2024

ALLTogether team science – development of a preclinical testing platform to improve outcomes for drug resistant T-cell Acute Lymphoblastic Leukaemia

Acute lymphoblastic leukaemia (ALL) is the most common cancer in children and can affect either B - or T cells. When T-cell leukaemia does not respond well to initial treatment (called resistant disease) or reoccurs (relapses), only two or three out of ten children will be cured. 
Research into better treatments for T-ALL has been difficult because the number of patients in each country is small. To overcome this, 14 European countries have joined to develop a single clinical trial for children, teenagers and young adults called ALLTogether1. Because this trial is so large, we now have a golden opportunity to study patients with T-ALL. This will help us understand why some T-ALLs do not respond to chemotherapy and help us find new drugs for patients with relapsed T-ALL.  
We aim to study changes in genes and proteins in unresponsive T-ALL cells after expansion of these cells in immune-deficient mice. Excitingly, we will use artificial intelligence to create computer simulations of the composition of the leukaemia to predict response to novel treatments. 
In addition, we will start testing leukaemia cells in the lab to see how combinations of already existing drugs will kill these leukaemic cells. We expect that our research will speed up drug development and personalised treatments in the future. Moreover, we will share this valuable resource of patient samples, gene and protein analyses, and new drug treatments with doctors and scientists in Europe (and beyond) to improve cure rates for patients with T-ALL
 

Detecting lingering cancer before it grows back

We are conducting research into whether cerebrospinal fluid (CSF), which is in the ventricles in the brain, contains labels (which we call biological  markers, or biomarkers for short) of brain cancer. Focussing on ependymoma, we have been looking for metabolite biomarkers, which are used in cell metabolism and may be released into the CSF from the tumour cells. Cell metabolism creates energy for the cell and builds components of the cell structure. Indeed we found some biomarkers of ependymoma in CSF samples taken from children by lumbar puncture, as the biomarker metabolites were found at a different level in ependymoma CSF compared to healthy CSF. These biomarkers were seen after surgery, when most tumour was removed, and should therefore be markers of lingering cancer cells. Lingering cancer cells are normally treated by

radiotherapy but the cancer grows back in half of the ependymoma patients. We wish to see if these nine biomarkers are found in CSF from relapsed ependymoma patients and then, across a time frame, how early on in treatment we can see these labels of relapse. If the biomarkers are found only in the patients who relapse, after the end of treatment and in their relapsed tumours, we could predict who will relapse and treat
them further. In future we could find these biomarkers by monitoring patients during and following radiotherapy for 2 years. This could lead to diagnosing relapse earlier than by using MRI scans, which could facilitate earlier treatment and perhaps a better outcome for the child. This addresses one of the Top 10 Children’s Cancer Priority Setting Partnership Research Priorities (number 5), which is "Why do children relapse, how can it be prevented, and what are the best ways to identify relapse earlier?" The metabolite biomarkers that we found point towards metabolic pathways that are potential new therapeutic targets for patients who need further therapy to prevent relapse. Those most abundant in ependymoma included metabolites in the export of acetyl- CoA from the mitochondrion (2 metabolites), in aerobic glycolysis (2 metabolites), and some amino acids (5 metabolites), whilst a sugar acid and an amino acid derivative were decreased in abundance (2 metabolites).
Therefore we might also address Research Priority number 1, "Can we find effective ... treatments for cancer including relapsed cancer?". Our proven method, liquid chromatography coupled to mass spectrometry targeted at our biomarkers, can effectively separate out, detect and quantify our biomarkers. We expect to deliver a test based on quantities of some of these biomarkers which can accurately determine if a patient is relapsing early on after the treatment for their primary tumour has ended.

Development of paediatric in vitro and in vivo models to study genetic and non genetic determinants
of cancer and foster new drug development.

Cancer is the leading disease-related cause of death in children 1-15 years old. Most deaths are due to cancer which becomes resistant to conventional therapies. Understanding why cancer develops, what mechanisms lead to relapse, how it changes over time and how it interacts with the immune system is crucial to developing new drugs to fight cancer. In vitro and in vivo models allow to study cancer, and also test new drugs. Different research questions require different models to study and be tested. This project aims at equipping the scientists in the institutions involved with several models, so that they can use the best model for each question and also validate the results in different models. We are particularly interested in three areas: 1. Generate PDX, which are known to recapitulate in vivo the human disease, and allow expansion of the tissue for further studies; 2. In vitro drug testing: we will grow tumour cells in vitro and we will test a battery of drugs that could help the child, to see if this approach could help the clinicians choose the treatment; 3. We will grow organoids and study how the immune system interacts with cancer.

The tools developed through this project will become available to other scientists through collaborations, so that we can maximize the impact of this project.

Expanding our understanding of CML-like Ph+ALL – are all patients at increased risk of relapse?

Acute lymphoblastic leukaemia (ALL) is a type of blood cancer that affects children and adults. It is an aggressive disease that needs intensive treatment with chemotherapy. One reason people may get leukaemia is that abnormalities develop in their genes. One such example of this is called the Philadelphia chromosome (Ph+). This is a bad risk ALL and is often associated with poor responses to and relapse following treatment. Ph+ is also seen in another type of leukaemia, called chronic myeloid leukaemia (CML). This type of leukaemia behaves very differently to Ph+ALL because the genetic abnormality is in an immature stem cell. Stem cells are the manufacturing system in the bone marrow that can mature into any of the three major and mature blood cells (white blood cells, red blood cells or platelets). In Ph+ALL, the genetic abnormality is in a mature white blood cell, termed a lymphoblast. It has always been assumed that CML and Ph+ALL are different diseases. However, recent scientific research from our research lab and others have suggested that in some children there is an overlap, and these cases have been termed ‘CML-like Ph+ALL’. This is a new type of leukaemia and research is needed to understand the disease better. However, we already know that these patients may have poorer outcomes to routine treatment and that they may need to be treated more like CML. In this project, we will investigate CML-like Ph+ALL in children. 

Exploring clonal evolution in rare (biclonal) aneuploid B-ALL cases

In children's leukemia, specifically in a type called B-cell acute lymphoblastic leukemia (B-ALL), the number of chromosomes within the cancer cells can vary. This variation, known as aneuploidy, is a common characteristic of this disease. When these leukemia cells have more chromosomes than normal, ranging from 52 to 67, it's called hyperdiploidy and is usually a sign that the patient might have a better chance of recovery. On the other hand, having fewer chromosomes than normal, less than 46, which is referred to as hypodiploidy, often indicates a tougher battle ahead with a poorer outlook. Our research focuses on a rare form of B-ALL where patients have both types of these abnormal cells at the same time—both the "extra chromosome" cells and the "fewer chromosome" cells. This situation provides a unique window into how different cancerous cells compete or cooperate within the same body, which can teach us a lot about how the disease progresses, how it might resist treatment, and why it sometimes comes back after treatment. Understanding the mix of these cancer cells in detail requires a deep dive into their genetic makeup, which is what we aim to do. By identifying the specific genetic changes that drive the growth and evolution of these cancer cells, we can learn more about how to fight them. To study this, we use a mouse model that can host human leukemia cells, allowing us to observe how the disease evolves in a living organism. This method will give us important insights into the unique biology of these rare mixed cases, potentially leading to better ways to treat and manage children's leukemia in the future. In simpler terms, we are exploring the genetic landscape of a unique and rare form of children's leukemia to uncover new knowledge about the disease that could lead to improved treatments. By studying how different cancer cells interact in the body, we hope to find better ways to tackle leukemia and give patients a brighter future.

Functional Biomarker Discovery in Paediatric Cancer: A Pathway to Personalised Therapy

Many childhood cancers have good outcomes, but patients with tumours that progress or relapse, like those  with neuroblastoma (NB) and rhabdomyosarcoma (RMS), often face poor outcomes due to limited  effective treatments. Understanding the biology of treatment resistance is crucial to improve risk  predictions and treatment options. In the meantime, these children need more individualized treatment  approaches to increase their chances of a cure. 

Recent evidence shows that paediatric cancer cells can adapt and survive treatment by changing their  behaviour through a process called cellular plasticity, without altering their DNA. Our lab has  developed molecular methods to study plasticity in individual cells over time, leading to an unprecedented understanding of plasticity and how it is a key driver of treatment resistance and  relapse in childhood cancer.  

We think translating plasticity knowledge into the clinic will help predict which patients are at risk of relapse  and develop innovative treatment options. To this end, we will aim to identify plasticity biomarkers in  early diagnostic/biopsies and metastatic/relapsed samples of clinically annotated paediatric cancer  patient samples using high-resolution single-cell technologies that probe the non-genetic space (the  epigenome). This will identify plasticity in patient’s samples and provide proof-of-evidence of their  predictive capability which could be further explored in larger cohorts. 

Through this proof-of-feasibility/principle study, we hope to develop a new methodology to tailor  treatment to each individual patient that could be tested in subsequent clinical trials.  
 

Identifying more efficient therapeutic targets for paediatric AML using CITE-seq

Acute myeloid leukaemia (AML) is an aggressive cancer of the blood system and is the second most common type of paediatric leukaemia. Despite a five-year survival rate of around 44%, treatment options are scarce and overall survival has shown modest gains with the increase of treatment intensity. In addition, cancer cells can evade or don’t respond to treatment, accounting for disease relapse in over 50% of patients (1). Besides new AML treatments largely relying on adult patient studies that might not translate to paediatric AML, recent advances have been only achieved by intensifying standard chemotherapy, increasing treatment-related toxicity and off-target effects (2). Thus, demonstrating the need for new targeted therapies. Cancer research has focused on understanding how the genetic elements (those encoded in our DNA) are
regulated by internal and external factors, such as hereditary factors and tobacco, respectively (3). However, there is a large gap in our fundamental knowledge of the most "druggable" targets in cancer, the proteins - molecules carrying the majority of cellular functions. Our most recent studies on protein biology identified a specific target that kills cancer cells while protecting healthy cells in adult AML, specifically, we targeted leukaemic stem cells (LSCs), the entities responsible for disease initiation and relapse, while protecting healthy stem cells, and offering a new therapeutic target with potentially lower treatment burden (4). Thus, this study proposes to characterise the genetic messenger and protein landscape of paediatric AML using novel state-of-art technology (CITE-seq), with the aim of improving disease prognosis by unravelling LSC evasion mechanisms and identification of therapeutic targets with reduced toxicity. This work will develop new strategies to study development and evasion of blood cancers, provide a platform of annotated LSC populations to the paediatric AML research community and allow our group to apply for larger grants in order to continue studying new targets that could offer more effective therapies for paediatric AML.

 Investigating the consequences of CEBP dysregulation in acute lymphoblastic leukaemia. 

Our genetic code is like a recipe book, we need instructions and ingredients to produce the cells within our body. The ingredients are called genes, and in healthy cells, enhancers provide the instructions that say which genes are to be switched on and when. In patients with a type of blood cancer called acute lymphoblastic leukaemia, some enhancers move location, switching on the wrong gene. These events are linked to a poor response to current treatment. When certain genes are switched on, they create proteins that bind to DNA. This binding can affect other genes, starting a “domino effect”. This contributes to cancer development. To understand how these proteins help the cancer cells become fitter than healthy cells, we need to learn which genes the proteins affect and whether this switches them on or off. Using this knowledge, we can test available drugs that might stop the “domino effect” in its tracks. If the drugs are effective, we should be able to slow the growth of the cancer cells and maybe even kill them. By finding specific treatments that only target cancer cells we can aim to reduce side-effects of treatment and improve the lives of those affected by cancer.

Near Infra-red fluorescence in Wilms tumour organoids

The day of surgery for removal of the cancer is one of the most stressful for children and families. This innovative project aims to improve Wilms tumour (WT) surgery and make that surgery easier, better, and safer. During surgery, the surgeon cannot see all the areas of disease and cancer cells may be left behind. Extra tissue may be taken to ensure all the tumour has been removed, but this can be unnecessary. Our team of world experts in fluorescent chemistry, surgery, organoids and genomics research will develop a dye that makes tumour cells glow so that during the operation surgeons can see exactly where the tumour is. 
Initially we will take samples from WT removed at Birmingham Children's Hospital and grow these in the lab into a mini-tumour (organoid). Then Indocyanine Green (ICG) and triazole-N-cyanine (TNC) dyes will be tested to see which ones the tumour will take up. WT does not readily take up ICG, so at best it may only show normal kidney instead of tumour. TNC glows like ICG but is brighter and travels within cells. This project aims to develop TNC into a WT specific dye so that the surgeon can see where the cancer cells are during surgery. Mr Pachl is uniquely positioned to implement the outcomes of this study at a regional, national and international level. Additionally his position as a consultant oncology surgeon at Birmingham Childrens Hospital will facilitate effective transition of this research from the lab directly to patients.
 

Overcoming tumour extracellular matrix barriers to effective CAR-T cell therapy for neuroblastoma.

Immunotherapy is a new approach to treat cancer that promises prolonged survival and in some cases may be curative. However many solid cancers like neuroblastoma tend to have low rates of response to immunotherapy, which we hypothesise results from a remodelling of the tissue structure into dense fibres of proteins that shield the tumour. In this project we propose to make modifications to the tumour tissue structure that we hypothesise will break down the tumours shield and therefore become more responsive to therapy. We will test this using a decellularized neuroblastoma model which maintains the tumour tissue architecture. Within this model we will test a cell-therapy called CAR-T therapy, where an immune cell called a T-cell is engineered to recognise and kill neuroblastoma cells. We will analyse the composition of neuroblastoma tissue and identify components that are inhibiting anti-tumour immunity. We will then engineer an ‘armed’ CAR-T cell, that is capable of degrading components of the tissue as an approach to improve CAR-T cell efficacy.

Targeting neurodevelopmental pathways in paediatric high-grade gliomas

Paediatric high-grade gliomas are fast-growing brain tumours that occur in children and adolescents. Unfortunately, no effective therapies are currently available for these patients and surgery is rarely possible because the tumour cells are intermingled with important healthy cells of the brain. In this project, we will use a combination of patient tissue samples and lab-grown tumours to investigate how malignant cells invade
healthy brain areas, and whether preventing this ability can alleviate tumour-related symptoms. Ultimately, we hope that the project will inform the development of new therapies that can effectively halt tumour growth without damaging healthy brain cells or any other part of the body.

The burden of rare pathogenic germline variants in neuroblastoma

Our team is on a mission to understand why some children are more likely to develop a type of cancer called neuroblastoma. Just like every person has unique features like eye color and height, we all have a unique set of genes that can affect our health. Sometimes, changes in our genes can make us more susceptible to certain diseases, including cancers. We are using a technique called whole exome sequencing that allows us to analyze all genes in one experiment. By comparing the genes of children with neuroblastoma to those of healthy individuals, we aim to uncover the hidden changes that may increase the risk of developing this disease. Our goal is to use this knowledge to guide families on how to better monitor and protect their children's health, potentially leading to earlier detection and more effective treatments for neuroblastoma.

The role of CaMK1D in the formation of the tumour microenvironment in paediatric diffuse large B cell
lymphomas

Lymphomas are types of blood cancer which affect children and are treated with multiple chemotherapy drugs in combination. Despite high chances of survival for many children, 10-15% will not respond to current treatments and are incurable. For children who are cured, the current treatments have both immediate and long-term side effects, which can be life-changing. There is an increasing need for new treatments which are more targeted, thereby reducing these side effects without reducing survival rates. In addition to cancerous cells various other cells surrounding tumour nest can play a critical role in the development and progression of lymphomas. However, the composition and general organisation of tissue which surrounds the tumour nest in lymphomas in children is currently unknown. In the proposed work we would like to address this by identifying different cells using specific molecular markers. We will also employ an advanced protocol to create a visual map of how different cell types are positioned relative to one another in cancerous tissues. Finally, we will examine how the abundant presence of a protein called CaMK1D affects the cellular architecture of the cancerous tissues.

The role of miR-93 in the survival of MLL-AF4+ acute lymphoblastic leukaemia blasts in the central nervous system niche

Leukaemia, a type of blood cancer, is the most common childhood cancer. Most children with leukaemia are cured. However, this is not the case for children who develop leukaemia under the age of one year. For these patients, those with infant leukaemia, the outcomes are much worse and, with current treatments, more than half will not survive. There are many features that are unique to infant leukaemia, which makes this disease hard to treat. One of these is the involvement of the central nervous system or CNS. CNS involvement refers to the presence of leukaemia in the structures that surround the brain and spinal cord. This is particularly common in patients with infant leukaemia and is often a site where leukaemia returns during or following treatment. By understanding how leukaemia cells survive and grow in this environment, we may be able to predict which children are at risk of leukaemia relapse and design better treatments.
Cancer cells often increase the activation of cell signalling pathways that promote growth and cell replication in order to continue to grow in an uninhibited manner. We have shown how one of these pathways, the PI3K pathway, appears to be more activated in leukaemia cells in the CNS environment using a mouse model of infant leukaemia. Our study implicates a possible regulator of this pathway, known as miR-93, in this process. When we reduced miR-93 levels, leukaemia cells were less able to survive in the CNS. To understand whether these differences could be important in patients, we aim to test patient leukaemia cells from the bone marrow and CNS for differences in miR-93 levels. If we can show that in patient samples, as in our mouse model, leukaemia cells from the CNS increase miR-93 levels, we can develop strategies to reduce miR-93 levels or reduce the effects of miR-93 as new treatments. 
 

Towards Understanding how Aberrant RAG Recombinase Activity Contributes to Relapse of Paediatric ALL

We recently discovered a completely new way in which errors in the production of antibody genes lead to leukaemia. Here, we will perform experiments to determine if inhibiting this reaction may slow progression to relapse.
The immune system needs to generate millions of antibodies every day to fight a vast number of potential infections. To create the huge number of different antibody genes required, gene segments are taken from one pool and mixed and matched with gene segments from a different pool by breaking and rejoining DNA.
We discovered a new way in which mistakes in this process can lead to the development of childhood leukaemia. This involves the piece of DNA that is “kicked out” of the genome every time a new antibody gene is produced. This piece of DNA can associate with recombinase enzymes that cut DNA to cause DNA breaks/mutations in a reaction we named “cut-and-run”. Crucially, we found that the breaks caused by cut-and-run correlate strongly with many of the mutations that are found in childhood leukaemias. Here, we will test the extent to which cut-and-run generates the mutations that lead to relapse.
Although five-year survival for many childhood leukaemias is very good, for children who relapse, the survival rate remains poor. Errors by the recombinase enzymes account for many of the mutations in leukaemias but the recombinase enzymes can trigger mutations in a number of different ways. Some cannot be inhibited but we believe that there are ways in which the cut-and-run reaction could be inhibited. Therefore, by determining the extent to which cut-and-run mutations are associated with progression to relapse, we can determine if more targeted treatments that prevent cut-and-run could slow or even block progression to relapse.

Understanding the Genetic Causes of Wilms Tumour

All humans have naturally occurring variation in their DNA. Most variants do not affect health but some can. Children that are born with a faulty variants in an anti-cancer gene are more likely to develop cancer in childhood. Our team compared variants between children who developed Wilms Tumor (WT) which the most common childhood kidney cancer, with people who did not have WT. We found genetic variants which are overrepresented in WT patients, and are now trying to understand how these variants can affect the expression of genes and potentially lead to cancer. We use next generation sequencing to read the DNA and study its epigenetics – these are the modifications made to the DNA to change how active it is. For example, one variant we have reduces expression of one gene but the same variants may also control the expression of its neighbouring gene. We hope to identify exactly which genetic variant changes which gene, and how this gene expression change leads to cancer. Understanding this variant-cancer connection can help identify children who are likely to develop cancer, and can inform tumour diagnosis and treatment planning.

Unraveling genes and mechanisms underlying relapse of T-cell acute lymphoblastic leukemia 

Acute lymphoblastic leukemia (ALL) is the most frequent cancer in children. ALL is treated by multi-drug chemotherapy, which cause serious side effects. Chemotherapy is ineffective in ~15% of children, which is related to drug resistance and relapse (disease recurrence). Relapsed ALL is very difficult to treat and often fatal. The majority of relapses occur in patients with T-ALL subtype (T-cell acute lymphoblastic leukemia).

Genetic prognostic markers of the risk of relapse are still missing in T-ALL. Since ALL is the most frequent childhood cancer, it is of highest importance to expand the knowledge about the mechanisms of leukemia recurrence.

Accurate identification of patients at high-risk of relapse, successful relapse prevention and treatment are currently one of the most important challenges in paediatric oncology. There is an urgent need to identify reliable predictors of relapse and to develop novel therapeutic strategies to prevent the relapse. This requires an in depth understanding of the mechanisms of disease progression and recurrence.

We hypothesize that the mechanisms contributing to T-ALL relapse include: drug-resistance, cell stemness (related to biological plasticity, which enable the cells to ‘escape’ the anticancer effects of therapy), senescence-like phenotype induced by chemotherapy (related to increased survival potential after recovery from the senescence-like state), clonal heterogeneity (meaning high diversity among leukemic cells, which
make them prone to evolve towards relapse).

We aim to provide novel knowledge on the genes and biological processes which drive the survival advantage of T-ALL cells and their evolution from diagnosis to relapse. We will use two state of the art methods: 1/ single- cell sequencing of the transcriptome (scRNA-seq) of T-ALL cells obtained from patients’ samples at diagnosis and at relapse to investigate clonal heterogeneity and evolution, 2/ genome-wide dropout screen using CRISPR/Cas9 method to identify genes essential for leukemic cells to survive. We will select several genes and verify their importance for T-ALL cells – we will activate or inactivate the expression of these genes in T- ALL cell lines and investigate, how these changes impact the ability of leukemic cells to proliferate and survive. Finally, we will use bioinformatics approaches and publicly available data generated from T-ALL samples to verify, if the alterations of genes’ expression observed in our study, are in fact related to the occurrence of T- ALL relapse in other groups of patients.

The ultimate goal of the project is to unravel the mechanisms of T-ALL relapse and to pave the way towards precise identification of high risk patients, which will facilitate the determination of the best treatment options to prevent leukemia relapse and improve the survival of T-ALL patients.

Unveiling the epigenetic landscape and therapeutic implications of epigenetic modifiers in human early T-cell leukemia initiation and progression

T-cell acute lymphoblastic leukemia (T-ALL) is a type of blood cancer affecting both children and adults worldwide, with approximately 30 new cases per million children and 10 new cases per million adults diagnosed each year. While current treatments cure around 80% of children, only
about 40% of adults achieve the same outcome. Even for those who are cured, the chemotherapy involved can cause significant illness and lead to long-term health complications. In particular, a subtype known as early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has a relapse
rate of approximately 60-74% and a high rate of poor response to conventional treatments, highlighting the critical need to explore alternative strategies to improve patient outcomes.

In ETP-ALL, several cases involve mutations that inappropriately activate a gene called EZH2. This gene is particularly relevant in cancer, as it is known to help tumor cells evade chemotherapy and drive disease relapse. Dr. Giambra and collaborators have recently optimized new methods
to functionally characterize ETP-ALL at the single-cell level. Using these experimental approaches, they aim to identify new markers specifically expressed in leukemia cell subsets of ETP-ALL patients. The primary goals of this research are to: 1) improve understanding of the biological
mechanisms that sustain and advance these aggressive cancer cells, both before and after drug treatments, and 2) optimize a form of “targeted” therapy for ETP-ALL which, in combination with conventional chemotherapy, may be more effective in curing patients with T-cell leukemia. These findings will also have the potential to inform treatment strategies for other types of cancer.

Validating an immunophenotype of chemotherapy resistance in childhood B-ALL – towards tailored
treatment and improved outcomes

B-acute lymphoblastic leukaemia (B-ALL) is the most common cancer in children. Although current cure ratesare around 90% they are achieved through treatment with highly toxic chemotherapy drugs which have long-term, debilitating side-effects. Furthermore, the outlook for the 10% of children whose disease returns is dismal, with only half surviving a further 5 years or more.

This research is focussing on identifying markers of chemotherapy resistance that can be successfully added to the existing panel of markers used in leukaemia diagnostics and to test the ability of this expanded panel to identify resistant cells within the leukaemia. Multiple samples will be used to help refine the panel to the most informative markers.