Whole-body imaging of lymphovascular niches identifies pre-metastatic roles of midkine.

Cutaneous melanoma is a type of cancer with an inherent potential for lymph node colonization, which is generally preceded by neolymphangiogenesis. However, sentinel lymph node removal does not necessarily extend the overall survival of patients with melanoma. Moreover, lymphatic vessels collapse and become dysfunctional as melanomas progress. Therefore, it is unclear whether (and how) lymphangiogenesis contributes to visceral metastasis. Soluble and vesicle-associated proteins secreted by tumours and/or their stroma have been proposed to condition pre-metastatic sites in patients with melanoma. Still, the identities and prognostic value of lymphangiogenic mediators remain unclear. Moreover, our understanding of lymphangiogenesis (in melanomas and other tumour types) is limited by the paucity of mouse models for live imaging of distal pre-metastatic niches. Injectable lymphatic tracers have been developed, but their limited diffusion precludes whole-body imaging at visceral sites. Vascular endothelial growth factor receptor 3 (VEGFR3) is an attractive 'lymphoreporter' because its expression is strongly downregulated in normal adult lymphatic endothelial cells, but is activated in pathological situations such as inflammation and cancer. Here, we exploit this inducibility of VEGFR3 to engineer mouse melanoma models for whole-body imaging of metastasis generated by human cells, clinical biopsies or endogenously deregulated oncogenic pathways. This strategy revealed early induction of distal pre-metastatic niches uncoupled from lymphangiogenesis at primary lesions. Analyses of the melanoma secretome and validation in clinical specimens showed that the heparin-binding factor midkine is a systemic inducer of neo-lymphangiogenesis that defines patient prognosis. This role of midkine was linked to a paracrine activation of the mTOR pathway in lymphatic endothelial cells. These data support the use of VEGFR3 reporter mice as a 'MetAlert' discovery platform for drivers and inhibitors of metastasis.

Dabrafenib, alone or in combination with trametinib, in BRAF V600-mutated pediatric Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare, heterogenous, neoplastic disorder primarily affecting children. BRAF mutations have been reported in >50% of patients with LCH. The selective BRAF inhibitor, dabrafenib, in combination with the MEK1/2 inhibitor, trametinib, has been approved in select BRAF V600-mutant solid tumors. Two open-label phase 1/2 studies were conducted in pediatric patients with BRAF V600-mutant, recurrent/refractory malignancies treated with dabrafenib monotherapy (CDRB436A2102; NCT01677741) or dabrafenib plus trametinib (CTMT212X2101; NCT02124772). The primary objectives of both studies were to determine safe and tolerable doses that achieve similar exposure to the approved doses for adults. Secondary objectives included safety, tolerability, and preliminary antitumor activity. Thirteen and 12 patients with BRAF V600-mutant LCH received dabrafenib monotherapy and in combination with trametinib, respectively. Investigator-assessed objective response rates per Histiocyte Society criteria were 76.9% (95% confidence interval [CI], 46.2-95.0) and 58.3% (95% CI, 27.7-84.8) in the monotherapy and combination studies, respectively. More than 90% of responses were ongoing at study completion. The most common treatment-related adverse events (AEs) were vomiting and increased blood creatinine with monotherapy and pyrexia, diarrhea, dry skin, decreased neutrophil count, and vomiting with combination therapy. Two patients each discontinued treatment with monotherapy and combination therapy because of AEs. Overall, dabrafenib monotherapy or in combination with trametinib demonstrated clinical efficacy and manageable toxicity in relapsed/refractory BRAF V600-mutant pediatric LCH, with most responses ongoing. Safety was consistent with that reported in other pediatric and adult conditions treated with dabrafenib plus trametinib.

Efficacy and Safety of Trametinib Monotherapy or in Combination With Dabrafenib in Pediatric BRAF V600-Mutant Low-Grade Glioma.

PURPOSE: BRAF V600 mutations occur in many childhood cancers, including approximately 20% of low-grade gliomas (LGGs). Here, we describe a phase I/II study establishing pediatric dosing and pharmacokinetics of trametinib with or without dabrafenib, as well as efficacy and safety in a disease-specific cohort with BRAF V600-mutant LGG; other cohorts will be reported elsewhere. METHODS: This is a four-part, phase I/II study (ClinicalTrials.gov identifier: NCT02124772) in patients age < 18 years with relapsed/refractory malignancies: trametinib monotherapy dose finding (part A) and disease-specific expansion (part B), and dabrafenib + trametinib dose finding (part C) and disease-specific expansion (part D). The primary objective assessed in all patients in parts A and C was to determine pediatric dosing on the basis of steady-state pharmacokinetics. Disease-specific efficacy and safety (across parts A-D) were secondary objectives. RESULTS: Overall, 139 patients received trametinib (n = 91) or dabrafenib + trametinib (n = 48). Trametinib dose-limiting toxicities in > 1 patient (part A) included mucosal inflammation (n = 3) and hyponatremia (n = 2). There were no dose-limiting toxicities with combination therapy (part C). The recommended phase II dose of trametinib, with or without dabrafenib, was 0.032 mg/kg once daily for patients age < 6 years and 0.025 mg/kg once daily for patients age ≥ 6 years; dabrafenib dosing in the combination was as previously identified for monotherapy. In 49 patients with BRAF V600-mutant glioma (LGG, n = 47) across all four study parts, independently assessed objective response rates were 15% (95% CI, 1.9 to 45.4) for monotherapy (n = 13) and 25% (95% CI, 12.1 to 42.2) for combination (n = 36). Adverse event-related treatment discontinuations were more common with monotherapy (54% v 22%). CONCLUSION: The trial design provided efficient evaluation of pediatric dosing, safety, and efficacy of single-agent and combination targeted therapy. Age-based and weight-based dosing of trametinib with or without dabrafenib achieved target concentrations with manageable safety and demonstrated clinical efficacy and tolerability in BRAF V600-mutant LGG.

Safety of combining dabrafenib plus trametinib in elderly BRAF V600 mutation-positive advanced melanoma patients: real-world data analysis of Spanish patients (ELDERLYMEL).

Efficacy and safety of dabrafenib and trametinib in metastatic melanoma have been demonstrated in two-phase III and one-phase I/II clinical trials. However, patients at least 75 years old (y.o.) were largely underrepresented. Additionally, the safety profile of dabrafenib and trametinib based on age is unknown. ELDERLYMEL is a retrospective noninterventional multicenter study, describing the effectiveness and safety of at least 75 y.o. patients compared with less than 75 y.o. patients with advanced BRAF V600-mutated melanoma treated with dabrafenib plus trametinib or dabrafenib monotherapy. A total of 159 patients were included, 130 less than 75 y.o. and 29 at least 75 y.o. Clinical features were similar between the groups, except in the number of comorbidities, number of metastatic sites, Eastern Cooperative Oncology Group (ECOG) performance status, and BRAF V600-mutation type. Five patients per group received dabrafenib monotherapy. There were no differences in adverse events (AEs) rate or grade between the groups. However, AE profiles were different between the groups, being pyrexia infrequent in patients at least 75 y.o. (13.8% vs. 42.3%; P = 0.005). Dabrafenib and trametinib dose intensities were lower in at least 75 y.o. patients ( P = 0.018 and P = 0.020), but there were no differences in effectiveness between the groups. Finally, in a multivariate analysis, sex (female) was the only variable independently associated with an increased risk of AE grade ≥3. Data from the ELDERLYMEL study demonstrate that dabrafenib plus trametinib is safe and effective in at least 75 y.o. patients with advanced BRAF V600-mutated melanoma without increasing toxicity. Additionally, we describe a different safety profile depending on age and sex.

Systems analysis identifies melanoma-enriched pro-oncogenic networks controlled by the RNA binding protein CELF1.

Melanomas are well-known for their altered mRNA expression profiles. Yet, the specific contribution of mRNA binding proteins (mRBPs) to melanoma development remains unclear. Here we identify a cluster of melanoma-enriched genes under the control of CUGBP Elav-like family member 1 (CELF1). CELF1 was discovered with a distinct prognostic value in melanoma after mining the genomic landscape of the 692 known mRBPs across different cancer types. Genome-wide transcriptomic, proteomic, and RNA-immunoprecipitation studies, together with loss-of-function analyses in cell lines, and histopathological evaluation in clinical biopsies, revealed an intricate repertoire of CELF1-RNA interactors with minimal overlap with other malignancies. This systems approach uncovered the oncogene DEK as an unexpected target and downstream effector of CELF1. Importantly, CELF1 and DEK were found to represent early-induced melanoma genes and adverse indicators of overall patient survival. These results underscore novel roles of CELF1 in melanoma, illustrating tumor type-restricted functions of RBPs in cancer.

RAB7 counteracts PI3K-driven macropinocytosis activated at early stages of melanoma development.

Derailed endolysosomal trafficking is emerging as a widespread feature of aggressive neoplasms. However, the oncogenic signals that alter membrane homeostasis and their specific contribution to cancer progression remain unclear. Understanding the upstream drivers and downstream regulators of aberrant vesicular trafficking is distinctly important in melanoma. This disease is notorious for its inter- and intra-tumoral heterogeneity. Nevertheless, melanomas uniformly overexpress a cluster of endolysosomal genes, being particularly addicted to the membrane traffic regulator RAB7. Still, the underlying mechanisms and temporal determinants of this dependency have yet to be defined. Here we addressed these questions by combining electron microscopy, real time imaging and mechanistic analyses of vesicular trafficking in normal and malignant human melanocytic cells. This strategy revealed Class I PI3K as the key trigger of a hyperactive influx of macropinosomes that melanoma cells counteract via RAB7-mediated lysosomal degradation. In addition, gain- and loss-of-function in vitro studies followed by histopathological validation in clinical biopsies and genetically-engineered mouse models, traced back the requirement of RAB7 to the suppression of premature cellular senescence traits elicited in melanocytes by PI3K-inducing oncogenes. Together, these results provide new insight into the regulators and modes of action of RAB7, broadening the impact of endosomal fitness on melanoma development.

RAB7 controls melanoma progression by exploiting a lineage-specific wiring of the endolysosomal pathway.

Although common cancer hallmarks are well established, lineage-restricted oncogenes remain less understood. Here, we report an inherent dependency of melanoma cells on the small GTPase RAB7, identified within a lysosomal gene cluster that distinguishes this malignancy from over 35 tumor types. Analyses in human cells, clinical specimens, and mouse models demonstrated that RAB7 is an early-induced melanoma driver whose levels can be tuned to favor tumor invasion, ultimately defining metastatic risk. Importantly, RAB7 levels and function were independent of MITF, the best-characterized melanocyte lineage-specific transcription factor. Instead, we describe the neuroectodermal master modulator SOX10 and the oncogene MYC as RAB7 regulators. These results reveal a unique wiring of the lysosomal pathway that melanomas exploit to foster tumor progression.

The human synMuv-like protein LIN-9 is required for transcription of G2/M genes and for entry into mitosis.

Regulated gene expression is critical for the proper timing of cell cycle transitions. Here we report that human LIN-9 has an important function in transcriptional regulation of G2/M genes. Depletion of LIN-9 by RNAi in human fibroblasts strongly impairs proliferation and delays progression from G2 to M. We identify a cluster of G2/M genes as direct targets of LIN-9. Activation of these genes is linked to an association between LIN-9 and B-MYB. Chromatin immunoprecipitation assays revealed binding of both LIN-9 and B-MYB to the promoters of G2/M regulated genes. Depletion of B-MYB recapitulated the biological outcome of LIN-9 knockdown, including impaired proliferation and reduced expression of G2/M genes. These data suggest a critical role for human LIN-9, together with B-MYB, in the activation of genes that are essential for progression into mitosis.

The AppA and PpsR proteins from Rhodobacter sphaeroides can establish a redox-dependent signal chain but fail to transmit blue-light signals in other bacteria.

The AppA protein of Rhodobacter sphaeroides has the unique ability to sense and transmit redox and light signals. In response to decreasing oxygen tension, AppA antagonizes the transcriptional regulator PpsR, which represses the expression of photosynthesis genes, including the puc operon. This mechanism, which is based on direct protein-protein interaction, is prevented by blue-light absorption of the BLUF domain located in the N-terminal part of AppA. In order to test whether AppA and PpsR are sufficient to transmit redox and light signals, we expressed these proteins in three different bacterial species and monitored oxygen- and blue-light-dependent puc expression either directly or by using a luciferase-based reporter construct. The AppA/PpsR system could mediate redox-dependent gene expression in the alphaproteobacteria Rhodobacter capsulatus and Paracoccus denitrificans but not in the gammaproteobacterium Escherichia coli. Analysis of a prrA mutant strain of R. sphaeroides strongly suggests that light-dependent gene expression requires a balanced interplay of the AppA/PpsR system with the PrrA response regulator. Therefore, the AppA/PpsR system was unable to establish light signaling in other bacteria. Based on our data, we present a model for the interdependence of AppA/PpsR signaling and the PrrA transcriptional activator.

Light-dependent regulation of photosynthesis genes in Rhodobacter sphaeroides 2.4.1 is coordinately controlled by photosynthetic electron transport via the PrrBA two-component system and the photoreceptor AppA.

Formation of the photosynthetic apparatus in Rhodobacter is regulated by oxygen tension and light intensity. Here we show that in anaerobically grown Rhodobacter cells a light-dependent increase in expression of the puc and puf operons encoding structural proteins of the photosynthetic complexes requires an active photosynthetic electron transport. The redox-sensitive CrtJ/PpsR repressor of photosynthesis genes, which was suggested to mediate electron transport-dependent signals, is not involved in this light-dependent signal chain. Our data reveal that the signal initiated in the photosynthetic reaction centre is transmitted via components of the electron transport chain and the PrrB/PrrA two-component system in Rhodobacter sphaeroides. Under blue light illumination in the absence of oxygen this signal leads to activation of photosynthesis genes and interferes with a blue-light repression mediated by the AppA photoreceptor and the PpsR transcriptional repressor in R. sphaeroides. Thus, light either sensed by a photoreceptor or initiating photosynthetic electron transport has opposite effects on the transcription of photosynthesis genes. Both signalling pathways involve redox-dependent steps that finally determine the effect of light on gene expression.

A eukaryotic BLUF domain mediates light-dependent gene expression in the purple bacterium Rhodobacter sphaeroides 2.4.1.

The flavin-binding BLUF domain functions as a blue-light receptor in eukaryotes and bacteria. In the photoreceptor protein photo-activated adenylyl cyclase (PAC) from the flagellate Euglena gracilis, the BLUF domain is linked to an adenylyl cyclase domain. The PAC protein mediates a photophobic response. In the AppA protein of Rhodobacter sphaeroides, the BLUF domain is linked to a downstream domain without similarity to known proteins. AppA functions as a transcriptional antirepressor, controlling photosynthesis gene expression in the purple bacterium R. sphaeroides in response to light and oxygen. We fused the PACalpha1-BLUF domain from Euglena to the C terminus of AppA. Our results show that the hybrid protein is fully functional in light-dependent gene repression in R. sphaeroides, despite only approximately 30% identity between the eukaryotic and the bacterial BLUF domains. Furthermore, the bacterial BLUF domain and the C terminus of AppA can transmit the light signal even when expressed as separated domains. This finding implies that the BLUF domain is fully modular and can relay signals to completely different output domains.

Inhibition of oncogenic transformation by mammalian Lin-9, a pRB-associated protein.

Genetic studies in Caenorhabditis elegans identified lin-9 to function together with the retinoblastoma homologue lin-35 in vulva differentiation. We have now identified a human homologue of Lin-9 (hLin-9) and provide evidence about its function in the mammalian pRB pathway. hLin-9 binds to pRB and cooperates with pRB in flat cell formation in Saos-2 cells. In addition, hLin-9 synergized with pRB and Cbfal to transactivate an osteoblast-specific reporter gene. In contrast, hLin-9 was not involved in pRB-mediated inhibition of cell cycle progression or repression of E2F-dependent transactivation. Consistent with these data, hLin-9 was able to associate with partially penetrant pRB mutants that do not bind to E2F, but retain the ability to activate transcription and to promote differentiation. hLin-9 can also inhibit oncogenic transformation, dependent on the presence of a functional pRB protein. RNAi-mediated knockdown of Lin-9 can substitute for the loss of pRB in transformation of human primary fibroblasts. These data suggest that hLin-9 has tumor-suppressing activities and that the ability of hLin-9 to inhibit transformation is mediated through its association with pRB.