Identification of PIDDosome activators as anti-cancer drug candidates

Genomic instability describes the tendency of cancer cells to alter the number of chromosomes and hence their genetic information. Genomic instability is fostering a phenomenon termed aneuploidy where the genetic information differs significantly form that of a healthy cell. Aneuploidy is a well-established hallmark of many cancers, such as those developing in the breast or lung and usually correlates with poor prognosis, more aggressive disease and poor treatment response. The molecular basis of genomic instability and aneuploidy is multifactorial but is usually linked to erroneous cell divisions, termed mitoses. Mitosis usually ensures that the duplicated genetic information is faithfully segregated to the two daughter cells arising during normal cell division. An essential component regulating this event is the so-called centrosome, a multi-protein structure that generates the poles of the mitotic spindle, is a filamentous structure containing tubulin-fibres and other proteins that orchestrates the exact separation of chromosomes during cell division among daughter cells. An accumulation of centrosomes, however, that can be triggered by various means can cause errors in chromosome segregation by the formation of multi-polar spindles, causing chromosomal instability leading to aneuploidy in following cell divisions. To avoid this problem, the cell has developed different safety mechanisms. One of them is the activation of p53, a key tumor suppressor, inactivated in more than 50% of all human cancers. How p53 is activated downstream of extra centrosomes is unclear but unravelling this at the molecular level should allow the development of novel anticancer therapeutics. Our own studies show for the very first time that the PIDDosome multi-protein complex is essential and sufficient to activate p53 under these conditions. Hence we propose to (i) identify the signals that connect extra centrosomes to PIDD and to explore (ii) the consequences of PIDDosome activation in preclinical models to validate the therapeutic potential and feasibility of such a strategy for anticancer therapy.

The PIDDosome is a multi-protein complex implicated in the activation of a cysteine-directed protease belonging to the caspase-family, i.e., Caspase-2. This complex comprises the p53- induced protein with a death domain (PIDD1), a bipartite adapter, RIP-associated ICH1/CED3-homologous protein with death domain (RAIDD) and pro-caspase-2 that, upon assembly, facilitates conversion of the pro-enzyme into its active form. Caspase-2 itself has been implicated in a series of biological responses, including ageing, the ER-stress response, inflammasome activation, autophagy, DNA damage and mitotic catastrophe, the latter referring to a set of tumor-suppressive cellular responses triggered by error-prone mitosis, including cell death and senescence, induced by largely undefined molecular cues. While our previous work has excluded a prominent role for Caspase-2 in DNA damage- induced apoptosis but simultaneously highlighted its role as tumor suppressor, live-cell imaging and biochemical analysis in our laboratory has recently revealed a key-role for the PIDDosome as regulator of cell fate upon tetraploidization (whole genome duplication, WGD). Mechanistically, we defined the increased centrosome number in tetraploid cells as a selective trigger of PIDDosome assembly. This starts a non-canonical p53 response via selective Caspase-2-mediated MDM2-cleavage, promoting either cellular senescence or cell death, depending on the cell type involved. While ordered tetraploidization or higher order WGD is critical for the terminal differentiation and function of selected cell types, including megakaryocytes, cardiomyocytes or hepatocytes, unscheduled tetraploidization, e.g. upon failed cytokinesis, is considered a first step to chromosomal instability (CIN), leading to aneuploidy-driven genomic instability, a hallmark of cancer. Within this project, we proposed to (i) screen for regulators of PIDDosome assembly and effectors and (ii) tested the consequences of excessive activation of Caspase-2 on normal physiology and malignant disease. This approach has identified candidates that can lead to the activation of Caspase-2 in cancer cells. Moreover, we believe to now understand better the potential consequences of forced PIDDosome formation in vivo that will together guide us in our aim to develop not only activators, but, based on our recent findings, also selective PIDDosome and/or caspase-2 inhibitors. This aim will be pushed further in collaboration with scientists at CeMM in Vienna, where we started to screen a 90K compound library as well as to generate bivalent small molecules that will promote the degradation of PIDDosome components for its inactivation. While activators may act a potential therapeutics in cancer, inhibitors are believed to find application