Role of FACT in normal development and cancer
The role of FACT in normal development and cancer
There more and more evidence demonstrating extensive reorganization of chromatin in cancer cells. Trying to identify factors, responsible for this reorganization, we established that increase in the expression and activity of histone chaperone FACT, is critical event in malignant transformation (Garcia H. et al, 2013). Based on literature FACT interacts with different components of nucleosomes to provide access of transcription, replication and DNA repair machineries to nucleosomal DNA, and at the same time to ensure nucleosome stability. With this quite basal function, FACT is surprisingly selectively expressed in mammals (Garcia H. et al, 2011). The highest level of FACT is observed during early embryonic development. Postnatal expression of both FACT subunits is limited to subpopulations of cells of lymphoid, reproductive and few other tissues. However their expression is reinstated in multiple types of tumors. Moreover, expression of FACT is essential for growth and survival of tumor, but not normal cells. Yet FACT is not mutated or amplified in tumors and its overexpression does not drive malignant transformation in the absence of oncogene.
Why stem and malignant tumor cells love to have a lot of FACT?
Embryonic stem cells (ESCs) and malignant tumor cells
are the highest FACT expressing cells, many folds higher
than any other cells in mammals. Moreover these two
categories of cells are extremely sensitive to FACT
inhibition, both types of cells die if FACT is depleted (Cao
S. et al, 2003, Garcia H. et al, 2013), while viability of
normal cells with lower expression of FACT is not
compromised. Tumor cells with moderate amount of
FACT slow down or stop proliferation in response to
FACT knockdown (Fleyshman D. etal, 2017). We are now
trying to understand what exactly FACT does in ESCs
and tumor cells. There are several possible scenarios
based on the known FACT activities observed in cell-free
systems and model organisms. FACT may ensure high
rate of replication and transcription, needed for these
categories of cells, it may reduce the level of replication stress. It may also be critical for maintaining of chromatin integrity via prevention of nucleosome loss when transcription and replication are too high. We are testing all these possibilities to understand what makes ESCs and malignant tumor cells so uniquely dependent on FACT.
How FACT level is regulated and what drives it up in cancer?
FACT consists of two subunits, SSRP1 and SPT16. They form complex and outside of the complex both subunits are very unstable (Safina A. et al, 2013). However, puzzle, which we observe all the time since we started working with FACT is that when we inhibit expression of SSRP1 subunit with different genetic means, the protein level of SPT16 subunits disappear quicker than of SSRP1. We proposed and demonstrated that SSRP1 mRNA is involved in the formation and stabilization of FACT complex. But the details of this mechanism are still far from the complete
understanding. In tumors the rate of mutations in both of
subunits are lower than background, suggesting that their
function is essential for tumors. This is also confirmed by
the high frequency of both subunits overexpression in
multiple types of tumors. Importantly in tumors there is
poor correlation between both subunits mRNA and protein
levels and increase in protein amount is more prominent,
suggesting that increase of FACT in cancer is not just due
to the induced expression of SSRP1 and SPT16 genes.
Working hypothesis that FACT is mostly regulated at the
level of protein stability, which depends on the presence of
certain regulatory factor, which we are trying to identify and to establish if it is responsible for driving up FACT levels in cancer. Understanding of this mechanism will show the ways how to develop FACT inhibitors for the treatment of cancer.
How FACT selects genes to assist transcription?
FACT distribution over genome identified using ChIP-sequencing technique is very nonrandom. FACT profile of enrichment in most cases precisely coincide with coding regions of some, but not all of expressed genes. FACT does not have sequence specific DNA binding domain or “reader” domain to recognize histone modifications. Thus, there is no standard way for FACT to spot its targets. We are working on the non-
canonical ways which
can be used by FACT to
find its “job assignment”.
This question is
important for the
understanding of another
layer of regulation of
the help of histone chaperones, i.e. via regulation of nucleosome stability over bodies of genes, which is completely unexplored right now.
Can FACT be safely inhibited at postnatal stage of the development and will this lead to the reduced incidence of cancer? Or maybe abolition of cancer at all?!
General knockout of SSRP1 subunit is lethal at 3.5dpc (Cao S. et al, 2003). However, low and rare expression of FACT postnatally suggested that its inactivation may not be so fatal after birth. With this in mind we generated mouse model of conditional knockout of SSRP1. Now using these mice we plan to define precisely what consequences for healthy tissues at different stages of mouse life FACT inactivation may have. This will help us to foresee what side effects can be expected from FACT inhibitors in clinic in adult and pediatric patients. We also plan to estimate how effective FACT inhibiting therapy will be for different types of tumors and as cancer preventive strategy, how long it should be used and whether tumors would re-emerge after stop of the therapy.
How to develop pharmacological inhibitor of FACT?
FACT is not enzyme, so there is no way to inhibit its
catalytic activity. FACT interacts with nucleosome via
multiple binding events, and we are not aware so far
which interaction is critical for FACT function in cancer.
Based on today’s state of knowledge we decided that
the best approach would be disruption of two subunits
interaction, since both of them become very unstable
upon this. We are perusing several paths to develop
FACT inhibitors. Each path has its own obstacles. First,
we tried to develop cell based readout model, which is our favorite way to look for the small molecules active in cells. However using available chemical libraries we did not find so far good inhibitors. Second, we tried to use structure-based approach. Crystal structure of human FACT is not available, but structure of SSRP1/SPT16 binding domains is available from fungi. So we first “humanized” FACT binding domains using sequence alignment and structure prediction tools and then tried to foresee which sites within dimerization domains of both subunits can be essential for their binding. Now we run in silico screenings and lab testing of small molecules for the inhibition of two subunits binding. Third approach is aimed to identify peptide which will be able to disrupt two subunits binding via screening of genetic libraries built from DNA fragments encoding SSRP1 and SPT16 binding domains.