Based on our current understanding, it is believed that the leukemic clones are perpetuated by a rare population of leukemia stem cells (LSC). These LSCs share many characteristics with normal hematopoietic stem cells (HSCs), such as phenotype, self-renewal potential, a quiescent cell cycle status and enhanced drug resistance. Whereas cytotoxic drugs eradicate actively cycling leukemic blasts, they often do not target LSCs embedded in the bone marrow niche. Recent studies indicate that the bone marrow stromal cells and the hematopoietic microenvironment in the bone marrow dictate stem cell quiescence, lack of motility and expression of anti-apoptotic proteins which leads to LSC survival and ultimately results in refractory leukemic disease or relapse. The disruption of specific LSC–stroma cell interactions might drive this leukemia-maintaining cell population into cell cycle and thus render them vulnerable to chemotherapy or targeted therapies.

In order to identify genes regulated upon leukemia–stroma cell interaction we performed global gene expression screens in stromal feeder layer cells and leukemic blasts co-cultured for different time periods. In comparison to mono-cultured blasts or normal HSCs, several differentially regulated genes such as receptors, ligands, extracellular matrix components or known proto-oncogenes were identified in the gene expression analysis of co-cultured leukemic blasts. We currently explore the role of several candidate genes and the associated signaling pathways. Promising target genes are further evaluated in vivo in murine leukemia models. In addition we are performing a comparative functional screen using a genome-scale CRISPR-Cas9 knockout library in leukemic blasts mono-cultured or co-cultured with stromal cells aiming to identify genes involved in stroma-mediated drug resistance.

Using a conditional Kras^G12D knock-in mouse model, we and others have demonstrated that expression of oncogenic Kras in hematopoietic progenitor cells causes an arrest at the DN2/3 stage during T-cell differentiation followed by the development of an aggressive T-cell lymphoblastic leukemia/lymphoma with long disease latency. Interestingly, 50% of analysed leukemia samples harboured Notch1-mutations, likely acquired during the Kras^G12D-mediated block in differentiation. These additional mutations may be acquired by replicative stress or increased reactive oxygen species (ROS) production. Alternatively, oncogenic RAS may directly affect DNA repair pathways, thereby causing misrepair and, due to concomitant resistance to apoptotic cell death, accumulation of genomic changes. In line with this hypothesis, recent studies indicated that DNA damage response and repair (DDR) can be disturbed within a defined malignant background. Using several in vitro and in vivo models, we analyse DNA repair pathways in KRAS^MUT and KRAS^WT cells. Upon induction of DNA double strand breaks, expression of oncogenic KRAS was associated with a shift to the preferential use of the error-prone alternative non-homologous end-joining (alt-NHEJ) pathway.

Interestingly, targeting components of this pathway significantly enhanced chemotherapy-induced apoptotic cell death. Of note, this effect was specific for leukemic cell harbouring KRAS-mutations. Currently, we systematically explore DNA damage repair pathways in the context of distinct oncogenes in several leukemia models and solid tumors.

Acute myeloid leukemia (AML) is characterized by aberrant signal transduction resulting in enhanced cell survival, proliferation and resistance to apoptotic cell death. Rewired signaling is caused by acquired genetic alterations and/or by overexpression/aberrant expression of signal transduction molecules. Whereas canonical signal transduction initiates transcription factor activation and target gene expression, recent reports have also shown profound effects on chromatin modification and regulation of DNA damage response and repair. Of note, disturbed signal transduction in cancer cells results in tumor specific alterations associated with aberrant self-renewal and stress response but also synthetic vulnerability. Against this background we aim to dissect the influence of defined alterations within signal transduction pathways on epigenetic modifications, DNA repair and resistance to genotoxic agents/small molecule inhibitors. To address this question, we take advantage of a broad spectrum of mouse models. In addition, we have created several isogenic cell culture models using siRNA mediated gene knockdown or CRISPR-Cas9-mediated knockout. Further, cancer cells will be treated with specific small molecule inhibitors targeting signal transduction proteins. The impact on DNA damage response and repair will be explored by applying qRT-based gene expression analyses and in vivo DNA repair assays. Based on these technologies we hope to identify novel treatment strategies combining inhibitors that target DNA damage repair pathways, epigenetic modifiers and signal transduction molecules.

Acute myeloid leukemia (AML) is characterized by increased proliferation, evasion of apoptotic stimuli and block of differentiation. Mutations in FMS-like tyrosine kinase 3 (FTL3) receptor such as the internal tandem duplicate (FLT3-ITD) are found in about 25% of de novo AML cases. This mutation causes constitutive active downstream signaling and allows cells to replicate at a high proliferation rate. The question we like to address is how cells maintain genomic integrity in such conditions.

Poly (ADP-ribose) polymerase (PARP) is a family of proteins that are involved in various genome maintenance processes and play a major role in correcting single strand breaks (SSB). Perturbing highly proliferating cells by inhibiting PARP can induce genetic instability by increasing the number of double strand breaks (DSB). The recruitment of DSB repair pathway proteins to the site of damage can be quantified by an accumulation of γH2AX foci.  

In this project we aim at shedding light in mechanisms involved in maintaining genomic stability, exploring pathways regulating DSB repair and exploit mechanisms that prevent induction of apoptosis in highly proliferating AML cells.