Activation of P53 pathway contributes to Xenopus hybrid inviability.

Hybrid incompatibility as a kind of reproductive isolation contributes to speciation. The nucleocytoplasmic incompatibility between Xenopus tropicalis eggs and Xenopus laevis sperm (te×ls) leads to specific loss of paternal chromosomes 3L and 4L. The hybrids die before gastrulation, of which the lethal causes remain largely unclear. Here, we show that the activation of the tumor suppressor protein P53 at late blastula stage contributes to this early lethality. We find that in stage 9 embryos, P53-binding motif is the most enriched one in the up-regulated Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) peaks between te×ls and wild-type X. tropicalis controls, which correlates with an abrupt stabilization of P53 protein in te×ls hybrids at stage 9. Inhibition of P53 activity via either tp53 knockout or overexpression of a dominant-negative P53 mutant or Murine double minute 2 proto-oncogene (Mdm2), a negative regulator of P53, by mRNA injection can rescue the te×ls early lethality. Our results suggest a causal function of P53 on hybrid lethality prior to gastrulation.

CD206+ M2-like macrophages protect against intervertebral disc degeneration partially by targeting R-spondin-2.

OBJECTIVE: This study aimed to explore the specific function of M2 macrophages in intervertebral disc degeneration (IDD). METHODS: Intervertebral disc (IVD) samples from normal (n = 4) and IDD (n = 6) patients were collected, and the expression of M2-polarized macrophage marker, CD206, was investigated using immunohistochemical staining. Nucleus pulposus cells (NPCs) in a TNF-α environment were obtained, and a mouse caudal IVD puncture model was established. Mice with Rheb deletions, specifically in the myeloid lineage, were generated and subjected to surgery-induced IDD. IDD-induced damage and cell apoptosis were measured using histological scoring, X-ray imaging, immunohistochemical staining, and TdT-mediated dUTP nick end labeling (TUNEL) assay. Finally, mice and NPCs were treated with R-spondin-2 (Rspo2) or anti-Rspo2 to investigate the role of Rspo2 in IDD. RESULTS: Accumulation of CD206 in human and mouse IDD tissues was detected. Rheb deletion in the myeloid lineage (RheBcKO) increased the number of CD206+ M2-like macrophages (mean difference 18.6% [15.7-21.6%], P < 0.001), decreased cell apoptosis (mean difference -15.6% [-8.9 to 22.2%], P = 0.001) and attenuated the IDD process in the mouse IDD model. NPCs treated with Rspo2 displayed increased extracellular matrix catabolism and apoptosis; co-culture with a conditioned medium derived from RheBcKO mice inhibited these changes. Anti-Rspo2 treatment in the mouse caudal IVD puncture model exerted protective effects against IDD. CONCLUSIONS: Promoting CD206+ M2-like macrophages could reduce Rspo2 secretion, thereby alleviating experimental IDD. Rheb deletion may help M2-polarized macrophages accumulate and attenuate experimental IDD partially by inhibiting Rspo2 production. Hence, M2-polarized macrophages and Rspo2 may serve as therapeutic targets for IDD.

Histone deacetylase 5-induced deficiency of signal transducer and activator of transcription-3 acetylation contributes to spinal astrocytes degeneration in painful diabetic neuropathy.

Diabetes patients with painful diabetic neuropathy (PDN) show severe spinal atrophy, suggesting pathological changes of the spinal cord contributes to central sensitization. However, the cellular changes and underlying molecular mechanisms within the diabetic spinal cord are less clear. By using a rat model of type 1 diabetes (T1D), we noted an extensive and irreversible spinal astrocyte degeneration at an early stage of T1D, which is highly associated with the chronification of PDN. Molecularly, acetylation of astrocytic signal transducer and activator of transcription-3 (STAT3) that is essential for maintaining the homeostatic astrocytes population was significantly impaired in the T1D model, resulting in a dramatic loss of spinal astrocytes and consequently promoting pain hypersensitivity. Mechanistically, class IIa histone deacetylase, HDAC5 were aberrantly activated in spinal astrocytes of diabetic rats, which promoted STAT3 deacetylation by direct protein-protein interactions, leading to the PDN phenotypes. Restoration of STAT3 signaling or inhibition of HDAC5 rescued astrocyte deficiency and attenuated PDN in the T1D model. Our work identifies the inhibitory axis of HDAC5-STAT3 induced astrocyte deficiency as a key mechanism underlying the pathogenesis of the diabetic spinal cord that paves the way for potential therapy development for PDN.

Kindlin-2 promotes rear focal adhesion disassembly and directional persistence during cell migration.

Cell migration involves front-to-rear asymmetric focal adhesion (FA) dynamics, which facilitates trailing edge detachment and directional persistence. Here, we show that kindlin-2 is crucial for FA sliding and disassembly in migrating cells. Loss of kindlin-2 markedly reduced FA number and selectively impaired rear FA sliding and disassembly, resulting in defective rear retraction and reduced directional persistence during cell migration. Kindlin-2-deficient cells failed to develop serum-induced actomyosin-dependent tension at FAs. At the molecular level, kindlin-2 directly interacted with myosin light chain kinase (MYLK, hereafter referred to as MLCK), which was enhanced in response to serum stimulation. Serum deprivation inhibited rear FA disassembly, which was released in response to serum stimulation. Overexpression of the MLCK-binding kindlin-2 F0F1 fragment (amino acid residues 1-167), which inhibits the interaction of endogenous kindlin-2 with MLCK, phenocopied kindlin-2 deficiency-induced migration defects. Inhibition of MLCK, like loss of kindlin-2, also impaired trailing-edge detachment, rear FA disassembly and directional persistence. These results suggest a role of kindlin-2 in promoting actomyosin contractility at FAs, leading to increased rear FA sliding and disassembly, and directional persistence during cell migration.

RSU-1 interaction with prohibitin-2 links cell-extracellular matrix detachment to downregulation of ERK signaling.

Cell-extracellular matrix (ECM) detachment is known to decrease extracellular signal-regulated kinase (ERK) signaling, an intracellular pathway that is central for control of cell behavior. How cell-ECM detachment is linked to downregulation of ERK signaling, however, is incompletely understood. We show here that focal adhesion protein Ras Suppressor 1 (RSU1) plays a critical role in cell-ECM detachment induced suppression of ERK signaling. We have identified prohibitin 2 (PHB2), a component of membrane lipid rafts, as a novel binding protein of RSU1, and mapped a major RSU1-binding site to PHB2 amino acids 150 to 206 in the C-terminal region of the PHB/SPFH (stomatin/prohibitin/flotillin/HflKC) domain. The PHB2 binding is mediated by multiple sites located in the N-terminal leucine-rich repeat region of RSU1. Depletion of PHB2 suppressed cell-ECM adhesion-induced ERK activation. Furthermore, cell-ECM detachment increased RSU1 association with membrane lipid rafts and interaction with PHB2. Finally, knockout of RSU1 or inhibition of RSU1 interaction with PHB2 by overexpression of the major RSU1-binding PHB2 fragment (amino acids 150-206) effectively suppressed the cell-ECM detachment induced downregulation of ERK signaling. Additionally, expression of venus-tagged wild-type RSU1 restored ERK signaling, while expression of venus-tagged PHB2-binding defective RSU1 mutant in which the N-terminal leucine-rich repeat region is deleted did not. Taken together, Our findings identify a novel RSU1-PHB2 signaling axis that senses cell-ECM detachment and links it to decreased ERK signaling.

Prefoldin subunit MM1 promotes cell migration via facilitating filopodia formation.

c-Myc modulator 1 (MM1), also known as PFDN5, is the fifth subunit of prefoldin. It was previously reported that MM1-based prefoldin promotes folding of actin during assembly of cytoskeleton, which plays key roles in cell migration. However, no evidence supports that MM1 affects cell migration. In the present study, we found that MM1 promotes cell migration in multiple cell lines. Further study revealed that MM1 promotes polymerization of ß-actin into filamentous form and increases both density and length of filopodia. Effects of MM1 on filopodia formation and cell migration depend on its prefoldin activity. Though c-Myc is repressed by MM1, simultaneous knock-down of c-Myc fails to rescue migration inhibition induced by MM1 ablation. Taken together, we here, for the first time, report that prefoldin subunit MM1 is involved in cell migration; this involvement of MM1 in cell migration is due to its prefoldin activity to boost polymerization of ß-actin during filopodia formation. Our findings may be helpful to elucidate the mechanism of cell migration and cancer metastasis.

Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.

Precise single-base editing in Xenopus tropicalis would greatly expand the utility of this true diploid frog for modeling human genetic diseases caused by point mutations. Here, we report the efficient conversion of C-to-T or G-to-A in X. tropicalis using the rat apolipoprotein B mRNA editing enzyme catalytic subunit 1-XTEN-clustered regularly interspaced short palindromic repeat-associated protein 9 (Cas9) nickase-uracil DNA glycosylase inhibitor-nuclear localization sequence base editor [base editor 3 (BE3)]. Coinjection of guide RNA and the Cas9 mutant complex mRNA into 1-cell stage X. tropicalis embryos caused precise C-to-T or G-to-A substitution in 14 out of 19 tested sites with efficiencies of 5-75%, which allowed for easy establishment of stable lines. Targeting the conserved T-box 5 R237 and Tyr C28 residues in X. tropicalis with the BE3 system mimicked human Holt-Oram syndrome and oculocutaneous albinism type 1A, respectively. Our data indicate that BE3 is an easy and efficient tool for precise base editing in X. tropicalis.-Shi, Z., Xin, H., Tian, D., Lian, J., Wang, J., Liu, G., Ran, R., Shi, S., Zhang, Z., Shi, Y., Deng, Y., Hou, C., Chen, Y. Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.

A double dealing tale of p63: an oncogene or a tumor suppressor.

As a member of tumor suppressor p53 family, p63, a gene encoding versatile protein variant, has been documented to correlate with cancer formation and progression, though it is rarely mutated in cancer patients. However, it has long been controversial on whether p63 is an oncogene or a tumor suppressor. Here, we comprehensively reviewed reports on roles of p63 in development, tumorigenesis and tumor progression. According to data from molecular cell biology, genetic models and clinic research, we conclude that p63 may act as either an oncogene or a tumor suppressor gene in different scenarios: TA isoforms of p63 gene are generally tumor-suppressive through repressing cell proliferation, survival and metastasis; ΔN isoforms, however, may initiate tumorigenesis via promoting cell proliferation and survival, but inhibit tumor metastasis and progression; effects of p63 on tumor formation and progression depend on the context of the whole p53 family, and either amplification or loss of p63 gene locus can break the balance to cause tumorigenesis.

In Situ Investigation of Defect-Free Copper Nanowire Growth.

The fabrication and placement of high purity nanometals, such as one-dimensional copper (Cu) nanowires, for interconnection in integrated devices have been among the most important technological developments in recent years. Structural stability and oxidation prevention have been the key issues, and the defect control in Cu nanowire growth has been found to be important. Here, we report the synthesis of defect-free single-crystalline Cu nanowires by controlling the surface-assisted heterogeneous nucleation of Cu atomic layering on the graphite-like loop of an amorphous carbon (a-C) lacey film surface. Without a metal-catalyst or induced defects, the high quality Cu nanowires formed with high aspect ratio and high growth rate of 578 nm/s. The dynamic study of the growth of heterogeneous nanowires was conducted in situ with a high-resolution transmission electron microscope. The study illuminates the new mechanism by heterogeneous nucleation control and laying the groundwork for better understanding of heterosurface-assisted nucleation of defect-free Cu nanowire on a-C lacey film.

Similarity in gene-regulatory networks suggests that cancer cells share characteristics of embryonic neural cells.

Cancer cells are immature cells resulting from cellular reprogramming by gene misregulation, and redifferentiation is expected to reduce malignancy. It is unclear, however, whether cancer cells can undergo terminal differentiation. Here, we show that inhibition of the epigenetic modification enzyme enhancer of zeste homolog 2 (EZH2), histone deacetylases 1 and 3 (HDAC1 and -3), lysine demethylase 1A (LSD1), or DNA methyltransferase 1 (DNMT1), which all promote cancer development and progression, leads to postmitotic neuron-like differentiation with loss of malignant features in distinct solid cancer cell lines. The regulatory effect of these enzymes in neuronal differentiation resided in their intrinsic activity in embryonic neural precursor/progenitor cells. We further found that a major part of pan-cancer-promoting genes and the signal transducers of the pan-cancer-promoting signaling pathways, including the epithelial-to-mesenchymal transition (EMT) mesenchymal marker genes, display neural specific expression during embryonic neurulation. In contrast, many tumor suppressor genes, including the EMT epithelial marker gene that encodes cadherin 1 (CDH1), exhibited non-neural or no expression. This correlation indicated that cancer cells and embryonic neural cells share a regulatory network, mediating both tumorigenesis and neural development. This observed similarity in regulatory mechanisms suggests that cancer cells might share characteristics of embryonic neural cells.

EphA7 regulates claudin6 and pronephros development in Xenopus.

Eph/ephrin molecules are widely expressed during embryonic development, and function in a variety of developmental processes. Here we studied the roles of the Eph receptor EphA7 and its soluble form in Xenopus pronephros development. EphA7 is specifically expressed in pronephric tubules at tadpole stages and knockdown of EphA7 by a translation blocking morpholino led to defects in tubule cell differentiation and morphogenesis. A soluble form of EphA7 (sEphA7) was also identified. Interestingly, the membrane level of claudin6 (CLDN6), a tetraspan transmembrane tight junction protein, was dramatically reduced in the translation blocking morpholino injected embryos, but not when a splicing morpholino was used, which blocks only the full length EphA7. In cultured cells, EphA7 binds and phosphorylates CLDN6, and reduces its distribution at the cell surface. Our work suggests a role of EphA7 in the regulation of cell adhesion during pronephros development, whereas sEphA7 works as an antagonist.

Targeted integration of genes in Xenopus tropicalis.

With the successful establishment of both targeted gene disruption and integration methods in the true diploid frog Xenopus tropicalis, this excellent vertebrate genetic model now is making a unique contribution to modelling human diseases. Here, we summarize our efforts on establishing homologous recombination-mediated targeted integration in Xenopus tropicalis, the usefulness, and limitation of targeted integration via the homology-independent strategy, and future directions on how to further improve targeted gene integration in Xenopus tropicalis.

Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis.

For the emerging amphibian genetic model Xenopus tropicalis targeted gene disruption is dependent on zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), which require either complex design and selection or laborious construction. Thus, easy and efficient genome editing tools are still highly desirable for this species. Here, we report that RNA-guided Cas9 nuclease resulted in precise targeted gene disruption in all ten X. tropicalis genes that we analyzed, with efficiencies above 45% and readily up to 100%. Systematic point mutation analyses in two loci revealed that perfect matches between the spacer and the protospacer sequences proximal to the protospacer adjacent motif (PAM) were essential for Cas9 to cleave the target sites in the X. tropicalis genome. Further study showed that the Cas9 system could serve as an efficient tool for multiplexed genome engineering in Xenopus embryos. Analysis of the disruption of two genes, ptf1a/p48 and tyrosinase, indicated that Cas9-mediated gene targeting can facilitate direct phenotypic assessment in X. tropicalis embryos. Finally, five founder frogs from targeting of either elastase-T1, elastase-T2 or tyrosinase showed highly efficient transmission of targeted mutations into F1 embryos. Together, our data demonstrate that the Cas9 system is an easy, efficient and reliable tool for multiplex genome editing in X. tropicalis.

Heat shock 70-kDa protein 5 (Hspa5) is essential for pronephros formation by mediating retinoic acid signaling.

Heat shock 70-kDa protein 5 (Hspa5), also known as binding immunoglobulin protein (Bip) or glucose-regulated protein 78 (Grp78), belongs to the heat shock protein 70 kDa family. As a multifunctional protein, it participates in protein folding and calcium homeostasis and serves as an essential regulator of the endoplasmic reticulum (ER) stress response. It has also been implicated in signal transduction by acting as a receptor or co-receptor residing at the plasma membrane. Its function during embryonic development, however, remains largely elusive. In this study, we used morpholino antisense oligonucleotides (MOs) to knock down Hspa5 activity in Xenopus embryos. In Hspa5 morphants, pronephros formation was strongly inhibited with the reduction of pronephric marker genes Lim homeobox protein 1 (lhx1), pax2, and ß1 subunit of Na/K-ATPase (atp1b1). Pronephros tissue was induced in vitro by treating animal caps with all-trans-retinoic acid and activin. Depletion of Hspa5 in animal caps, however, blocked the induction of pronephros as well as reduced the expression of retinoic acid (RA)-responsive genes, suggesting that knockdown of Hspa5 attenuated RA signaling. Knockdown of Hspa5 in animal caps resulted in decreased expression of lhx1, a transcription factor directly regulated by RA signaling and essential for pronephros specification. Co-injection of Hspa5MO with lhx1 mRNA partially rescued the phenotype induced by Hspa5MO. These results suggest that the RA-Lhx1 signaling cascade is involved in Hspa5MO-induced pronephros malformation. This study shows that Hspa5, a key regulator of the unfolded protein response, plays an essential role in pronephros formation, which is mediated in part through RA signaling during early embryonic development.

Histone methyltransferase Dot1L plays a role in postembryonic development in Xenopus tropicalis.

Histone methylations have been implicated to play important roles in diverse cellular processes. Of particular interest is the methylation of histone H3K79, which is catalyzed by an evolutionarily conserved methyltransferase, disruptor of telomeric silencing (Dot1)-like (Dot1L). To investigate the role of Dot1L during vertebrate development, we have generated a Dot1L-specific transcription activator-like effector nuclease (TALEN) nuclease to knockdown endogenous Dot1L in Xenopus tropicalis, a diploid species highly related to the well-known developmental model Xenopus laevis, a pseudotetraploid amphibian. We show that the TALEN was extremely efficient in mutating Dot1L when expressed in fertilized eggs, creating essentially Dot1L knockout embryos with little H3K79 methylation. Importantly, we observed that Dot1L knockdown had no apparent effect on embryogenesis because normally feeding tadpoles were formed, consistent with the lack of maternal Dot1L expression. On the other hand, Dot1L knockdown severely retarded the growth of the tadpoles and led to tadpole lethality prior to metamorphosis. These findings suggest that Dot1L and H3K79 methylation play an important role for tadpole growth and development prior to metamorphosis into a frog. Our findings further reveal interesting similarities and differences between Xenopus and mouse development and suggest the existence of 2 separate phases of vertebrate development with distinct requirements for epigenetic modifications.

Heritable CRISPR/Cas9-mediated targeted integration in Xenopus tropicalis.

Xenopus tropicalis is an emerging vertebrate genetic model. A gene knock-in method has not yet been reported in this species. Here, we report that heritable targeted integration can be achieved in this diploid frog using a concurrent cleavage strategy mediated by the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system. The key point of the strategy is the addition of a Cas9/guide RNA cleavage site in the donor vector, allowing simultaneous cutting of the chromosomal target site and circular donor DNA in vivo. For the 3 distinct loci tested, all showed efficient targeted integration that was verified by both germ-line transmission and Southern blot analyses. By designing the target sites in introns, we were able to get precise editing of the tyrosinase coding sequence and green fluorescent protein expression from endogenous n-tubulin promoter and enhancers. We were unable to detect off-target effects with the T7 endonuclease I assay. Precise editing of protein coding sequences in X. tropicalis expands the utility of this diploid frog, such as for establishing models to study human inherited diseases.

CXADR-like membrane protein (CLMP) in the rat ovary: stimulation by human chorionic gonadotrophin during the periovulatory period.

CXADR-like membrane protein (CLMP) is a novel cell-cell adhesion molecule. The present study investigated the spatiotemporal expression pattern of CLMP and its regulation in the rat ovary during the periovulatory period. Real-time polymerase chain reaction analysis revealed that Clmp mRNA was rapidly stimulated in intact ovaries by 4h after human chorionic gonadotrophin (hCG) treatment. In situ hybridisation analysis demonstrated that Clmp mRNA expression was stimulated in theca cells at 4h after hCG and remained elevated until 12h. Clmp mRNA was also upregulated in granulosa cells and was present in forming corpora lutea. Our data indicate that the protein kinase A but not the protein kinase C pathway regulates the expression of Clmp mRNA in granulosa cells. Phosphatidylinositol 3 kinase and p38 kinase are also involved in regulating Clmp mRNA expression. The stimulation of Clmp mRNA by hCG requires new protein synthesis. Furthermore, inhibition of epidermal growth factor receptor activation significantly inhibited Clmp mRNA expression, whereas inhibition of prostaglandin synthesis or progesterone action had no effect. The stimulation of CLMP in the rat ovary may be important in cell adhesion events during ovulation and luteal formation such as maintaining the structure and communication of ovarian follicular and luteal cells.

Homeoprotein hhex-induced conversion of intestinal to ventral pancreatic precursors results in the formation of giant pancreata in Xenopus embryos.

Liver and ventral pancreas develop from neighboring territories within the endoderm of gastrulae. ventral pancreatic precursor 1 (vpp1) is a marker gene that is differentially expressed in a cell population within the dorsal endoderm in a pattern partially overlapping with that of hematopoietically expressed homeobox (hhex) during gastrulation. In tail bud embryos, vpp1 expression specifically demarcates two ventral pancreatic buds, whereas hhex expression is mainly restricted to the liver diverticulum. Ectopic expression of a critical dose of hhex led to a greatly enlarged vpp1-positive domain and, subsequently, to the formation of giant ventral pancreata, putatively by conversion of intestinal to ventral pancreatic precursor cells. Conversely, antisense morpholino oligonucleotide-mediated knockdown of hhex resulted in a down-regulation of vpp1 expression and a specific loss of the ventral pancreas. Furthermore, titration of hhex with a dexamethasone-inducible hhex-VP16GR fusion construct suggested that endogenous hhex activity during gastrulation is essential for the formation of ventral pancreatic progenitor cells. These observations suggest that, beyond its role in liver development, hhex controls specification of a vpp1-positive endodermal cell population during gastrulation that is required for the formation of the ventral pancreas.

Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs).

Transcription activator-like effector nucleases (TALENs) are an approach for directed gene disruption and have been proved to be effective in various animal models. Here, we report that TALENs can induce somatic mutations in Xenopus embryos with reliably high efficiency and that such mutations are heritable through germ-line transmission. We modified the Golden Gate method for TALEN assembly to make the product suitable for RNA transcription and microinjection into Xenopus embryos. Eight pairs of TALENs were constructed to target eight Xenopus genes, and all resulted in indel mutations with high efficiencies of up to 95.7% at the targeted loci. Furthermore, mutations induced by TALENs were highly efficiently passed through the germ line to F(1) frogs. Together with simple and reliable PCR-based approaches for detecting TALEN-induced mutations, our results indicate that TALENs are an effective tool for targeted gene editing/knockout in Xenopus.

Bortezomib interferes with C-KIT processing and transforms the t(8;21)-generated fusion proteins into tumor-suppressing fragments in leukemia cells.

The boronic acid dipeptide bortezomib inhibits the chymotrypsin-like activity of the 26S proteasome and shows significant therapeutic efficacy in multiple myeloma. However, recent studies suggest that bortezomib may have more complex mechanisms of action in treating cancer. We report here that the endocytosis and lysosomal degradation of the receptor tyrosine kinase C-KIT are required for bortezomib- but not tyrosine kinase inhibitor imatinib-caused apoptosis of t(8;21) leukemia and gastrointestinal stromal tumor cells, suggesting that C-KIT may recruit an apoptosis initiator. We show that C-KIT binds and phosphorylates heat shock protein 90ß (Hsp90ß), which sequestrates apoptotic protease activating factor 1 (Apaf-1). Bortezomib dephosphorylates pHsp90ß and releases Apaf-1. Although the activated caspase-3 is not sufficient to cause marked apoptosis, it cleaves the t(8;21) generated acute myeloid leukemia 1-eight twenty one (AML1-ETO) and AML1-ETO9a fusion proteins, with production of cleavage fragments that perturb the functions of the parental oncoproteins and further contribute to apoptosis. Notably, bortezomib exerts potent therapeutic efficacy in mice bearing AML1-ETO9a-driven leukemia. These data show that C-KIT-pHsp90ß-Apaf-1 cascade is critical for some malignant cells to evade apoptosis, and the clinical therapeutic potentials of bortezomib in C-KIT-driven neoplasms should be further explored.