after subcutaneous injection into immunodeficient NRG mice, or during in vitro colony forming assays ( Fig. 5a ). Doxycycline-induced knockdown of ARF6 significantly reduced tumor growth in vivo ( Fig. 5b ), demonstrating that ARF6 has a role in tumor progression that is independent of the ARF6-mediated adaptive immune suppression we observed in immunocompetent mice 12 . In vitro , MTG013 cells were increasingly resistant to rising concentrations of Dab + Tram ( Fig. 5c ), likely a result of progressive relief of an ERK negative feedback loop 5 and reactivation of MAPK signaling 48 . From these Dab + Tram dose responses, we chose a low and a high dose Dab + Tram regimen to test in combination with knockdown ( Fig. 5d-f ) or pharmacologic inhibition of ARF6 ( Fig. 5g, g, h ). Change in viability was measured over 48 hours of treatment. By itself, silencing ARF6 caused incomplete but significant loss of viability similar to Dab + Tram ( Fig. 5e ). Thus, inhibition of MAPK or ARF6 were equally cytostatic, but cell viability persisted above the baseline viability at time zero, indicating a low level of tumor cell survival (illustrated in Fig. 5d ). Importantly, silencing of ARF6 re-sensitized MTG013 cells to Dab + Tram ( Fig. 5e ). Specifically, when ARF6 knockdown was combined with Dab + Tram, there was a pronounced cytotoxic effect, where cell viability after 48 hours of treatment was less than time zero, and we observed this trend with both low and high combination doses of Dab + Tram ( Fig. 5e ). Consistently, silencing of ARF6 increased apoptosis induced by Dab + Tram ( Fig. 5f ). Like genetic depletion of ARF6, prevention of ARF6 activation with the ARF6 GEF inhibitor SecinH3 36 ( Fig. 5g ), or direct inhibition of ARF6 with NAV-2729 13 ( Fig. 5h ), decreased viability after Dab + Tram. NAV-2729 also significantly improved sensitivity to Dab + Tram during a 14- day colony outgrowth assay ( Fig. 5i ). Overall, the concordance between these orthogonal methods of ARF6 inhibition demonstrates reproducible efficacy in reversing clinically acquired MAPK inhibitor resistance. Unlike MTG013, MTG030 cells have an increased copy number of MAP2K1 ( Table S1 ), which encodes for the BRAF substrate and effector protein MEK1. In addition, HRAS is amplified. These genetic changes may explain why these PDX melanoma cells were tolerant of Dab + Tram ( Fig. 5j ). In fact, intermediate to high doses of Dab + Tram enhanced tumor cell viability/growth in the first 48 hours of treatment ( Fig. 5k , middle and right panels), and these cells appeared to be more resistant to MAPKi than MTG013 ( Fig. 5c ). The ARF6 GEF inhibitor, SecinH3, prevented the immediate burst in viability after Dab + Tram ( Fig. 5k) . Direct inhibition of ARF6 with NAV-2729 was cytotoxic when combined with low to intermediate doses of Dab + Tram ( Fig. 5l , left and middle panels). Similar to SecinH3, NAV-2729 prevented the burst of enhanced viability that occurred with high dose Dab + Tram ( Fig. 5l , right panel). With longer treatments (14 days), Dab + Tram reduced tumor colony formation, however, a low level of resistant tumor colonies persisted ( Fig. 5m ), and this was significantly diminished by knockdown of ARF6 ( Fig. 5m, S3h ). Hence, these data suggest that targeting ARF6 may render melanomas with resistance mutations more vulnerable to MAPK inhibitors. DISCUSSION We have shown that the small GTPase ARF6 helps maintain BRAF V600E protein expression through a post-transcriptional regulatory mechanism that stimulates BRAF V600E translation. Without ARF6-GTP, BRAF V600E protein levels gradually decline. Notably, ATP-competitive kinase inhibitors such as vemurafenib can reduce BRAF V600E protein levels by preventing the HSP90 co-chaperone protein CDC37 from binding BRAF 49 . In this context, our findings suggest that cancer cells activate ARF6 in a positive feedback loop to maintain BRAF V600E protein expression during kinase inhibition. Understanding how protein translation is deregulated in disease is important for the development of effective treatment approaches 50 . Messenger RNA translation occurs in cyclical bursts in mammalian cells 51 . Thus, a dynamic cycle of activation - deactivation of ARF6 might help stimulate pulsatile surges in BRAF V600E synthesis to maintain steady-state levels, particularly when BRAF inhibitors are present and trigger ARF6 activation. While more work is needed to understand the mechanistic underpinnings and the potential extent of ARF6 regulation of protein expression, our data suggests that sustained inhibition of ARF6 can diminish BRAF V600E levels and help overcome established resistance to MAPK targeted therapy ( Fig. 6 ). Our findings suggest that targeting ARF6 inhibits a stress-adaption pathway that gives rise to DTP cells. ARF6 mediated survival both during growth factor scarcity and MAPK targeted therapy. In the latter scenario, ARF6 was rapidly activated after initiation of RAFi treatment and mediated adaptive recovery of MAPK signaling. ARF6-GTP facilitated survival during the first few days of MAPKi therapy and enabled the eventual emergence of drug-resistant growth. Overall, our data support the hypothesis that DTP cells can be drug-induced 3 , 4 and provide mechanistic insights into how this phenomenon might occur in BRAF mutant cancers. Our findings not only help explain how BRAF-mutant melanoma survives the acute phases of MAPK inhibition, they also highlight an emerging theme of pro-invasive small GTPases that link mechanisms of tumorigenesis to drug resistance. Like ARF6, the small GTPase RAC1 facilitates invasion 52 , tumorigenesis 52 , 53 and resistance to MAPK targeted therapy 53 , 54 . Recently, RAC1 was shown to be activated by MEK inhibition 52 . Interestingly, RAC1 was activated in human melanoma cells between 8–16 hours after the initiation of treatment with trametinib. In contrast, ARF6 was activated within 1–2 hours of BRAF inhibition ( Fig. 4a-b ). The difference in kinetics could be due to the choice of MAPKi (MEK vs. BRAF), the use of different cell lines, or possibly due to distinct upstream mechanisms that result in serial activation of these small GTPases; ARF6 followed by RAC1. Unlike ARF6, however, activation of RAC1 was not reported to signal through the MAPK pathway 53 . Like RAC1 and ARF6, RhoA also has a role in MAPKi resistance, upstream of the focal adhesion kinase (FAK)-PI3K-AKT pathway 47 . To the best of our knowledge, RAC1 and RhoA have never been shown to regulate BRAF oncoprotein expression, which may be unique to ARF6. ARF6-dependent survival may also help explain why tumor-specific deletion of Arf6 significantly diminished tumor development and progression in BRAF V600E PTEN WT melanoma models 12 . While impaired tumor formation and sluggish growth were attributable to ARF6-dependent suppression of the adaptive immune response in that model 12 , our current findings suggest that ARF6 might also render tumor cells more resistant to apoptotic death incited by immune attack. More work is needed to understand ARF6-mediated tumor survival, including during immune-mediated tumor killing. By interrogating ARF6 in vitro and in immunodeficient mice, we removed the influence of adaptive immunity and discovered an unanticipated role for ARF6 in tumor cell survival. While our findings support a mechanism whereby ARF6 activation fortifies BRAF V600E protein synthesis, other ARF6 mechanisms may be at play. For example, we have previously shown that ARF6-GTP upregulated PI3K expression and AKT-signaling in melanoma while inhibition of ARF6 reduced PI3K and AKT activation 8 . In this current study, ARF6 was critical for tumor growth acceleration caused by loss of PTEN. Together these data support that ARF6 regulates the PI3K-AKT axis and as such, it is possible that ARF6 modulates PI3K-AKT driven anti-apoptotic signaling. Lastly, because ARF6 mediates internalization 55 and recycling 56 of integrins (i.e. focal adhesion turnover), ARF6 activity might be linked to FAK-dependent resistance to MAPK targeted therapy in melanoma 47 . Independent of these possibilities, our data reveal a previously unknown vulnerability in oncogenic BRAF signaling, ARF6, which may be exploitable for addressing DTP cell survival and targeted therapy resistance. METHODS Mouse husbandry, genotyping and RCAS virus delivery in vivo . Animal studies were performed in accordance with a protocol approved by the University of Utah Institutional Animal Care and Use Committee (IACUC). Generation of the Dct::TVA ; Braf V600E ; Cdkn2a f/f , Dct::TVA; Braf V600E ; Cdkn2a f/f ; Arf6 f/f , and Dct::TVA; Braf V600E ; Cdkn2a f/f ; Pten f/f murine models have been described previously 8 , 12 . The flank tumor incidence, onset, growth rate and overall survival were measured and calculated as described previously 12 . Both male and female animals were used in this study and were equally distributed across experimental groups. Prior analysis confirmed that sex does not influence tumor formation, tumor size, or survival onset in our model (PMID: 39098861, PMID: 33098202) For the PDX cell line (MTG013) model, all animal studies were approved by the University of Utah IACUC and were performed in accordance with relevant guidelines and regulations by the Huntsman Cancer Institute (HCI) Preclinical Research Resource (PRR) laboratory. 10 females and 10 males of six to eight-week-old NOD rag gamma (NGR, NOD- Rag1 null IL2rg null , NOD rag gamma, NOD-RG) mice, Jackson Laboratory stock 7799, were injected subcutaneously with 5 × 10 5 cells in matrigel. Mice were treated with or without Dox chow (Envigo: Global 18% Protein Rodent Diet with 625ppm doxycycline. Cat# TD.01306.) five days after injection. Mice were monitored for health weekly, and tumor size was measured twice weekly using digital calipers; the tumor volume was calculated using the following formula: (length × width 2 /2). Cell lines Authentication of all human melanoma cell lines were periodically confirmed by STR profiling in the University of Utah Genomics core facility using the Promega (Madison, WI) GenePrint 10 system, or by ATCC. A375, LOX-IMVI, UACC.62, were provided by Dr. M. VanBrocklin, HCI. A2058 cells were purchased from the ATCC (Cat# CRL11147D). SKMEL28 cells were provided by Dr. D. Grossman, HCI. A2058 and A375 were maintained in DMEM-high glucose (ThermoFisher Scientific, Cat# 11995073) supplemented with 10% v/v FBS (Atlas Biologicals, Cat# F-0500-DR), 1% v/v penicillin-streptomycin-glutamine (ThermoFisher Scientific, Cat# 10378016). LOX-IMVI, SKMEL28, and UACC.62 cells were maintained in PRMI1640-high glucose media (ThermoFisher Scientific, Cat# A1049101) supplemented with 10% v/v FBS, 1% v/v penicillin-streptomycin-glutamine. Early passage, patient-derived MTG013/HCICM-013 and MTG030/HCI-CM030 melanoma cells were obtained from the HCI PRR laboratory. These primary cells were derived from tumor that was obtained from two distinct patients who provided written informed consent according to a tissue collection and usage protocols IRB 89989 and 10924, approved by the University of Utah Institutional Review Board. Access to these biospecimens is available through the HCI PRR lab. Patient-derived human melanoma cells were maintained in Mel2 media, which consists of 80% v/v MCDB 153 media (Sigma, Cat# M7403-10X1L), 20% v/v Leibovitz’s L-15 Media (ThermoFisher Scientific, Cat# 11415064), 2% v/v FBS, 1.68mM CaCI 2 , 1x Insulin-Transferrin-Selenium-Ethanolamine (ITS-X)(Fisher Scientific, Cat# 51500056), 5ng/mL EGF(Sigma, Cat# E-4127), 15ug/mL Bovine Pituitary Extract (ThermoFisher, Cat# 13028014), 1% v/v Penicillin-Streptomycin (ThermoFisher Scientific, Cat# 15070063). Early passage murine tumor cell lines were derived from primary melanoma tumors induced in Dct::TVA ; Braf V600E ; Cdkn2a f/f mices 8 , 12 . Cell line 5588 = ARF6 WT . Cell line 20000 = ARF6 NULL . Cell line 6431 expresses ectopic ARF6 Q67L . Cells were cultured with DMEM/ F12 HEPES (ThermoFisher Scientific, Cat # 37075) containing 10% v/v FBS, 1% v/v penicillin-streptomycin-glutamine, 1% v/v MEM Non-Essential Amino Acids Solution (ThermoFisher Scientific, Cat #11140050) under standard conditions at 37°C in a humidified atmosphere, 5% CO 2 . DF-1 and A375-TVA cells were provided by S. Holmen (HCI). DF-1 cells were maintained in DMEM-high glucose supplemented with 10% FBS, 0.5% v/v gentamicin (ThermoFisher Scientific, Cat# 15710072), and maintained at 39°C, with 5% CO2. A375-TVA cells were maintained in DMEM-high glucose supplemented with 10% FBS and 0.5% v/v gentamicin at 37°C with 5% CO2 and were used to verify RCAS/Cre expression in DF-1 cells. Human colorectal carcinoma HT-29 cells were purchased from ATCC (Cat# HTB-38) and were maintained in ATCC-formulated McCoy’s 5a Medium Modified (ATCC, Cat# 30-2007), 10%v/v FBS, 1%v/v penicillin-streptomycin-glutamine. Human glioma DBTRG-05MG cells were purchased from ATCC (Cat# CRL-2020) and were maintained in ATCC-formulated RPMI-1640 Medium (Cat# 30-2001), 10%v/v FBS, 30mg/L L-proline (Sigma-Aldrich, Cat# 81709-10G), 35mg/L L-cystine (ThermoFisher Scientific, Cat# J63745.14), 3.57g/L HEPES (ThermoFisher Scientific, Cat# 15630080), 15mg/L hypoxanthine (Sigma-Aldrich, Cat# H9636-1G), 1mg/L adenosine triphosphate (Sigma-Aldrich, Cat# A6419-1G), 10mg/L adenine (Sigma-Aldrich, Cat# A2786-5G), 1mg/L thymidine (Sigma-Aldrich, Cat# T1895-1G), and 1%v/v penicillin-streptomycin-glutamine. Cells were incubated at 37°C in a humidified atmosphere with 5% CO 2 . RNA interference Transient silencing of endogenous ARF6 was performed by sequential transfection of siRNA ( ARF6 , Qiagen Cat# 1027417; GeneGlobe S02757286), and compared to AllStars Negative Control siRNA (Qiagen, Cat# 1027181) at a final concentration of 40nM using Lipofectamine™ RNAiMAX transfection reagent (ThermoFisher Scientific, Cat# 13778150). Briefly, cells were seeded in a 6-well plate and first transfected with 40nM siRNA mixed with 7.5µL of Lipofectamine™ RNAiMAX transfection reagent. After 24 hours, transfections were repeated under the same conditions. Cells were collected 24 h after the second transfection for cell viability and western blot analyses. For conditional ARF6 silencing with short hairpin RNA (shRNA), MTG013 and MTG030 cells were stably transduced with a replication-incompetent retrovirus (piSMART-hEF1a-GFP-shARF6, see Key Resource Table) and cultured under 1µM puromycin selection. In vitro , stably transduced cell lines were treated with 1.0 µM doxycycline. Western blot and ARF6-GTP-pulldown Cells were lysed using Pierce® IP Lysis buffer (ThermoFisher Scientific, Cat # 87788) with 1X Halt™ Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific, Cat# 78442). Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Cat# 23227). Cell lysates were boiled with SDS sample buffer. Proteins from the cell lysates were separated by SDS polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (ThermoFisher Scientific, Cat# 88518). The PVDF membranes were blocked with TBST (10 mM Tris-HCl, 150 mM NaCl, and 0.1% v/v Tween-20) containing 5% w/v skim milk and incubated with primary antibodies. After washing in TBST, membranes were incubated with HRP-conjugated secondary antibodies and then washed with TBST before developing with Western Lightning™ Plus Chemiluminescence Reagent (PerkinElmer, Cat# NEL103001EA) or SuperSignal™ West Dura Extended Duration Substrate (ThermoFisher Scientific, Cat# 37075). Luminescent signal was detected using the Azure c300 or c600 (Azure Biosystems). ImageJ (NIH, Bethesda, MD, USA) was used to quantify the intensity of bands on the blots. Images were adjusted equally for brightness and contrast using ImageJ or Adobe Photoshop (Adobe Inc.). ARF6-GTP pull-downs were performed using GGA3 PBD Agarose beads (Cell Biolabs, Cat# STA-419) as previously described 11 . Briefly, cells were treated with chemical compounds for the indicated time. After treatment, cells were lysed with pulldown lysis buffer (Cell Biolabs, Cat# 240102) including 1X Halt™ Protease and Phosphatase Inhibitor Cocktail. Lysates were centrifuged, supernatants were added to GGA3-conjugated beads and agitated for 1 hour at 4°C. Beads were washed in ARF6-pulldown lysis buffer and prepared for western blot analysis. Cell viability assay Cell viability was detected by CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Cat# G7571). Briefly, 2000 cells/well were seeded in 96 well plates overnight. The next day, cell viability was measured before treatment (0-hour time point). After 48 or 72 hours of treatment, media were removed and replaced with the CellTiter-Glo® Reagent. Luminescence was measured by Perkin Elmer EnVision Multi-Mode Plate Reader. Apoptosis Assay Apoptosis was detected by RealTime Glo™ Annexin V Apoptosis Assay (Promega; Cat# JA1000). Briefly, 10,000 cells/well were seeded in 96 well plates overnight. The next day, cells were treated with serum starvation or chemical compounds to induce apoptosis plus apoptosis detection reagent. Annexin V luminescence was measured by Perkin Elmer EnVision Multi-Model Plate Reader. Drug tolerant colony formation Human melanoma cells A375 were seeded at 10,000 cells per well in 6 well plates and treated with vemurafenib, dabrafenib, trametinib, and/or QS11 for 30 days. For colony formation assay with early passage, patient-derived MTG013 cells were seeded at 200,000 cells per well in 6 well plates and treated with dabrafenib, trametinib, and/or NAV-2729 for 14 days. For patient-derived MTG030 cells were treated with dabrafenib, trametinib, and/or doxycycline. Drugs were refreshed every 2–3 days. After 14 or 30 days of drug treatment, cells were fixed with methanol and stained with 0.5% crystal violet stains. Plates were scanned with LICOR Odyssey® DLx scanner. Colony Area or intensity was measured by ImageJ 57 . Representative images were captured with a Nikon Automated Widefield Microscope. Cloning, viral transduction and generation of stable cell lines The pTRIPZ lentviral system (used for cloning pTRIPZ-ARF6 WT -V5 and pTRIPZ-ARF6 Q67L -V5) was gifted from Dr. Todd W. Ridky 58 . ARF6 WT -V5 and ARF6 Q67L -V5 were inserted into the p-TRPIZ vector using the In-Fusion Snap Assembly system (Takarabio, Cat# 638945). HEK-293T cells were co-transfected with 2nd generation lentivirus packaging vectors (5µg pCMV-Gag/Pol, Addgene, Cat#35614; 1µg pCMV-VSVG, Addgene, Cat# 8454) and 5µg of expression constructs (including piSMART-hEF1a-TurboGFP-sh ARF6 , see Key Resource Table) using Lipofectamine™ 3000 Transfection Reagent (Thermo Scientific, Cat# L3000008). Viral supernatants were harvested 48 hours and 72 hours post-transfection and filtered through a 0.45 µm filter. Filtered viral supernatants were applied to target cell lines together with 10 µg/ml of Polybrene (Sigma Cat# TR-1003). After infection, cells were placed in fresh media for three days before selection with 1µM puromycin for 14 days. Doxycycline dose response treatments confirmed ectopic expression or knockdown efficiency. Stable cell lines were maintained in 1µm puromycin. Adenoviral ARF6 Q67L was created by Vector Biolabs as previously described 11 . Cells were infected with 10 7 pfu/mL virus and incubated for 24 hours prior to experimentation. Proteomics Protein extraction and reverse-phase protein array of frozen mouse tumors were performed by the MD Anderson Cancer Center Functional Proteomic RPPA Core Facility. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was isolated from A375 cells after doxycycline-induced expression of ARF6 Q67L . Cells were untreated or treated with 1µM doxycycline for 4, 8, 24, or 48 hours, then collected and stored in RNAlater (ThermoFisher Scientific, Cat# AM7024). RNA was extracted using RNeasy Plus kit (Qiagen, Cat# 74034) according to manufacturer's instructions. Extracted RNA from each sample was converted into cDNA using SuperScript IV VILO (SSIV VILO) Master Mix (ThermoFisher Scientific, Cat# 11756050). qRT-PCR was performed in triplicate for each sample using PowerUp™ SYBR™ Green Master Mix (ThermoFisher Scientific, Cat# A25780) on the QuantStudio™ 6 Flex Real-Time PCR System (ThermoFisher Scientific) in 96-well plates. Primers used for qRT-PCR are shown in the Key Resource Table. The specificity of the amplicons was assessed by melting curve analyses. Relative mRNA expression of each gene was calculated using the number of cycles needed to reach the crossing threshold of detection (CT) and normalized to the expression of GAPDH . Immunofluorescence Murine tumors were embedded and frozen in Tissue-Tek® O.C.T. compound. Tissues were sectioned 6–10µm thick using a cryostat. The tissue was fixed to the slides with acetone followed by three rinses in "PBSA" (1× PBS + 0.1% sodium azide). Slides were permeabilized with 1% bovine serum albumin (BSA) + 0.1% Saponin solution, followed by blocking in PBSA + 3% v/v BSA for 60 minutes. After blocking, the slides were incubated with the primary antibody overnight at 4°C. The next day, the slides were washed with PBSA and incubated with secondary antibody for 1 hour at room temperature, then washed again with PBSA After washing the slides before counterstaining with DAPI for 30 minutes at room temperature, followed by a 5-minute wash in PBSA, and mounting in 40% w/v polyvinyl pyrrolidone + 4% v/v glycerol + 0.1% sodium azide dissolved in 1 mol/L Tris, pH 8.0. Images were collected on an Olympus Fluoview1000 scanning laser confocal microscope at 1,200x magnification. Quantification of fluorescent signals on mouse tumor tissue was performed in ImageJ. Final signal intensity for BRAF, pMEK and pERK was calculated by total green signal count divided by the number of nuclei (DAPI stained). Statistical Analysis Details of each statistical analysis are included in the figure legends. Statistical tests were performed using Prism software (GraphPad). Quantitative values are shown with or represented as the mean of at least three biologic replicates. Declarations Data Availability The data generated herein are available from the corresponding author upon request. Ethics and Inclusion Statement This research included local researchers throughout the research process, is locally relevant and has been determined in collaboration with local partners. Roles and responsibilities were agreed amongst collaborators ahead of the research and with capacity in mind. This research was not severely restricted or prohibited by local stakeholders. This research was approved by a local ethics review committee and complied with animal welfare regulations, environmental protection and biorisk-related regulations and the local research setting was sufficient to conduct the research described. No stigmatization, incrimination, discrimination, nor personal risk to participants or researchers was involved in this research. The researchers and facilities will not benefit monetarily from sharing biological materials, including if transferred out of the country of origin. Local and regional research relevant to this study have been included in citations. Authors’ Contributions Conceptualization, A.H.G; project administration, A.H.G., J.W.; investigation, J.W., P.G., T.J., D.M.B., A.R., L.K.S., R.K.W., A.H.G.; data curation, E.C.W.; methodology, Y.W., M.M., S.L.H., R.L.J-T., A.H.G.; validation, P.G.; resources, J.K.H.T., T.L., E.A.S., R.L.J-T., S.L.H., V.G.Y., M.A.D.; supervision, A.H.G., R.K.W., R.L.J-T., Y.W.; writing-original draft, A.H.G, J.W.; writing-reviewing & editing, A.H.G, J.W., Y.W., J.K.H.T., P.G., T.J., D.M.B.; funding acquisition, A.H.G. Acknowledgements We thank Diana Lim and Nikita Abraham for preparation of scientific graphics; the HCI Preclinical Research Resource Lab, University of Utah (UU) Flow Cytometry Core, UU Genomics Core, UU HSC Cell Imaging Core; MD Anderson Cancer Center Functional Proteomics Core. This project was supported by funding from NIH/NCI P30CA042014 (HCI), and by funding in support of A.H.G. including American Cancer Society 133649-RSG-19-019-01-CSM, NIH/NCI K08CA188563, NIH/NCI R37CA230630, the UU Department of Pathology, and the Earl A. Chiles Research Institute at the Providence Cancer Institute of Oregon. S.L.H. is supported by NIH/NCI R01CA121118. M.A.D. is supported by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, the AIM at Melanoma Foundation, the NIH/NCI P50CA221703, the American Cancer Society, the Melanoma Research Alliance, Cancer Fighters of Houston, the Anne and John Mendelsohn Chair for Cancer Research, and philanthropic contributions to the Melanoma Moon Shots Program of MD Anderson. References Hanahan, D. Hallmarks of Cancer: New Dimensions. Cancer Discov 12, 31–46 (2022). Pu, Y. et al. Drug-tolerant persister cells in cancer: the cutting edges and future directions. Nat Rev Clin Oncol 20, 799–813 (2023). He, J. et al. 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Supplementary Files FigS1071425.tif Supplementary Figure 1 FigS2070925.tif Supplementary Figure 2 FigS3070925.tif Supplementary Figure 3 KeyResourcetable1.docx Key Resource Table Cite Share Download PDF Status: Published Journal Publication published 28 Apr, 2026 Read the published version in Oncogene → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Grossmann","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-7665-1403","institution":"Providence Cancer Institute of Oregon","correspondingAuthor":true,"prefix":"","firstName":"Allie","middleName":"H.","lastName":"Grossmann","suffix":""}],"badges":[],"createdAt":"2025-07-15 20:20:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7133814/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7133814/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41388-026-03805-w","type":"published","date":"2026-04-28T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89972423,"identity":"edfc7d5f-fdc0-4c65-83af-5c2eb93acfe5","added_by":"auto","created_at":"2025-08-27 05:44:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3249920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eARF6 is sufficient to control oncogenic BRAF\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eprotein levels through protein translation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Relative amount of MAPK signaling proteins in tumor cells derived from \u003cem\u003eBraf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/ Cdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice detected by Reverse Phase Protein Array, two-tailed t-test. n= 3 replicates per cell line.\u003cstrong\u003e b-h,\u003c/strong\u003e Western Blot for indicated proteins\u003cstrong\u003e. b,\u003c/strong\u003e murine melanoma cells derived from \u003cem\u003eBraf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/ Cdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice. n=3 biological independent experiments \u003cstrong\u003ec,\u003c/strong\u003e dox-inducible ectopic expressed ARF6\u003csup\u003eQ67L\u003c/sup\u003e. n=3 biological independent experiments \u003cstrong\u003ed,\u003c/strong\u003e Western blot for indicated proteins in UACC.62 and A375 cells with or without adenoviral-mediated ectopic expression of ARF6\u003csup\u003eQ67L\u003c/sup\u003e, control= empty vector. \u003cstrong\u003ee,\u003c/strong\u003e 4μM QS11 for 48h in A2058, HT-29, and DBTRG-05MG. 2μM QS11 for 24h other cell lines \u003cstrong\u003ef, \u003c/strong\u003e2μM QS11. \u003cstrong\u003eg,\u003c/strong\u003e 17-AAG and QS11 for 24h in A375 with doxycycline-inducible ectopic expressed ARF6\u003csup\u003eQ67L\u003c/sup\u003e. \u003cstrong\u003eh,\u003c/strong\u003e 20μg/ml cycloheximide (CHX) in A375 with doxycycline-inducible ectopic expressed ARF6\u003csup\u003eQ67L\u003c/sup\u003e. BRAF\u003csup\u003eV600E\u003c/sup\u003e protein quantification at 48h. n=3 biological independent experiments. \u003cstrong\u003ei,\u003c/strong\u003e Quantitative RT-PCR for \u003cem\u003eBRAF\u003c/em\u003e mRNA in A375 with doxycycline-inducible ectopic expressed ARF6\u003csup\u003eQ67L\u003c/sup\u003e,\u003csup\u003e \u003c/sup\u003en=3 biological independent experiments. \u003cstrong\u003eb, c, h,\u003c/strong\u003e Two-tailed ratio paired t-test.\u003c/p\u003e","description":"","filename":"Fig1070925.png","url":"https://assets-eu.researchsquare.com/files/rs-7133814/v1/e099addbbcc3220ba52d0f13.png"},{"id":89971182,"identity":"e4a4a9ff-b262-4b49-b0a9-f66b90d0f7a4","added_by":"auto","created_at":"2025-08-27 05:36:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5558042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eARF6 is necessary to control oncogenic BRAF\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eprotein levels.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Representative immunofluorescence images of cryo-embedded frozen tumor tissues from \u003cem\u003eBraf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/ Cdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice, 1200X magnification. \u003cstrong\u003eb-e,\u003c/strong\u003e Western blot for indicated proteins. \u003cstrong\u003eb,\u003c/strong\u003e murine melanoma cells derived from \u003cem\u003eBraf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/ Cdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice, n=3 biological independent experiments. \u003cstrong\u003ec,\u003c/strong\u003e 10μM SecinH3, BRAF\u003csup\u003eV600E\u003c/sup\u003e protein quantification at 48h, n=3 biological independent experiments. \u003cstrong\u003ed,\u003c/strong\u003e 5μM NAV-2729. \u003cstrong\u003ee,\u003c/strong\u003e A375 cells with or without adenoviral-mediated ectopic expression of ARF6\u003csup\u003eT27N\u003c/sup\u003e, control= empty vector. \u003cstrong\u003ea,\u003c/strong\u003e Two-tailed unpaired t-test. \u003cstrong\u003eb, e,\u003c/strong\u003e Two-tailed ratio paired t-test.\u003c/p\u003e","description":"","filename":"Fig2070925.png","url":"https://assets-eu.researchsquare.com/files/rs-7133814/v1/9538425552ec4c42c1e9e554.png"},{"id":89971178,"identity":"97ec81fa-e0c6-41ab-ba0d-a4d8e97d1734","added_by":"auto","created_at":"2025-08-27 05:36:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2920099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eARF6 promotes tumor survival and accelerated disease progression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Apoptotic protein profile of tumor cells derived from \u003cem\u003eBraf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/ Cdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice detected by Reverse Phase Protein Array, two-tailed t-test. n= 3 replicates per cell line. \u003cstrong\u003eb,\u003c/strong\u003e Western Blot for indicated proteins in murine melanoma cells derived from \u003cem\u003eBraf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/ Cdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice, n=3 biological independent experiments. Two-tailed ratio paired t-test. \u003cstrong\u003ec, \u003c/strong\u003eApoptosis detection, measured at 48h, dox-inducible ectopic expressed ARF6\u003csup\u003eWT\u003c/sup\u003e and ARF6\u003csup\u003eQ67L \u003c/sup\u003ein A375 cells, n=4 replicates per condition, One-way ANOVA with multiple comparisons. \u003cstrong\u003ed,\u003c/strong\u003e Apoptosis detection, 4μM QS11, measured at 48h, n=5 for A375 and n=3 for A2058 replicates per condition, Two-tailed unpaired t-test. \u003cstrong\u003ee,\u003c/strong\u003e Cell viability detection, measured at 72h, n=5 replicates per condition. Two-tailed unpaired t-test. \u003cstrong\u003ef,\u003c/strong\u003e Rate of tumor growth measured from the time of initial detection in \u003cem\u003eDct::TVA;Braf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eCdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice, n= 24 \u003cem\u003ePten\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e, n=14 \u003cem\u003ePten\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice, two-tailed t-test with Welch’s correction. \u003cstrong\u003eg, \u003c/strong\u003eRate of tumor growth measured from the time of initial detection in \u003cem\u003eDct::TVA;Braf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eCdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ePten\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice, \u003cem\u003en\u003c/em\u003e = 14 \u003cem\u003eArf6\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003en\u003c/em\u003e = 22 \u003cem\u003eArf6\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice, two-tailed t-test with Welch’s correction. \u003cstrong\u003eh, \u003c/strong\u003eSurvival of mice (before primary tumor reached 2 cm) after Cre injection (day 0) within 130 days, \u003cem\u003en\u003c/em\u003e = 14 \u003cem\u003eArf6\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003en\u003c/em\u003e = 18 \u003cem\u003eArf6\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice, Log-rank (Mantle-Cox) test. Solid line withing data points = mean. \u003cstrong\u003ei,\u003c/strong\u003e Apoptotic protein profile of tumors from \u003cem\u003eDct::TVA;Braf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eCdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ePten\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice (n=6 mice per group) detected by Reverse Phase Protein Array, two-tailed t-test.\u003c/p\u003e","description":"","filename":"Fig3070925.png","url":"https://assets-eu.researchsquare.com/files/rs-7133814/v1/5128f80a4159723446d0374c.png"},{"id":89971189,"identity":"63fec363-b4c0-4d75-b1b9-2b9bdf63b9c8","added_by":"auto","created_at":"2025-08-27 05:36:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4498937,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eARF6 activation protects against MAPKi-induced apoptosis and promotes the development of MAPKi-resistance cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-b,\u003c/strong\u003e Total ARF6 and ARF6-GTP pulldown in A375, 5μM vemurafenib for 4h, Dabrafenib treatment for 4h, PF-07799933 treatment for 2h in 0%FBS media. \u003cstrong\u003eb,\u003c/strong\u003e 5μM vemurafenib. \u003cstrong\u003ec-f,\u003c/strong\u003e Apoptosis detection.\u003cstrong\u003e c,\u003c/strong\u003e 1μM Vemurafenib, dox-inducible ectopic expressed ARF6\u003csup\u003eWT\u003c/sup\u003e and ARF6\u003csup\u003eQ67L \u003c/sup\u003ein A375, apoptosis measured at 48h, Ctrl= no doxycycline, n=5 replicates per condition.\u003csup\u003e \u003c/sup\u003e\u003cstrong\u003ed,\u003c/strong\u003e 1μM Vemurafenib, 4μM QS11 for A375, n=4 replicates per condition, apoptosis measured at 48h, 2μM Vemurafenib, 4μM QS11 for UACC.62, n=3 replicates per condition, apoptosis measured at 24h. \u003cstrong\u003ee,\u003c/strong\u003e 1.25μM Dabrafenib, 0.0625μM Trametinib, dox-inducible ectopic expressed ARF6\u003csup\u003eWT\u003c/sup\u003e and ARF6\u003csup\u003eQ67L\u003c/sup\u003e in A375, apoptosis measured at 48h, Ctrl= no doxycycline, n=3 replicates per condition.\u003csup\u003e \u003c/sup\u003e\u003cstrong\u003ef,\u003c/strong\u003e 1.25μM Dabrafenib, 0.0625μM Trametinib, 4μM QS11, apoptosis measured at 48h, n=3 replicates per condition. \u003cstrong\u003eg,\u003c/strong\u003e Western Blot for indicated proteins. 1μM Vemurafenib, 4μM QS11 in A375. 2μM Vemurafenib, 4μM QS11 in UACC.62. \u003cstrong\u003eh-i,\u003c/strong\u003e Colony outgrowth assay in A375. \u003cstrong\u003eh,\u003c/strong\u003e 1μM Vemurafenib, 4μM QS11, for 30 days. \u003cstrong\u003ei, \u003c/strong\u003e250nM Dabrafenib, 12.5nM Trametinib, 2μM QS11, 4μM QS11, for 30 days. \u003cstrong\u003ec-f,\u003c/strong\u003e One-way ANOVA with multiple comparisons. \u003cstrong\u003eh-i\u003c/strong\u003e, Two-tailed unpaired t-test. n=4 biological independent experiments.\u003c/p\u003e","description":"","filename":"Fig4070925.png","url":"https://assets-eu.researchsquare.com/files/rs-7133814/v1/db8e336e9dd271fcd2b31760.png"},{"id":89971181,"identity":"404d6730-4cce-4f60-a2dd-15061c8bc9d9","added_by":"auto","created_at":"2025-08-27 05:36:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6655028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eARF6 inhibition sensitizes MAPKi-resistant cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Schematics of \u003cem\u003ein vivo\u003c/em\u003eand \u003cem\u003ein vitro\u003c/em\u003e experiments with patient-derived xenograft cell lines. \u003cstrong\u003eb,\u003c/strong\u003eRate of tumor growth measurements started six days after initial engraftment of MTG013 cells [stably transduced with doxycycline-induced short hairpin RNA (shRNA) for \u003cem\u003eARF6\u003c/em\u003e]\u003cem\u003e \u003c/em\u003ein NRG mice, n=10 controls, n=10 fed doxycycline chow (\u003cem\u003eshARF6\u003c/em\u003e), two-tailed t-test with Welch’s correction. \u003cstrong\u003ec, e, g, h, k, l, \u003c/strong\u003eCell viability detection measured at 48 hours patient-derived cell lines (see Supplementary Table 1).\u003cstrong\u003e c, \u003c/strong\u003eDose response to Dabrafenib plus Trametinib (Dab+Tram) in MTG013, n= 5 replicates per condition. \u003cstrong\u003ed,\u003c/strong\u003e Schematic of cell viability assay. \u003cstrong\u003ee-f\u003c/strong\u003e, Doxycycline-induced \u003cem\u003eshARF6\u003c/em\u003e. \u003cstrong\u003ee,\u003c/strong\u003en=4 replicates per condition. \u003cstrong\u003ef, \u003c/strong\u003eApoptosis detection. n= 3 replicates per condition. \u003cstrong\u003eg, h, i, k, l\u003c/strong\u003e, pharmacologic inhibition of ARF6. \u003cstrong\u003eg,\u003c/strong\u003en= 5 replicates per condition. \u003cstrong\u003eh,\u003c/strong\u003e n= 5 replicates per condition. \u003cstrong\u003ej,\u003c/strong\u003eDose response of Dab+Tram in MTG030. n= 5 replicates per condition. \u003cstrong\u003ek,\u003c/strong\u003en=4 replicates per condition. \u003cstrong\u003el,\u003c/strong\u003e n=4 replicates per condition. \u003cstrong\u003ei, m\u003c/strong\u003e, Colony outgrowth assay in MTG013 and MTG030 for 14 days. \u003cstrong\u003ei,\u003c/strong\u003e MTG013 treated with 5 μM Dabrafenib and 0.25μM Trametinib and/or 1.25 μM NAV-2729. n=4 biological independent experiments. \u003cstrong\u003em,\u003c/strong\u003e MTG030 cells treated with 5 μM Dabrafenib and 0.25μM Trametinib, Ctrl= no doxycycline. n=4 biological independent experiments\u003cstrong\u003e. e, f, g, h, j, k, l,\u003c/strong\u003e One-way ANOVA with multiple comparisons. \u003cstrong\u003ei, m,\u003c/strong\u003e Two-tailed unpaired t-test.\u003c/p\u003e","description":"","filename":"Fig5070925.png","url":"https://assets-eu.researchsquare.com/files/rs-7133814/v1/b275b1bf296924d72190b280.png"},{"id":89973812,"identity":"0c967e78-5db7-4421-95ee-022db0565ae8","added_by":"auto","created_at":"2025-08-27 05:52:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2006621,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed model of ARF6-dependent drug tolerant persister cell survival.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePharmacologic inhibition of BRAF\u003csup\u003eV600E\u003c/sup\u003e induces ARF6 activation, triggering an adaptive stress response pathway that fortifies BRAF oncoprotein synthesis, reactivation of the MAPK pathway and DTP cell survival. Combined inhibition of ARF6 and MAPK signaling limits drug tolerance and enhances tumor cell death.\u003c/p\u003e","description":"","filename":"Fig6070925.png","url":"https://assets-eu.researchsquare.com/files/rs-7133814/v1/f879e768b3b5153d4be537db.png"},{"id":108078040,"identity":"2380e32e-664a-497b-a168-4b4986202b8f","added_by":"auto","created_at":"2026-04-29 07:12:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27695240,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7133814/v1/d27c7bf7-ff3b-4efa-9357-d57dec0fccbf.pdf"},{"id":89971173,"identity":"519a7bb4-cb57-423d-94b5-e033c043d236","added_by":"auto","created_at":"2025-08-27 05:36:14","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1853576,"visible":true,"origin":"","legend":"Supplementary Figure 1","description":"","filename":"FigS1071425.tif","url":"https://assets-eu.researchsquare.com/files/rs-7133814/v1/305801b1bc90296496df64f7.tif"},{"id":89971172,"identity":"09658172-7822-4c61-9be7-efbfa77dcc17","added_by":"auto","created_at":"2025-08-27 05:36:14","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":906920,"visible":true,"origin":"","legend":"Supplementary Figure 2","description":"","filename":"FigS2070925.tif","url":"https://assets-eu.researchsquare.com/files/rs-7133814/v1/d89d2536ac172d7179a83e25.tif"},{"id":89971188,"identity":"0aec5614-2fa2-4539-b9df-4218275c3a0b","added_by":"auto","created_at":"2025-08-27 05:36:15","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2933028,"visible":true,"origin":"","legend":"Supplementary Figure 3","description":"","filename":"FigS3070925.tif","url":"https://assets-eu.researchsquare.com/files/rs-7133814/v1/b92881d2bacaeac888c6dc72.tif"},{"id":89971176,"identity":"ede873c4-1ae4-4e30-9ceb-f9f195608eb1","added_by":"auto","created_at":"2025-08-27 05:36:14","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":23631,"visible":true,"origin":"","legend":"Key Resource Table","description":"","filename":"KeyResourcetable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7133814/v1/fff4f86250f6c27faa6fbb0f.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eRapid activation of ARF6 after RAF inhibition augments BRAFV600E and promotes therapy resistance\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAvoiding cell death is fundamental to the progressive acquisition of hallmark cancer behaviors\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and to the survival of drug tolerant persister (DTP) cells that give rise to therapy resistance\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Proposed origins of DTP cells include clonal selection of pre-existing drug-resistant cells, and drug-induction of a reversible DTP state that enables the outgrowth of fixed resistance\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The molecular mechanisms of DTP cell emergence are complex and incompletely understood.\u003c/p\u003e\u003cp\u003eMitogen activated protein kinase (MAPK) signaling is hyperactivated in many cancer types and directly opposes cell death by inactivating pro-apoptotic proteins of the intrinsic pathway\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In cutaneous melanoma, where most cases harbor somatic mutations in \u003cem\u003eBRAF, NRAS\u003c/em\u003e or \u003cem\u003eNF1\u003c/em\u003e that cause aberrant MAPK signaling\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, resistance to MAPK targeted therapy remains a significant clinical challenge\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe detected abnormally elevated levels of ARF6-GTP, the active form of the small GTPase ARF6, in melanoma\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. ARF6 activation in melanoma can occur through extracellular signals such as HGF\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, WNT5a\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and Interferon-g\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e; or by altered expression of guanine exchange factors (GEFs) or GTPase activating proteins (GAPs) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. HGF and WNT5a signaling also mediate resistance to MAPK targeted therapy in melanoma\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. We showed that ARF6-GTP promotes tumor development, progression, and acceleration of metastasis in murine models of BRAF\u003csup\u003eV600E\u003c/sup\u003e melanoma\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Functionally, ARF6 activation enhances tumor cell invasion and adaptive immune suppression\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Hence, ARF6 mediates the acquisition of at least two hallmark malignant behaviors\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Whether ARF6 is involved in DTP cell biology is unknown.\u003c/p\u003e\u003cp\u003eARF6 is a ubiquitously expressed protein critical for endomembrane trafficking and actin cytoskeleton remodeling\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and has diverse physiologic roles across multiple organ systems\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. To expand our understanding of ARF6 function in cancer, we interrogated proteomic alterations induced by ARF6 activation and discovered that ARF6 dynamically regulates expression of the BRAF oncoprotein in melanoma and other cancer types, impacting tumor cell survival, including during MAPK targeted therapy.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eARF6 augments a dynamic pool of oncogenic BRAF protein\u003c/h2\u003e\u003cp\u003eIn early passage murine melanoma cell lines with homozygous \u003cem\u003eBRAF\u003c/em\u003e\u003csup\u003eV600E\u003c/sup\u003e mutation, derived from our genetically engineered murine melanoma models\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, proteomic analysis showed higher levels of BRAF\u003csup\u003eV600E\u003c/sup\u003e protein, and increased phosphorylated MEK1, ERK, RSK and Jun, in cells expressing constitutively active ARF6-GTP (ARF6\u003csup\u003eQ67L\u003c/sup\u003e) compared to ARF6\u003csup\u003eWT\u003c/sup\u003e (\u003cb\u003eFig.\u0026nbsp;1a\u003c/b\u003e). In contrast, p38 MAPK-JNK signaling was unaltered (\u003cb\u003eFig.\u0026nbsp;1a\u003c/b\u003e). ARF6-GTP-induced BRAF\u003csup\u003eV600E\u003c/sup\u003e expression was confirmed by Western blot (\u003cb\u003eFig.\u0026nbsp;1b\u003c/b\u003e). These findings align with our previously published genomic data from this tumor model showing upregulation of genes in the MAPK cascade in bulk tumor transcriptomes\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Based on these findings, we hypothesized that ARF6 controls MAPK signaling by regulating oncogenic BRAF expression. In pursuit of this, we interrogated human melanoma cells and found that doxycycline-induced, ectopically expressed ARF6-GTP, in the form of ARF6\u003csup\u003eQ67L\u003c/sup\u003e (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea\u003c/b\u003e), or adenoviral delivered ARF6\u003csup\u003eQ67L\u003c/sup\u003e, augmented endogenous BRAF\u003csup\u003eV600E\u003c/sup\u003e expression in human melanoma cells (\u003cb\u003eFig.\u0026nbsp;1c-d\u003c/b\u003e). Consistent with genetic activation of ARF6, pharmacological activation of ARF6 with QS11 (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb\u003c/b\u003e), an inhibitor of ARF GTPase Activating Protein 1\u003csup\u003e31\u003c/sup\u003e, increased BRAF\u003csup\u003eV600E\u003c/sup\u003e protein expression in human melanoma, colorectal carcinoma and glioma cell lines (\u003cb\u003eFig.\u0026nbsp;1e, S1c\u003c/b\u003e). BRAF\u003csup\u003eV600E\u003c/sup\u003e protein levels rose quickly after treatment with the ARF6 agonist QS11, as early as two hours, and continued to accumulate over 48 hours (\u003cb\u003eFig.\u0026nbsp;1f\u003c/b\u003e). These data demonstrate that sustained ARF6 activation is sufficient to acutely increase endogenous BRAF\u003csup\u003eV600E\u003c/sup\u003e protein.\u003c/p\u003e\u003cp\u003eThe BRAF\u003csup\u003eV600E\u003c/sup\u003e oncoprotein is stabilized by the chaperone protein HSP90\u003csup\u003e32, 33\u003c/sup\u003e, limiting proteasome-mediated degradation. Unlike HSP90, ARF6 prevents lysosome mediated degradation of proteins through endosomal recycling\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Thus, we asked if BRAF\u003csup\u003eV600E\u003c/sup\u003e might be degraded by the lysosome. Blocking lysosomal degradation by Bafilomycin A1 failed to increase BRAF\u003csup\u003eV600E\u003c/sup\u003e protein (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed\u003c/b\u003e). Thus, it is unlikely that ARF6 regulates oncogenic BRAF expression through endolysosomal trafficking. ARF6-GTP did not alter HSP90 protein expression (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ee\u003c/b\u003e). Nevertheless, the ARF6-regulated pool of BRAF\u003csup\u003eV600E\u003c/sup\u003e was HSP90 dependent because inhibition of HSP90 with 17-AAG prevented accumulation of BRAF\u003csup\u003eV600E\u003c/sup\u003e protein after QS11 treatment (\u003cb\u003eFig.\u0026nbsp;1g\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eSurprisingly, ARF6 mediated BRAF\u003csup\u003eV600E\u003c/sup\u003e expression was dependent on protein translation. Specifically, inhibition of protein translation with cycloheximide prevented the accumulation of BRAF\u003csup\u003eV600E\u003c/sup\u003e protein upon ARF6 activation \u003cb\u003e(Fig.\u0026nbsp;1h).\u003c/b\u003e Activation of ARF6 failed to alter \u003cem\u003eBRAF\u003c/em\u003e mRNA levels in A375 melanoma cells, which harbor a homozygous \u003cem\u003eBRAF\u003c/em\u003e\u003csup\u003eV600E\u003c/sup\u003e mutation \u003cb\u003e(Fig.\u0026nbsp;1i)\u003c/b\u003e, demonstrating that ARF6-mediated upregulation of BRAF\u003csup\u003eV600E\u003c/sup\u003e occurred without altering \u003cem\u003eBRAF\u003c/em\u003e oncogene expression. These data confirm that ARF6 activation is sufficient to increase BRAF\u003csup\u003eV600E\u003c/sup\u003e protein levels and suggest a previously unknown role for ARF6 in regulating protein translation.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eARF6 is necessary for maintenance of the BRAF protein\u003c/h3\u003e\n\u003cp\u003eIn contrast to ARF6 activation, deletion of \u003cem\u003eArf6\u003c/em\u003e in BRAF\u003csup\u003eV600E\u003c/sup\u003e murine melanoma tumors\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e reduced total BRAF\u003csup\u003eV600E\u003c/sup\u003e levels and downstream phosphorylated MEK (p-MEK) and ERK (p-ERK), detected by immunofluorescence \u003cem\u003ein situ\u003c/em\u003e (\u003cb\u003eFig.\u0026nbsp;2a, S1f\u003c/b\u003e). Consistently, silencing of \u003cem\u003eArf6\u003c/em\u003e downregulated BRAF\u003csup\u003eV600E\u003c/sup\u003e and p-MEK detection in murine melanoma cells (\u003cb\u003eFig.\u0026nbsp;2b\u003c/b\u003e). To test whether inactivation of ARF6 (ARF6-GDP) could produce the same effect, we treated human melanoma cells with SecinH3, an ARF6 guanine exchange factor inhibitor that reduces ARF6-GTP levels \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eg\u003c/b\u003e) and reduces spontaneous metastasis of human BRAF\u003csup\u003eV600E\u003c/sup\u003e melanoma xenograft tumors\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In human melanoma cells, SecinH3 significantly reduced BRAF\u003csup\u003eV600E\u003c/sup\u003e protein within 48 hours of treatment (\u003cb\u003eFig.\u0026nbsp;2c\u003c/b\u003e). NAV-2729, a direct inhibitor of ARF6 GTPase function\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eh\u003c/b\u003e), also reduced BRAF\u003csup\u003eV600E\u003c/sup\u003e protein after 48 hours (\u003cb\u003eFig.\u0026nbsp;2d\u003c/b\u003e). Finally, ectopic expression of inactive ARF6 (ARF6-GDP), in the form of ARF6\u003csup\u003eT27N\u003c/sup\u003e, reduced BRAF\u003csup\u003eV600E\u003c/sup\u003e protein (\u003cb\u003eFig.\u0026nbsp;2e\u003c/b\u003e), suggesting that ARF6 activation is necessary to maintain expression of endogenous BRAF\u003csup\u003eV600E\u003c/sup\u003e. Together these data demonstrate that ARF6 may be necessary to maintain steady state levels of the BRAF\u003csup\u003eV600E\u003c/sup\u003e oncoprotein and suggest that targeted inhibition of ARF6 might be an alternative approach to reducing BRAF\u003csup\u003eV600E\u003c/sup\u003e oncoprotein expression.\u003c/p\u003e\n\u003ch3\u003eARF6-GTP promotes tumor survival by protecting against apoptosis\u003c/h3\u003e\n\u003cp\u003eBecause MAPK signaling opposes the intrinsic apoptotic signaling pathway\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, we reasoned that ARF6-mediated fluctuations in BRAF\u003csup\u003eV600E\u003c/sup\u003eprotein might be linked to survival. Proteomic clues to ARF6-mediated survival were evident in murine melanoma cell lines cultured in full serum (\u003cb\u003eFig.\u0026nbsp;3a-b\u003c/b\u003e). Compared to ARF6\u003csup\u003eWT\u003c/sup\u003e, cells expressing ARF6\u003csup\u003eQ67L\u003c/sup\u003e showed significantly increased levels of the anti-apoptotic protein MCL-1 and phosphorylation (inactivation) of BAD at residue S112 (pS112)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, as well as decreased levels of pro-apoptotic proteins BAX and FOXO3 (\u003cb\u003eFig.\u0026nbsp;3a\u003c/b\u003e). ARF6 dependent expression of MCL-1 and FOXO3 were confirmed by Western blot (\u003cb\u003eFig.\u0026nbsp;3b\u003c/b\u003e). ERK signaling has been reported to increase MCL-1\u003csup\u003e37\u003c/sup\u003e and decrease FOXO3\u003csup\u003e38\u003c/sup\u003e protein levels\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Thus, our data suggest that ARF6 activation might promote tumor cell survival through ERK-mediated anti-apoptotic signaling.\u003c/p\u003e\u003cp\u003eTo test whether ARF6 activation could protect against apoptosis, we deployed a doxycycline-inducible system to express either ectopic ARF6\u003csup\u003eQ67L\u003c/sup\u003e or ARF6\u003csup\u003eWT\u003c/sup\u003e in human melanoma cells (\u003cb\u003eFig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea, S2a\u003c/b\u003e). Doxycycline alone did not alter viability of A375 parental cells, (\u003cb\u003eFig. S2b\u003c/b\u003e), while doxycycline-induced ARF6\u003csup\u003eQ67L\u003c/sup\u003e significantly reduced apoptosis caused by serum withdrawal (\u003cb\u003eFig.\u0026nbsp;3c\u003c/b\u003e). In contrast, doxycycline-induced ectopic expression of ARF6\u003csup\u003eWT\u003c/sup\u003e did not alter apoptosis caused by serum withdrawal (\u003cb\u003eFig.\u0026nbsp;3c\u003c/b\u003e), suggesting that the active form of ARF6 is required for the survival benefit. Consistent with ARF6\u003csup\u003eQ67L\u003c/sup\u003e, pharmacological activation of ARF6 with QS11 protected against apoptosis caused by serum starvation (\u003cb\u003eFig.\u0026nbsp;3d\u003c/b\u003e). QS11 alone failed to alter cell viability during steady-state conditions, when cells were cultured in full serum (\u003cb\u003eFig. S2c\u003c/b\u003e), indicating that the compound does not stimulate proliferation. Overall, these data demonstrate that ARF6 activation can protect against apoptosis during growth signal deprivation.\u003c/p\u003e\u003cp\u003eGiven that ARF6 can regulate both PI3K-AKT\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and BRAF\u003csup\u003eV600E\u003c/sup\u003e -MAPK signaling (\u003cb\u003eFig.\u0026nbsp;1\u003c/b\u003e) and apoptosis upon serum withdrawal (\u003cb\u003eFig.\u0026nbsp;3c-d\u003c/b\u003e), we asked if ARF6 supports the viability of BRAF-mutant human cancer cells grown in full serum. Consistent with this, \u003cem\u003eARF6\u003c/em\u003e silencing led to significantly reduced viability in multiple human melanoma cell lines (\u003cb\u003eFig.\u0026nbsp;3e\u003c/b\u003e). Similarly, treatment with NAV-2729, a direct inhibitor of ARF6 GTPase function\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, reduced ARF6-GTP levels (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eh\u003c/b\u003e) and decreased cell viability in most of the human melanoma cells tested (\u003cb\u003eFig. S2d\u003c/b\u003e), although not as effectively as \u003cem\u003eARF6\u003c/em\u003e silencing (\u003cb\u003eFig.\u0026nbsp;3e\u003c/b\u003e). These data demonstrate that ARF6 can optimize survival during normal growth conditions.\u003c/p\u003e\n\u003ch3\u003eARF6 is required for accelerated tumor progression caused by PTEN loss\u003c/h3\u003e\n\u003cp\u003eIn parallel with the MAPK pathway, survival signaling can also originate from the PI3K-AKT pathway\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e and we previously reported that activation of ARF6 enhanced PI3K expression and PI3K-AKT signaling\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePTEN\u003c/em\u003e loss of function mutations activate the PI3K-AKT pathway, are frequently detected in cutaneous melanoma\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, cooperate with mutant BRAF or NRAS to drive melanomagenesis\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, and accelerate primary tumor growth in genetically engineered \u003cem\u003eDct::TVA, Braf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eCdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e murine melanoma models induced in epidermal melanocytes of the ear pinnae\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Like pinnae tumors, deletion of \u003cem\u003ePten\u003c/em\u003e dramatically accelerated the growth of BRAF\u003csup\u003eV600E\u003c/sup\u003e melanoma induced in the flank (\u003cb\u003eFig.\u0026nbsp;3f\u003c/b\u003e). To test the necessity of ARF6 in this highly aggressive model, we crossed \u003cem\u003eArf6\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eArf6\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e) mice with the \u003cem\u003eDct::TVA, Braf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eCdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003ePten\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice. In this model, tumor-specific loss of \u003cem\u003eArf6\u003c/em\u003e significantly reduced tumor growth to a level equivalent to \u003cem\u003ePten\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e tumors (measured from the time of tumor formation, \u003cb\u003eFig.\u0026nbsp;3g\u003c/b\u003e), and prolonged overall survival despite the absence of PTEN (\u003cb\u003eFig.\u0026nbsp;3h\u003c/b\u003e). Unlike \u003cem\u003ePten\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e mice\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, loss of ARF6 did not reduce overall tumor incidence in \u003cem\u003ePten\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e mice (\u003cb\u003eFig. S2e\u003c/b\u003e), demonstrating that loss of PTEN is sufficient to overcome the weakened tumor initiation phenotype we previously observed with \u003cem\u003eArf6\u003c/em\u003e knockout. Nevertheless, loss of ARF6 significantly delayed tumor onset in \u003cem\u003ePten\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e mice (\u003cb\u003eFig. S2e\u003c/b\u003e). Consistent with the \u003cem\u003ePten\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e tumor cell lines (\u003cb\u003eFig.\u0026nbsp;3b\u003c/b\u003e), tumors from \u003cem\u003ePten\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e; \u003cem\u003eArf6\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e mice showed increased levels of pro-apoptotic proteins BAK and BIM (\u003cb\u003eFig.\u0026nbsp;3i\u003c/b\u003e), suggesting enhanced apoptosis signaling in the absence of ARF6. Given that \u003cem\u003eArf6\u003c/em\u003e deletion prevented primary tumor acceleration caused by PTEN loss (\u003cb\u003eFig.\u0026nbsp;3f-g\u003c/b\u003e), there is a component of ARF6-dependent survival that is necessary for, and/or functions independently of the PI3K pathway. Indeed, ARF6-dependent survival may also originate from rheostatic control of BRAF\u003csup\u003eV600E\u003c/sup\u003e expression (\u003cb\u003eFigs.\u0026nbsp;1\u0026ndash;2\u003c/b\u003e) and downstream, MAPK-mediated anti-apoptotic signaling.\u003c/p\u003e\u003cp\u003e\u003cb\u003eARF6 is activated by RAF inhibition, protects against MAPK inhibitor-induced apoptosis, and potentiates resistance to MAPK inhibition\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBecause ARF6 can regulate BRAF\u003csup\u003eV600E\u003c/sup\u003e protein expression (\u003cb\u003eFigs.\u0026nbsp;1\u0026ndash;2\u003c/b\u003e), we asked if BRAF inhibition alters ARF6 activation. Remarkably, class I BRAF inhibitors, vemurafenib or dabrafenib, increased ARF6-GTP levels (\u003cb\u003eFig.\u0026nbsp;4a\u003c/b\u003e). This occurred both in the presence and absence of serum and is reproducible in independent BRAF\u003csup\u003eV600E\u003c/sup\u003e cell lines (\u003cb\u003eFig.\u0026nbsp;4a\u003c/b\u003e and \u003cb\u003eS3a\u003c/b\u003e). Notably, the pan-mutant BRAF inhibitor PF-07799933, which inhibits BRAF mutant monomers and dimers and has antitumor activity in treatment refractory patients\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, also increased ARF6-GTP levels in human melanoma (\u003cb\u003eFig.\u0026nbsp;4a\u003c/b\u003e). Importantly, ARF6 activation occurred rapidly after BRAF inhibition, as early as one hour (\u003cb\u003eFig.\u0026nbsp;4a-b\u003c/b\u003e), suggesting that ARF6 activation functions in an acute adaptive response pathway to BRAF-targeted therapy.\u003c/p\u003e\u003cp\u003eBecause ARF6 was rapidly activated upon RAF inhibition and ARF6-GTP promoted survival upon serum withdrawal (\u003cb\u003eFigs.\u0026nbsp;4a-b, 3c-d\u003c/b\u003e), we asked whether ARF6 activation can facilitate survival during MAPK inhibitor (MAPKi) treatment. Indeed, genetic activation of ARF6 dramatically reduced apoptosis after 48 hours of vemurafenib (\u003cb\u003eFig.\u0026nbsp;4c\u003c/b\u003e), whereas silencing of \u003cem\u003eArf6\u003c/em\u003e significantly increased apoptosis induced by vemurafenib (\u003cb\u003eFig S3b\u003c/b\u003e), consistent with a role for ARF6 in early tumor cell survival during targeted therapy. Overexpression of wildtype ARF6 also decreased vemurafenib-induced apoptosis, but to a lesser extent than ARF6\u003csup\u003eQ67L\u003c/sup\u003e (\u003cb\u003eFig.\u0026nbsp;4c\u003c/b\u003e). Similar to ARF\u003csup\u003eQ67L\u003c/sup\u003e, pharmacological activation of ARF6 with QS11 almost completely abrogated vemurafenib induced apoptosis (\u003cb\u003eFig.\u0026nbsp;4d\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eCombination RAF\u0026thinsp;+\u0026thinsp;MEK inhibition is the preferred choice of MAPKi therapy in BRAF\u003csup\u003eV600E\u003c/sup\u003e melanoma patients, due to superior clinical outcomes compared to single agent RAF inhibition\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Thus, we interrogated ARF6 in this context. A375 melanoma cells are highly sensitive to both single-agent RAF inhibition and combination RAF\u0026thinsp;+\u0026thinsp;MEK inhibition in short-term cultures (\u003cb\u003eFig S3c-d\u003c/b\u003e). In contrast, A2058 melanoma cells are resistant to vemurafenib (\u003cb\u003eFig S3c\u003c/b\u003e), possibly due to a \u003cem\u003eMAP2K1\u003c/em\u003e P124S mutation\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, but remain sensitive to the combination of dabrafenib\u0026thinsp;+\u0026thinsp;trametinib (Dab\u0026thinsp;+\u0026thinsp;Tram) (\u003cb\u003eFig S3d\u003c/b\u003e). Importantly, genetic or pharmacologic activation of ARF6 reduced Dab\u0026thinsp;+\u0026thinsp;Tram sensitivity in these cell lines by significantly reducing apoptosis (\u003cb\u003eFig.\u0026nbsp;4e-f\u003c/b\u003e). These combined data suggest that the consequence of ARF6 activation upon BRAF inhibition (\u003cb\u003eFig.\u0026nbsp;4a-b\u003c/b\u003e) might be the emergence of resistance.\u003c/p\u003e\u003cp\u003eBecause ARF6 activation can fortify BRAF\u003csup\u003eV600E\u003c/sup\u003e protein (\u003cb\u003eFig.\u0026nbsp;1\u003c/b\u003e), we reasoned that ARF6 might facilitate recovery of MAPK signaling after RAF inhibition. Indeed, ARF6 activation by QS11 resulted in a markedly faster recovery of phosphorylated ERK (pERK) after vemurafenib treatment (\u003cb\u003eFig.\u0026nbsp;4g and Fig. S3e\u003c/b\u003e). Additional evidence that ARF6-GTP boosted MAPK recovery manifested in ERK-mediated inhibition of the apoptotic proteins BAD and BIM\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Unlike the control, QS11 significantly recovered ERK-mediated phosphorylation (inhibition) of BAD 24\u0026ndash;48 hours after vemurafenib (\u003cb\u003eFig.\u0026nbsp;4g and S3e\u003c/b\u003e). Furthermore, downregulation of BIM was more pronounced with QS11 (\u003cb\u003eFig.\u0026nbsp;4g and S3e\u003c/b\u003e). These findings demonstrate that ARF6 activation can potentiate MAPK reactivation and anti-apoptotic signaling after BRAF inhibition.\u003c/p\u003e\u003cp\u003eTo test if ARF6-GTP promotes the emergence of DTP cells, leading to therapy resistance, we quantified colony formation during vemurafenib (\u003cb\u003eFig.\u0026nbsp;4h\u003c/b\u003e) or Dab\u0026thinsp;+\u0026thinsp;Tram treatment (\u003cb\u003eFig.\u0026nbsp;4i\u003c/b\u003e). Activation of ARF6 with QS11 significantly increased drug-resistant colony formation in both conditions (\u003cb\u003eFig.\u0026nbsp;4h-i, S3f-g\u003c/b\u003e). Hence, our overall data supports that ARF6 is activated in the early phases of adaptive resistance, acutely responding to diminished MAPK signaling, and facilitating the survival of drug-tolerant persister cells in melanoma.\u003c/p\u003e\n\u003ch3\u003eARF6 inhibition sensitizes patient-derived, MAPK inhibitor-resistant melanoma cells\u003c/h3\u003e\n\u003cp\u003eBecause ARF6 activation significantly reduced tumor cell death after MAPKi (\u003cb\u003eFig.\u0026nbsp;4c-f\u003c/b\u003e), we asked whether inhibition of ARF6 could sensitize melanoma to clinically acquired or innate MAPKi resistance. For this, we pivoted to early-passage, patient-derived xenograft (PDX) melanoma cell lines (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig.\u0026nbsp;5a\u003c/b\u003e). We recently reported that the \u003cem\u003eMET\u003c/em\u003e gene is amplified in MTG013/CM013 PDX cells\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, which may explain the patient\u0026rsquo;s history of disease progression through vemurafenib treatment because HGF-MET signaling is a common mechanism of reactivation of MAPK signaling after RAFi\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Similar to the patient\u0026rsquo;s clinical outcome (progression through vemurafenib), MTG013 PDXs are resistant to high dose Dab\u0026thinsp;+\u0026thinsp;Tram\u003csup\u003e47\u003c/sup\u003e. We transduced these PDX cells with a doxycycline-inducible shRNA construct to conditionally knockdown \u003cem\u003eARF6\u003c/em\u003e expression after subcutaneous injection into immunodeficient NRG mice, or during \u003cem\u003ein vitro\u003c/em\u003e colony forming assays (\u003cb\u003eFig.\u0026nbsp;5a\u003c/b\u003e). Doxycycline-induced knockdown of ARF6 significantly reduced tumor growth \u003cem\u003ein vivo\u003c/em\u003e (\u003cb\u003eFig.\u0026nbsp;5b\u003c/b\u003e), demonstrating that ARF6 has a role in tumor progression that is independent of the ARF6-mediated adaptive immune suppression we observed in immunocompetent mice\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eIn vitro\u003c/em\u003e, MTG013 cells were increasingly resistant to rising concentrations of Dab\u0026thinsp;+\u0026thinsp;Tram (\u003cb\u003eFig.\u0026nbsp;5c\u003c/b\u003e), likely a result of progressive relief of an ERK negative feedback loop\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and reactivation of MAPK signaling\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. From these Dab\u0026thinsp;+\u0026thinsp;Tram dose responses, we chose a low and a high dose Dab\u0026thinsp;+\u0026thinsp;Tram regimen to test in combination with knockdown (\u003cb\u003eFig.\u0026nbsp;5d-f\u003c/b\u003e) or pharmacologic inhibition of ARF6 (\u003cb\u003eFig.\u0026nbsp;5g, g, h\u003c/b\u003e). Change in viability was measured over 48 hours of treatment. By itself, silencing \u003cem\u003eARF6\u003c/em\u003e caused incomplete but significant loss of viability similar to Dab\u0026thinsp;+\u0026thinsp;Tram (\u003cb\u003eFig.\u0026nbsp;5e\u003c/b\u003e). Thus, inhibition of MAPK or ARF6 were equally cytostatic, but cell viability persisted above the baseline viability at time zero, indicating a low level of tumor cell survival (illustrated in \u003cb\u003eFig.\u0026nbsp;5d\u003c/b\u003e). Importantly, silencing of ARF6 re-sensitized MTG013 cells to Dab\u0026thinsp;+\u0026thinsp;Tram (\u003cb\u003eFig.\u0026nbsp;5e\u003c/b\u003e). Specifically, when \u003cem\u003eARF6\u003c/em\u003e knockdown was combined with Dab\u0026thinsp;+\u0026thinsp;Tram, there was a pronounced cytotoxic effect, where cell viability after 48 hours of treatment was less than time zero, and we observed this trend with both low and high combination doses of Dab\u0026thinsp;+\u0026thinsp;Tram (\u003cb\u003eFig.\u0026nbsp;5e\u003c/b\u003e). Consistently, silencing of ARF6 increased apoptosis induced by Dab\u0026thinsp;+\u0026thinsp;Tram (\u003cb\u003eFig.\u0026nbsp;5f\u003c/b\u003e). Like genetic depletion of ARF6, prevention of ARF6 activation with the ARF6 GEF inhibitor SecinH3\u003csup\u003e36\u003c/sup\u003e (\u003cb\u003eFig.\u0026nbsp;5g\u003c/b\u003e), or direct inhibition of ARF6 with NAV-2729\u003csup\u003e13\u003c/sup\u003e (\u003cb\u003eFig.\u0026nbsp;5h\u003c/b\u003e), decreased viability after Dab\u0026thinsp;+\u0026thinsp;Tram. NAV-2729 also significantly improved sensitivity to Dab\u0026thinsp;+\u0026thinsp;Tram during a 14- day colony outgrowth assay (\u003cb\u003eFig.\u0026nbsp;5i\u003c/b\u003e). Overall, the concordance between these orthogonal methods of ARF6 inhibition demonstrates reproducible efficacy in reversing clinically acquired MAPK inhibitor resistance.\u003c/p\u003e\u003cp\u003eUnlike MTG013, MTG030 cells have an increased copy number of \u003cem\u003eMAP2K1\u003c/em\u003e (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), which encodes for the BRAF substrate and effector protein MEK1. In addition, \u003cem\u003eHRAS\u003c/em\u003e is amplified. These genetic changes may explain why these PDX melanoma cells were tolerant of Dab\u0026thinsp;+\u0026thinsp;Tram (\u003cb\u003eFig.\u0026nbsp;5j\u003c/b\u003e). In fact, intermediate to high doses of Dab\u0026thinsp;+\u0026thinsp;Tram enhanced tumor cell viability/growth in the first 48 hours of treatment (\u003cb\u003eFig.\u0026nbsp;5k\u003c/b\u003e, middle and right panels), and these cells appeared to be more resistant to MAPKi than MTG013 (\u003cb\u003eFig.\u0026nbsp;5c\u003c/b\u003e). The ARF6 GEF inhibitor, SecinH3, prevented the immediate burst in viability after Dab\u0026thinsp;+\u0026thinsp;Tram (\u003cb\u003eFig.\u0026nbsp;5k)\u003c/b\u003e. Direct inhibition of ARF6 with NAV-2729 was cytotoxic when combined with low to intermediate doses of Dab\u0026thinsp;+\u0026thinsp;Tram (\u003cb\u003eFig.\u0026nbsp;5l\u003c/b\u003e, left and middle panels). Similar to SecinH3, NAV-2729 prevented the burst of enhanced viability that occurred with high dose Dab\u0026thinsp;+\u0026thinsp;Tram (\u003cb\u003eFig.\u0026nbsp;5l\u003c/b\u003e, right panel). With longer treatments (14 days), Dab\u0026thinsp;+\u0026thinsp;Tram reduced tumor colony formation, however, a low level of resistant tumor colonies persisted (\u003cb\u003eFig.\u0026nbsp;5m\u003c/b\u003e), and this was significantly diminished by knockdown of \u003cem\u003eARF6\u003c/em\u003e (\u003cb\u003eFig.\u0026nbsp;5m, S3h\u003c/b\u003e). Hence, these data suggest that targeting ARF6 may render melanomas with resistance mutations more vulnerable to MAPK inhibitors.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWe have shown that the small GTPase ARF6 helps maintain BRAF\u003csup\u003eV600E\u003c/sup\u003e protein expression through a post-transcriptional regulatory mechanism that stimulates BRAF\u003csup\u003eV600E\u003c/sup\u003e translation. Without ARF6-GTP, BRAF\u003csup\u003eV600E\u003c/sup\u003e protein levels gradually decline. Notably, ATP-competitive kinase inhibitors such as vemurafenib can reduce BRAF\u003csup\u003eV600E\u003c/sup\u003e protein levels by preventing the HSP90 co-chaperone protein CDC37 from binding BRAF\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In this context, our findings suggest that cancer cells activate ARF6 in a positive feedback loop to maintain BRAF\u003csup\u003eV600E\u003c/sup\u003e protein expression during kinase inhibition. Understanding how protein translation is deregulated in disease is important for the development of effective treatment approaches\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Messenger RNA translation occurs in cyclical bursts in mammalian cells\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Thus, a dynamic cycle of activation - deactivation of ARF6 might help stimulate pulsatile surges in BRAF\u003csup\u003eV600E\u003c/sup\u003e synthesis to maintain steady-state levels, particularly when BRAF inhibitors are present and trigger ARF6 activation. While more work is needed to understand the mechanistic underpinnings and the potential extent of ARF6 regulation of protein expression, our data suggests that sustained inhibition of ARF6 can diminish BRAF\u003csup\u003eV600E\u003c/sup\u003e levels and help overcome established resistance to MAPK targeted therapy (\u003cb\u003eFig.\u0026nbsp;6\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eOur findings suggest that targeting ARF6 inhibits a stress-adaption pathway that gives rise to DTP cells. ARF6 mediated survival both during growth factor scarcity and MAPK targeted therapy. In the latter scenario, ARF6 was rapidly activated after initiation of RAFi treatment and mediated adaptive recovery of MAPK signaling. ARF6-GTP facilitated survival during the first few days of MAPKi therapy and enabled the eventual emergence of drug-resistant growth. Overall, our data support the hypothesis that DTP cells can be drug-induced\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and provide mechanistic insights into how this phenomenon might occur in BRAF mutant cancers.\u003c/p\u003e\u003cp\u003eOur findings not only help explain how BRAF-mutant melanoma survives the acute phases of MAPK inhibition, they also highlight an emerging theme of pro-invasive small GTPases that link mechanisms of tumorigenesis to drug resistance. Like ARF6, the small GTPase RAC1 facilitates invasion\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, tumorigenesis\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e and resistance to MAPK targeted therapy\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Recently, RAC1 was shown to be activated by MEK inhibition\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Interestingly, RAC1 was activated in human melanoma cells between 8\u0026ndash;16 hours after the initiation of treatment with trametinib. In contrast, ARF6 was activated within 1\u0026ndash;2 hours of BRAF inhibition (\u003cb\u003eFig.\u0026nbsp;4a-b\u003c/b\u003e). The difference in kinetics could be due to the choice of MAPKi (MEK vs. BRAF), the use of different cell lines, or possibly due to distinct upstream mechanisms that result in serial activation of these small GTPases; ARF6 followed by RAC1. Unlike ARF6, however, activation of RAC1 was not reported to signal through the MAPK pathway\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Like RAC1 and ARF6, RhoA also has a role in MAPKi resistance, upstream of the focal adhesion kinase (FAK)-PI3K-AKT pathway\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. To the best of our knowledge, RAC1 and RhoA have never been shown to regulate BRAF oncoprotein expression, which may be unique to ARF6.\u003c/p\u003e\u003cp\u003eARF6-dependent survival may also help explain why tumor-specific deletion of \u003cem\u003eArf6\u003c/em\u003e significantly diminished tumor development and progression in BRAF\u003csup\u003eV600E\u003c/sup\u003e PTEN\u003csup\u003eWT\u003c/sup\u003e melanoma models\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. While impaired tumor formation and sluggish growth were attributable to ARF6-dependent suppression of the adaptive immune response in that model\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, our current findings suggest that ARF6 might also render tumor cells more resistant to apoptotic death incited by immune attack. More work is needed to understand ARF6-mediated tumor survival, including during immune-mediated tumor killing.\u003c/p\u003e\u003cp\u003eBy interrogating ARF6 \u003cem\u003ein vitro\u003c/em\u003e and in immunodeficient mice, we removed the influence of adaptive immunity and discovered an unanticipated role for ARF6 in tumor cell survival. While our findings support a mechanism whereby ARF6 activation fortifies BRAF\u003csup\u003eV600E\u003c/sup\u003e protein synthesis, other ARF6 mechanisms may be at play. For example, we have previously shown that ARF6-GTP upregulated PI3K expression and AKT-signaling in melanoma while inhibition of ARF6 reduced PI3K and AKT activation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In this current study, ARF6 was critical for tumor growth acceleration caused by loss of PTEN. Together these data support that ARF6 regulates the PI3K-AKT axis and as such, it is possible that ARF6 modulates PI3K-AKT driven anti-apoptotic signaling. Lastly, because ARF6 mediates internalization\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e and recycling\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e of integrins (i.e. focal adhesion turnover), ARF6 activity might be linked to FAK-dependent resistance to MAPK targeted therapy in melanoma\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Independent of these possibilities, our data reveal a previously unknown vulnerability in oncogenic BRAF signaling, ARF6, which may be exploitable for addressing DTP cell survival and targeted therapy resistance.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cb\u003eMouse husbandry, genotyping and RCAS virus delivery\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e Animal studies were performed in accordance with a protocol approved by the University of Utah Institutional Animal Care and Use Committee (IACUC). Generation of the \u003cem\u003eDct::TVA\u003c/em\u003e; \u003cem\u003eBraf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eCdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eDct::TVA; Braf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eCdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eArf6\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003eDct::TVA; Braf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eCdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003ePten\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e murine models have been described previously\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The flank tumor incidence, onset, growth rate and overall survival were measured and calculated as described previously\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Both male and female animals were used in this study and were equally distributed across experimental groups. Prior analysis confirmed that sex does not influence tumor formation, tumor size, or survival onset in our model (PMID: 39098861, PMID: 33098202)\u003c/p\u003e\u003cp\u003e For the PDX cell line (MTG013) model, all animal studies were approved by the University of Utah IACUC and were performed in accordance with relevant guidelines and regulations by the Huntsman Cancer Institute (HCI) Preclinical Research Resource (PRR) laboratory. 10 females and 10 males of six to eight-week-old NOD rag gamma (NGR, NOD-\u003cem\u003eRag1\u003c/em\u003e\u003csup\u003enull\u003c/sup\u003e\u003cem\u003eIL2rg\u003c/em\u003e\u003csup\u003enull\u003c/sup\u003e, NOD rag gamma, NOD-RG) mice, Jackson Laboratory stock 7799, were injected subcutaneously with 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells in matrigel. Mice were treated with or without Dox chow (Envigo: Global 18% Protein Rodent Diet with 625ppm doxycycline. Cat# TD.01306.) five days after injection. Mice were monitored for health weekly, and tumor size was measured twice weekly using digital calipers; the tumor volume was calculated using the following formula: (length \u0026times; width\u003csup\u003e2\u003c/sup\u003e/2).\u003c/p\u003e\n\u003ch3\u003eCell lines\u003c/h3\u003e\n\u003cp\u003eAuthentication of all human melanoma cell lines were periodically confirmed by STR profiling in the University of Utah Genomics core facility using the Promega\u003c/p\u003e\u003cp\u003e(Madison, WI) GenePrint 10 system, or by ATCC. A375, LOX-IMVI, UACC.62, were provided by Dr. M. VanBrocklin, HCI. A2058 cells were purchased from the ATCC (Cat# CRL11147D). SKMEL28 cells were provided by Dr. D. Grossman, HCI. A2058 and A375 were maintained in DMEM-high glucose (ThermoFisher Scientific, Cat# 11995073) supplemented with 10% v/v FBS (Atlas Biologicals, Cat# F-0500-DR), 1% v/v penicillin-streptomycin-glutamine (ThermoFisher Scientific, Cat# 10378016). LOX-IMVI, SKMEL28, and UACC.62 cells were maintained in PRMI1640-high glucose media (ThermoFisher Scientific, Cat# A1049101) supplemented with 10% v/v FBS, 1% v/v penicillin-streptomycin-glutamine.\u003c/p\u003e\u003cp\u003eEarly passage, patient-derived MTG013/HCICM-013 and MTG030/HCI-CM030 melanoma cells were obtained from the HCI PRR laboratory. These primary cells were derived from tumor that was obtained from two distinct patients who provided written informed consent according to a tissue collection and usage protocols IRB 89989 and 10924, approved by the University of Utah Institutional Review Board. Access to these biospecimens is available through the HCI PRR lab. Patient-derived human melanoma cells were maintained in Mel2 media, which consists of 80% v/v MCDB 153 media (Sigma, Cat# M7403-10X1L), 20% v/v Leibovitz\u0026rsquo;s L-15 Media (ThermoFisher Scientific, Cat# 11415064), 2% v/v FBS, 1.68mM CaCI\u003csub\u003e2\u003c/sub\u003e, 1x Insulin-Transferrin-Selenium-Ethanolamine (ITS-X)(Fisher Scientific, Cat# 51500056), 5ng/mL EGF(Sigma, Cat# E-4127), 15ug/mL Bovine Pituitary Extract (ThermoFisher, Cat# 13028014), 1% v/v Penicillin-Streptomycin (ThermoFisher Scientific, Cat# 15070063).\u003c/p\u003e\u003cp\u003eEarly passage murine tumor cell lines were derived from primary melanoma tumors induced in \u003cem\u003eDct::TVA\u003c/em\u003e; \u003cem\u003eBraf\u003c/em\u003e\u003csup\u003e\u003cem\u003eV600E\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eCdkn2a\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mices\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Cell line 5588\u0026thinsp;=\u0026thinsp;ARF6\u003csup\u003eWT\u003c/sup\u003e. Cell line 20000\u0026thinsp;=\u0026thinsp;ARF6\u003csup\u003eNULL\u003c/sup\u003e. Cell line 6431 expresses ectopic ARF6\u003csup\u003eQ67L\u003c/sup\u003e. Cells were cultured with DMEM/ F12 HEPES (ThermoFisher Scientific, Cat # 37075) containing 10% v/v FBS, 1% v/v penicillin-streptomycin-glutamine, 1% v/v MEM Non-Essential Amino Acids Solution (ThermoFisher Scientific, Cat #11140050) under standard conditions at 37\u0026deg;C in a humidified atmosphere, 5% CO\u003csub\u003e2\u003c/sub\u003e. DF-1 and A375-TVA cells were provided by S. Holmen (HCI). DF-1 cells were maintained in DMEM-high glucose supplemented with 10% FBS, 0.5% v/v gentamicin (ThermoFisher Scientific, Cat# 15710072), and maintained at 39\u0026deg;C, with 5% CO2. A375-TVA cells were maintained in DMEM-high glucose supplemented with 10% FBS and 0.5% v/v gentamicin at 37\u0026deg;C with 5% CO2 and were used to verify RCAS/Cre expression in DF-1 cells.\u003c/p\u003e\u003cp\u003eHuman colorectal carcinoma HT-29 cells were purchased from ATCC (Cat# HTB-38) and were maintained in ATCC-formulated McCoy\u0026rsquo;s 5a Medium Modified (ATCC, Cat# 30-2007), 10%v/v FBS, 1%v/v penicillin-streptomycin-glutamine. Human glioma DBTRG-05MG cells were purchased from ATCC (Cat# CRL-2020) and were maintained in ATCC-formulated RPMI-1640 Medium (Cat# 30-2001), 10%v/v FBS, 30mg/L L-proline (Sigma-Aldrich, Cat# 81709-10G), 35mg/L L-cystine (ThermoFisher Scientific, Cat# J63745.14), 3.57g/L HEPES (ThermoFisher Scientific, Cat# 15630080), 15mg/L hypoxanthine (Sigma-Aldrich, Cat# H9636-1G), 1mg/L adenosine triphosphate (Sigma-Aldrich, Cat# A6419-1G), 10mg/L adenine (Sigma-Aldrich, Cat# A2786-5G), 1mg/L thymidine (Sigma-Aldrich, Cat# T1895-1G), and 1%v/v penicillin-streptomycin-glutamine. Cells were incubated at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eRNA interference\u003c/h2\u003e\u003cp\u003eTransient silencing of endogenous ARF6 was performed by sequential transfection of siRNA (\u003cem\u003eARF6\u003c/em\u003e, Qiagen Cat# 1027417; GeneGlobe S02757286), and compared to AllStars Negative Control siRNA (Qiagen, Cat# 1027181) at a final concentration of 40nM using Lipofectamine\u0026trade; RNAiMAX transfection reagent (ThermoFisher Scientific, Cat# 13778150). Briefly, cells were seeded in a 6-well plate and first transfected with 40nM siRNA mixed with 7.5\u0026micro;L of Lipofectamine\u0026trade; RNAiMAX transfection reagent. After 24 hours, transfections were repeated under the same conditions. Cells were collected 24 h after the second transfection for cell viability and western blot analyses.\u003c/p\u003e\u003cp\u003eFor conditional \u003cem\u003eARF6\u003c/em\u003e silencing with short hairpin RNA (shRNA), MTG013 and MTG030 cells were stably transduced with a replication-incompetent retrovirus (piSMART-hEF1a-GFP-shARF6, see Key Resource Table) and cultured under 1\u0026micro;M puromycin selection. \u003cem\u003eIn vitro\u003c/em\u003e, stably transduced cell lines were treated with 1.0 \u0026micro;M doxycycline.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot and ARF6-GTP-pulldown\u003c/h2\u003e\u003cp\u003eCells were lysed using Pierce\u0026reg; IP Lysis buffer (ThermoFisher Scientific, Cat # 87788) with 1X Halt\u0026trade; Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific, Cat# 78442). Protein concentrations were determined using the Pierce\u0026trade; BCA Protein Assay Kit (ThermoFisher Scientific, Cat# 23227). Cell lysates were boiled with SDS sample buffer. Proteins from the cell lysates were separated by SDS polyacrylamide gel electrophoresis (SDS\u0026ndash;PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (ThermoFisher Scientific, Cat# 88518). The PVDF membranes were blocked with TBST (10 mM Tris-HCl, 150 mM NaCl, and 0.1% v/v Tween-20) containing 5% w/v skim milk and incubated with primary antibodies. After washing in TBST, membranes were incubated with HRP-conjugated secondary antibodies and then washed with TBST before developing with Western Lightning\u0026trade; Plus Chemiluminescence Reagent (PerkinElmer, Cat# NEL103001EA) or SuperSignal\u0026trade; West Dura Extended Duration Substrate (ThermoFisher Scientific, Cat# 37075). Luminescent signal was detected using the Azure c300 or c600 (Azure Biosystems). ImageJ (NIH, Bethesda, MD, USA) was used to quantify the intensity of bands on the blots. Images were adjusted equally for brightness and contrast using ImageJ or Adobe Photoshop (Adobe Inc.).\u003c/p\u003e\u003cp\u003eARF6-GTP pull-downs were performed using GGA3 PBD Agarose beads (Cell Biolabs, Cat# STA-419) as previously described\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Briefly, cells were treated with chemical compounds for the indicated time. After treatment, cells were lysed with pulldown lysis buffer (Cell Biolabs, Cat# 240102) including 1X Halt\u0026trade; Protease and Phosphatase Inhibitor Cocktail. Lysates were centrifuged, supernatants were added to GGA3-conjugated beads and agitated for 1 hour at 4\u0026deg;C. Beads were washed in ARF6-pulldown lysis buffer and prepared for western blot analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCell viability assay\u003c/h2\u003e\u003cp\u003eCell viability was detected by CellTiter-Glo\u0026reg; Luminescent Cell Viability Assay (Promega, Cat# G7571). Briefly, 2000 cells/well were seeded in 96 well plates overnight. The next day, cell viability was measured before treatment (0-hour time point). After 48 or 72 hours of treatment, media were removed and replaced with the CellTiter-Glo\u0026reg; Reagent. Luminescence was measured by Perkin Elmer EnVision Multi-Mode Plate Reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eApoptosis Assay\u003c/h2\u003e\u003cp\u003eApoptosis was detected by RealTime Glo\u0026trade; Annexin V Apoptosis Assay (Promega; Cat# JA1000). Briefly, 10,000 cells/well were seeded in 96 well plates overnight. The next day, cells were treated with serum starvation or chemical compounds to induce apoptosis plus apoptosis detection reagent. Annexin V luminescence was measured by Perkin Elmer EnVision Multi-Model Plate Reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eDrug tolerant colony formation\u003c/h2\u003e\u003cp\u003eHuman melanoma cells A375 were seeded at 10,000 cells per well in 6 well plates and treated with vemurafenib, dabrafenib, trametinib, and/or QS11 for 30 days. For colony formation assay with early passage, patient-derived MTG013 cells were seeded at 200,000 cells per well in 6 well plates and treated with dabrafenib, trametinib, and/or NAV-2729 for 14 days. For patient-derived MTG030 cells were treated with dabrafenib, trametinib, and/or doxycycline. Drugs were refreshed every 2\u0026ndash;3 days. After 14 or 30 days of drug treatment, cells were fixed with methanol and stained with 0.5% crystal violet stains. Plates were scanned with LICOR Odyssey\u0026reg; DLx scanner. Colony Area or intensity was measured by ImageJ\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Representative images were captured with a Nikon Automated Widefield Microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eCloning, viral transduction and generation of stable cell lines\u003c/h2\u003e\u003cp\u003eThe pTRIPZ lentviral system (used for cloning pTRIPZ-ARF6\u003csup\u003eWT\u003c/sup\u003e-V5 and pTRIPZ-ARF6\u003csup\u003eQ67L\u003c/sup\u003e-V5) was gifted from Dr. Todd W. Ridky\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. ARF6\u003csup\u003eWT\u003c/sup\u003e-V5 and ARF6\u003csup\u003eQ67L\u003c/sup\u003e-V5 were inserted into the p-TRPIZ vector using the In-Fusion Snap Assembly system (Takarabio, Cat# 638945). HEK-293T cells were co-transfected with 2nd generation lentivirus packaging vectors (5\u0026micro;g pCMV-Gag/Pol, Addgene, Cat#35614; 1\u0026micro;g pCMV-VSVG, Addgene, Cat# 8454) and 5\u0026micro;g of expression constructs (including piSMART-hEF1a-TurboGFP-sh\u003cem\u003eARF6\u003c/em\u003e, see Key Resource Table) using Lipofectamine\u0026trade; 3000 Transfection Reagent (Thermo Scientific, Cat# L3000008). Viral supernatants were harvested 48 hours and 72 hours post-transfection and filtered through a 0.45 \u0026micro;m filter. Filtered viral supernatants were applied to target cell lines together with 10 \u0026micro;g/ml of Polybrene (Sigma Cat# TR-1003). After infection, cells were placed in fresh media for three days before selection with 1\u0026micro;M puromycin for 14 days. Doxycycline dose response treatments confirmed ectopic expression or knockdown efficiency. Stable cell lines were maintained in 1\u0026micro;m puromycin.\u003c/p\u003e\u003cp\u003eAdenoviral ARF6\u003csup\u003eQ67L\u003c/sup\u003e was created by Vector Biolabs as previously described\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Cells were infected with 10\u003csup\u003e7\u003c/sup\u003e pfu/mL virus and incubated for 24 hours prior to experimentation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eProteomics\u003c/h2\u003e\u003cp\u003eProtein extraction and reverse-phase protein array of frozen mouse tumors were performed by the MD Anderson Cancer Center Functional Proteomic RPPA Core Facility.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative reverse transcription polymerase chain reaction (qRT-PCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was isolated from A375 cells after doxycycline-induced expression of ARF6\u003csup\u003eQ67L\u003c/sup\u003e. Cells were untreated or treated with 1\u0026micro;M doxycycline for 4, 8, 24, or 48 hours, then collected and stored in RNAlater (ThermoFisher Scientific, Cat# AM7024). RNA was extracted using RNeasy Plus kit (Qiagen, Cat# 74034) according to manufacturer's instructions. Extracted RNA from each sample was converted into cDNA using SuperScript IV VILO (SSIV VILO) Master Mix (ThermoFisher Scientific, Cat# 11756050). qRT-PCR was performed in triplicate for each sample using PowerUp\u0026trade; SYBR\u0026trade; Green Master Mix (ThermoFisher Scientific, Cat# A25780) on the QuantStudio\u0026trade; 6 Flex Real-Time PCR System (ThermoFisher Scientific) in 96-well plates. Primers used for qRT-PCR are shown in the Key Resource Table. The specificity of the amplicons was assessed by melting curve analyses. Relative mRNA expression of each gene was calculated using the number of cycles needed to reach the crossing threshold of detection (CT) and normalized to the expression of \u003cem\u003eGAPDH\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\u003cp\u003eMurine tumors were embedded and frozen in Tissue-Tek\u0026reg; O.C.T. compound. Tissues were sectioned 6\u0026ndash;10\u0026micro;m thick using a cryostat. The tissue was fixed to the slides with acetone followed by three rinses in \"PBSA\" (1\u0026times; PBS\u0026thinsp;+\u0026thinsp;0.1% sodium azide). Slides were permeabilized with 1% bovine serum albumin (BSA)\u0026thinsp;+\u0026thinsp;0.1% Saponin solution, followed by blocking in PBSA\u0026thinsp;+\u0026thinsp;3% v/v BSA for 60 minutes. After blocking, the slides were incubated with the primary antibody overnight at 4\u0026deg;C. The next day, the slides were washed with PBSA and incubated with secondary antibody for 1 hour at room temperature, then washed again with PBSA After washing the slides before counterstaining with DAPI for 30 minutes at room temperature, followed by a 5-minute wash in PBSA, and mounting in 40% w/v polyvinyl pyrrolidone\u0026thinsp;+\u0026thinsp;4% v/v glycerol\u0026thinsp;+\u0026thinsp;0.1% sodium azide dissolved in 1 mol/L Tris, pH 8.0. Images were collected on an Olympus Fluoview1000 scanning laser confocal microscope at 1,200x magnification. Quantification of fluorescent signals on mouse tumor tissue was performed in ImageJ. Final signal intensity for BRAF, pMEK and pERK was calculated by total green signal count divided by the number of nuclei (DAPI stained).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eDetails of each statistical analysis are included in the figure legends. Statistical tests were performed using Prism software (GraphPad). Quantitative values are shown with or represented as the mean of at least three biologic replicates.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data generated herein are available from the corresponding author upon request.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eEthics and Inclusion Statement\u003c/h2\u003e\u003cp\u003e This research included local researchers throughout the research process, is locally relevant and has been determined in collaboration with local partners. Roles and responsibilities were agreed amongst collaborators ahead of the research and with capacity in mind. This research was not severely restricted or prohibited by local stakeholders. This research was approved by a local ethics review committee and complied with animal welfare regulations, environmental protection and biorisk-related regulations and the local research setting was sufficient to conduct the research described. No stigmatization, incrimination, discrimination, nor personal risk to participants or researchers was involved in this research. The researchers and facilities will not benefit monetarily from sharing biological materials, including if transferred out of the country of origin. Local and regional research relevant to this study have been included in citations.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eAuthors\u0026rsquo; Contributions\u003c/h2\u003e\u003cp\u003eConceptualization, A.H.G; project administration, A.H.G., J.W.; investigation, J.W., P.G., T.J., D.M.B., A.R., L.K.S., R.K.W., A.H.G.; data curation, E.C.W.; methodology, Y.W., M.M., S.L.H., R.L.J-T., A.H.G.; validation, P.G.; resources, J.K.H.T., T.L., E.A.S., R.L.J-T., S.L.H., V.G.Y., M.A.D.; supervision, A.H.G., R.K.W., R.L.J-T., Y.W.; writing-original draft, A.H.G, J.W.; writing-reviewing \u0026amp; editing, A.H.G, J.W., Y.W., J.K.H.T., P.G., T.J., D.M.B.; funding acquisition, A.H.G.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eWe thank Diana Lim and Nikita Abraham for preparation of scientific graphics; the HCI Preclinical Research Resource Lab, University of Utah (UU) Flow Cytometry Core, UU Genomics Core, UU HSC Cell Imaging Core; MD Anderson Cancer Center Functional Proteomics Core. This project was supported by funding from NIH/NCI P30CA042014 (HCI), and by funding in support of A.H.G. including American Cancer Society 133649-RSG-19-019-01-CSM, NIH/NCI K08CA188563, NIH/NCI R37CA230630, the UU Department of Pathology, and the Earl A. Chiles Research Institute at the Providence Cancer Institute of Oregon. S.L.H. is supported by NIH/NCI R01CA121118. M.A.D. is supported by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, the AIM at Melanoma Foundation, the NIH/NCI P50CA221703, the American Cancer Society, the Melanoma Research Alliance, Cancer Fighters of Houston, the Anne and John Mendelsohn Chair for Cancer Research, and philanthropic contributions to the Melanoma Moon Shots Program of MD Anderson.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHanahan, D. 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ColonyArea: an ImageJ plugin to automatically quantify colony formation in clonogenic assays. \u003cem\u003ePLoS One\u003c/em\u003e 9, e92444 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcNeal, A.S. \u003cem\u003eet al.\u003c/em\u003e CDKN2B Loss Promotes Progression from Benign Melanocytic Nevus to Melanoma. \u003cem\u003eCancer Discov\u003c/em\u003e 5, 1072\u0026ndash;1085 (2015).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7133814/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7133814/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe intrinsic ability of cancer cells to evade death underpins tumorigenesis, progression, metastasis and the survival of drug-tolerant persister (DTP) cells. Herein, we discovered that when activated, the small GTPase ARF6 plays a central role in tumor survival by facilitating expression of the BRAF\u003csup\u003eV600E\u003c/sup\u003e oncoprotein. Tumor-specific \u003cem\u003eArf6\u003c/em\u003e deletion caused a significant reduction in BRAF\u003csup\u003eV600E\u003c/sup\u003e protein and MAPK signaling and prevented rapid tumor progression. In the context of targeted therapy, BRAF inhibition induced swift activation of ARF6, driving a positive feedback loop that restored MAPK-driven anti-apoptotic signaling, facilitated DTP cell survival during the early phases of treatment and contributed to drug-tolerant growth. In patient-derived melanoma cells with innate or clinically acquired resistance to MAPK inhibitors, ARF6 inhibition enhanced sensitivity to combined BRAF\u0026thinsp;+\u0026thinsp;MEK inhibition. Collectively, these findings elucidate an ARF6-dependent mechanism of BRAF oncoprotein synthesis that may be exploited in BRAF\u003csup\u003eV600E\u003c/sup\u003e driven cancers as a therapeutic vulnerability.\u003c/p\u003e","manuscriptTitle":"Rapid activation of ARF6 after RAF inhibition augments BRAFV600E and promotes therapy resistance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 05:36:09","doi":"10.21203/rs.3.rs-7133814/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f33b5a0d-9290-4196-a430-460f93a07499","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53586438,"name":"Health sciences/Oncology/Cancer/Cancer therapy/Cancer therapeutic resistance"},{"id":53586439,"name":"Biological sciences/Cancer/Cancer models"}],"tags":[],"updatedAt":"2026-04-29T07:11:33+00:00","versionOfRecord":{"articleIdentity":"rs-7133814","link":"https://doi.org/10.1038/s41388-026-03805-w","journal":{"identity":"oncogene","isVorOnly":false,"title":"Oncogene"},"publishedOn":"2026-04-28 04:00:00","publishedOnDateReadable":"April 28th, 2026"},"versionCreatedAt":"2025-08-27 05:36:09","video":"","vorDoi":"10.1038/s41388-026-03805-w","vorDoiUrl":"https://doi.org/10.1038/s41388-026-03805-w","workflowStages":[]},"version":"v1","identity":"rs-7133814","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7133814","identity":"rs-7133814","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}