Although it seems counterintuitive that shutting down DNA repair mechanisms in cancer could actually improve prognosis, that’s exactly the concept that Germano et al. present in a letter published in Nature. They propose that when mismatch repair (MMR) mechanisms are disrupted in colorectal tumors, new mutations, and thus new potential neoantigens, continually arise, renewing the tumors’ susceptibility to attack by the host immune system.

In the clinic, colorectal cancers (CRCs) are often early onset and progress rapidly, yet they have a peculiarly favorable prognosis and respond well to immune checkpoint blockade. Previously, researchers hypothesized that the high mutational burden in these cancer might lead to an increased number of neoantigens, making such tumors vulnerable to attack by the immune system. Across multiple settings, however, mutation burden alone has not been a reliable predictor of prognosis. To study whether MMR mechanisms actively contributed to the clinical features of CRCs, Germano et al. turned their focus to the MLH1 protein, an essential protein in MMR complexes.

Using the CRISPR/Cas9 system, the researchers inactivated Mlh1 in CT26 and MC38 cancer cell lines, which caused MMR deficiency. The MMR-deficient cancer cell lines proliferated well in vitro and when injected into immunocompromised mice. However, when injected (either subcutaneously or orthotopically) into immunocompetent syngeneic mice, they failed to grow or grew very poorly in locations where the MMR-proficient cancer cell lines grew well, indicating that the immune system was involved in tumor suppression. Treatment with anti-CD8 antibody abrogated tumor control, indicating that CD8⁺ T cells were instrumental in the effect.

To mimic a clinical setting of established tumors being treated with checkpoint inhibitors, the researchers grew MMR-deficient or -proficient tumors in immunocompromised mice and transplanted fragments into immunocompetent syngeneic mice. When the transplanted tumors reached target size, some were treated with a combination of anti-PD-1 and anti-CTLA-4. While MMR-proficient tumors grew well and were insensitive to checkpoint therapy, MMR-deficient tumors grew more slowly, responded to checkpoint therapy, and had higher CD8⁺ T cell infiltration.

RNA sequencing of tumors revealed that while MMR-proficient tumors showed a stable number of mutations over time, MMR-deficient tumors had an increasing number of mutations, indicating that new mutations, and thus new predicted neoantigens, were continually emerging over time. In theory, the emergence of new neoantigens would allow the immune system to spot and attack new targets as they arise, rather than becoming ineffective once immunoediting has occurred.

In an attempt to exploit MMR-deficiency as a possible immunotherapeutic strategy, Germano et al. turned to temozolomide (TMZ), an FDA approved chemotherapeutic agent known to trigger DNA damage that can result in inactivation of mismatch repair. Using MC38 tumor cells, the team successfully generated TMZ-resistant cells carrying a de novo Mlh1 deletion which was not present in the MMR-proficient parental cells before TMZ treatment. This indicated that mismatch repair had successfully been inactivated; confirming this result, the altered MC38 cells were unable to form tumors in immunocompetent mice.

To determine whether this effect of TMZ would be relevant in humans, the researchers analyzed a collection of colorectal cancer cell lines and generated TMZ-resistant lines from the subset that were initially sensitive to TMZ. In line with data from mouse models, human CRC cells with altered MMR mechanisms showed high levels of neoantigens, which increased over time. The researchers were also able to obtain tissue samples from five patients who had achieved partial responses or prolonged stabilization following TMZ-based chemotherapy. These samples revealed that after treatment, three of the five patients’ cancers upregulated MGMT, the main enzyme responsible for repairing TMZ-damaged DNA. The other two maintained low MGMT levels and were found to have a high mutational burden and mutations in an MMR-related gene that were not present before TMZ therapy.

Germano et al. conclude that silencing mismatch repair genes dynamically increases the number of de novo mutations and neoantigens in cancer cells, which can result in continually expanding and enhanced immune responses over time.

by Lauren Hitchings