Non-genetic heterogeneity in response to anti-mitotic chemotherapeutics

The "anti-mitotics" are a class of drugs originally isolated from natural sources that are used frequently during cancer chemotherapy. The name of this class derives from the fact that they all block mitosis, the process of cell division, in turn leading to cell death. A leading example is paclitaxel (Taxol) originally isolated from the pacific yew tree and widely used to treat breast and ovarian cancer.

These traditional chemotherapy drugs give even greater patient benefit when used in combination with modern therapies; for example, simultaneous use of anti-mitotics with Herceptin (trastuzumab) has very quickly become the preferred treatment following surgical removal of an invasive breast cancer. This combination appears to be very effective at killing the micrometastases left behind following surgery that would otherwise lead to reappearance of the cancer. Consequently, the anti-mitotics will continue to be important chemotherapy drugs for the foreseeable future.

These drugs are however far from perfect. One problem is that they have a rather unique side affect, peripheral neuropathy, which can lead to loss of sensation in the fingers and toes. If an individual aims to "beat' their cancer, with the hope of many more good-quality years, this poses a very real dilemma; when this side affect appears, the immediate reaction of the oncology team is reduce the dose, but will that reduce the effectiveness of the treatment? Sometimes, patients even opt out of anti-mitotics specifically to avoid this side effect. Therefore, finding new drugs that offer similar benefit but with reduced side affects is a worthwhile cause. Indeed, extensive R&D efforts within academia, biotech and the pharmaceutical industry, efforts approximating $10 billion, has resulted in an extensive array of 2nd generation anti-mitotic drugs, for example, inhibitors of the Eg5 motor protein and Aurora/Plk kinase inhibitors.

Unlike the original, naturally occurring anti-mitotics, these new drugs have been synthesized to inhibit mitosis-specific proteins so that they kill divding cells without harming non-dividing nerve cells. Many of these drugs are excellent; they inhibit the proteins they were designed to with great potency and specificity, and many are well tolerated when administered to patients. And importantly, the peripheral neuropathies associated with traditional anti-mitotic drugs do not seem to occur. However, the anti-cancer effects of these new drugs has thus far been rather disappointing.

The reasons for this are unclear but it is important to note that these 2nd generation drugs have not yet been tested in clinical settings similar to those where the traditional anti-mitotics are known to be most effective. This is because it is difficult to test new drugs in settings where the current standard of care is already very good. Nevertheless, there is a very real danger that these excellent new drugs will be cast aside. This will of course be a real shame in terms of the extensive human endeavour and research funding that resulted in these drugs being created. Moreover, it may mean that we fail to realise an opportunity whereby peripheral neuropathies are no longer a complication when treating breast cancer.

One of the factors contributing to this state of affairs is that we actually don't know how anti-mitotic drugs kill cells. My lab recently discovered that cancer cells exposed to anti-mitotics can either undergo death in mitosis or they can "slip" out of mitosis and survive. Since then we have identified a gene that controls this difference: when we inhibit it, the balance is shifted in favour of slippage. The overall goal of this proposal is to follow up this discovery with the intention of seeing if we can tip the balance the other way, i.e. in favour of death. This may then open up new opportunities for combination strategies that sensitise cancer cells not only to traditional anti-mitotics, but also to the 2nd generation drugs.

Genetically identical daughter cells can exhibit different fates when exposed to anti-mitotic drugs. This non-genetic heterogeneity arises due to the stochasticity of biochemical reactions, generating cells with identical genomes but different proteomes. We have discovered that inhibiting the transcription factor c-Myc modulates this fate choice. Our hypothesis is that Myc regulates transcriptional subprograms that have downstream effects on protein networks that influence cell fate decisions operating in mitosis. Because Myc is itself dynamic, stochastic variation in Myc activity could contribute to heterogeneity in response to anti-mitotics. We have designed three work packages to test these hypotheses:

WORK PACKAGE #1. Using tried and tested approaches, we will probe Myc as a modulator of mitotic cell fate. We will use RNAi, dominant negatives, overexpression and small molecule inhibitors to alter Myc function, then use time-lapse microscopy to monitor mitosis and apoptosis in order to generate cell fate profiles measuring the frequency of "death in mitosis" vs. "slippage".

WORK PACKAGE #2. We will generate cell lines expressing Myc biosensors, e.g. using plasmids and/or BACs with reporters downstream of Myc target promoters. Stable cell lines harbouring the transgenes will be used for pedigree analyses; sister cells will be monitored using time-lapse fluorescence microscopy to measure endogenous Myc activity, challenged with anti-mitotic drug, and their fate determined.

WORK PACKAGE #3. The links between Myc and mitotic cell fate are largely unexplored, but we hypothesize that inhibition of Myc does not effect mitosis per se, rather the ability to apoptose while in mitosis. To test this, and to determine which aspect of apoptosis account for the fate shift, we will take two complementary approaches: a top-down descriptive approach, namely focused gene expression profiling, and a bottom-up functional approach, namely mitochondrial BH3 profiling.

SCIENTIFIC IMPACT. In addition to the Academic Beneficiaries outlined above, our results will also have implications for translational scientists in both academia and the pharma industry focusing on improving anti-mitotic chemotherapy drugs. In particular, inhibition of Myc delays mitotic cell death and promotes slippage. One possibility therefore is that by manipulating pathways downstream of Myc, it may be possible to design strategies that accelerate mitotic cell death and thereby sensitize cells to anti-mitotic drugs. Similarly, by gaining more insight into the mechanisms that determine relative sensitivity and resistance to anti-mitotic drugs, longer term outcomes of this research may allow clinicians to stratify patients into cohorts that are likely to benefit from the inclusion of anti-mitotic drugs in their chemotherapy regimens.

ECONOMIC AND SOCIETAL IMPACT. The potential of my research to impact in this manner is illustrated by our track record. Having established a world-leading reputation in the field of mitosis research, our expertise is frequently sought by pharmaceutical companies involved in cell cycle drug discovery, exemplified by prior collaborations with AstraZeneca, Millennium Pharmaceuticals and GSK. Our collaboration with AstraZeneca in particular had a significant impact on their Aurora inhibitor program, and resulted in the commercialization of ZM447439, which has become the de facto standard Aurora B inhibitor, used widely in the mitosis community as a research tool. More recently, we described an Mps1 inhibitor AZ3146 which is now also commercially available and is emerging as a very useful tool for the field. In order to continue to have impact in this way we need to maintain our position as a leader in the field of mitosis research.

PUBLIC ENGAGEMENT. Wider benefit will come from engaging with the wider public. Again, our potential is illustrated by our track record. I have developed a reputation for good public speaking and I consequently get invited to deliver public lectures and talk to lay audiences about our scientific progress. For example, in May 2009 I gave a talk to an audience of Manchester University Alumni and in February 2010 I was invited to deliver the annual Percival Lecture organised by the Manchester Litt and Phil Society. In March 2013, Dr Caroline Topham, the named PDRA, hosted a "lab tour" during International Women's Day. During the course of this project, I will continue to capitalise on similar opportunities as and when they arise.

RESEARCH CAREER DEVELOPMENT. The impact of my research also manifests through the career progression of young scientists who leave my lab with an excellent training in molecular cell biology. This is highlighted by four of my former PhD students securing post-doc positions in top US labs, with two also securing EMBO Long-term fellowships. One of these has now moved on to secure their own tenure-track academic Faculty post.