ApoptoMDS

Keeping “bad” cells alive to prevent leukemia Changes in apoptotic signalling are a major factor in the development of bone marrow failure, which can leave people susceptible to developing leukemia. Researchers in the ApoptoMDS project are exploring a new hypothesis around the progression of bone marrow failure to leukemia, as Dr Miriam Erlacher explains.

A certain proportion of cells in our bodies die every day through a process called apoptosis, which is the focus of a lot of attention in research, with scientists investigating a number of questions around the relationship between apoptotic signalling and specific diseases. This is a major area of interest to Dr Miriam Erlacher, a paediatrician at the Freiburg University Medical Centre, who is investigating different bloodborne diseases, including myelodysplastic syndromes (MDS), juvenile myelomonocytic leukemia (JMML) and acute leukemia. “MDS and acute leukemia can arise de novo from the bone marrow. They can also occur as a secondary event, in patients with inherited bone marrow failure syndromes,” she outlines.

“Fanconi anaemia, a type of bone marrow failure syndrome, causes problems with blood cell formation. Patients with Fanconi anaemia face two types of problems. Firstly, patients with bone marrow failure do not have enough cells. Then it also leaves people more susceptible to developing certain types

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of cancers and blood-borne diseases, such as MDS and leukemia. The relationship between these two types of problems is unclear.”

When Hannahan and Weinberg established their ‘hallmark of cancer’ model, they included cell death resistance as an essential factor for a tumour to emerge. “Typically, when a cell gets into a pre-malignant stage, the cell realises that it should die, and then it undergoes apoptosis,” outlines Dr Erlacher.

Cells need to survive such stress signals for a malignancy to develop, so a cancer cell would be apoptosis-resistant. “The conventional model says that cancer can only be avoided if all pre-malignant cells die,” says Dr Erlacher.

The team in the ApoptoMDS project are now exploring a very different hypothesis however, that apoptosis within tissue does not unambiguously prevent cancer formation, but rather can promote tumorigenesis. “It’s actually better to have many pre-malignant cells, as killing too many pre-malignant cells puts a high proliferative and selection pressure on the

remaining cells, which leads to further malignant progression when coupled to genetic instability,” explains Dr Erlacher.

Unexpected findings in a mouse model This hypothesis has its roots in an experimental mouse model developed earlier in Dr Erlacher’s career, in collaboration with Dr Andreas Villunger at the Medical University of Innsbruck, who supervised her PhD. “We wanted to understand whether apoptosis of DNA damaged cells was sufficient to prevent leukemogenesis induced by repeated cycles of irradiation. We hypothesized that inhibiting apoptosis by genetic deletion of the proapoptotic gene PUMA would lead to more rapid leukemia development. But surprisingly, PUMA deficient mice did not develop any T cell lymphomas, while all other mouse strains rapidly developed lymphomas,” she outlines.

“We could show that this was due to a better survival of cells, and as a consequence, reduced proliferation and selection stress.

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We concluded that it is better that damaged and maybe pre-malignant - cells survive. This reduces the necessity of proliferation and the selection pressure.”

Bone marrow failure model Since leaving her position in Innsbruck, Dr Erlacher has started working as a paediatric haematologist, a role in which she is investigating whether this hypothesis that has been described also holds true for some diseases in humans. While mice repeatedly subjected to irradiation develop T cells lymphoma, human patients treated with chemo- or radiotherapy are at risk of developing secondary MDS. “I realized that T cell lymphoma in mice and MDS induced by chemotherapy might have many things in common,” says Dr Erlacher. Due to the nature of her job as a paediatric haematologist and oncologist, Dr Erlacher and her team decided not to focus on treatment-related MDS, but on other, similar diseases: MDS and leukemia occurring secondary to bone marrow failure.

“We are now working to test whether the hypothesis holds true in such cases,” she says.

Researchers in the ApoptoMDS project are testing this hypothesis in a mouse model of bone marrow failure called dyskeratosis congenita, using genetic means to inhibit apoptosis in mice.

Dyskeratosis congenita differs from Fanconi anaemia. “Patients with dyskeratosis congenita have cells with very short telomeres. The telomeres get shorter and

shorter, and once they are critically short, the cells either die, or they stop proliferating and undergo senescence,” explains Dr Erlacher. “Cells from patients with Fanconi anaemia accumulate DNA damage, and as a consequence die or stop proliferating.”

This can eventually lead on to the development of leukemia, yet it is difficult to predict the rate of progression. The idea is that in an empty bone marrow, the surviving cells have to strongly proliferate to compensate for cell loss. “Under this situation, a malignant cell can arise and outgrowth can occur. So the very first step is to inhibit bone marrow failure,” says Dr Erlacher.

First of all, you would like to try and delay bone marrow failure, then as a consequence you expect that leukemia develops later. So it’s a kind of delaying mechanism.

The goal in research at this stage is not to completely eradicate bone marrow failure, but rather to extend the period over which individuals don’t experience any problems.

“First of all, you would like to try and delay bone marrow failure, then as a consequence you expect that leukemia develops later. So it’s a kind of delaying mechanism,” says Dr Erlacher. Fundamental research into apoptotic mechanisms is central to this wider goal; Dr Erlacher and her colleagues are comparing healthy cells with cells from mice with bone marrow failure, aiming to gain deeper

a point that Dr Erlacher plans to investigate, using samples collected from patients at the Freiburg University Medical Centre. “We have a lot of material, collected over the last twenty years or so. We can then compare the results from mouse models with human cells,” she says.

There are significant challenges around this work, in particular investigating the factors that might pre-dispose an individual patient to developing bone marrow failure and then MDS, which is an important part of the project’s agenda. While mice can be bred

MDS

First malignancy Repeated bone marrow injury by chemo - and/or radiotheraphy

Driver mutation

Damaged Stem Cell Bone marrow failure syndromes

insights. “We compare the cells in vivo, in the bone marrow of mice. And we compare the cells ex vivo, in vitro,” she continues.

The wider aim here is not just to measure rates of apoptosis in these mice, but to also inhibit it in bone marrow cells. From this, researchers can then look to assess whether inhibiting apoptosis helps to delay the development of bone marrow failure, and then leukemia later on. “We already have some evidence that bone marrow failure occurs later on, so the mice are much less sick and they do not die so quickly. But we do not know yet whether this inhibits leukemogenesis,” outlines Dr Erlacher. This is

Apoptosis

FANCONI ANEMIA: DNA damage

Further mutation

Premalignant Cell Proliferation pressure

MDR-AML

Clonal advantage/ differentiation block

AML Cells Mutation conferring Apoptosis resistance

DYSKERATOSIS CONGENITA: short telomeres

Cytopenia

Dysplasia + Cytopenia

[modified: How cell death shapes cancer, Labi and Erlacher, Cell Death Dis, 2015]

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ApoptoMDS Hematopoietic stem cell Apoptosis in bone marrow failure and MyeloDysplastic Syndromes: Friend or foe?

Project Objectives

Inherited bone marrow failure syndromes (e.g.

Fanconi anemia or dyskeratosis congenita) predispose to malignancies such as myelodysplastic syndroms and acute leukemia. It is postulated that increased susceptibility to apoptosis in hematopoietic stem and progenitor cells (HSPCs) contributes to hematopoietic failure. We hypothesize that HSPC apoptosis does not only lead to bone marrow aplasia but paves the way also for leukemogenesis. It is the aim of ApoptoMDS to identify deregulated apoptosis pathways. The team is testing whether inhibiting apoptosis is sufficient to improve HSPC function, mitigate bone marrow failure and prevent leukemia. This will open doors for novel therapeutic approaches to expand the less severe symptomatic period for affected patients.

Project Funding

Funding scheme: ERC-StG-2014 - ERC Starting Grant. Total cost: EUR 1 372 525 EU contribution: EUR 1 372 525

Contact Details

Project Coordinator, Dr. Miriam Erlacher, MD PhD Division of Pediatric Hematology and Oncology Department of Pediatrics and Adolescent Medicine Freiburg University Medical Center Mathildenstr. 1 79106 Freiburg T: +49 (0)761/270 43010 E: miriam.erlacher@uniklinik-freiburg.de W: https://www.uniklinik-freiburg.de/index. php?id=15941&L=1 Miriam Erlacher, MD PhD

Miriam Erlacher, MD PhD, is attending physician in the Division of Pediatric Hematology and Oncology, University Medical Center Freiburg. She is most interested in leukemia, bone marrow failure syndromes and other syndromes with predisposition to myeloid malignancies.

Her ApoptoMDS project brings together her research interests in apoptosis signaling and her clinical interests

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with a particular defect, allowing researchers to analyse them even before they get sick, it is more difficult to look at the very early, initial stages of disease in human patients.

“We usually get the first material when a patient actually gets sick. So it’s very difficult to analyse the first stages of disease,” explains Dr Erlacher. Researchers have been able to test children from families with a history of a particular genetic problem however, from which some new insights can be drawn.

“Sometimes we can analyse patients before they get sick,” says Dr Erlacher.

Leukemogenesis A second part of the project centres around studying the relationship between MDS and leukemia. Researchers aim to build a deeper understanding of how the disease develops, from which point scientists could then potentially look at intervening and developing new therapeutic approaches.

“The old-fashioned way of understanding leukemogenesis is that inhibiting apoptosis in pre-malignant cells will lead to rapid development of leukemia, because we prevent cells from dying when they get malignant. We hope that by inhibiting apoptosis, we can help delay the development of bone marrow failure and, later on, also leukemia,” says Dr Erlacher.

This also relates to patients with what is called treatment-induced MDS. “This is very similar to secondary MDS in patients with bonemarrow failure,” continues Dr Erlacher.

This is not a syndrome that patients have from birth, but rather it develops as a result of chemotherapy treatment. Many bone marrow cells die following chemotherapy, which can leave patients vulnerable. “When patients are treated for breast cancer, they undergo chemotherapy. In every cycle the bone marrow cells die, they undergo apoptosis. After every cycle the cells have to proliferate, and then they are again killed,”

explains Dr Erlacher. Preventing bone marrow cells from dying during treatment could help to delay or inhibit the development of treatment-induced MDS, believes Dr Erlacher.

“We should look to inhibit apoptosis. This is not caused by an actual cell defect, but rather apoptosis is caused by treatment, by the chemotherapeutics,” she says.

Inhibiting cell death The wider objective in this research is to demonstrate that inhibiting cell death in these syndromes can delay the development of myelosuppression and also prevent further progression on to leukemia. This could potentially open up new therapeutic approaches to prevent MDS, yet there are still some hurdles to overcome in these terms.

“The difficulty with translating this finding to clinics is that we would have to find a drug or other product that inhibits apoptosis.

However, currently companies tend to prioritise finding drugs to kill cells,” explains Dr Erlacher. “When we have such drugs, we would aim to target them at the bone marrow. We don’t want to keep all cells alive, we would want just to keep the bone marrow cells alive while the chemotherapy has to remain effective in treating the tumour cells.”

This could be an effective way of protecting the bone marrow cells, either from intrinsic defects in bone marrow failure syndromes or from the effects of chemotherapy, preventing the development of secondary MDS. A deep understanding of the fundamental mechanisms behind the progression of a disease is essential to the development of these types of drugs, and this is the priority in research for Dr Erlacher at this stage.

“The first step is to understand the diseases, and then you can look to see what can be translated,” she outlines. “I’m interested in translation, but I think we need robust basic research before we can consider translation.”

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