Deena Iskander, Clinical Research Fellow Investigation of blood cell production and the effect of steroids in Diamond-Blackfan Anaemia Deena Iskander, Clinical Research Fellow Centre for Haematology, Faculty of Medicine, Hammersmith Hospital, Imperial College London Paediatric Haematology and Bone Marrow Transplantation Unit, St Mary’s Hospital, Imperial College Healthcare NHS Trust, London, UK Imperial Molecular Pathology Laboratory, Hammersmith Hospital, Imperial College Healthcare Trust DBA Family Weekend, May 2016 Thank you for giving me the opportunity to present my work to you today entitled… I started this work in 2013 as a CRF sponsored by major chaitrty supporting haematology research- Bloodwise

Plan Background to project Aims Experimental design Results Conclusions Further work Today I will introduce the background…

Diamond-Blackfan Anaemia Inherited bone marrow failure disease 5-10 per million babies born Other blood count abnormalities e.g. low white blood cell count Low haemoglobin: ANAEMIA requiring blood transfusions IBMFS i.e. born with it, invilves bone marrow but syndrome because also other features Rare Anemia and other blood count abnormalties 60% patients have Physical changes Problems developing throughout life e.g. slow growth, infections Physical changes arising at birth e.g. heart problems, kidney problems, joint problems Vlachos A et al. Expert Rev Hematol, 2014 Narla A & Ebert B, Blood 2010

DBA is a caused by a change in the genetic code -a ‘mutation’- that leads to half the normal amount of one ribosomal protein Small ribosomal subunit Large ribosomal subunit This is a picture of a ribosome in a human body cell. A ribosome is a protein structure responsible for making all the proteins that a cell needs so ribosomes are essential for every single cell in the human body. Ribosomes themselves are made up of around 80 proteins in the large and small subunits DBA is caused…. This stops ribosomes from working normally proteins needed for normal function of the the body’s organs are missing Slavov et al Cell Reports 2015 Mutation inherited from one parent in 35% patients and arises ‘out of the blue’ in 65%

The same mutation can cause different effects in different people dad mum 11y son Preterm birth with poor growth and low birth weight Diagnosed at birth (mother known to have DBA) RPL11 mutation Transfusions needed for first few years Steroids aged 5y– initial response but loss of response by 10y Second steroid trial no response Resistant to steroids 30y old mum Diagnosed aged 7y RPL11 mutation Blood transfusions needed in pregnancy Treated with steroids Responsive to steroids- no further blood transfusions needed Interestingly however having the same mutation can lead to very different effects in the same people This picture shows an ‘imaginary’ family similar to some of the families we have seen. Square represents male, circle a female and the shape being coloured in reflects patients that are affected by a disease Some of family details changed

DBA is characterised by absent or reduced red blood cell precursors in the bone marrow DBA BM Non-DBA child BM This brings me onto the classical findings in DBA at presentation, Although patients have a severe anaemia, anaemia can result from many different diseases so the next step in diagnosis is to perform a bone marrow examination in which we look at bone marrow under the microscope This shows absent or significantly reduced erythroid precursors in the bone marrow so in the non DBA BM you see these cells highlighted with a red arrow but they are missing in the DBA maroow This observation led is to consider why red blood cells fail to develop in DBA

How red blood cells develop in the bone marrow precursor Red blood cell ‘parents’ Stem cells Many of you will have heard of stem cells. Basically these are the most immature blood cells that have the power to develop into different blood cell types As stem cells mature, shown by arrows in this diagram, the cells become more specialised so stem cells become cells that can only mature into red cells. We call these red blood cell parents or progenitors. These go on to form more mature red blood cell precursors then finally red blood cells While we can see precursor cells and red blood cells in the BM and blood respectively stem cells and parent cells are invisble. We cant see them with a microspope. In mice scientists have found a way of capturing red blood cell parents from bone marrow using protein labels and this has allowed them to study these cells So we asked a similar question in human cells, that is /…’what is…’ Importantly both stem cells and What is the identity of the ‘parents’/progenitors of red cells in humans? ‘Invisible’

Different blood cell defects are implicated in DBA Absent progenitors in 7 relapsed patients with DBA (Nathan D JCI 1978) Normal progenitor numbers with impaired development of progenitors to red blood cells (Lipton J et al Blood 1989) In terms of how this all links to DBA, different blood cell defects are…. These old papers point to problems with red blood cell progenitors but we wanted to investigate this further with modern techniques so we asked…. What is the origin of red blood cell failure in DBA?

Management of DBA Diagnosis Steroid responsive 80% Dose weaned/stopped Prednisolone steroid tablets - high dose Blood transfusions /Bone marrow transplantation Steroid responsive 80% Steroid resistant 20% Diagnosis Dose weaned/stopped Steroid resistant 40% We hoped that by answering these questions ultimately we will be able to improve the management of DBA Here is a flow chart to show current management 80% people get better on steroids and no longer need blood transfusions Ultimately only 40% patients can use steroids in the long-term

How do steroids work in DBA? Act directly on DNA Increase haemoglobin Increase the number of times that red blood cell progenitors divide, leading to more and more red blood cells Act directly on DNA stimulating some genes and blocking other genes Increas Hb in people with DBA and without DBA So our final question is….. How do steroids affect red blood cell progenitors in DBA?

Aims a) Define the identities of red blood cell progenitors in human bone marrow b) Describe early blood cell development in DBA 2 Understand how steroids improve anaemia in DBA by studying the cells and genes that are affected by steroids in human red blood cell progenitors

Experimental design Investigation of Bone marrow from patients with DBA and healthy children after consent Samples labelled anonymously once they arrive in the lab Clinical information accompanying each sample stored confidentially Isolation of cells of interest Looing at the cells directly isolated from bone marrow Growing the cells in a ‘soup’ that promotes red blood cell development Investigation of the markers on the outside of red blood cell progenitor populations How the cells grow What genes the cells express

Patients with DBA compared with healthy children Non-DBA (18 people) DBA (15 people) Average age (range) 6y7m (1y6m-43y) 6y (10m-17y) Reason for bone marrow Donating their bone marrow for a bone marrow transplant 13 for follow-up, 1 for low blood counts, 1 for diagnosis of DBA Treatment NA 10 on regular blood transfusions (2 steroids not yet tried, 8 resistant to steroid) 5 steroid responsive I am going to show you some of our results from patients with DBA and helathy controls but first here are some of the clinical detials. Importantly the patients and controls have similar ages.

Diagnosis of DBA by DNA sequencing I don’t wish to talk at length about diagnosis as my colleague Sandra will explain this work further but in all patients in this study the diagnosis of DBA supported by clinical features Also Using our in house custom designed Next generation sequencing of all 80 RP genes we have achieved a good pick up rate of 69% as shown in this pie chart which demonstrates the frequencies of patients with mutations in particular RP genes. A ribosomal protein mutation likely to be causing DBA has been identified by sequencing in 69% (68/99) patients tested. 1 Gerrard G, Valgañón M, En Foong H et al. British Journal Haematol. 2013

Results Aim 1a: What is the identity of the ‘parents’/progenitors of red cells?

Characterisation of red blood cell progenitors using protein labels on their surface x10 Early red cell progenitors x10 Late red cell progenitors A B Human bone marrow has 2 different red blood cell progenitor types which we call early and late, depending on the type of cell colonies that grow from these progenitors: When we looked at human BM we found 2 different groups of cells with different protein labels on their surface so we hypothesised: Hypotheses: A cells are early red blood cell progenitors B cells are late red blood cell progenitors Iskander D et al, Blood, 2015

Indeed we found that these newly defined progenitors look different….. B N=3 …..and express different genes A B P VALUES NOT SIGNIFICANT May-Grünwald Giemsa staining of flow-sorted EEP and LEP from control adult BM. mRNA expression levels of CD36, GATA-1 and GATA-2 determined by quantitative real-time PCR in purified GMP, MEP, EEP, LEP and erythroblasts (Lin-CD34-CD36hiCD71+GlyA+ EB), derived from 3 control BM samples. Transcript levels are normalised to GAPDH and expressed relative to the corresponding gene expression in MEP. As expected, GMP do not express CD36 and GATA-1 but express a low level of GATA-2. Iskander D et al, Blood, 2015

Conclusions I: Normal blood cell production We have identified the early and late red blood cell progenitor populations in the bone marrow of healthy children. The same populations were also found in adult bone marrow. The above findings have since been corroborated by other groups. (Mori Y, PNAS, 2015) This is important because it means we can capture these cells from human bone marrow samples and study them in health and diseases, which should lead to understanding these diseases better.

Results II Aim 1b: Describe early blood cell development in DBA Aim 2: Understand how steroids improve anaemia in DBA

DBA progenitors do not grow normally x10 Non-DBA DBA Remember what red blood cells from normal red blood cell progenitors look like. We found that DBA bone marrow gave us fewer red blood cells and they looked paler and smaller Iskander D et al, Blood, 2015 N=4 CON N=5 DBA

Transfusion-dependent (TD) versus steroid-responsive (SR) patients NON-DBA When we looked at this in more detail, we found that while in healthy children there were lots of red blood cell progenitors, in patients with DBA there were hardly any early or late - which explains why these patients need blood transfusions. Steroids fixed this problem by restoring the presence of red blood cell progenitors. Iskander D et al, Blood, 2015

Conclusions II: DBA blood cell production In transfusion-dependent DBA There are fewer red blood cell progenitors and they don’t work normally Red blood cell progenitor abnormalities are restored in steroid-responsive DBA

Summary of progress and future work We have identified the early and late red blood cell progenitor populations in the bone marrow of healthy children. We have used these definitions to identify the places in red cell production where things are going wrong in DBA. We will continue to study these cells with the aim of identifying why things go wrong in DBA and how steroids can help. The overall aim of this work is to find new approaches and gene targets that we can use to treat DBA. Change aim 1 in normal an in DBA 1a and 1b 2- functional

Acknowledgements The patients and their families- All of you! Tassos Karadimitris Irene Roberts Beth Psaila, Valentina Caputo, Aris Chaidos, Katerina Goudevenou, Andi Roy Qais Al-Oqaily, Neha Bhatnagar, Joana Costa, Maialen Lasa, Kalliopi Makarona, David O’Connor, Kyriake Petivi, Kanagaraju Ponnusamy, Antonella Rotolo, Nikolaos Trasanidis Mauritius Kleijnen, David Pitcher Holger Auner, Sandra Loaiza, Katarzyna Parzych Niklas Feldhahn, Bryant Boulianne Mark Layton, Hammersmith Hospital NIHR BRC Imaging and FACS Facility, Imperial College Josu de la Fuente Paediatric Haematology team, St Mary’s Hospital Yvonne Harrington, SpRs John Goldman Centre for Cellular Therapy Imperial Molecular Pathology lab Letizia Foroni, Jamshid Khorashad, Gareth Gerrard, Hui en Foong , Sandra Hing The patients and their families- All of you! 11s gsese se 6