Most GISTs are caused by activating mutations of the KIT or platelet derived growth factor receptor A (PDGFRA) tyrosine kinases, which makes them amenable to successful treatment with the small molecule kinase inhibitor imatinib mesylate (Gleevec). It is well known, that imatinib inhibits the activation of normal and mutated KIT, PDGFRA as well as a number of other protein kinases. However, despite high initial response rates, durable, complete responses are unfortunately rare and the majority of GIST patients acquire resistance to imatinib over time. Although the inhibitory action of imatinib on the KIT and PDGFRA kinases is well established, the mechanistic events by which kinase-inhibition leads to clinical responses on a cellular basis are not known in detail. However, it is important to identify the biochemical mediators of imatinib-induced GIST cell death in order to develop innovative strategies. Such strategies atttempt to induce more complete responses, overcome imatinib resistance, and to thus enable more effective disease control with an aim towards cure.
Working with a GIST cell line model that was originally developed in Dr. Jonathan Fletcher’s lab (GIST882)(1), we were interested in the biochemical players in GIST cell death (apoptosis). We made an important observation when treating these cells with imatinib: The inhibition of the KIT or PDGFRA kinases was a very early effect. Two hours after starting the treatment, a complete inhibition of the kinase activity could be demonstrated. The same was true for the inhibition of the immediate “downstream” kinases of KIT and PDGFRA, such as AKT and MAPK (2). However, following the cells closely over several days, we noticed that a significant increase of GIST cell apoptosis did not occur until two to three days after the start of imatinib treatment (3). This indicates that there is a substantial lag period between imatinib action (complete kinase inhibition) and the onset of apoptosis.
This pattern of a delayed onset of cell death after start of a cytotoxic treatment reminded us of what had been reported for “conventional” chemotherapeutic agents, which often work by inducing DNA damage. We therefore decided to examine the expression levels of molecules that are involved in the response to DNA damage after treating GIST cells with imatinib. One of the proteins that we were interested in was histone H2AX.
Let me give you a quick overview about histones and the DNA damage response. Histones are proteins that are mainly involved in the compaction of DNA. As you know, the DNA molecules in each and every cell in our body would be very long if they were stretched out (approximately 2 meters) and have to be compacted to make them fit into the nuclei of our cells. This is achieved by wrapping the string of DNA around so-called nucleosomes (“beads-on-a-string” configuration). Nucleosomes are comprised of histones containing two molecules each of histone H2A, H2B, H3 and H4. Histone H2A (amongst others) can occur in several related forms (variants), one of which is histone H2AX. Histone H2AX is randomly distributed throughout the nucleosomes instead of H2A. Although histones have mainly been known as structural proteins, it has recently emerged that they also have other functions. Just a few years ago, histone H2AX has been identified to be a key player in the response to DNA damage. H2AX is rapidly phosphorylated (activated) after DNA damage and it then functions to recruit other repair proteins to the site of damage (4,5).
When treating GIST882 cells with imatinib, we found a massive increase of H2AX with levels beginning to rise already after eight hours, meaning that this occurred during the lag period between kinase inhibition and onset of apoptosis (3). Interestingly, we found that not only the phosphorylated form of H2AX increased, but also the non-activated form. In addition to that, we noticed that the majority of H2AX was not strictly localized to the chromatin (DNA plus nucleosomes) anymore, but was free inside the nucleus and rest of the cell (cytoplasm). These findings pointed to a potentially unknown function of H2AX that is not necessarily coupled to DNA damage-response and a possible causative role in the onset of apoptosis after imatinib treatment in GIST cells.
We therefore performed various follow- up experiments to prove this hypothesis. We first engineered GIST882 cells to overexpress (make more) histone H2AX and found that this led to increased cell death. When we reduced histone H2AX levels in GIST882 cells (using a technique called small interfering RNA, or siRNA), the cells were protected from apoptosis when treated with imatinib. No changes could be detected when looking at histone H2A (and not the variant H2AX) meaning that histone H2AX has specific functions that it does not share with H2A. Taken together, the outcome of our experiments suggests that the increase in histone H2AX after imatinib treatment has a causative role in killing GIST cells and hence the therapeutic response to imatinib.
To corroborate our data, we performed a series of further studies. We first were interested in the pathway that leads to increased H2AX levels in GIST cells after imatinib. When we treated GIST882 with various compounds that inhibit signaling cascades downstream of KIT, we were only able to induce increased H2AX levels with inhibitors of the PI3K/AKT pathway, whereas an inhibitor of the MAPK pathway did not have an effect. These findings are in line with the notion that the PI3K/AKT pathway is more important for GIST cell survival (6). We also found that GIST cells are able to downregulate levels of H2AX using the protein degradation machinery of the cell (ubiquitinproteasome system). This means that GIST cells are able to routinely get rid of what can potentially kill them. Only when they are treated with imatinib, this pathway is inactivated and H2AX levels rise again. Further experiments (in collaboration with Dr. Jonathan Fletcher) showed that Gleevec-resistant GIST cell lines were not able to increase their soluble H2AX levels after imatinib treatment, showing that it is indeed the action of imatinib that causes H2AX levels to rise. Lastly, we asked whether our findings can also be recapitulated in vivo. In collaboration with Dr. Cristina Antonescu and Dr. Peter Besmer, we stained paraffin-embedded tissue sections of GISTs that developed in mice harboring a germline-activating KIT mutation (7). An increased number of cells expressing histone H2AX were found in mice that were treated with imatinib further corroborating our results.
Finally, we addressed the question of how increased levels of soluble histone H2AX in a cell could induce apoptosis. When we examined cells overexpressing H2AX in more detail, we found that their nuclei showed a clumped appearance of their chromatin, also called chromatin aggregation. It is known that histones can bind unspecifically to DNA because of their negative electric charge. It has also been reported that an excess of histones can lead to impaired gene transcription, and we were able to show that excessive amounts of H2AX can abrogate transcription in an in vitro assay. We can then hypothesize that excess soluble histone H2AX can cause chromatin aggregation and impaired gene transcription, thereby sensitizing the cells to undergo apoptosis. Moreover, GIST cells appear to be particularly sensitive to reduced gene transcription when compared to cells that are not malignant. This was also shown in a recent study that used the CDK2/ transcriptional inhibitor flavopiridol (8).
Taken together, our study highlights the role of histone-mediated cytotoxicity in GIST cell death induced by imatinib and establishes an unexpected role of H2AX in this process. However, given that KIT activates several downstream signaling pathways, it is possible that this is not the only mechanism contributing to GIST cell apoptosis.
We believe that our results have various implications for GISTs and cancer therapy in general. First, the observation that H2AX upregulation is critical for GIST cell sensitivity to imatinib suggests novel therapeutic approaches in which H2AX induction might be accomplished by alternative mechanisms thereby countering imatinib resistance. Second, the observation that PI3K inhibition leads to induction of H2AX expression provides a novel mechanistic basis for anti-apoptotic roles of PI3K, and suggests that PI3K as a promising therapeutic target in GIST, either combined with KIT inhibition or alone. Third, our findings suggest that H2AX upregulation by tyrosine kinase oncoprotein inhibitors may restore tumor sensitivity to conventional chemo- or radiotherapy. Further understanding of how oncogenic protein kinases overcome anti-cancer barriers during tumor evolution is likely to improve the therapeutic and preventive use of targeted small molecule inhibitors.
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