Screening Identifies Mechanisms of Drug Resistance to PI3K Inhibitors

One of the most important focuses in personalized oncology therapeutics is on the ability to understand mechanisms of treatment resistance, with the aim of selecting more effective treatments. Underlying this is the need to identify tumor mutations that are inherently resistant to a specific treatment modality. The complexity of the pathways and networks of pathways in cancer makes it necessary to test a large number of combinations of gene-drug interactions; in the future, genomic methods combined with clinical investigation should greatly improve patient treatment outcomes. 

In an online-first article in Nature Chemical Biology, Sebastian Nijman of the CeMM—Research Center for Molecular Medicine— of the Austrian Academy of Sciences in Vienna and his colleagues describe the development of a chemical genetic approach that has identified mechanisms that can lead to resistance to PI3K inhibitors.

As Professor Nijman explained to the cancernetwork, "Even for [targeted drugs] that have been designed to act specifically on cells carrying a certain cancer mutation, only a fraction of the patients respond. The reasons for this are often unclear. But obviously, if one could know beforehand who would respond and who would not, this could be a great advantage. Patients that would not benefit could be saved the side effects of treatment and given an alternative treatment. Or perhaps one could extend the duration of response by designing a combination therapy. So what is needed is being able to predict response to treatment. Our study had this aim and we found one such 'mechanism of resistance.'"

The researchers developed a way to measure the cellular fitness of isogenic human cell lines in order to quantitatively assess functional drug-gene interactions. As the authors explain, “this method assists the systematic assessment of the impact of cancer aberrations on proliferation in response to a collection of drugs.”

The actual screen used an isogenic breast cancer cell line to which a genetic alteration was introduced linked to a molecular "barcode." The barcode is a non-transcribed short sequence of DNA that can be easily identified and quantitated. After addition of the drug, measuring the abundance of the barcode DNA via PCR in the population of cells served as a proxy for the measure of cellular fitness/growth.

The researchers tested the interaction of 87 drugs with 70 gene mutations (either knock-down or overexpression) in the breast cancer cells, and this resulted in data from over 6000 drug-gene pairs. The genes were selected based on literature and database searches and included important genes linked to breast cancer, such as HER2, BRCA1, BRCA2, c-MYC, NOTCH1, and PTEN. The cell line selected had a normal karyotype and is responsive to most signaling pathways present in normal breast epithelial cells. The study identified previously known interactions, confirming the utility of the approach. 

Importantly, the study also identified a new mechanism of resistance to a group of PI3K inhibitors that are currently being investigated in clinical trial. Many breast tumors harbor activating mutations in the PI3K pathway. The screen identified that the intracellular active domain of NOTCH1 confers resistance to the dual PI3K-mTOR (mammalian target of rapamycin) inhibitor BEZ-235. Further testing with other cancer cell lines suggested that NOTCH1 activation generally uncouples proliferation from the PI3K-mTOR pathway: "We have evidence that other cancer types may show the same mechanism of resistance" Nijman confirmed. NOTCH1 appears to override the need for the PI3K-mTOR pathway through increased c-MYC transcription. The published work demonstrates that transcriptional activation of c-MYC via NOTCH1 is sufficient to confer the resistance to BEZ235. This result was validated by inhibiting c-MYC expression with RNAi in the NOTCH1 upregulated cells.

NOTCH activation is known to occur in a subset of breast cancers and is associated with tumor progression and poor prognosis. MYC amplification is a relatively frequent event in breast cancer. BEZ235 is being developed by Novartis and is currently in phase I and II clinical trials for patients with solid tumors, including advanced breast cancers, as both a monotherapy and in combination with chemotherapy and other targeted agents. 

In terms of next steps, Professor Nijman says that his laboratory is still "digesting the many hits that came out of the screen." The researchers are also continuing to develop the technology and performing more screens.  Nijman added, "What excites me is that using our approach, [which] was not guaranteed to work, we have made some very interesting and clinically relevant discoveries. I believe that this is only the beginning!"

Novel DNA-Silencing Function of BRCA1 Discovered

It is well established that mutations in brca1 and brca2 are linked to hereditary ovarian and breast cancer; individuals with a single mutation in BRCA1 have a higher risk of developing these cancers, and these mutations can lead to shorter versions of the BRCA1 protein; prevent the protein from being made; cause a change in an amino acid that changes the function of the BRCA1 protein; or result in a deletion of a large segment of the protein.

Professor of Molecular Biology Inder M. Verma, of the Laboratory of Genetics at the Salk Institute for Biological Studies, and colleagues have now identified that "maintenance of global heterochromatin integrity" is a novel function of BRCA1. The authors propose, in an article published in Nature on September 7, 2011, that this DNA-silencing function is linked to the role of BRCA1 as a tumor suppressor. Heterochromatin is a tightly packed, mostly repetitive, nontranslated DNA sequence associated with a distinct chromatin structure that is commonly associated with gene silencing. The research suggests that deficiency in BRCA1 may cause aberrant expression of noncoding DNA that can lead to genomic instability that then promotes tumor development.

While the tumor suppressor BRCA1 has been implicated in preserving genomic stability, researchers have not yet been able to identify the biochemical activity of BRCA1 and its role in maintaining genomic integrity.

Inder Verma and colleagues have uncovered a role for BRCA1 in silencing sections of noncoding DNA, preserving the structure of heterochromatin. They show that abnormal transcription of these so-called satellite repeat regions occurs in mouse tissues and human breast cancer samples deficient in BRCA1. From these findings the authors propose that the role of BRCA1 in maintaining heterochromatin-mediated DNA silencing and genomic stability accounts for its tumor suppression functions.

Using knockout mice as a model system, the authors observed alternation of epigenetic regulation in the brain of BRCA1 knockout mice. Specifically, BRCA1 loss resulted in the derepression of tandem repeats of satellite DNA and a reduction of condensed DNA regions in the mouse genome. Additionally, the mice had a loss of ubiquitylation of histone H2A at satellite repeats. The researchers demonstrated that BRCA1 is necessary for the repression of heterochromatin by reexpressing human BRCA1 in the knockout mice.

The generation of a mammary gland-specific deletion of BRCA1 also resulted in the disruption of heterochromatin DNA in female mice, and the silencing function of BRCA1 was not limited to mouse cells, as human HeLa cells also exhibited the disregulation of constitutively silenced heterochromatin.

The DNA-silencing function of BRCA1 in humans was validated by showing the derepression of satellite transcripts in human breast tumors in BRCA mutation carriers.

The authors further showed that in the presence of wild-type BRCA1, increasing the expression of satellite DNA transcripts (derepressed heterochromatic DNA), resulted in a phenotype that partly mimics the loss of BRCA1. This indicates that the presence of constitutively silenced heterochromatin acts to prevent DNA damage and the onset of genomic instability. The high overexpression of satellite repeats has been observed in epithelial cancers.

Although the authors note that neither the function nor the pathological significance of these satellite transcripts is known, this research suggests that satellite transcripts can contribute to the evolution of cancer through the onset of genomic instability. This study further illuminates the role of BRCA1 in preventing this type of genomic instability.