12q13.13

ACVRLK1,ALK-1,ALK1,HHT,HHT2,ORW2,SKR3,TSR-I

Solid tumours

As a receptor mainly expressed on the surface of endothelial cells, ALK1 overexpression and unbalances in its signalling are implicated in many solid tumours, despite the origin and specific features of the latter. Thus, they will be discussed in a single paragraph.
In a study (Hu-Lowe et al., 2011) performed on 3000 human tumour specimens representing more than 100 tumour types, ALK1 resulted particularly expressed in the vasculature of prostate cancers, malignant melanomas of the skin, follicular cancers of the tyroid, renal clear cell cancers and endometrioid ovarian cancers.
A reduced expression of ACVRL1 by qRT-PCR and immunohistochemistry was demonstrated in nasopharingeal carcinomas by Zhang et al., 2012.
An increased ALK1 expression in papillary thyroid carcinomas with bone formation was increased if compared to that in normal thyroid tissue and tumors without bone formation, as assessed using immunohistochemistry and quantitative real-time polymerase chain reaction (Na et al., 2013).
In Head and Neck Squamous Cell Carcinomas (HNSCC), using immunohistochemistry and qRT-PCR, Chien et al. found a correlation between a high ACVRL1 expression and an advanced T classification, a positive N classification, an advanced TNM stage, the presence of lymphovascular invasion, an extracapsular spread of lymph node metastasis and a poorer prognosis (Chien et al., 2013).
As a therapeutic target, anti-ALK1 drugs (both in the form of an Fc-fusion protein acting as a soluble receptor for BMP9 and of an anti-ALK1 monoclonal antibody) are under investigation in phase I and phase II clinical trials in a wide range of solid tumours (Vecchia et al., 2013). Phase II studies clinical trials encompass particularly squamous cell carcinoma of the head and neck, endometrial cancer, epithelial ovarian cancer, fallopian tube cancer and primary peritoneal carcinoma for ACE-041 (also known as Dalantercept, the Fc-receptor fusion protein). Dalantercept displayed promising antitumour activity particularly in patients with advanced refractory cancer (Bendell et al., 2014). PF-03446962 (the anti-ALK1 monoclonal antibody), is up to now studied in phase II clinical trials particularly in malignant mesoteliomas of the pleura and transitional cell carcinomas of the bladder. A recent study showed that PF-03446962 has no activity as a single drug in refractory urothelial cancer as is thus suggested, for this kind of cancer, only as a combination therapy with other agents against the VEGF receptor axis (Necchi et al., 2014). Both Dalantercept and PF-03446962 are currently under investigation in phase II trials particularly in advanced and refractory hepatocarcinomas.
As assessed by Hosman et al., 2013, mutations in ACVRL1 gene, as the ones observed in HHT2 patients, seem to reduce the prevalence of some types of solid tumours and account for the unexpected good life expectancy of HHT patients older than 60 years of age. Although it is important to take with care the results of the study due to the methodology used for the assessment (for the statistical and logistic difficulties to perform a longitudinal study in a rare disease, the authors used a questionnaire, inevitably biased), HHT patients older than 60 presented an apparent reduction in lung, liver and colorectal cancer compared to controls. This could potentially be related to the ALK1 haploinsuffiency present in ALK1 HHT mutations, opposite to the overexpression usually showed in cancers. On the other hand, colorectal cancer was instead more frequent in younger HHT patients, particularly in the subgroup with SMAD4 mutations and juvenile polyposis.

Hereditary hemorrhagic telangiectasia type 2 (HHT2)

Hereditary Hemorrhagic Telangiectasia (HHT), or Rendu-Osler-Weber disease, is a vascular dysplasia inherited as an autosomal dominant trait (Shovlin, 2010; McDonald et al., 2011). It affects approximately 1 in 5-8000 individuals (Faughnan et al., 2011) with regional differences due to founder effects (Westermann et al., 2003, Lesca et al., 2008). The clinical diagnosis of HHT is based on the presence of at least three of the following "Curaçao criteria" (Shovlin et al., 2000): (1) spontaneous, recurrent epistaxis; (2) mucocutaneous telangiectases at characteristic sites as nose, lips, oral cavity, finger tips and gastrointestinal (GI) mucosa; (3) visceral arteriovenous malformations (AVMs) in lungs, liver, GI, brain and spinal cord; (4) family history of first-degree relative in whom HHT has been diagnosed using these criteria. Significant clinical variability was observed in HHT (Lesca et al., 2007; Govani and Shovlin, 2009), with both intra- and interfamilial variations in age-of-onset, localization of lesions, and severity of complications, whereas it usually shows a high penetrance. HHT is usually not apparent at birth, but evolves with age into a recognizable phenotypic pattern. Spontaneous recurrent nosebleeds are the most common and usually earliest clinical manifestation. HHT telangiectases develop and get worse with age. Complete penetrance was found to be by 40 years of age (Porteous et al., 1992). HHT patients show approximately 15-50% of pulmonary AVMs (PAVMs), 32-78% of liver AVMs (HAVMS) and approximately 23% will harbor AVMs in the brain (CAVMs). Although 80% of patients with HHT have gastric or small intestinal telangiectases, only 25-30% of patients will develop symptomatic GI bleeding which usually does not present until the fifth or sixth decades of life (Faughnan et al., 2011).
HHT arises from heterozygous mutations in ENG (HHT1, OMIM #187300) coding for ENDOGLIN (ENG) (McAllister et al., 1994) and ACVRL1 (HHT2, OMIM #600376) coding for ALK1 (Johnson et al., 1996), Type III and Type I TGF-β receptors, respectively. Certain HHT2 patients develop a Pulmonary Artery Hypertension (PAH)-like syndrome, suggesting that ACVRL1 mutations are also likely to be involved in PAH (Trembath et al., 2001; Olivieri et al., 2006). A subset of patients with juvenile polyposis, carrying mutations in SMAD4/MADH4 (JPHT, OMIM #175050), can also develop HHT (Gallione et al., 2004). Recently, mutations in BMP9 were reported in three unrelated families affected by a vascular-anomaly syndrome presenting with phenotypic overlap with HHT (Wooderchak-Donahue et al., 2013). Additional as-yet-unknown HHT genes have been suggested by linkage analysis in two affected kindred on chromosome 5 and on chromosome 7 (Cole et al., 2005; Bayrak-Toydemir et al., 2006). Molecular genetic testing of the three known genes detects mutations in approximately 85% of patients. As reported above, the mutated genes encode proteins that mediate signaling by TGF-β family.
More than 375 ACVRL1 variants are present in the international HHT mutation database and more than 185 are demonstrated to be pathogenic for HHT.
TGF-β ligands regulate angiogenesis through their actions either on endothelial cells (EC) and/or mural cell, demonstrating that they play important roles in both activation (via ALK1) and resolution (via ALK5) phases of angiogenesis. It has been reported that BMP9, rather than BMP10, might be the specific ALK1 ligand and activator of the Smad1/5/8 signaling pathway in endothelial cells and that they are potent inhibitors of EC migration and growth (David et al., 2007). Previous studies have suggested the synergy between Notch and TGF-β, and that Notch signaling modulates the balance between TGF-β/ALK1 and TGF-β/ALK5 signaling pathways (Fu et al., 2009).

ALK1-1 and pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a severe and rare disease affecting small pulmonary arteries, with progressive remodeling leading to elevated pulmonary vascular resistance and right ventricular failure, and is a major cause of progressive right-sided heart failure and premature death (Trembath et al., 2001). PAH is defined as the sustained elevation of mean pulmonary artery pressure (PA) above 25 mmHg at rest or 30 mmHg during exercise (Rabinovitch, 2012). The histopathology is marked by vascular proliferation/fibrosis, remodeling, and vessel obstruction (Chan and Loscalzo, 2008).
In the second World Symposium held in Evian, France, in 1998, was proposed a clinical classification for pulmonary hypertension. The first category was defined PAH and includes two subgroups, the first incorporates both the idiopathic form (IPAH) that the inherited (HPAH) of the disease. The second subgroup includes a number of conditions associated with various diseases (APAH), including connective tissue diseases, human immunodeficiency virus infection, congenital heart disease, and portal hypertension (Simonneau et al., 2004; Machado et al., 2009).
Heterozygous mutations in the transforming growth factor-β receptor (TGF-β receptor) super family have been genetically linked to PAH and likely play a causative role in the development of disease. Particularly, mutations in the bone morphogenetic factor receptor type 2 (BMPR2) gene account for approximately 70% of all familial pedigrees of PAH (HPAH) and 10-30% of idiopathic PAH cases (IPAH) (Chan and Loscalzo, 2008; Machado et al., 2009).
Much less commonly (5%) two other members of the TGF-β superfamily are also recognized as uncommon causes of PAH: activine A receptor type II-like kinase 1 (ALK1) and, at significant lower frequency, endoglin (ENG) (Harrison et al., 2003). Heterozygous mutations of these genes cause the autosomal dominant vascular disorder hereditary haemorrhagic teleangiectasia (HHT) (Shovlin, 2010). In fact, in a small proportion of HHT patients, was observed a form of pulmonary arterial hypertension that is associated with a model of precapillary pulmonary hypertension that is histopathologically indistinguishable from idiopathic form of PAH. Since the publication by Trembath et al. in 2001 (Trembath et al., 2001), who first reported patients with a mutation in the gene ACVRL1 with clinical features of both PAH and HHT, subsequently, have been recognized several other mutations in the ALK1 gene that seem to predispose patients with HHT development of PAH. This observation was further confirmed by other studies (Olivieri et al., 2006) and extensively discussed by Machado et al. (Machado et al., 2009). The exact prevalence of PAH in the HHT population has not been systematically evaluated, but most authors agree that it is a rare complication found in less than 1% of HHT patients (Cottin et al., 2007). In rare cases, ACVRL1 mutations have been reported to cause IPAH or HPAH without HHT (Harrison et al., 2003).
Both ALK1 and BMPR2 belong to the family of TGF-β receptors, they have different specific ligands but share a common intracellular pathway based on the activation of the SMAD proteins 1/5/8 (Faughnan et al., 2009). The formation of an heteromeric complex with BMPR2 and ALK1 could at least in part explain why any dysregulation of this pathway may promote pulmonary endothelial and/or smooth muscle cell dysfunction and proliferative characteristic of PAH, in subjects carrying mutation either in BMPR2 or in ACVRL1 gene.
Mutations identified in several studies on ALK1 associated with PAH are all likely to disrupt activation of this intracellular pathway and the majority of these comprise missense mutations. Particularly, mutations in exon 10 of ACVRL1 are relevant because they occur in functional domains of the receptor within a conserved carboxyl-terminal region of ALK1 (the non-activating non-down regulating box) NANDOR BOX (Faughnan et al., 2009; Machado et al., 2009). Of note, the NANDOR BOX, located from codon 479 to 489, is necessary for regulation of TGF-β signaling, accordingly any alteration may have effects on TGF-β-induced receptor signaling (Girerd et al., 2010).
Moreover, recent studies in animal models have shown that Alk1 heterozygous mice spontaneously develop signs of pulmonary hypertension in the early months of life, and with increasing age show more occluded vessels and pulmonary vascular remodeling, indicating a progression of the disease. These mice had also higher ROS levels in adult lungs contributing to PAH development compared to control mice. Whereas Bmpr2 heterozygous mouse model requires additional factors, such as hypoxia and serotonin or inflammation, to elicit a pulmonary hypertensive phenotype (Jerkic et al., 2011).
Finally, Girerd B. et al. hypothesized that mutated ACVRL1 status might be associated with distinct PAH phenotypes, as compared with patients PAH without ALK1 mutations. The authors analyzed clinical, functional characteristic, hemodynamic features and outcomes for patients with PAH carrying ACVRL1 mutation. Of notice, these patients were significantly younger at diagnosis (P

Hematological malignancies

Roughly 80% of non-Hodgkins lymphomas and 60% of Hodgin lymphomas express ALK1 in their vasculature (Hu-Lowe et al., 2011). The expression of ALK1 in haematological cancers was further confirmed in an exploratory study on patients affected with Acute Myeloid leukemia (AML) (Otten et al., 2011). Using qRT-PCR, ALK1 was demonstrated to be expressed by 82% of patients samples (pretherapeutic bone marrow or peripheral blood from 93 patients with newly diagnosed AML). Furthermore, formalin-fixed, paraffin-embedded trephine bone marrow specimens from two arbitrarily selected patients with AML and from two patients with non-leukemic reactive changes were analyzed for ALK-1 expressions by immunohistochemistry. Endothelial cells from two AML patients and those with reactive disorders were strongly positive and a fraction of AML blasts stained positively for ALK-1 in AML bone marrows, whereas normal hematopoietic cells were negative.
Anyway, ALK1 alterations, opposite to those in ALK5, seemed not to have a significant impact on survival. Furthermore, the prevalence of haematological cancers in HHT patients as assessed by Hosman et al., 2013 showed no difference compared to controls.