Summary

Colorectal cancer is one of the most common and fatal cancers, accounting for approximately 10% of new cancer diagnoses and 9% of cancer - related deaths. Although the 5 - year relative overall survival rate for local disease (stage I - III) ranges from 68% to 90%, the survival rate for stage IV metastatic colorectal cancer (mCRC) is unsatisfactory, at approximately 11% to 14%.

Most metastatic colorectal cancer (mCRC) has mismatch repair (MMR) capability and is unresponsive to immunotherapy, while MMR - deficient (dMMR) tumors are usually responsive to immune checkpoint blockade ^[1].

Immune checkpoint inhibitors rely on high tumor mutation associated with MMR mechanism defects and provide significant survival benefits for patients with DMMR/MSI - H mCRCs, but this population represents only 4% of the MCRC population. Most patients with pMMR/MSS mCRC have immunologic rejection and intrinsic resistance to immune checkpoint inhibitors ^[2]. Therefore, turning a cold tumor into a hot tumor and achieving immune sensitization would transform a pMMR/MSS mCRC patient into an immunotherapeutic beneficiary.

Studies have suggested that Temozolomide can induce hypermutation in pMMR/MSS and MGMT - silent MCrCs, thus realizing the immune sensitization of MCrCs ^[3].

01. Temozolomide action mechanism

Temozolomide, an oral alkylating agent approved for use in patients with glioblastoma (GBM), methylates DNA at position O6 of guanine. During DNA replication, this methyl adduct causes the mismatching of guanine with thymine, leading to genomic instability and eventually cell death.

Acquired resistance to Temozolomide may be associated with a high mutation rate in the tumor, usually characterized by a specific gene scar with a C > T transformation and often secondary mutations in the MMR gene, especially the MSH6 mutation, which is observed in Temozolomide - sensitive mCRC.

O6 - methylguanine DNA methyltransferase (MGMT) is able to remove the methyl adduct from O6 guanine by covalent transfer, effectively repairing the genetic change.

If the methylation of the MGMT promoter leads to the silencing of the gene, the protein expressed by the gene will be decreased, resulting in decreased activity of MGMT and its reduced repair effect on DNA, thus making tumor cells more sensitive to alkylating agents such as Temozolomide ^[3]. Methylation of the MGMT promoter is present in approximately 40% of CRC patients, but the overall response rate to Temozolomide in these patients is less than 10%, and patients need to have loss of MGMT protein expression to benefit from this.

02. Temozolomide - induced conversion of pMMR to dMMR

In the absence of MGMT expression, the resulting base mismatches activate the mismatch repair (MMR) pathway. The most toxic DNA base adduct induced by Temozolomide is O6 - meG. Although Temozolomide induces multiple other base adducts, these are less important in killing primary TMZ - sensitive tumors due to the high toxicity of O6 - meG. O6 - meG can pair not only with cytosine but also with thymine. The O6 - meG adduct mismatched with thymine is identified by the mismatch repair (MMR) pathway during the first round of DNA replication after Temozolomide treatment. MMR can only correct mismatched bases in a newly synthesized strand. Therefore, MMR excises the thymine while preserving the original lesion O6 - meG. Since O6 - meG is not repaired, it still pairs with thymine when the gap is filled. The repetition of this process is referred to as an "invalid loop" of the MMR. The resulting persistent single - stranded DNA (SSDNA) gap interferes with the second round of DNA replication, eventually leading to DNA double - strand breaks, cell stagnation, and death.

MGMT methylation is found in approximately 40% of MCRCs, with an objective response rate (ORR) of 10% in patients with MGMT - methylated MCRCs for Dacarbazine and its oral analog, Temozolomide.

Acquired resistance to Temozolomide may be associated with a high mutation rate in the tumor, usually characterized by a specific gene scar with a C > T transformation and often secondary mutations in the MMR gene, especially the MSH6 mutation, which is observed in Temozolomide - sensitive mCRC. The new MSH6p.T1219I mutation was detected in 94% of MSS mCRC patients treated with Temozolomide, proving that only drug treatment can change the MMR state of cells, and the targeted DNA repair process can cause mutation evolution of pMMR tumors.

Previous studies have shown that Temozolomide can induce hypermutation in pMMR/MSS and MGMT - silent MCrCs, leading to the immune sensitization of MCrCs.

▲Temozolomide and DNA repair mechanism ^[4]

03. MAYA study: Temozolomide combined with Nivolumab and low - dose Ipilimumab in the treatment of MGMT - silent metastatic colorectal cancer MSS

The high mutation rate induced by the initiation of therapy with Temozolomide provides a theoretical basis for the immune sensitization of pMMR/MSS and MGMT - silent mCRC.

To clinically verify this hypothesis, researchers designed a MAYA proof - of - concept trial ^[5].

A Multicenter, Single - Arm Phase II Clinical Trial to Evaluate the Efficacy and Safety of an Immuno - Sensitizing Strategy, Initiated with Temozolomide and Followed by Combination of Low - Dose Ipilimumab and Nivolumab for the Treatment of Microsatellite Stability (MSS) and O6 - meG - DNA methyltransferase (MGMT) - Silent Metastatic Colorectal Cancer (mCRC).

A total of 204 RAS/BRAF wild - type subjects were included in the experiment, and 135 subjects received the first phase of treatment. Of these, 102 cases (76%) discontinued due to death or disease progression after Temozolomide initiation, and 33 cases (24%) were under control and started Phase 2 treatment, which formed the final study population. The median follow - up was 23.1 months, and the 8 - month incidence of progression - free survival (PFS) was 36%. The median PFS and overall survival were 7.0 and 18.4 months, respectively, with an overall response rate of 45%. Grade 3 - 4 immune - related adverse events included rash (6%), colitis (3%), and pituitary inflammation (3%). No unexpected adverse events or treatment - related deaths were reported.

▲ Kaplan - Meier curve of (A)PFS and (B)OS of patients under treatment in Part II

04. Future prospects

The MAYA trial reached the primary endpoint, providing proof - of - concept evidence that a regimen of Temozolomide coadministered with low - dose Ipilimumab and Nivolumab following initiation may have durable clinical benefits in pMMR/MSS and MGMT - silent mCRC patients. Clinical trials have demonstrated that Temozolomide can induce dMMR states and eventually "heat" immunologically "cold tumors". That's why Temozolomide, a traditional chemotherapy drug, has regained its potential. Acquired resistance to Temozolomide is often accompanied by hypermutations and frequent secondary mutations in the MMR gene, especially the MSH6 gene. On the contrary, dMMR and hypermutations are likely the result of chemotherapy - induced mutations and methylation of the MGMT promoter. So, malignant tumors that are sensitive to Temozolomide treatment (often receiving Temozolomide "start - up treatment" for a long time) with silence of pMMR/MSS and MGMT may be induced into a dMMR state, which will be one of the most potential directions of immune sensitization in the long term, and it is likely to affect future clinical practice.

References

[1] Nucleic Acids Res.51, 3722–3734.

[2] J Clin Oncol 36:773-779, 2018

[3] Cancer Res. 2005 Jul 15; 65(14):6394-400.

[4] Front Cell Dev Biol. 2024 Jul 1; 12:1436563

[5] J Clin Oncol. 2022 May 10; 40(14):1562-1573

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