Cell death control on extended mitotic arrest

The BCL2 family determines the commitment of cells to apoptosis and is frequently de-regulated in malignant disease. Overexpression of BCL2, or its siblings, such as MCL1, can facilitate cancer initiation and progression while hindering the efficacy of cytotoxic chemotherapy. This sparked the interest for the development of BCL2 inhibitors that recently recorded promising results in pre-clinical and clinical trials. Meanwhile, cytotoxic drugs targeting the microtubule cytoskeleton such as taxanes and vinca alkaloids remain essential therapeutic ingredients to combat a variety of solid and hematologic malignancies, despite their problematic side effects, demanding improvement of therapeutic regimens. Microtubule targeting agents (MTAs) act by disturbing the normal mitotic cell division and thereby triggering apoptosis by rather poorly understood mechanisms. MCL1 was shown to undergo degradation in the course of mitotic arrest and this recently qualified as the decisive event initiating apoptosis upon MTA treatment. To this end we recently identified the BCL2 family member, NOXA, as a critical factor required for mitotic MCL1 degradation and cell death. Whereas the NOXA-dependent MCL1 degradation seems to be a ubiquitous phenomenon in cancer cell lines, the underlying mechanism and its relevance for the action of antimitotic drugs and its potential as a predictive marker for therapy responses in cancer treatment remain to be established. Here we propose a project aiming to define the mechanisms used by NOXA to trigger MCL1 degradation upon mitotic arrest and to assess the potential of these proteins to be used as biomarkers for therapy responses to MTA treatment in breast cancer, in order to define more effective targeted therapies to improve patient care. To this end, we plan to use a panel of breast cancer cell lines to assess the importance of the NOXA- MCL1 axis in defining their susceptibility to antimitotic chemotherapy, alone, or in combination with novel BCL2 inhibitors (BH3-mimetics), that are already well advanced in clinical trials. Furthermore, we willengineer candidate breast cancer cell lines by disrupting the endogenous NOXA locus for genetic complementation assays and use those cell lines to assess the importance and the potential of NOXA- modulation for cancer onset, progression and treatment in vitro and in vivo. Finally, bioinformatics analyses of available expression data in cancer genome databases and independent mRNA as well as protein expression analysis in primary patient samples with clinical follow-up will be harnessed to assess the potential biomarker value of the NOXA- MCL1 axis, or its regulators, for the response of patients to MTA-based therapy. Taken together, the completion of this project will advance our understanding of how anti-mitotic drugs trigger cancer cell death and synergize with BCL2 inhibitors that will help in turn to design superior patient-tailored combination therapies and define novel predictive tools to more precisely forecast treatment success in breast cancer.

Microtubule targeting agents (MTAs), such as the natural compound, Taxol, and its dervatives, act by disturbing normal cancer cell division and thereby trigger cell death. How MTA treatment elicits cell death in cancer cells, most notably breast cancer, remains rather poorly understood. Certain cell death-inhibitory proteins belonging the BCL2 family were shown to be depleted when cells are treated with MTAs. To this end we previously identified the pro-apoptotic protein, NOXA, along with BIM, as critical factors required for cancer cell death induced by microtubule poisons. NOXA can bind and neutralize the cell death inhibitory molecule, MCL1, and we contributed to the understanding how this process in regulated in different cancer cell lines. Degradation of MCL1 protein, along with the inhibition of related anti-apoptotic proteins, such as BCLX, seems to be an ubiquitous phenomenon in cancer cell lines preceding cancer cell death. The underlying molecular mechanisms and their relevance for the action of microtubule targeting agents, as well as their predictive value for therapy responses in cancer treatment remained to be established at the time this project was initiated. Here, we defined a novel mechanism of mitotic cell death initiation where NOXA is needed to eliminate anti-apoptotic MCL1 in response to treatment with microtubule targeting agents. Direct interaction of these molecules was defined to be key for their joint destruction during cell cycle arrest. This process involved a class of molecules that tag others for degradation in a cellular unit that disassembles proteins, called the proteasome. For this event to happen, a third molecule, MARCH5, that adds a molecular tag to MCL1 and NOXA, making them visible to be destroyed together. This finding points out a way how cancer cells can be rendered more sensitive to microtubule targeting agents, as inhibition of MARCH5 increased cell death susceptibility of cancer cells. Moreover, we were able to show that NOXA is rate limiting for the response of a hard to treat subset of breast cancer cells, referred to as triple negative, to microtubule targeting agents and that these agents synergize best with a novel class of molecules that block the pro-survival function of BCLX, while inhibitors of BCL2, already in clinical use, proved less effective. Finally, our analysis of primary breast cancer patient samples and bioinformatics analyses showed that NOXA expression levels have predictive value with high levels correlating with improved overall survival and superior response to microtubule targeting agents. Taken together, the completion of this project was designed to advance our understanding of how anti-mitotic drugs trigger cancer cell death and synergize with BCL2 inhibitors to help design superior patient-tailored combination therapies. Our study should help to define novel predictive tools to more precisely forecast treatment success in breast cancer patients.