BEAT IT projects

Epigenetic control of apoptosis and genome integrity in B cell transformation

Verena Labi's Lab (Medical University of Innsbruck)

Context: Aberrant epigenetic remodeling poses a risk for blood cancer. The epigenetic TET enzymes (TET1-3) oxidize 5-methylcytosine to 5-hydroxymethylcytosine, an intermediate in DNA demethylation and a stable epigenetic mark. Thus, TET enzymes facilitate DNA demethylation and maintain chromatin accessibility. Work from our lab had shown that TET’s settle B lymphocyte identity. TET2 loss-of-function (LOF) in humans, in the presence or absence of somatic Tet2 mutations inherited from aberrant hematopoietic stem cells, is prevalent in the preneoplastic condition of clonal hematopoiesis and blood cancers. Despite our understanding of TET2 LOF as founder hit and accelerator of oncogene-driven blood cancer, the mechanisms by which TET2 LOF fosters the formation and survival of (pre)malignant cells are incompletely understood.

Preliminary Work: Using mouse models and in vitro culture systems, we have established that TET2 loss boosts MYC-driven B cell transformation. Whereas premalignant cells present with increased DNA damage and enhcanced apoptosis, TET2 LOF tumors show increased expression of the pro-survival BCLX protein. However, it remains unclear whether and how TET2 LOF directs the rewiring of cell death networks during transformation.

Significance: Both, overexpression of the MYC oncoprotein and reduced TET activity are hallmarks of leukemias and lymphomas. This project seeks to explore how TET2 undermines MYC-driven transformation, and to define whether rewiring of BCL2-mediated apoptosis or other cell death networks is causally affected by TET2 LOF. Thus, we will integrate autochthonous mouse models, genomics, epigenomics and apoptosis profiling. These combined data sets should yield testable dependencies towards the end of the funding period which will be exploited in preclinical follow up studies. Beyond MYC-driven blood cancer, TET2 LOF in hematopoietic stem cells predisposes to all-cause mortality in humans. Thus, identifying targetable vulnerabilities of TET2 LOF will serve healthy aging.

Approach: Our objective is to understand how epigenetic perturbation by TET2 LOF reprograms B cells using (i) B cells in a healthy state, (ii) TET2 deficient B cells (perturbed state), (iii) TET2 deficient B cells expressing MYC or BCL2 (premalignant state), and (iv) TET2 deficient MYC or BCL2-driven lymphoma B cells (malignant state).

Minority MOMP as a driver of B cell mutagenesis and transformation

Joel Riley's Lab (Medical University of Innsbruck)

Context: Despite the advances in the treatment of many forms of cancer, options for patients with leukaemia and lymphoma remain limited. Most cancer therapies work by inducing cell death, specifically apoptosis, in the cancer cell, resulting in tumour regression. The major pathway of apoptosis in cancer cell death is via the mitochondria, where mitochondrial outer membrane permeabilisation (MOMP) results in the release of pro-apoptogenic factors into the cytoplasm, rapidly activating caspases and committing the cell to die. For years the prevailing dogma was that all mitochondria in a cell undergo MOMP, however in recent years this has been challenged. It is not understood that when a cell encounters a sub-lethal stress a small number of mitochondria in a cell can permeabilise, resulting in a small amount of caspase activity, insufficient to kill the cell, but sufficient to cause CAD-dependent DNA damage, genomic instability, and ultimately drive transformation. However, whether minority MOMP contributes to cancer development in B cell lymphomas remains unclear.

Preliminary Work: Our previous work has shown that cells treated in vitro with a sub-lethal stress (for example, repeated treatment with low dose BH3 mimetics) can undergo transformation. Furthermore, cells treated with repeated sub-lethal doses of BH3 mimetics establish tumours more readily in mice compared to control cells. In the pro-B cell line Baf3, we can induce minority MOMP using these same sub-lethal conditions, confirming that cells of haematopoietic lineages can also undergo minority MOMP. However, whether B cells undergo minority MOMP in vivo, or in response to therapy, remains unclear. Furthermore, the consequences of minority MOMP in cells experiencing oncogenic stress (such as MYC overexpression) has not yet been explored.

Significance: This project seeks to explore how minority MOMP contributes to cancer development ab initio, and whether therapy can induce minority MOMP, potentially explaining why patients relapse after anti-cancer treatment. Combining advanced sequencing technologies, we will gain an overall picture of the genomic, karyotypic, epigenetic and mutational landscapes of B cell lymphomas as they develop over time. Armed with this data, we will begin to identify and validate targetable vulnerabilities in B cell cancers with the aim to improve current treatment offerings.

Approach: Using well-characterised models of B cell cancer (EµMYC, EµBCL2) combined with loss of CAD we will sequence (i) healthy B cells, (ii) perturbed CAD-deleted B cells, (iii) premalignant CAD-deleted B cells expressing MYC or BCL2, and (iv) malignant CAD-deleted MYC or BCL2-driven lymphoma B cells.

Cell death as a barrier against chromosomal instability and aneuploidy in B cells

Andreas Villunger's Lab (Medical University of Innsbruck)

Context: Given the noted deleterious effects of genomic instability on the fitness of normal cells it has been a matter of debate if chromosomal instability (CIN) and aneuploidy can be causal for malignant transformation 1, 2. Clearly, cells experiencing CIN and aneuploidy are under high selective pressure and are often unfit to thrive until eventually sampling an aberrant karyotype which benefits survival and is genomically stable 1, 2. Therefore, the aneuploidy seen in blood cancer or other malignancies may be secondary to transformation or genomic changes that promote aneuploidy tolerance, best documented for p53 deficiency 3. Consistently, forcing aneuploidy by centrosome accumulation was shown to suffice to promote spontaneous malignancies, including lymphomas that showed impaired p53 function 4, 5. Similar results have been noted in response to a weakening of the spindle assembly checkpoint (SAC), which speeds up T cell lymphomagenesis, but only on a p53 compromized background 6. Regardless whether cause or consequence, what secures the survival of cells with aneuploid karyotypes under such conditions in relation to the BCL2 network or alternative cell death regulators remains to be uncovered.

Hypothesis: We hypothesize that CIN drives the selection of cell type dependent aneuploid karyotypes that create specific alterations in core components of the BCL2 network and mitochondrial priming to secure cell survival early during transformation. These adaptive BCL2 network changes will need to be readjusted during cancer progression or during therapy and will thus create distinct targetable vulnerabilities that can eventually be exploited, e.g., by the use of BH3 mimetics and drivers of CIN, such as microtubule targeting agents or inhibitors of mitotic kinases.

Approach: Within the framework of this team effort, we aim to understand the nature and underpinnings of BCL2 network adaptations and alterations in mitochondrial priming during CIN-driven transformation. We propose to tackle this challenge by following up an unbiased CRISPR/Cas9 based screen used for hypothesis formulation and studying a well-defined reductionist preclinical model of oncogene-driven B cell lymphomagenesis in combination with a genetic driver of CIN in a multi-omics approach.

Significance: Defining adaptive changes in cell survival networks and priming states in cells facing chronic CIN and ultimately presenting stable aneuploidies will help to define new opportunities for the rational design of early detection and treatment strategies preventing blood cancer formation or progression.

Cell death network rewiring during transformation

Francesca Finotello's Lab (University of Innsbruck)

Research Context: High-throughput sequencing technologies can generate multi-omics data across different molecular layers, such as the transcriptome, genome, exome, and epigenome of cells. Unsupervised bioinformatic methods like Multi-Omics Factor Analysis (MOFA) can integrate this multimodal data and efficiently disentangle the major axes of variations that underlie complex biological processes. However, these methods cannot incorporate prior knowledge on the relationships between the various molecular components that orchestrate complex biological processes like malignant transformation.

Rationale and Aim: Molecular perturbations that lead to malignant transformation have a broad impact on the genome, transcriptome, and epigenome of the affected cells. The profound rewiring of these different but interconnected layers can be only marginally captured via the analysis of single data modalities. We aim at developing a computational framework for the integrative analysis of mouse multi-omic data that reconstructs interpretable and dynamic cell interactomes. By applying this approach to multi-omics data generated in lymphomagenesis mouse models by our consortium, we aim at characterizing genome-wide network rewiring during malignant B cell transformation.

Own Work: We have already devised a strategy to integrate prior knowledge on cell regulatory interactions (e.g. between transcription factors and their target genes). By appliying this apporach to a compendium of transcriptomics data from human lung tumors, we could identify an interpretable multimodal signature of transcription factors, pathways, and cell types associated with anticancer immune responses. These preliminary results underline the capability of our prior-knowledge-informed approach of extracting interpretable, multimodal information from RNAseq data describing the major axes of variation and their underlying mechanisms.

Approach: Building upon these promising results, we will apply this approach to mouse multimodal data and further extend it to integrate annotations on molecular interactions to reconstruct context-specific regulatory networks. This novel method will be key to reconstruct the multi-omic rewiring underlying B cell transformation in mice, and to identify sub-networks that are shared or peculiar to the tested perturbations, holding great potential to pinpoint actionable targets for cancer prevention and therapy.