LEUKEMOGENESISAPL · An in-vivo Screen for the Identification of Leukemia-Promoting Factors

An in-vivo Screen for the Identification of Leukemia-Promoting Factors

Final Report on Project “LeukemogenesisAPL“ In our project, we have investigated very early events in leukemogenesis in the “model disease” Acute Promyelocytic Leukemia (APL). APL is caused by a leukemia-initiating fusion protein, called PML-RAR. We found that this protein disrupts an important anti-tumor mechanism known as “Cellular Senescence”. “Cellular senescence” (“cell aging”) is an inborn mechanism that enables cells to undergo systemic changes leading to irreversible inactivation. It is generally thought to represent a first-line defense against cancerous changes. We also found that in APL cells, cellular senescence involves the assembly of a protein complex that regulates the organization of cellular DNA, which is known to occur in various ways and determines the activity of the genes located in a particular stretch of DNA. DNA is packaged by a set of small proteins (“histones”) bound to it. Protein-packaged DNA is referred to as “Chromatin”. Loose packaging is typical for active genes, whereas inactive (“repressed”) genes are more tightly packaged into the structure known as heterochromatin. The activation/repression state of genes by means of chromatin compaction on a scale of the entire cellular genome is often referred to as “chromatin landscape”. • We have found that cellular senescence involves the activity of a protein complex, the PML/Daxx/ATRX complex (termed PAX-complex) that regulates this chromatin landscape by incorporating a variant of the packaging proteins, the histone variant “H3.3”. It becomes incorporated into chromatin by the PAX complex, which forms at the nuclear domains defined by the PML protein. The PML-RAR fusion protein disrupts these domains, preventing PAX assembly. This failure of assembly causes failure to enter senescence, possibly due to defects in H3.3 incorporation into the chromatin, which primes the cells for irreversible inactivation. Cells with inactivated senescence will be highly sensitive towards further changes promoting leukemogenesis and tumorigenesis in general. • We have developed an ex-vivo method to study and quantify the activity of the PML-RAR protein and other oncogenic factors. This laboratory tool can minimize the number of animal experiments. • We have also found, that the activity of the PML part of PML-RAR needs to be analyzed in a same-species context for its biological effects. This observation underlines that caution should be applied, when studying human cancer-inducing proteins in the mouse as a model organism. IMPACT: • As the PAX complex is endowed with enzymatic activity (ATPase), it is a potential target for the development of novel anticancer therapies, which work through induction of senescence, without the need of genotoxic drugs. • Our ex vivo assay has the potential to reduce animal experiments in leukemia research. TARGET GROUP(S): • Scientific Researchers, • Biomedical community, • pharmaceutical industry • animal experimentation decision makers SUMMARY AND MODEL: Left: Disruption of the PAX-complex (staining: Daxx, green) by expression of PML-RAR (staining: RAR, red). White arrows mark the physiological PAX complex in non-PMLRAR-expressing cells. Nuclei are stained in blue. The lower (PML-RAR-positive) cell shows no Daxx foci, but a dispersed (“disrupted”) Daxx-staining Right: Model: PML-RAR prevents the shift of H3.3 towards heterochromatin, which is necessary for establishment of Cellular Senescence.

Data: CORDIS, © European Union