Design Of Rational Treatments Research Articles

Face-valid phenotypes in a mouse model of the most common mutation in EEF1A2-related neurodevelopmental disorder.

De novo heterozygous missense mutations in EEF1A2, encoding neuromuscular translation-elongation factor eEF1A2, are associated with developmental and epileptic encephalopathies. We used CRISPR/Cas9 to recapitulate the most common mutation, E122K, in mice. Although E122K heterozygotes were not observed to have convulsive seizures, they exhibited frequent electrographic seizures and EEG abnormalities, transient early motor deficits and growth defects. Both E122K homozygotes and Eef1a2-null mice developed progressive motor abnormalities, with E122K homozygotes reaching humane endpoints by P31. The null phenotype is driven by progressive spinal neurodegeneration; however, no signs of neurodegeneration were observed in E122K homozygotes. The E122K protein was relatively stable in neurons yet highly unstable in skeletal myocytes, suggesting that the E122K/E122K phenotype is instead driven by loss of function in muscle. Nevertheless, motor abnormalities emerged far earlier in E122K homozygotes than in nulls, suggesting a toxic gain of function and/or a possible dominant-negative effect. This mouse model represents the first animal model of an EEF1A2 missense mutation with face-valid phenotypes and has provided mechanistic insights needed to inform rational treatment design.

Network Approaches for Precision Oncology.

The growth of multi-omic tumour profile datasets along with knowledge of genome regulatory networks has created an unprecedented opportunity to advance precision oncology. Achieving this goal requires computational methods that can make sense of and combine heterogeneous data sources. Interpretability and integration of prior knowledge is of particular relevance for genomic models to minimize ungeneralizable models, promote rational treatment design, and make use of sparse genetic mutation data. While networks have long been used to capture genomic interactions at the levels of genes, proteins, and pathways, the use of networks in precision oncology is relatively new. In this chapter, I provide an introduction to network-based approaches used to integrate multi-modal data sources for patient stratification and patient classification. There is a particular emphasis on methods using patient similarity networks (PSNs) as part of the design. I separately discuss strategies for inferring driver mutations from individual patient mutation data. Finally, I discuss challenges and opportunities the field will need to overcome to achieve its full potential, with an outlook towards a clinic of the future.

Foundational concepts in the biology of bacterial keratitis

Bacterial infections of the cornea, or bacterial keratitis (BK), are notorious for causing rapidly fulminant disease and permanent vision loss, even among treated patients. In the last sixty years, dramatic upward trajectories in the frequency of BK have been observed internationally, driven in large part by the commercialization of hydrogel contact lenses in the late 1960s. Despite this worsening burden of disease, current evidence-based therapies for BK - including broad-spectrum topical antibiotics and, if indicated, topical corticosteroids - fail to salvage vision in a substantial proportion of affected patients. Amid growing concerns of rapidly diminishing antibiotic utility, there has been renewed interest in urgently needed novel treatments that may improve clinical outcomes on an individual and public health level. Bridging the translational gap in the care of BK requires the identification of new therapeutic targets and rational treatment design, but neither of these aims can be achieved without understanding the complex biological processes that determine how bacterial corneal infections arise, progress, and resolve. In this chapter, we synthesize the current wealth of human and animal experimental data that now inform our understanding of basic BK pathophysiology, in context with modern concepts in ocular immunology and microbiology. By identifying the key molecular determinants of clinical disease, we explore how novel treatments can be developed and translated into routine patient care.

As Hopes Have Flown Before: Toward the Rational Design of Treatments for Alcohol Use Disorder

As Hopes Have Flown Before: Toward the Rational Design of Treatments for Alcohol Use Disorder

Identification of somatic mutations and neoantigens to predict development of autoimmune adverse events to immune therapy in melanoma.

19 Background: Immunotherapy has quickly altered the treatment paradigm for advanced melanoma. Prior studies using whole exome sequencing (WES) in immunotherapy have shown limited correlation with clinical response to immunotherapy, but not an evaluation of autoimmune side effects. Understanding mechanisms of immune-related adverse events (irAE) at a genomic level may lead to improved rational treatment design and better patient selection. Methods: DNA was extracted from 87 tumor samples from 49 patients with metastatic melanoma treated with ipilimumab (anti-CTLA-4) or with nivolumab or pembrolizumab (anti-PD-1) immunotherapy. WES was performed with exome capture on an Illumina HiSeq sequencer. Statistical analysis was performed with Chi-squared testing. Computational neoantigen analysis has been performed on the sequencing data. Results: 27 patients with advanced or metastatic melanoma were treated with only ipilimumab, 11 with only anti-PD1, and 11 patients were treated with both anti-CLTA-4 and anti-PD1 either sequentially or in combination. irAE evaluated included cutaneous, colitis, endocrinopathies, hepatitis, and pneumonitis. In 10 patients who developed colitis, 14 mutated genes were enriched in their tumor samples (p=0.01-0.046) compared with patients without colitis. Analyzing all patients with any irAE versus no irAE treated with anti-CTLA-4, anti-PD1, or both (either sequential or combination) revealed 7 mutated genes enriched in patient samples who developed irAE (p=0.02-0.05). Preliminary neoantigen analysis has identified possible irAE associated neoantigens and further analysis of additional samples are ongoing. Overall mutational burden of the tumor samples did not correlate with development of irAE. Conclusions: Autoimmune side effects are frequent and often serious side effects of immunotherapy for melanoma. Genetic analysis may provide insight in prediction of these side effects and help direct therapeutic decisions.

Animal genomics in natural reservoirs of infectious diseases.

Natural virus reservoirs such as wild bats, birds, rodents and non-human primates are generally non-model organisms that have, until recently, presented limited opportunities for in-depth study. Next-generation sequencing provides a way to partially circumvent this limitation, since the methods required for data acquisition and analysis are largely species-independent. Comparative genomics and other 'omics' provide new opportunities to study the structure and function of various biological systems of wild species that are otherwise out of reach. Genomes of natural reservoir hosts can help to identify dominant pathways of virus-host interaction and to reveal differences between susceptible and resistant organisms, populations and species. This is of great scientific interest and may also provide a resource for the rational design of treatments for viral diseases in humans and livestock. In this way, we will 'learn from nature' and one day apply this knowledge to create disease-resistant livestock or develop novel therapeutic and prevention strategies. Reservoir host genomics will also open up possibilities for developing novel vaccines for wildlife, aid in the development of new diagnostic platforms, and have broad implications for developmental and evolutionary biology. In this review, the authors focus on natural reservoir hosts of viral pathogens, although most of the discussion points should be equally applicable to natural reservoirs of pathogenic bacteria, fungi or other parasites.

Cancer stem cell-associated microRNAs: searching for markers and targets in hepatocellular carcinoma.

Rational design of treatments with increasing selectivity for tumor cells has been the goal of cancer research for many decades. Advances in the knowledge of cell composition, function and regulation have been rapidly applied in the search for differences between normal and cancer cells that could provide new targets for treatment or diagnosis. Scientific and technological innovations are now opening unprecedented opportunities in this field.

The concentration game: differential effects of bioactive signaling in 2D and 3D culture.

Traumatic injuries to the central nervous system, such as traumatic brain injury, spinal cord injury and stroke, have a high prevalence, enormous financial costs and lack clinical treatments that restore neurological function (Ma et al., 2014). These injuries trigger a series of secondary biochemical and cellular responses that ultimately lead to cellular death and the maintenance of an unsupportive extracellular matrix (ECM) for tissue regeneration (Silva et al., 2014). Artificial ECM or scaffolds represent a way to alter this unsupportive environment to improve the efficacy of stem cell therapies and enhance neural tissue regeneration (Figure 1). Scaffold use could lead to greater improvements in neurological function (such as improved bladder control, increased dexterity and body control, etc.) than observed with the implantation of cells alone. To date, the inclusion of basic scaffolds with stem cell therapy treatments have shown increased efficacy in rodent models (Yasuda et al., 2010). More advanced scaffolds could better mimic the chemical, physical and mechanical properties of the ECM to promote cellular survival, adhesion, proliferation and differentiation. Altering the injured ECM to mitigate the barriers to axon invasion, myelination and cellular maturation further and lead to even greater gains in neurological function.

Abstract A01: Exploring phosphatase and tensin homolog (PTEN) loss as a potential predictive marker for response to everolimus in patients with pancreatic neuroendocrine tumors (PNETs)

Abstract Introduction: Identification of patients with exquisite sensitivity and/or durable responses to targeted therapies may lead to improved patient selection and allow for more rational treatment designs. Loss of PTEN tumor suppressor gene function, usually due to deletion, leads to activation of phosphatidylinositol 3-kinase/Akt and mammalian target of rapamycin (mTOR) signaling. The RADIANT-3 trial of everolimus in advanced PNETs demonstrated a significant prolongation of progression-free survival (PFS) from 4.6 months with placebo versus 11 months with everolimus. Despite the improvement in PFS, the response rate was only 5% among patients receiving everolimus. The index case for our study was an exceptional responder who had a significant radiological response as well as a PFS of 24 months, which were both better than expected from the literature. This led to the hypothesis that there may be mutational changes in genes affecting the mTOR pathway that could predict sensitivity to mTOR inhibitors. In this study, we specifically explored the role of PTEN loss as a potential predictive marker. Methods and Materials: Between 2010 and 2014, patients with well-differentiated unresectable and metastatic PNETs treated at the University of Miami/Sylvester Comprehensive Cancer Center and Jackson Memorial Hospital with everolimus were identified. Eight patients had pathology specimens available for testing. PTEN loss detected by Fluorescence In Situ Hybridization (FISH) was carried out using a commercially available probe for cytoband 10q23. Patients' response to everolimus was evaluated through June 2014. The primary outcome was PFS. PTEN expression by immunohistochemistry (IHC) will also be performed and the results will be compared to those obtained by FISH. Results: The median age was 60 years (range 45-78). 50% of the patients were females and 50% were males. Two patients had gastrinomas, 1 patient had an insulinoma, and five patients had non-functional PNETs. All patients had unresectable metastases to the liver. In addition to sandostatin LAR, the patients received everolimus starting at a dose of 10mg daily. Three patients were found to have deletion of PTEN. Of those, one patient did not tolerate everolimus and the PFS for the other two was 8 and 24 months respectively. Detection of PTEN loss by FISH yielded no results in 2 patients due to insufficient tumor left in the specimen. PFS in these 2 patients was 24 and 4 months respectively. Testing is ongoing in the last 3 patients and the PFS for these patients is 3, 10 and 13 months. PTEN expression by IHC is also ongoing and will be reported at the meeting. Conclusion: The index case for this study had a PTEN deletion and had a partial response to treatment and prolonged disease control for 2 years with everolimus. Testing is ongoing in additional cases to determine if there is a consistent correlation between PTEN loss by FISH and PFS. Citation Format: Moh'd Khushman, Joyce Slingerland, Yao-Shan Fan, Monica Garcia-Buitrago, Ernesto Bustinza, Maria Restrepo, Daniel Sussman, Caio Rocha-Lima, Peter Hosein. Exploring phosphatase and tensin homolog (PTEN) loss as a potential predictive marker for response to everolimus in patients with pancreatic neuroendocrine tumors (PNETs). [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A01.

Rational Design and Adaptive Management of Combination Therapies for Hepatitis C Virus Infection.

Recent discoveries of direct acting antivirals against Hepatitis C virus (HCV) have raised hopes of effective treatment via combination therapies. Yet rapid evolution and high diversity of HCV populations, combined with the reality of suboptimal treatment adherence, make drug resistance a clinical and public health concern. We develop a general model incorporating viral dynamics and pharmacokinetics/ pharmacodynamics to assess how suboptimal adherence affects resistance development and clinical outcomes. We derive design principles and adaptive treatment strategies, identifying a high-risk period when missing doses is particularly risky for de novo resistance, and quantifying the number of additional doses needed to compensate when doses are missed. Using data from large-scale resistance assays, we demonstrate that the risk of resistance can be reduced substantially by applying these principles to a combination therapy of daclatasvir and asunaprevir. By providing a mechanistic framework to link patient characteristics to the risk of resistance, these findings show the potential of rational treatment design.

Exploring phosphatase and tensin homolog (PTEN) loss via immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) as a potential predictive marker for response to everolimus in patients (pts) with neuroendocrine tumors (NET).

333 Background: Identification of pts with exquisite sensitivity and/or durable responses to targeted therapies may lead to improved patient selection and allow for more rational treatment designs. Exceptional responders to everolimus in NET including pancreatic (PNET) were observed in our cohort of pts. PTEN is a key negative regulator of the phosphatidylinositol 3-kinase(PI3K)/Akt and mammalian target of rapamycin (mTOR) pathway. Loss of PTEN tumor suppressor gene function, usually due to deletion, leads to PI3K/Akt/mTOR pathway activation. Inthis study, we explored the role of PTEN as a potential predictive marker of everolimus in pts with NET including PNET. Methods: Between 2010 and 2014, pts with well-differentiated unresectable and metastatic gastrointestinal NET treated at our institution with everolimus were identified. 17 patients had pathology specimens available for testing. PTEN loss detection by FISH was carried out using a commercially available probe for cytoband 10q23, and by IHC using a commercially available antibody. Patients’ response to everolimus was evaluated through August 2014. The primary outcome was PFS and PTEN status was correlated with PFS for any potential association. Results: The median age was 59 years (range 45-78); 7 were male; 8 had PNET (2 gastrinomas, 1 insulinoma and 5 non-functional); 7 had small bowel NET and 2 unknown primary. All pts received everolimus starting at 10mg daily and octreotide LAR. Of the pts with PNETs, 3 had PTEN loss by FISH. Of those, one did not tolerate everolimus. The PFS for the other two pts was 8 and 24 months respectively. Among the 4 pts with intact PTEN; PFS was 14, 26, 3 and 12 months. 1 patient had insufficient tumor for testing. PTEN FISH is ongoing in the 9 non-pancreatic NET pts. PTEN expression by IHC is also ongoing and will be reported at the meeting. Conclusions: Testing for PTEN loss by FISH is feasible. Due to the small sample size, the role of PTEN loss could not be defined as a predictive marker in PNET. Testing on additional cases is ongoing and will be presented at the meeting.

Abstract 1724: Genomic mechanisms of exquisite sensitivity and acquired resistance to everolimus in a patient with anaplastic thyroid carcinoma

Abstract Understanding genetic mechanisms of sensitivity and resistance to targeted anticancer therapies may improve patient selection and rational treatment designs. One approach to increase this understanding involves the study of exceptional responders: rare patients with unexpected exquisite sensitivity or durable responses to therapy. We identified an exceptional responder on a study of the allosteric mTOR inhibitor everolimus in thyroid cancer: a 57-yr-old woman with refractory metastatic anaplastic thyroid carcinoma (ATC), a highly aggressive neoplasm with no adequate therapies and a median survival of 5 months. After beginning treatment with everolimus, the patient experienced a near complete response that lasted for 18 months, followed by progressive disease, which was then re-biopsied. To date, mechanisms of clinical resistance to mTOR inhibition have not been described. We performed whole exome sequencing (WES) of both pre-treatment and drug resistant tumor tissue to look for the underlying mechanisms of exquisite sensitivity and acquired resistance to everolimus. WES of the pre-treatment tumor revealed a somatic nonsense mutation in TSC2, a tumor suppressor gene whose inactivation is known to activate the mTOR pathway and result in sensitivity to mTOR inhibition in some cancers. WES of the drug resistant tumor additionally revealed a mutation in mTOR (mTOR-F2108L) not detected in the pre-treatment tumor. Structural modeling demonstrated that this mutation occurs in the FKBP12-rapamycin binding domain of mTOR and is predicted to prevent binding of the drug to the protein. Overexpressing mTOR-F2108L in HEK-293T cells resulted in significant resistance to rapamycin compared to cells expressing wild type (wt) mTOR. In cells expressing the mutant mTOR, rapamycin did not decrease phosphorylation of S6K1, a downstream target of mTOR, compared with cells expressing wt mTOR. Notably, cells expressing mTOR-F2108L remained sensitive to the direct TOR inhibitor torin, suggesting a therapeutic approach to overcome resistance in this patient. In summary, we add ATC to the growing list of cancers found to be exquisitely sensitive to everolimus when activating mTOR pathway mutations are present. Moreover, we present the first reported, to our knowledge, mechanism of acquired resistance to everolimus identified in patients. The fact that this occurs via a binding domain mutation that blocks allosteric mTOR inhibition suggests that followup therapy with direct TOR inhibitors may still have benefit in some patients who develop resistance to everolimus. The use of precision medicine approaches in ATC to screen for alterations in the mTOR pathway may help identify subsets of patients who would benefit from targeted therapies directed against mTOR. Moreover, the use of serial biopsies to profile patients who develop resistance to everolimus could dictate optimal followup treatment in ATC and other cancers. Citation Format: Nikhil Wagle, Brian C. Grabiner, Eliezer M. Van Allen, Ali Amin-Mansour, Scott C. Carter, Nathanael Gray, Justine A. Barletta, Scott J. Swanson, Daniel Ruan, David J. Kwiatkowski, Glenn J. Hanna, Robert I. Haddad, David Sabatini, Pasi A. Janne, Levi A. Garraway, Jochen H. Lorch. Genomic mechanisms of exquisite sensitivity and acquired resistance to everolimus in a patient with anaplastic thyroid carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1724. doi:10.1158/1538-7445.AM2014-1724

Activating mTOR Mutations in a Patient with an Extraordinary Response on a Phase I Trial of Everolimus and Pazopanib

Understanding the genetic mechanisms of sensitivity to targeted anticancer therapies may improve patient selection, response to therapy, and rational treatment designs. One approach to increase this understanding involves detailed studies of exceptional responders: rare patients with unexpected exquisite sensitivity or durable responses to therapy. We identified an exceptional responder in a phase I study of pazopanib and everolimus in advanced solid tumors. Whole-exome sequencing of a patient with a 14-month complete response on this trial revealed two concurrent mutations in mTOR, the target of everolimus. In vitro experiments demonstrate that both mutations are activating, suggesting a biologic mechanism for exquisite sensitivity to everolimus in this patient. The use of precision (or "personalized") medicine approaches to screen patients with cancer for alterations in the mTOR pathway may help to identify subsets of patients who may benefit from targeted therapies directed against mTOR.

Timing of post-influenza bacterial superinfection with MRSA dictates the outcome of MRSA pneumonia in mice (P4027)

Abstract Secondary bacterial pneumonias caused by S. aureus, S. pneumoniae and H. influenzae are a common occurrence following influenza infection and are responsible for significant morbidity and mortality worldwide. Others have shown in murine models that 5-8 days after influenza challenge the susceptibility to secondary bacterial infections increases; though the mechanism of this increased susceptibility is still elusive. Here, we report that 2-3 days after influenza challenge (before the onset of clinical symptoms, i.e. body weight loss) mice actually have increased resistance to secondary infection with methicillin resistant S. aureus (MRSA). Importantly, despite this early protection from MRSA, the response to bacteria early in the influenza infection results in increased viral growth and subsequently death of mice. The mechanism of this increased resistance to secondary MRSA pneumonia early after influenza is dependent on the transient presence of IL-13 and on type I IFN signaling, as mice deficient in these cytokine or cytokine receptor do not show increased resistance to secondary MRSA pneumonia 2-3 days after influenza infection, and injection of exogenous IL-13 to mice co-infected with MRSA pneumonia on day 7 increases resistance to bacteria. Thus, depending on the time of secondary infection in relation to influenza, the disease phenotype contracted by mice is greatly different, awareness of which is important for the design of rational treatments in clinics.

Fragile X syndrome: causes, diagnosis, mechanisms, and therapeutics

Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and is also linked to other neurologic and psychiatric disorders. FXS is caused by a triplet expansion that inhibits expression of the FMR1 gene; the gene product, FMRP, regulates mRNA metabolism in the brain and thus controls the expression of key molecules involved in receptor signaling and spine morphology. While there is no definitive cure for FXS, the understanding of FMRP function has paved the way for rational treatment designs that could potentially reverse many of the neurobiological changes observed in FXS. Additionally, behavioral, pharmacological, and cognitive interventions can raise the quality of life for both patients and their families.

Defective mitochondrial translation differently affects the live cell dynamics of complex I subunits

Complex I (CI) of the oxidative phosphorylation system is assembled from 45 subunits encoded by both the mitochondrial and nuclear DNA. Defective mitochondrial translation is a major cause of mitochondrial disorders and proper understanding of its mechanisms and consequences is fundamental to rational treatment design. Here, we used a live cell approach to assess its consequences on CI assembly. The approach consisted of fluorescence recovery after photobleaching (FRAP) imaging of the effect of mitochondrial translation inhibition by chloramphenicol (CAP) on the dynamics of AcGFP1-tagged CI subunits NDUFV1, NDUFS3, NDUFA2 and NDUFB6 and assembly factor NDUFAF4. CAP increased the mobile fraction of the subunits, but not NDUFAF4, and decreased the amount of CI, demonstrating that CI is relatively immobile and does not associate with NDUFAF4. CAP increased the recovery kinetics of NDUFV1-AcGFP1 to the same value as obtained with AcGFP1 alone, indicative of the removal of unbound NDUFV1 from the mitochondrial matrix. Conversely, CAP decreased the mobility of NDUFS3-AcGFP1 and, to a lesser extent, NDUFB6-AcGFP1, suggestive of their enrichment in less mobile subassemblies. Little, if any, change in mobility of NDUFA2-AcGFP1 could be detected, suggesting that the dynamics of this accessory subunit of the matrix arm remains unaltered. Finally, CAP increased the mobility of NDUFAF4-AcGFP1, indicative of interaction with a more mobile membrane-bound subassembly. Our results show that the protein interactions of CI subunits and assembly factors are differently altered when mitochondrial translation is defective.

Developing combination immunotherapies for type 1 diabetes: recommendations from the ITN–JDRF Type 1 Diabetes Combination Therapy Assessment Group

Like many other complex human disorders of unknown aetiology, autoimmune-mediated type 1 diabetes may ultimately be controlled via a therapeutic approach that combines multiple agents, each with differing modes of action. The numerous advantages of such a strategy include the ability to minimize toxicities and realize synergies to enhance and prolong efficacy. The recognition that combinations might offer far-reaching benefits, at a time when few single agents have yet proved themselves in well-powered trials, represents a significant challenge to our ability to conceive and implement rational treatment designs. As a first step in this process, the Immune Tolerance Network, in collaboration with the Juvenile Diabetes Research Foundation, convened a Type 1 Diabetes Combination Therapy Assessment Group, the recommendations of which are discussed in this Perspective paper.

Virtual Physiological Human Research Initiative: The Future for Rational HIV Treatment Design?

The clinical management of HIV-positive patients receiving existing drug regimens is performed largely as a clinician’s best guess, based on available viral and patient data. The development of new antiretroviral therapies for HIV is at an impasse. How can we progress beyond this? ViroLab, an example of a new ‘style’ of research project, is attempting to answer this question by stepping into the realm of prediction. A method is in development that can model and predict the efficacy of antiretroviral treatment and may enhance clinical decision support. If extended, this method can potentially target any other pathology where a ligand–substrate interaction is concerned. This type of research falls within the remit of a new initiative: the Virtual Physiological Human (VPH). The potential impact of VPH on current and future rational treatment design is discussed.

Malignant Melanoma in the 21st Century: The Emerging Molecular Landscape

Malignant melanoma presents a substantial clinical challenge. Current diagnostic methods are limited in their ability to diagnose early disease and accurately predict individual risk of disease progression and outcome. The lack of adequate approaches to properly define disease subgroups precludes rational treatment design and selection. Better tools are urgently needed to provide more accurate and personalized melanoma patient management. Recent progress in the understanding of the molecular aberrations that underlie melanoma oncogenesis will likely advance the diagnosis, prognosis, and treatment of melanoma. The emerging pattern of molecular complexity in melanoma tumors mirrors the clinical diversity of the disease and highlights the notion that melanoma, like other cancers, is not a single disease but a heterogeneous group of disorders that arise from complex molecular changes. Understanding of molecular aberrations involving important cellular processes, such as cellular signaling networks, cell cycle regulation, and cell death, will be essential for better diagnosis, accurate assessment of prognosis, and rational design of effective therapeutics. Defining an individual patient's unique tumor characteristics may lead to personalized prediction of outcomes and selection of therapy. We review the emerging molecular landscape of melanoma and its implications for better management of patients with melanoma.

Biophysics and structure–function relationship of T-type Ca 2+ channels

T-type channels are distinguished among voltage-gated Ca2+ channels by their low voltage thresholds for activation and inactivation, fast inactivation and small single channel conductance in isotonic Ba2+. Detailed biophysical and pharmacological characterization of native T-type channels indicated that these channels represent a heterogeneous family. Cloning of three family members (CaV3.1-3.3) confirmed these observations and allowed the study of the structure-function relationship of these channels. T-type channels are likely heterotetrameric structures consisting of a single polypeptide of four homologous domains (I-IV), each one containing six transmembrane spans (S1-S6), and cytoplasmic N- and C-termini. Structure-function studies have revealed that fast macroscopic inactivation of CaV3.1 is modulated by specific residues in the proximal C-terminus and in the transmembrane domain IIIS6. The particular gating properties within the T-type channel subfamily are determined by several parts of the protein, whereas differences with respect to high-voltage-activated Ca2+ channels are mostly determined by domains I, II and III. Several gating properties are affected by alternative splicing, C-terminal truncations and mutations associated to idiopathic epilepsy. Intriguingly, the aspartate residues of the EEDD locus of the selectivity filter not only determine the permeation properties and the block by Cd2+ and protons, but also activation and deactivation. Mutagenesis has also revealed that the outermost arginines of the S4 segment of domain IV influence the activation of CaV3.2, though no specific voltage-sensing amino acid has yet been properly identified. The selective modulation of CaV3.2 by G-proteins, CaMKII and PKA is determined by the II-III linker and the high-affinity inhibition of CaV3.2 by Ni2+ relies on a histidine residue in the IS3-S4 linker. Certainly, more structure-function studies are needed for a better understanding of T-type channel physiology and the rational design of treatments against T-type channel-related pathologies.