Rag GTPases Research Articles

Genetic dissection of early endosomal recycling highlights a TORC1‐independent role for Rag GTPases

Endocytosed cell surface membrane proteins rely on various recycling pathways for their return to the plasma membrane1. Although endosome‐to‐plasma membrane recycling is critical for many cellular processes2, much of the required machinery is unknown. We discovered that yeast also has a recycling route to the cell surface that bypasses the Golgi. Using an engineered synthetic reporter that exclusively follows this pathway, we performed a genetic screen to reveal a cohort of novel and conserved regulators of direct endosome to cell surface recycling. One critical component was the Rag GTPases, Gtr1 and Gtr2, which worked downstream of the exchange factor Vam6 and upstream of the Gtr1 effector Ltv1. Although the Rag GTPases are known to activate TORC1 on the vacuole/lysosome3–5, we find the Vam6>Gtr1‐Gtr2>Ltv1 recycling pathway is independent of TORC1, thus establishing a distinct role for these GTPases in early endosome recycling.Support or Funding InformationRO1GM058202

A Non‐Canonical Function of Leucyl tRNA Synthetase Negatively Regulates Skeletal Myogenesis

Aside from its house‐keeping function in protein synthesis, leucyl tRNA synthetase (LeuRS) has a non‐canonical function in regulating cell growth via the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) pathway by sensing amino acid availability. mTOR also governs skeletal myogenesis, but its signaling network is distinct from that in cell growth regulation. We set out to investigate a potential role of LeuRS in skeletal myogenesis. Knockdown of LeuRS enhanced myoblast differentiation in vitro, whereas overexpression of LeuRS inhibited myogenesis. In a mouse model of injury‐induced adult muscle regeneration, local knockdown of LeuRS enhanced regeneration. Hence, LeuRS acts as a negative regulator of myogenesis both in vitro and in vivo. However, inhibition of the translational function of LeuRS by leucinol severely impaired myogenesis, consistent with the notion that protein synthesis is required for the myogenic process. These observations suggest a non‐translation role for LeuRS in suppressing myogenesis. Indeed, we found leucine‐binding but not tRNA charging activity of LeuRS to be necessary for its overexpression effect on myogenesis, suggesting that LeuRS may act as a leucine sensor in its function to ensure homeostasis of myogenesis. To probe the mechanism underlying this non‐canonical function of LeuRS, we examined the involvement of Rag GTPases, mediators of LeuRS activation of mTORC1 in cell growth. Constitutively active Rag reversed the effect of LeuRS knockdown on myogenesis and, conversely, Rag knockdown overcame the inhibitory effect of LeuRS overexpression. Rag knockdown also enhanced muscle regeneration in vivo, mirroring the effect of LeuRS knockdown. Taken together, our findings reveal for the first time a function for LeuRS in modulating the homeostasis of myogenesis. This new LeuRS function is mediated by a Rag‐mTORC1 pathway that negatively regulates the myogenic IRS1‐PI3K‐Akt pathway.Support or Funding InformationKeck Foundation, NIH/NIAMS R01AR048914

Abstract IA02: Regulation of growth by the mTOR pathway

Abstract mTOR is the target of the immunosuppressive and anti-cancer drug rapamycin and the central component of a nutrient- and hormone-sensitive signaling pathway that regulates cell growth and proliferation. We now appreciate that this pathway becomes deregulated in many human cancers and has key roles in the control of metabolism, cell and organ size, and aging. We have identified two distinct mTOR-containing proteins complexes, one of which regulates growth through S6K and another that regulates cell survival and metabolism through Akt. These complexes, called mTORC1 and mTORC2, define both rapamycin-sensitive and insensitive branches of the mTOR pathway. Over the last few years we have focused on how nutrients, particularly amino acids, regulate mTORC1. We have identified a complex signaling pathway that relays amino acid availability to mTORC1 through the heterodimeric Rag GTPases. We recently discovered several of the amino acid sensors for the pathway. I will discuss new results from our lab on the regulation and functions of mTORC1. Citation Format: David M. Sabatini. Regulation of growth by the mTOR pathway. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA02.

KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1.

The mechanistic target of rapamycin complex 1 kinase (mTORC1) is a central regulator of cell growth that responds to diverse environmental signals and is deregulated in many human diseases, including cancer and epilepsy1–3. Amino acids are a key input, and act through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation4. Multiple protein complexes regulate the Rag GTPases in response to amino acids, including GATOR1, a GTPase activating protein for RagA, and GATOR2, a positive regulator of unknown molecular function. Here, we identify a four-membered protein complex (KICSTOR) composed of the KPTN, ITFG2, C12orf66, and SZT2 gene products as required for amino acid or glucose deprivation to inhibit mTORC1 in cultured cells. In mice lacking SZT2, mTORC1 signaling is increased in several tissues, including in neurons in the brain. KICSTOR localizes to lysosomes; binds to GATOR1 and recruits it, but not GATOR2, to the lysosomal surface; and is necessary for the interaction of GATOR1 with its substrates, the Rag GTPases, and with GATOR2. Interestingly, several KICSTOR components are mutated in neurological diseases associated with mutations that lead to hyperactive mTORC1 signaling5–10. Thus, KICSTOR is a lysosome-associated negative regulator of mTORC1 signaling that, like GATOR1, is mutated in human disease11,12.

SZT2 dictates GATOR control of mTORC1 signalling.

Mechanistic target of rapamycin complex 1 (TORC1) integrates nutrient signals to control cell growth and organismal homeostasis across eukaryotes. The evolutionarily conserved GATOR complex regulates mTORC1 signalling through Rag GTPases, and GATOR1 displays GTPase activating protein (GAP) activity for RAGA and RAGB (RAGA/B) and GATOR2 has been proposed to be an inhibitor of GATOR1. Furthermore, the metazoan-specific SESN proteins function as guanine nucleotide dissociation inhibitors (GDIs) for RAGA/B, and interact with GATOR2 with unknown effects. Here we show that SZT2 (seizure threshold 2), a metazoan-specific protein mutated in epilepsy, recruits a fraction of mammalian GATOR1 and GATOR2 to form a SZT2-orchestrated GATOR (SOG) complex with an essential role in GATOR- and SESN-dependent nutrient sensing and mTORC1 regulation. The interaction of SZT2 with GATOR1 and GATOR2 was synergistic, and an intact SOG complex was required for its localization at the lysosome. SZT2 deficiency resulted in constitutive mTORC1 signalling in cells under nutrient-deprived conditions and neonatal lethality in mice, which was associated with failure to inactivate mTORC1 during fasting. Hyperactivation of mTORC1 in SZT2-deficient cells could be partially corrected by overexpression of the GATOR1 component DEPDC5, and by the lysosome-targeted GATOR2 component WDR59 or lysosome-targeted SESN2. These findings demonstrate that SZT2 has a central role in dictating GATOR-dependent nutrient sensing by promoting lysosomal localization of SOG, and reveal an unexpected function of lysosome-located GATOR2 in suppressing mTORC1 signalling through SESN recruitment.

Nudix-type motif 2 contributes to cancer proliferation through the regulation of Rag GTPase-mediated mammalian target of rapamycin complex 1 localization

Lysosomal localization of mammalian target of rapamycin complex 1 (mTORC1) is a critical step for activation of the molecule. Rag GTPases are essential for this translocation. Here, we demonstrate that Nudix-type motif 2 (NUDT2) is a novel positive regulator of mTORC1 activation. Activation of mTORC1 is impaired in NUDT2-silenced cells. Mechanistically, NUDT2 binds to Rag GTPase and controls mTORC1 translocation to the lysosomal membrane. Furthermore, NUDT2-dependent mTORC1 regulation is critical for proliferation of breast cancer cells, as NUDT2-silenced cells arrest in G0/G1 phases. Taken together, these results show that NUDT2 is a novel complex formation enhancing factor regulating mTORC1-Rag GTPase signaling that is crucial for cell growth control.

Novel tRNA function in amino acid sensing of yeast Tor complex1.

TOR complex1 (TORC1), a master regulator of cell growth, is regulated by amino acids. Amino acids are fundamental nutrients, and 20 species of amino acids building proteins are not interchangeable with each other. Therefore, TORC1 should sense each amino acid individually. Mammalian mTORC1 is controlled by Rag GTPases and their regulators. However, Rag factors are dispensable for amino acid sensing by TORC1 in the budding yeast, suggesting an alternative mechanism of TORC1 regulation. Here, genetic investigation discovered the involvement of (aminoacyl-)tRNA ((aa-)tRNA) in TORC1 regulation. Biochemical TORC1 assay also showed that tRNA directly inhibits TORC1 kinase activity. Reducing cellular tRNA molecule desensitizes TORC1 inactivation by nitrogen starvation invivo. Based on these results, I propose a model of the TORC1 regulatory mechanism in which free tRNA released from protein synthesis under amino acid starvation inhibits TORC1 activity. Therefore, TORC1 uses tRNA-mediated mechanism and Rag factors in parallel to sense intracellular amino acids.

Dynamics of mTORC1 activation in response to amino acids.

Amino acids are essential activators of mTORC1 via a complex containing RAG GTPases, RAGULATOR and the vacuolar ATPase. Sensing of amino acids causes translocation of mTORC1 to lysosomes, an obligate step for activation. To examine the spatial and temporal dynamics of this translocation, we used live imaging of the mTORC1 component RAPTOR and a cell permeant fluorescent analogue of di-leucine methyl ester. Translocation to lysosomes is a transient event, occurring within 2 min of aa addition and peaking within 5 min. It is temporally coupled with fluorescent leucine appearance in lysosomes and is sustained in comparison to aa stimulation. Sestrin2 and the vacuolar ATPase are negative and positive regulators of mTORC1 activity in our experimental system. Of note, phosphorylation of canonical mTORC1 targets is delayed compared to lysosomal translocation suggesting a dynamic and transient passage of mTORC1 from the lysosomal surface before targetting its substrates elsewhere.

Structural insight into the arginine-binding specificity of CASTOR1 in amino acid-dependent mTORC1 signaling.

The mechanistic Target Of Rapamycin Complex 1 (mTORC1) is central to the cellular response to changes in nutrient signals such as amino acids. CASTOR1 is shown to be an arginine sensor, which plays an important role in the activation of the mTORC1 pathway. In the deficiency of arginine, CASTOR1 interacts with GATOR2, which together with GATOR1 and Rag GTPases controls the relocalization of mTORC1 to lysosomes. The binding of arginine to CASTOR1 disrupts its association with GATOR2 and hence activates the mTORC1 signaling. Here, we report the crystal structure of CASTOR1 in complex with arginine at 2.5 Å resolution. CASTOR1 comprises of four tandem ACT domains with an architecture resembling the C-terminal allosteric domains of aspartate kinases. ACT1 and ACT3 adopt the typical βαββαβ topology and function in dimerization via the conserved residues from helices α1 of ACT1 and α5 of ACT3; whereas ACT 2 and ACT4, both comprising of two non-sequential regions, assume the unusual ββαββα topology and contribute an arginine-binding pocket at the interface. The bound arginine makes a number of hydrogen-bonding interactions and extensive hydrophobic contacts with the surrounding residues of the binding pocket. The functional roles of the key residues are validated by mutagenesis and biochemical assays. Our structural and functional data together reveal the molecular basis for the arginine-binding specificity of CASTOR1 in the arginine-dependent activation of the mTORC1 signaling.

The Loss of Lam2 and Npr2-Npr3 Diminishes the Vacuolar Localization of Gtr1-Gtr2 and Disinhibits TORC1 Activity in Fission Yeast.

In mammalian cells, mTORC1 activity is regulated by Rag GTPases. It is thought that the Ragulator complex and the GATOR (GAP activity towards Rags) complex regulate RagA/B as its GDP/GTP exchange factor (GEF) and GTPase-activating protein (GAP), respectively. However, the functions of components in these complexes remain elusive. Using fission yeast as a model organism, here we found that the loss of Lam2 (SPBC1778.05c), a homolog of a Ragulator component LAMTOR2, as well as the loss of Gtr1 or Gtr2 phenocopies the loss of Npr2 or Npr3, homologs of GATOR components Nprl2 or Nprl3, respectively. These phenotypes were rescued by TORC1 inhibition using pharmacological or genetic means, and the loss of Lam2, Gtr1, Gtr2, Npr2 or Npr3 disinhibited TORC1 activity under nitrogen depletion, as measured by Rps6 phosphorylation. Consistently, overexpression of GDP-locked Gtr1S20L or GTP-locked Gtr2Q60L, which suppress TORC1 activity in budding yeast, rescued the growth defect of Δgtr1 cells or Δgtr2 cells, respectively, and the loss of Lam2, Npr2 or Npr3 similarly diminished the vacuolar localization and the protein levels of Gtr1 and Gtr2. Furthermore, Lam2 physically interacted with Npr2 and Gtr1. These findings suggest that Lam2 and Npr2-Npr3 function together as a tether for GDP-bound Gtr1 to the vacuolar membrane, thereby suppressing TORC1 activity for multiple cellular functions.

The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids

The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1.

Differential Regulation of mTORC1 by Amino Acids

Organisms sense nutrient fluctuations and respond accordingly to control cell growth. When this process goes awry, the result can be detrimental, often leading to cancer. Central to this process is the mechanistic target of rapamycin (mTOR), a conserved kinase that is the main component of a protein complex termed mTOR complex 1 (mTORC1). mTORC1 activation is frequently observed in human cancers, and therapeutics that target mTORC1 have proven efficacious in clinical trials. Amino acids are the most potent stimuli for mTORC1 activation; however, the detailed molecular mechanisms are obscure, and importantly, the specific amino acids that regulate mTORC1 are unclear. Recently, we discovered that the amino acids, leucine and glutamine activate mTORC1 through two distinct signaling pathways involving the small G‐proteins RagA/B and the adenosine diphosphate ribosylation factor‐1 (Arf1), respectively. Importantly, we identified Arf1 as an essential component that specifically couples glutamine signaling to mTORC1 activation at the lysosome and independent of RagA/B. This is important because cancer cells have a high dependence on glutamine for growth and proliferation. Our results uncover a signaling cascade to mTORC1 activation independent of the Rag GTPases and suggest that mTORC1 is differentially regulated by specific amino acids.Support or Funding InformationCancer Prevention & Research Institute of Texas (CPRIT)

Conserved regulators of Rag GTPases orchestrate amino acid-dependent TORC1 signaling.

The highly conserved target of rapamycin complex 1 (TORC1) is the central component of a signaling network that couples a vast range of internal and external stimuli to cell growth, proliferation and metabolism. TORC1 deregulation is associated with a number of human pathologies, including many cancers and metabolic disorders, underscoring its importance in cellular and organismal growth control. The activity of TORC1 is modulated by multiple inputs; however, the presence of amino acids is a stimulus that is essential for its activation. Amino acid sufficiency is communicated to TORC1 via the highly conserved family of Rag GTPases, which assemble as heterodimeric complexes on lysosomal/vacuolar membranes and are regulated by their guanine nucleotide loading status. Studies in yeast, fly and mammalian model systems have revealed a multitude of conserved Rag GTPase modulators, which have greatly expanded our understanding of amino acid sensing by TORC1. Here we review the major known modulators of the Rag GTPases, focusing on recent mechanistic insights that highlight the evolutionary conservation and divergence of amino acid signaling to TORC1.

The CASTOR Proteins Are Arginine Sensors for the mTORC1 Pathway

Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of ~30 μM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway.

Rag GTPase in amino acid signaling.

Rag small GTPases were identified as the sixth subfamily of Ras-related GTPases. Compelling evidence suggests that Rag heterodimer (RagA/B and RagC/D) plays an important role in amino acid signaling toward mechanistic target of rapamycin complex 1 (mTORC1), which is a central player in the control of cell growth in response to a variety of environmental cues, including growth factors, cellular energy/oxygen status, and amino acids. Upon amino acid stimulation, active Rag heterodimer (RagA/B(GTP)-RagC/D(GDP)) recruits mTORC1 to the lysosomal membrane where Rheb resides. In this review, we provide a current understanding on the amino acid-regulated cell growth control via Rag-mTORC1 with recently identified key players, including Ragulator, v-ATPase, and GATOR complexes. Moreover, the functions of Rag in physiological systems and in autophagy are discussed.

PRL-3 activates mTORC1 in Cancer Progression.

PRL-3, a metastasis-associated phosphatase, is known to exert its oncogenic functions through activation of PI3K/Akt, which is a key regulator of the rapamycin-sensitive mTOR complex 1 (mTORC1), but a coherent link between PRL-3 and activation of mTOR has not yet been formally demonstrated. We report a positive correlation between PRL-3 expression and mTOR phospho-activation in clinical tumour samples and mouse models of cancer and demonstrate that PRL-3 increased downstream signalling to the mTOR substrates, p70S6K and 4E-BP1, by increasing PI3K/Akt-mediated activation of Rheb-GTP via TSC2 suppression. We also show that PRL-3 increases mTOR translocation to lysosomes via increased mTOR binding affinity to Rag GTPases in an Akt-independent manner, demonstrating a previously undescribed mechanism of action for PRL-3. PRL-3 also enhanced matrix metalloproteinase-2 secretion and cellular invasiveness via activation of mTOR, attributes which were sensitive to rapamycin treatment. The downstream effects of PRL-3 were maintained even under conditions of environmental stress, suggesting that PRL-3 provides a strategic survival advantage to tumour cells via its effects on mTOR.

NLK phosphorylates Raptor to mediate stress-induced mTORC1 inhibition.

The mechanistic target of rapamycin (mTOR) is a central cell growth controller and forms two distinct complexes: mTORC1 and mTORC2. mTORC1 integrates a wide range of upstream signals, both positive and negative, to regulate cell growth. Although mTORC1 activation by positive signals, such as growth factors and nutrients, has been extensively investigated, the mechanism of mTORC1 regulation by stress signals is less understood. In this study, we identified the Nemo-like kinase (NLK) as an mTORC1 regulator in mediating the osmotic and oxidative stress signals. NLK inhibits mTORC1 lysosomal localization and thereby suppresses mTORC1 activation. Mechanistically, NLK phosphorylates Raptor on S863 to disrupt its interaction with the Rag GTPase, which is important for mTORC1 lysosomal recruitment. Cells with Nlk deletion or knock-in of the Raptor S863 phosphorylation mutants are defective in the rapid mTORC1 inhibition upon osmotic stress. Our study reveals a function of NLK in stress-induced mTORC1 modulation and the underlying biochemical mechanism of NLK in mTORC1 inhibition in stress response.

Amino acid-dependent NPRL2 interaction with Raptor determines mTOR Complex 1 activation

We assign a new function to a tumor suppressor NPRL2 that activates the mTOR complex 1 (mTORC1) activity. The positive regulation of mTORC1 activity by NPRL2 is mediated through NPRL2 interaction with Raptor. While NPRL2 interacts with Rag GTPases, RagD in particular, to interfere with mTORC1 activity in amino acid scarcity, NPRL2 interacts with Raptor in amino acid sufficiency to activate mTORC1. A reciprocal relationship exists between NPRL2 binding to Rag GTPases and Raptor. NPRL2 majorly locates in the lysosomal membranes and has a higher binding affinity to the dominant negative mutant heterodimer of RagAGDP/RagDGTP that inactivates mTORC1. However, the binding affinity of NPRL2 with Raptor is much less pronounced in cells expressing the dominant negative mutant heterodimer of RagAGDP/RagDGTP than in cells expressing the dominant positive mutant heterodimer, RagAGTP/RagDGDP. The positive effect of NPRL2 on TORC1 pathway was also evidenced in Drosophila animal model. Here, we propose a ‘seesaw’ model in which the interactive behavior of NPRL2 with Raptor determines mTORC1 activation by amino acid signaling in animal cells.

Amino Acids Stimulate TORC1 through Lst4-Lst7, a GTPase-Activating Protein Complex for the Rag Family GTPase Gtr2.

Rag GTPases assemble into heterodimeric complexes consisting of RagA or RagB and RagC or RagD in higher eukaryotes, or Gtr1 and Gtr2 in yeast, to relay amino acid signals toward the growth-regulating target of rapamycin complex 1 (TORC1). The TORC1-stimulating state of Rag GTPase heterodimers, containing GTP- and GDP-loaded RagA/B/Gtr1 and RagC/D/Gtr2, respectively, is maintained in part by the FNIP-Folliculin RagC/D GAP complex in mammalian cells. Here, we report the existence of a similar Lst4-Lst7 complex in yeast that functions as a GAP for Gtr2 and that clusters at the vacuolar membrane in amino acid-starved cells. Refeeding of amino acids, such as glutamine, stimulated the Lst4-Lst7 complex to transiently bind and act on Gtr2, thereby entailing TORC1 activation and Lst4-Lst7 dispersal from the vacuolar membrane. Given the remarkable functional conservation of the RagC/D/Gtr2 GAP complexes, our findings could be relevant for understanding the glutamine addiction of mTORC1-dependent cancers.

Molecular genetics and clinical features of Birt-Hogg-Dubé syndrome.

Birt-Hogg-Dubé (BHD) syndrome is an inherited renal cancer syndrome in which affected individuals are at risk of developing benign cutaneous fibrofolliculomas, bilateral pulmonary cysts and spontaneous pneumothoraces, and kidney tumours. Bilateral multifocal renal tumours that develop in BHD syndrome are most frequently hybrid oncocytic tumours and chromophobe renal carcinoma, but can present with other histologies. Germline mutations in the FLCN gene on chromosome 17 are responsible for BHD syndrome--BHD-associated renal tumours display inactivation of the wild-type FLCN allele by somatic mutation or chromosomal loss, confirming that FLCN is a tumour suppressor gene that fits the classic two-hit model. FLCN interacts with two novel proteins, FNIP1 and FNIP2, and with AMPK, a negative regulator of mTOR. Studies with FLCN-deficient cell and animal models support a role for FLCN in modulating the AKT-mTOR pathway. Emerging evidence links FLCN with a number of other molecular pathways and cellular processes important for cell homeostasis that are frequently deregulated in cancer, including regulation of TFE3 and/or TFEB transcriptional activity, amino-acid-dependent mTOR activation through Rag GTPases, TGFβ signalling, PGC1α-driven mitochondrial biogenesis, and autophagy. Currently, surgical intervention is the only therapy available for BHD-associated renal tumours, but improved understanding of the FLCN pathway will hopefully lead to the development of effective forms of targeted systemic therapy for this disease.