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Targeting DNA repair pathways, sparking anti cancer immunity

Periodic Reporting for period 1 - TARGET (Targeting DNA repair pathways, sparking anti cancer immunity)

Reporting period: 2021-10-01 to 2023-03-31

TARGET project aims at testing for the first time the hypothesis that therapeutic inactivation of DNA repair pathways in cancer cells can be exploited for patient benefit by reawakening an anti-tumor immune response against cancer cells. Colorectal cancer (CRC) is widely recognized as one of the leading causes of cancer-related mortality, ranking second in frequency and lethality worldwide. Despite recent advancements in targeted and immunological therapies, the overall survival of metastatic CRC (mCRC) patients remains poor, mainly due to acquired resistance to systemic therapies and the immune-coldness of most CRC with proficient mismatch repair pathways. Accordingly, approximately 95% of mCRC patients are immune refractory, and do not benefit from immune checkpoint blockades. Notably, molecular defects in the mismatch repair (MMR) machinery are present in 5% of metastatic mCRC and 15% of all CRC stages. MMR deficient (MMRd) tumors known as microsatellite unstable (MSI) show better prognosis and favorable clinical outcomes when compared to microsatellite stable (MSS) tumors of the same histology. This is due to the alterations in the MMR system that cause high levels of molecular heterogeneity, genomic instability, and increased tumor mutational burden. Importantly, MMRd tumors display remarkable response to therapies based on immune checkpoint inhibitors likely due to the accumulation of mutations, which unleashes adaptive immunity and triggers immunosurveillance (Fig. 1).
This led to the unprecedented ‘pan cancer’ approval of immune therapeutic regimens based on tests to detect MMR deficiency and, in June 2020, to the approval of immune therapy for any solid tumor with high tumor mutational burden.
However, several aspects of this concept remain to be elucidated:
- What are the bases of the extraordinarily long-lasting responses of patients with MMRd tumors?
- Are there other DNA repair defects able to increase immune surveillance and response to immunotherapy?
- Can we pharmacologically inhibit DNA repair proteins to promote the production of tumor neoantigens allowing the immune system to detect cancer cells?

This unconventional, but possibly high gain approach, builds on the concept that the immune system can identify and selectively target tumor cells carrying DNA alterations and represent the final aim of this project.

By using a multidisciplinary approach and the exploitation of both patient-derived organoids and animal models (Fig. 2), we aim at systematically:
- assess whether and how inactivation of DNA repair pathways triggers anticancer immunity and restrict cancer;
- identify DNA repair pathways which, when disabled, reawaken the immune system;
- discover and develop inhibitors of DNA repair proteins able to induce significant increase of immunogenic neoantigens and tumor immunity;
- establish how DNA repair inhibitors and immune modulators can be combined in cancer treatments.
TARGET contributes to defining the connection between DNA repair mechanisms and immune surveillance in CRC. Based on our previous findings, we hypothesize that the alterations in the DNA damage repair (DDR) pathway might be an “eat me signal” detected by immune cells in the presence of immune checkpoint blockades. Exploiting the extensive collection of cell models in our laboratory, we aim to induce cancer vulnerability weakening the efficiency of DDR pathways. To this end, we developed a loss of function CRISPR CAS9 murine library encompassing the mammalian complement of 500 genes involved in almost all DDR pathways. To study how the loss of function of DDR genes triggers an anticancer immune response, we are exploiting murine CRC cell lines and syngeneic mouse models.

Since the majority of CRC patients are immune refractory, we are currently studying whether among these patients some of them could be eligible for immune based therapies. As a matter of fact, recent findings concerning immunohistochemical staining and molecular profiling of CRC tumors pinpointed the coexistence of MSS/MMRp and MSI/MMRd cancers cells in the same tumor lesion in almost 1% of CRC patients (Fig. 3). Intrigued by these findings, we generated a preclinical model of MMR heterogeneity, blending Mlh1+/+ and Mlh1-/- colorectal cancer murine cells at different ratio. Notably, tumor growth delay and tumor rejections occurred in Mlh1+/+ Mlh1-/- mixed tumors and increased when the percentages of Mlh1-/- cells was augmented in the mixed population. Overall, the observation that a MMRp tumor harboring a small fraction of MMRd cells can trigger an effective antitumor immune response might have implications for the rational design of clinical trials for tumors recalcitrant to immunotherapies

Tumors may evade immune control mainly owing to alterations in the antigen presenting machinery (APM). Several studies have reported that molecular defects in the major complex of histocompatibility I (MHC I) and in the protein Beta 2 Microglobulin (B2M) represent possible mechanisms of acquired resistance to immune checkpoint blockade in melanoma and lung tumors. We functionally evaluated the impact of B2M loss in immune evasion and resistance to immune checkpoint blockades in colorectal, pancreatic and breast cancer murine cell lines. Although the antigen presentation was compromised, the growth of MMRd murine cell lines was severely impaired by the administration of immune checkpoint blockades.
Although the APM was compromised, we provide evidence that patients bearing MMRd cancers other than colorectal may receive benefit from immune checkpoint blockades. The analysis of tumor microenvironment revealed that CD4+ T cells were pivotal in establishing an effective cancer immune response but only in the context of MMRd tumors. Overall, these data highlight the role of tumor associated effector CD4+ T cells in pre-clinical models and patients with impaired APM (Fig. 4).
One of the current greatest challenges for translational cancer research is to understand how to switch a cold tumor (immune refractory) into hot tumor (immune responsive). We previously reported that inactivation of DNA MMR leads to MSI status and generates hypermutated cancers with increased number of neoantigens. We also reported that treatment of mouse and human CRC cells with temozolomide (TMZ) leads to MMR deficiency, increasing tumor mutational burden (TMB) and response to immune checkpoint blockades. These preclinical data led to design ARETHUSA, a proof-of-concept two steps clinical trial (Fig. 5). During the first step the TMZ treatment was used both with curative intent and to trigger an hypermutation status in MSS mCRC patients. In the second step the anti PD-1 agent pembrolizumab was deployed only if patients develop a TMB > 20 mut/Mb upon progression to TMZ. Furthermore, in patients receiving a prolonged treatment of TMZ, alterations in MMR emerged. Interestingly, we found that a subset of the patients whose tumors displayed the acquisition of MMR deficiency and increased TMB, achieved disease stabilization upon pembrolizumab treatment.

Overall, we provide evidence that the inactivation of genes involved in DNA repair can be achieved pharmacologically with TMZ treatment, while offering potential clinical benefit to mCRC patients refractory to immune based treatments.
MMR deficient tumors respond to antiPD-1 despite the antigen presenting machinery APM is compromised
ARETHUSA trial: mutational signature and TMB evaluation were represented for TMZ treated patients
Aims of TARGET: color code for risk assessment: green low, yellow intermediate and orange high risk
MMR heterogeneity characterize a small percentage of colorectal cancers
DNA repair deficiency in cancer induces neoantigen formation promoting a prolific immune response