Elucidating the role of GCN2 in the pathogenesis of pulmonary vascular disease

Pulmonary hypertension is a term that describes a group of diseases which have in common the following feature: increased blood pressure levels in the blood vessels leading into and out of the lungs, which leads to heart failure and finally death, if left untreated. There are two main types of pulmonary hypertension. In the first type, the obstruction is at the level of the arteries feeding the lungs ('pulmonary arterial hypertension') and in the second type the obstruction occurs either at or below the level of the veins draining the lungs ('pulmonary venous hypertension'). Recently there has been a discovery of a new inherited problem which can cause a rare form of pulmonary hypertension, pulmonary veno-occlusive disease (PVOD), which combines both arterial and venous features. This problem consists of mutations in the gene for a protein called general control nonderepressible 2, or GCN2. The GCN2 protein is crucially important in a reaction known as the integrated stress response, which is usually activated in periods of protein starvation. There has been no previous hint that problems with this protein can affect the heart or lung circulation; so this is a completely new area of research.

Inherited pulmonary arterial hypertension has previously been linked with problems in another gene which creates a protein known as bone morphogenetic protein receptor type II (BMPR2). Patients with this problem have a deficiency of BMPR2. Finding this genetic problem has led to new ways to treat pulmonary arterial hypertension such as increasing BMPR2 levels or by activating the remaining BMPR2. These are currently being tested in mice. It is our hope that the discovery of GCN2 will drive the same process for pulmonary veno-occlusive disease specifically and pulmonary hypertension in general. Currently there is no specific treatment for either PVOD or pulmonary venous hypertension other than a transplant, which very few patients are eligible for.

I plan to examine how exactly GCN2 deficiency affects the heart and lungs. To do this, we will use several models of the disease, such as fruit flies and mice lacking the GCN2 gene, and blood samples and lung blood vessel cells by patients with GCN2 mutations donated at the time of transplant. We will initially compare mice that are completely lacking this gene with normal mice and see whether GCN2-deficient mice develop pulmonary hypertension. If they don't, we will see whether crossing these mice with mice lacking BMPR2 will 'uncover' the disease. So far we have discovered that GCN2-deficient mice develop mild pulmonary hypertension and this is further exacerbated by adding in BMPR-2 deficiency. Our next steps are to see whether GCN2 is a protective factor and if replacing and or activating GCN2 will be protective against pulmonary hypertension in mice

We also know from the fly studies that the GCN2 and BMPR2 pathways overlap. For example, activating the GCN2 pathway reduces the reaction to stimulation of BMPR2 in fly wings and in fly cells. I will examine if this is also true in cells taken from human lung blood vessels. If BMPR2 is downstream of GCN2-related pathways then perhaps activating BMPR2 will be an alternative way of treating pulmonary hypertension associated with GCN2 deficiency.

Once we understand how GCN2 deficiency leads to development of pulmonary hypertension in mice, we will check if these theories are supported in experiments using blood and tissue samples donated by pulmonary hypertension patients. Eventually we aim to develop new treatments for GCN2-associated pulmonary hypertension and to test them in clinical trials in patients.

Pulmonary veno-occlusive disease (PVOD) is a severe form of pulmonary arterial hypertension (PAH), which carries an appalling prognosis. Most patients die within 2-3 years of diagnosis if not transplanted. Very little is known about PVOD, and patients are treated empirically with medications validated in PAH. Recently, mutations in eukaryotic translation initiation factor 2-alpha kinase 4 (EIF2AK4, also known as general control nonderepressible 2 or GCN2) were described in PVOD patients. GCN2 controls the response to amino acid deficiency by phosphorylating the translation initiation factor eIF2-alpha to trigger the Integrated Stress Response (ISR). GCN2 has not been implicated previously in cardio-pulmonary disease and represents a completely novel therapeutic target. I hypothesise that GCN2 deficiency promotes the development of PVOD via dysregulated BMP and inflammatory signalling.
I shall test this hypothesis in three aims:
a) Defining the effects of GCN2 deficiency on a cellular level.
b) Determining the mechanism linking GCN2 and BMP signalling.
c) Determining the consequences of deranged GCN2 signalling in mice and exploring the possibility of similar mechanisms in patients.

I plan to use Drosophila and cell models to rapidly screen for points of interaction between GCN2 and BMPR2 pathways. I will then validate these pathways in mice and in patient-derived serum and tissue samples. I have established colonies of gcn2-/- and bmpr2R899X/+ mice and have crossed these mice to produce a doubly-deficient line. After phenotyping these mice from a structural, physiological, metabolic and inflammatory basis I intend to expose them to chronic activation of the ISR, both by itself and in conjunction with known PAH-causing stimuli such as hypoxia and chronic LPS. I will also phenotype and follow-up patients with GCN2 mutations from the UK-PAH cohort. Eventually I plan to modulate GCN2-dependent pathways to treat pulmonary vascular disease.

Potential academic, economic, and societal beneficiaries of this research include the following:

a) scientists and researchers in pulmonary vascular medicine and the integrated stress response,
b) the wider scientific community,
c) physicians involved in the care of pulmonary vascular disease patients,
d) patients with pulmonary vascular disease,
e) pharmacological companies
f) A-level and university students interested in science, and
g) the lay public.

In the short- and medium-term I envision that the beneficiaries will be scientists and researchers in the fields of pulmonary vascular medicine and the integrated stress response. The discoveries made during the course of this research, and the novel rodent models constructed will be presented at national and international meetings and published in peer-reviewed journals. This will contribute to the net knowledge about pulmonary vascular disease. As an example of previous engagement efforts with fellow scientists, researchers, and physicians; I have previously chaired the plenary session on 'Novel science, innovative imaging and a clinical update in pulmonary hypertension' at the British Thoracic Society Winter Meeting 2016. I have also been invited to speak at the Royal College of Physicians Cardiovascular Biology workshop on pulmonary vascular disease which is planned for September 2017.

I also plan to engage A-level students, medical students and junior doctors interested in science. I have previously addressed final-year medical students in the University of Cambridge on careers in academic medicine and received very good feedback in response. I will be involved in the CIMR's participation in both the Cambridge Science Festival and the Nuffield Science Bursary. The latter programme allows A-level students to work alongside researchers towards a project that is then presented in a public forum.

I will also engage with patients with pulmonary vascular disease and their carers via the open days hosted by the Pulmonary Hypertension Association UK in conjunction with Papworth Hospital NHS Trust. The British Heart Foundation in conjunction with the Morrell group also hosts regular seminars where British Heart Foundation volunteers and members of the lay public are invited to discuss research findings and to contribute their opinions about the direction of research.

In the long-term the beneficiaries will include patients with known GCN2 mutations and pulmonary veno-occlusive disease. The rarity of these diseases has precluded much clinical knowledge being gathered about them and one of the aims of this project will be to address that deficit. Once we have more knowledge about the clinical progression of these diseases we can better advise and treat patients. If we succeed in identifying specific biomarkers of GCN2-associated pulmonary vascular disease, then we will also have more objective means of tracking disease progression.Finally, although not within the remit of this particular application, there is the hope that any disease-causing mechanisms identified may one day be translated into clinical interventions for patients with GCN2-associated pulmonary vascular disease and eventually for patients with pulmonary venous hypertension. If we were to achieve this, beneficiaries would then include scientists specializing in heart failure and in the hypoxic lung diseases such as emphysema and pulmonary fibrosis, pharmacological companies that we would collaborate with to try and bring this end to fruition, and patients with pulmonary venous hypertension such as that due to left heart disease and hypoxic lung disease.