Two Wrongs Make It More Wrong: On Double Mutations

It is becoming increasingly well known that about 25% of CRPC tumors harbor some form of a mutation in BRCA1, 2, ATM or other such genes. Colin Pritchard and others have done some really excellent work on this topic by giving us genomic snapshots of the disease (you can see me interview him in St Gallen on this topic here on UroToday: link).1 The efficacy of Parp inhibitors in this setting is being tested in a number of trials at the moment. 

What is less known is that these mutations, many of them at least, can be undone. Yes, undone. This is the manner in which tumor cells become resistant to parp inhibitors. They mutate again, essentially undoing the original mutation. Its cancers double negative, or two wrongs making it right- if by “right” we mean restoring tumor cell survival. 

In late April, two groups (One of which I am a member) simultaneously published findings demonstrating the presence of secondary “reversion” mutations in CRPC patients who developed clinical resistance to parp inhibitor therapies. This had previously been shown in patients with Ovarian cancer also treated with parp inhibitors, but these are the first reports in prosate cancer. 2, 3

It works like this: The standard mutations in BRCA2 produces a stop codon that results in a shortened BRCA2 mRNA transcript a nonfunctional BRCA2 protein. This is the situation where parp manages DNA Repair and parp inhibitors are clinically active, by inducing synthetic lethality.  For those of you who, like me, get some of the new terminology a little confused, allow me to define synthetic lethality – which is used a lot in the literature now. The term applies to the situation when two genetic events, that are innocuous when occurring in isolation, result in cell death when they occur.4 If BRCA is not functioning, Parp will do the job, if parp is inhibited, there is no DNA repair mechanism, and the cell dies. Parp inhibitors will not work if BRCA is not mutated, and BRCA mutations aren’t quite so dangerous when parp is competent. They both need to be impaired for the lethality to occur.

However, over time, in the genetically unstable background of malignancy, long-term exposure to Parp inhibitors enables the selected outgrowth of tumor cells with additional mutations. In cases in which these secondary mutations occur within the BRCA gene and eliminates the stop codon, the wild-type BRCA2 reading frame is essentially ‘fixed’ and the functionality of BRCA2, specifically homologous recombination,  is restored. This was described in 2008 by Lord and Ashworth (now the Director of the Helen Diller Family Cancer Center where I work).5

What does this mean and what does it tell us about cancer biology in general?

Well first, it is an example par excellance, of a concept and phrase that I have had memorized since my training, and use frequently: Treatment mediated selection pressure. A reversion mutation is unlikely to exist in nature, because there weren’t selection forces that drove the survival of clones that, by chance, developed these mutations. That selection pressure, of course, is the parp inhibitor. While reversion mutations probably do not exist in nature in high number they probably are not entirely new either. In fact, they have probably existed for about 50-60 years, which is the age of the modern chemotherapy era. Although they weren’t discovered, it is likely that they have been driving some resistance to chemotherapy for decades – and possibly to radiation for a century. (This is pure conjecture on my part which is one of the fun things about writing in a non-peer review setting).

Alkylating agent chemotherapy and radiation therapy both induce single strand breaks in DNA, which is how they are generally lethal to cancer cells. Parp, in its natural role, repairs single strand DNA repair (base exision repair) and therefore radiation in a DNA repair deficient setting would theoretically also be effective, but could be overcome by a reversion mutation. Platinum chemotherapy, on the other hand, induces DNA adducts, not single strand breaks,  but is useful in the setting of BRCA mutation as well, because of the impaired homologous recombination mechanism. Theoretically, then, reversion mutations could, and should, develop in the setting of platinum therapy of a BRCA mutant cancer. There is a nice review of this from 2015 in Molecular Cell.6

Finally, I should point out a useful technical feature of these two papers that described the emergence of reversion mutations in patient specimens: they were discovered without doing tumor biopsies. Both investigators utilized whole exome sequencing circulating-free DNA  (cf DNA sometimes called circulating tumor DNA, or ct DNA). The  advantage of the cf DNA approach is several fold: For one, the patient doesn’t have to undergo the discomfort of a biopsy, Also, we don’t have to worry about missing tumor, bone crush artifact or other factors that can diminish the precision of the tumor biopsy. Third, and I think this is important, if we can use a circulating approach we can overcome the worry about tumor heterogeneity. If there are two tumor foci, and one contains a mutation and the other one doesn’t (which can happen) then you just have to hope you have biopsied the correct one, so to speak. If, on the other hand, the DNA is circulating, then you can, with one tube of blood, capture the whole tumor DNA ‘soup’ and make your diagnosis. 

The challenge, of course, is making sure the test has enough sensitivity to discover all this, and that you have the expertise on hand to interpret the results. Who knows what additional curious findings await us as we dive deeper and deeper into the unstable genetics that drive tumor survival in the face of the treatments we apply. 

Treatment mediated selection pressure demands our respect. 

Written by: Charles Ryan, MD, B.J. Kennedy Chair in Clinical Medical Oncology, Director and Professor of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, Minnesota

Published Date: June 7th, 2017

References:
1. Pritchard, C.C., et al., Inherited DNA-Repair Gene Mutations in Men with Metastatic Prostate Cancer. N Engl J Med, 2016. 375(5): p. 443-53.
2. Quigley, D., et al., Analysis of Circulating Cell-free DNA Identifies Multi-clonal Heterogeneity of BRCA2 Reversion Mutations Associated with Resistance to PARP Inhibitors. Cancer Discov, 2017.
3. Goodall, J., et al., Circulating Free DNA to Guide Prostate Cancer Treatment with PARP Inhibition. Cancer Discov, 2017.
4. Mateo, J., et al., DNA Repair in Prostate Cancer: Biology and Clinical Implications. Eur Urol, 2017. 71(3): p. 417-425.
5. Ashworth, A., Drug resistance caused by reversion mutation. Cancer Res, 2008. 68(24): p. 10021-3.
6. O'Connor, M.J., Targeting the DNA Damage Response in Cancer. Mol Cell, 2015. 60(4): p. 547-60.