Does chemotherapy kill cancer cells by inducing DNA double stranded breaks? Maybe not

Scientists, you may have heard for most of your career that DNA double stranded breaks (DSBs) are the most toxic DNA lesion a cell confronts, and that chemotherapy works by making DSBs. 

The DSB mantra has been the cornerstone of cancer biology and established as scientific dogma ever since the mid-1990s. This is when the hereditary breast and ovarian cancer genes BRCA1 and BRCA2 were identified. Cancer cells with BRCA gene mutations were exquisitely sensitive to chemotherapy. Supporting the DSB model, the BRCA genes were found to make protein products that function in the accurate repair of DSBs by a pathway known as homologous recombination (HR).  Accordingly, HR deficiency (HRD) became the key feature thought to confer chemotherapy sensitivity and has been the benchmark for identifying treatable tumors. Coming full circle, restored HR was found in tumors that gained resistance to chemotherapy.

As the traditional tale goes, chemotherapy is deemed effective because it makes DSBs and the reason normal cells live whereas cancer cells die is largely attributed to DSBs being repaired in normal but not in cancer cells. Cancer cell death is thought to stem from DSBs activating cell death pathways or undergoing permanent cell cycle arrest. In another scenario, without DNA repair, cancer cells are expected to “glue” their broken DNA back together without any precision causing a tangled mess of chromosomes that would be incompatible with cell growth.

From this understanding, it also followed that DSBs seed the birth of cancer. Without accurate DNA repair, a wayward BRCA-mutant cell could shuffle its genome extensively to gain a malignancy threshold sufficient to drive breast or ovarian tumors.  On the flip side, a rapidly developing fetus with too many unrepairable DSBs would not be able to survive beyond an embryonic stage consistent with the lack of live born BRCA null mice.

That chemotherapy works by making DSBs is one of those aphorisms that gets repeated in numerous articles and reviews. But I’m here to tell you something different.

Could there be more to the story?

For the past several decades or so, there have been hints that the BRCA proteins may be doing more than we give them credit for. The research is still emerging, but data indicate that BRCA proteins are essential for ensuring high-quality DNA replication, the process by which DNA is duplicated.  DNA replication faces many challenges that could slow or terminate the DNA copying process. For example, DNA replication is especially vulnerable to making mistakes when it is devouring all the nucleotides as in a developing embyo. In addition to loss of its building blocks, replication could falter when DNA polymerases copying a DNA template are confronted by oddities such as chemical modifications, twisted DNA structures or attached proteins. It’s like a train piling up to a broken track and impeded from moving forward unless the track is repaired. However, research shows that it is also problematic when the train continues over an impediment. This can happen when the cell uses special polymerases to circumvent the block in an effort to continue DNA replication at all costs. However, pushing through and over obstacles with special polymerases can lead to mutations. Likewise, “jumping” over obstacles by employing polymerases that manage to restart replication on the other side of a broken track can lead to gaps in the DNA copy. A run-away train of either type could ultimately jeopardize the DNA quality.

Evidence for a critical role for the BRCA proteins in preventing DNA with gaps has been mounting for years. The first evidence that BRCA proteins were critical for DNA replication came in 2010, when a team led by Vincenzo Costanzo found by electron microscope that BRCA-deficient frog extracts displayed DNA with small gaps (Hashimoto et al., 2010).  A few years later, Massimo Lopes found that BRCA’s working partner, RAD51, also was critical to prevent these gaps (Zellweger et al., 2015). In 2013, Dr. Angelos Constantinou found DNA replication was unhinged, meaning that without the central Fanconi anemia protein, FANCD2 that operates with the BRCA proteins, DNA replication failed to properly be restrained (Lossaint et al., 2013). He further demonstrated that unrestrained DNA replication correlated with cells showing higher levels of single-stranded DNA (ssDNA) and postulated that this under-replicated DNA reduced cell fitness. Inspired by these studies, we examined a series of BRCA-mutant breast and ovarian cancer cells and found that a common feature was also unrestrained replication that led to gap formation (Panzarino et al., 2020). More strikingly, we observed in a series of cell tissue culture models and human tumors that gaps were always suppressed when chemoresistance developed. Most importantly, we found that DSBs and the genes regulating their repair did not accurately predict tumor response, but gaps and the genes that regulate their formation or repair clearly did. Collectively, these findings redefine how chemotherapy works, gaps are the killing lesion, not DSBs (Panzarino et al., 2020).

Moving past the DSB model, not so easy

At this point, the question is less about whether gaps exist; it is more about whether they are just precursors to the more well respected DSB. Linking a ssDNA gap to an eventual DSB is one way to connect the old dogma to the new findings. However, it also allows many leading scientists to resist the new research as mere distraction from the more serious DSB. And, while there is a beehive full of scientists focused on this new gap biology with subsequent research confirming the relevance of gaps alone to tumor killing, it is not clear that the buzz is being heard by the greater scientific community, especially clinicians. The dogma is entrenched; it could take years to peel back this sentiment from the textbooks and clinical guidelines. Since the 1970s, radiologists were made aware that ionizing radiation made ssDNA nicks and gaps in DNA but were told that they were of little biological consequence, only the DSBs were deemed relevant to killing tumor.  Even mainstream grants, papers, and well-respected scientists continue telling the old story without a trace of concern they are spewing antiquated concepts.

The hostility to the gap model is not entirely surprising. To rethink how chemotherapy works is to open the door to questioning a whole host of things that cancer researchers know to be “true,” including how the blockbuster drug PARP inhibitor works. Here too, our data show that DSBs are not relevant but that gaps are the key determinant by which PARP inhibitor kills BRCA tumors (Cong et al., 2021). After two years of antagonism, the gap model is gaining traction at least among a younger scientists perhaps more comfortable thinking outside the box perhaps as not members of the “club”. Either way, the concept that gaps, not DSBs, mediate chemotherapy killing and underlies BRCA deficiency phenotypes also called “BRCAness” is what we are saying as often and loudly as possible. 

While the debate will continue, the existence of the gaps has the potential to profoundly change our understanding of how chemotherapy works and explain a host of other outcomes associated with BRCA mutations that includes the unviability of mouse embryos and developmental defects in patients with Fanconi anemia. The future will reveal whether many of our current successful drug combinations enact their selective tumor killing by uniquely making gaps in tumor DNA. Ideally a new class of cancer drugs will be designed that are more potent but less toxic to patients because they solely topple cancer cells by elevating DNA gaps and/or activating the pathways that eliminate tumors with gapped filled DNA (Cantor, 2021). 

*Inspired by a NYT Opinion Essay by Rachel Gross in April 2022

Cantor, S.B. (2021). Revisiting the BRCA-pathway through the lens of replication gap suppression: "Gaps determine therapy response in BRCA mutant cancer". DNA Repair (Amst) 107, 103209.

Cong, K., Peng, M., Kousholt, A.N., Lee, W.T.C., Lee, S., Nayak, S., Krais, J., VanderVere-Carozza, P.S., Pawelczak, K.S., Calvo, J., et al. (2021). Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency. Mol Cell.

Hashimoto, Y., Ray Chaudhuri, A., Lopes, M., and Costanzo, V. (2010). Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis. Nat Struct Mol Biol 17, 1305-1311.

Lossaint, G., Larroque, M., Ribeyre, C., Bec, N., Larroque, C., Decaillet, C., Gari, K., and Constantinou, A. (2013). FANCD2 binds MCM proteins and controls replisome function upon activation of s phase checkpoint signaling. Molecular cell 51, 678-690.

Panzarino, N.J., Krais, J.J., Cong, K., Peng, M., Mosqueda, M., Nayak, S.U., Bond, S.M., Calvo, J.A., Doshi, M.B., Bere, M., et al. (2020). Replication Gaps Underlie BRCA-deficiency and Therapy Response. Cancer Res.

Zellweger, R., Dalcher, D., Mutreja, K., Berti, M., Schmid, J.A., Herrador, R., Vindigni, A., and Lopes, M. (2015). Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells. J Cell Biol 208, 563-579.