For the first time, the BRCA1 mutation is introduced to syngeneic mice line using the gene editing tool, CRISPR/Cas9. Here’s how we did it.
The National Cancer Institute estimates about 12 percent of women in the general population will be diagnosed with breast cancer sometime in their lives. By contrast, 55 to 65 percent of women who inherit a harmful BRCA1 mutation and around 45 percent of women who inherit a harmful BRCA2 mutation will develop breast cancer by age 70 years. Women who inherit one of these harmful mutations also have an elevated risk of developing ovarian cancer.
Since the mutations were discovered over 20 years ago, the field has developed genetic tests that can detect BRCA1 and BRCA2 mutations in women, and drugs that may be able to help lower the risk of breast cancer in women carrying one of these harmful variants. There is also a new class of targeted therapies now available for women with metastatic breast and ovarian cancer whose BRCA genes are damaged. The drugs, called PARP-inhibitors because they block Poly-ADP-ribose polymerase enzymes, help to keep cancer cells with the BRCA mutation from dividing and growing.
Yet there is still so much we do not understand about the BRCA mutations. One important question, given the popularity of the current immunotherapies, is whether BRCA mutations are sensitive to immuno-oncology drugs that have taken the oncology field by storm. An obvious way to begin to answer this question is by interrogating animal models with intact immune system—so-called syngeneic mouse models—who express one of these BRCA mutations. Unfortunately, there are no such immune-competent murine models that carry this mutation.
Until now. About 1½ years ago, our Early Discovery group in Leiden set about creating a BRCA1 model, with the help of the gene editing technology CRISPR/Cas9. Much of the excitement around this new biological tool relates to its potential to treat human diseases or edit human embryos, but it is also expediting the creation of new transgenic animal models. Our lab and others have employed CRISPR to generate customized models that are now being used to study a number of different diseases.
Creating a BRCA1 breast cancer model was not so straightforward, however, because the gene is extremely hard to knock out in certain breast cancer cell lines. We completely failed to create a KO version in our PDX-derived cell lines (tumors transplanted from patients into mice to study their cancers and their response to drugs). But our Leiden scientists did succeed with an EMT6 breast cancer cell line.
To do this, they took the wild-type EMT6 cells and engineered a new EMT6 cell line by silencing the BRCA mutation using CRISPR. To our knowledge, this was the first time any laboratory had successfully introduced this homozygous mutation—meaning it possesses two identical alleles of the mutation—into a syngeneic mouse line using CRISPR/Cas9.
What did we find?
With two different EMT6 cell lines—one expressing the BRCA1 mutation and one without—we had the essential players required to test new drugs and drug combinations designed to target the BRCA1 mutation and DNA repair pathways. Our sites in Freiburg and Morrisville—the hub for our Oncology Discovery team—used the cell lines to see whether the BRCA mutation was impacting the efficacy of PARP inhibitors. Our evidence turned up several: Enhanced sensitivity to the drug varied depending upon which PARP inhibitor you used. In vitro 2D as well as 3D experiments showed enhanced activity of a variety of PARP inhibitors in the mutated cell line. These results could be reproduced in different mouse experiments.
We also used the cell lines to see whether PARP inhibitors and checkpoint inhibitors, previously shown to induce tumor responses in a syngeneic mouse model of ovarian cancer, elicited the same responses in a breast cancer mouse model. To determine this, the wild-type and KO BRCA1 cell lines were implanted subcutaneously in syngeneic mouse, and then exposed to various PARP inhibitors and checkpoint inhibitors. We found that basic tumor growth was about the same for both cell lines. We also observed that the models responded to checkpoint inhibitors, though the wild-type model reacted more strongly to CTLA-4 inhibitor, while the KO model responded more to the PD-1 inhibitor. This was in line with observations from other groups dealing with BRCA1 KO lines from different histologies.
We are now offering the CRISPR generated EMT6/BRCA1 KO model for commercial use in breast cancer studies, and plan to expand the portfolio to include other breast cancer lines, including patient-derived xenografts of breast cancers. While we still have much to learn about BRCA mutations, CRISPR could help make the work go a lot faster.
Review all 14 posters that Charles River presented during AACR 2018 by visiting our website, where you can also learn more about our oncology capabilities. You can also view Eureka’s full coverage of AACR 2018 here.