Periodic Reporting for period 2 - TARGET (Targeting DNA repair pathways, sparking anti cancer immunity)
Okres sprawozdawczy: 2023-04-01 do 2024-09-30
TARGET 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 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. Importantly, the vast majority (95%) of mCRC patients do not benefit from immune checkpoint blockade (ICB). Notably, molecular defects in the mismatch repair (MMR) machinery are present in 5% of metastatic mCRCs 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. Importantly, MMRd tumors display remarkable response to therapies based on immune checkpoint inhibitors likely due to the accumulation of mutations, which triggers immunosurveillance (Fig. 1). This led to the ‘pan cancer’ approval of immune therapeutic regimens for MMR deficient solid tumor. Based on these premises TARGET was conceived to address these questions:
- 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?
Using a multidisciplinary approach and the exploitation of both patient-derived organoids and animal models (Fig. 2), TARGET aims at systematically:
- assess whether and how inactivation of DNA repair pathways triggers anticancer immunity and restricts 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.
- 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?
Using a multidisciplinary approach and the exploitation of both patient-derived organoids and animal models (Fig. 2), TARGET aims at systematically:
- assess whether and how inactivation of DNA repair pathways triggers anticancer immunity and restricts 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.
As planned, exploiting our extensive collection of cell models, we studied cancer vulnerabilities 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 exploited murine CRC cell lines and syngeneic mouse models.
Since the majority of CRC patients are immune refractory, we assessed whether disabling DDR genes could make their tumors eligible for immune-based therapies. Recent findings show 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 evidences, we generated a preclinical model of MMR heterogeneity, mixing Mlh1+/+ and Mlh1-/- colorectal cancer murine cells at different ratio. Notably, tumor growth delay and tumor rejections occurred in Mlh1+/+ and Mlh1-/- mixed tumors and increased when the percentages of Mlh1-/- cells augmented in the mixed population. The observation that a MMRp tumor harboring a small fraction of MMRd cells can trigger an effective antitumor immune response has implications for the rational design of clinical trials for tumors recalcitrant to immune checkpoint blockade (ICB).
Tumors can evade immune surveillance through alterations in the antigen presenting machinery (APM). Several studies reported that molecular defects in the major complex of histocompatibility I (MHC I) and in the protein Beta 2 Microglobulin (B2M) represent mechanisms of acquired resistance to ICB in melanoma and lung tumors. We evaluated the impact of B2M loss in immune evasion and resistance to ICB in colorectal, pancreatic and breast cancer murine cell lines. Although the antigen presentation was compromised, the growth of MMRd murine cell lines was still severely impaired by the administration of ICB. 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 (Fig. 4).
The impact of the non-coding DNA (98% of the entire genome) on the immunogenicity of MMRd tumors is largely unknown. As part of TARGET we recently found that non-canonical transcripts, can be sources of immunogenic peptides in murine CRC cells (Rospo et al., Genome Medicine, 2024). We initially identified the non-coding derived neoantigens that bound the MHC class I complex and then identified those that were lost after immune pressure in immune-competent mice (the same neoantigens were preserved in immune-compromised mice). Finally, we validated the immunogenicity of the peptides through the measurement of IFN-γ release from splenocytes co-cultured with synthetic peptides. Overall, we provided proof-of-concept that in MMRd tumors, the non-coding part of the genome can effectively contribute to defining the immunogenic properties of these tumor types.
As conceived, TARGET relies on the support of NeoPhore (https://www.neophore.com/(odnośnik otworzy się w nowym oknie)) that aims to discover whether the DNA MMR machinery could be modulated with a small molecule inhibitor, thereby enabling studies on the time-dependent sequelae of MMR deficiency. In collaboration with NeoPhore we found that resorcinol fragment-derived inhibitor 1 (NP1867) binds irreversibly with high affinity and inactivation rate to PMS2 through adduction of Cys73 in the ATP binding site, as demonstrated by protein-ligand X-ray crystallography. In vitro and cell-based selectivity profiling of NP1867 revealed minimal off-target activity. Importantly, in cell-based mechanistic and functional assays, the inhibition of PMS2 by NP1867 was observed.
Since the majority of CRC patients are immune refractory, we assessed whether disabling DDR genes could make their tumors eligible for immune-based therapies. Recent findings show 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 evidences, we generated a preclinical model of MMR heterogeneity, mixing Mlh1+/+ and Mlh1-/- colorectal cancer murine cells at different ratio. Notably, tumor growth delay and tumor rejections occurred in Mlh1+/+ and Mlh1-/- mixed tumors and increased when the percentages of Mlh1-/- cells augmented in the mixed population. The observation that a MMRp tumor harboring a small fraction of MMRd cells can trigger an effective antitumor immune response has implications for the rational design of clinical trials for tumors recalcitrant to immune checkpoint blockade (ICB).
Tumors can evade immune surveillance through alterations in the antigen presenting machinery (APM). Several studies reported that molecular defects in the major complex of histocompatibility I (MHC I) and in the protein Beta 2 Microglobulin (B2M) represent mechanisms of acquired resistance to ICB in melanoma and lung tumors. We evaluated the impact of B2M loss in immune evasion and resistance to ICB in colorectal, pancreatic and breast cancer murine cell lines. Although the antigen presentation was compromised, the growth of MMRd murine cell lines was still severely impaired by the administration of ICB. 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 (Fig. 4).
The impact of the non-coding DNA (98% of the entire genome) on the immunogenicity of MMRd tumors is largely unknown. As part of TARGET we recently found that non-canonical transcripts, can be sources of immunogenic peptides in murine CRC cells (Rospo et al., Genome Medicine, 2024). We initially identified the non-coding derived neoantigens that bound the MHC class I complex and then identified those that were lost after immune pressure in immune-competent mice (the same neoantigens were preserved in immune-compromised mice). Finally, we validated the immunogenicity of the peptides through the measurement of IFN-γ release from splenocytes co-cultured with synthetic peptides. Overall, we provided proof-of-concept that in MMRd tumors, the non-coding part of the genome can effectively contribute to defining the immunogenic properties of these tumor types.
As conceived, TARGET relies on the support of NeoPhore (https://www.neophore.com/(odnośnik otworzy się w nowym oknie)) that aims to discover whether the DNA MMR machinery could be modulated with a small molecule inhibitor, thereby enabling studies on the time-dependent sequelae of MMR deficiency. In collaboration with NeoPhore we found that resorcinol fragment-derived inhibitor 1 (NP1867) binds irreversibly with high affinity and inactivation rate to PMS2 through adduction of Cys73 in the ATP binding site, as demonstrated by protein-ligand X-ray crystallography. In vitro and cell-based selectivity profiling of NP1867 revealed minimal off-target activity. Importantly, in cell-based mechanistic and functional assays, the inhibition of PMS2 by NP1867 was observed.
We had previously reported that inactivation of DNA MMR leads to MSI status and generates hypermutated cancers with increased number of neoantigens. We also found that treatment of mouse and human CRC cells with temozolomide (TMZ) leads to MMR deficiency, increasing tumor mutational burden (TMB) and response to ICB. Altogether 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, these results indicate that pharmacological agents can be used to disable DNA repair pathways, and this can lead to clinical benefit in mCRC patients refractory to immune based treatments.
Overall, these results indicate that pharmacological agents can be used to disable DNA repair pathways, and this can lead to clinical benefit in mCRC patients refractory to immune based treatments.