Frameshift-derived neoantigens constitute immunotherapeutic targets for patients with microsatellite-instable haematological malignancies: Frameshift peptides for treating MSI+ blood cancers
Introduction
Functional inactivation of the DNA mismatch repair (MMR) system highly increases spontaneous mutation rates. This defect is recognised as microsatellite instability (MSI), a form of DNA-instability changing the length of extended mono- or polynucleotide tracts by insertions or deletions of single repeat units.1, 2 Almost all tumours of patients affected by the Lynch syndrome as well as 10–15% of sporadic human cancers display MSI – particularly colorectal, endometrial and gastric cancers. In contrast, MSI seemed to be a rare phenomenon in many other tumour entities, including primary leukaemia.3 Increasing evidence indicates that certain haematological malignancies are especially prone to MSI. Prominent MSI has been detected in post-transplant lymphoproliferative disorders, therapy-related acute myeloid leukaemia/myelodysplastic syndromes (tAML/MDS) that develop secondary to (cancer)-chemotherapy as well as in HIV infection-related lymphomas.4, 5, 6 All the latter conditions have either transient or prolonged heavy immunosuppression in common. Even a direct link between immunocompetency and raise of MSI+ neoplasia has been suggested and experimentally proven to explain these findings.6 MSI in coding regions of genes leads to frameshift mutations (FSMs) and the production of truncated proteins7 that in most cases have lost their (tumour suppressive) function. These genes will be positively selected in the multistep process of carcinogenesis. MSI-target genes are thus not only involved in tumour initiation but also cancer progression and potentially even metastasis.8 On the other hand, these C-terminally modified proteins may be the Achilles’ heel of MSI+ tumours. They are not expressed in normal cells and have therefore strong intrinsic antigenicity. Remarkably, an increasing number of MSI-induced frameshift (FS)-derived neoantigens was found to harbour epitopes readily recognised by T cells.9, 10, 11, 12, 13, 14
In principle, immunotherapy is considered as a treatment option for patients with haematological malignancies and a growing number of promising therapeutic targets have been identified.15, 16, 17
Here, we show that MSI-induced frameshift peptides (FSPs) represent specific antigens of MSI+ leukaemias. This finding substantially broadens the repertoire of target structures for leukaemia immunotherapy and thus shall be taken into consideration for subsequent developing T cell-based vaccines and cellular therapies.
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Target cell lines, gDNA preparation and fragment analysis
All tumour cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) or from the German Cancer Research Center (DKFZ, Heidelberg, Germany) tumour bank and grown in RPMI 1640 medium (+10% foetal calf serum, 2 mmol/L l-glutamine). Cell lines specified in Table 1 were used for cytotoxicity analysis. An extracellular staining protocol as well as detection of hMLH1 and hMSH2 proteins within target cells can be found as online Supplementary
HMLH1 and hMSH2 gene expression in leukaemia and lymphoma (L/L) cell lines
A panel of MSI+ L/L cell lines has been included into this study (Table 1). First, expression of hMLH1 and hMSH2 proteins was analysed by immunoblotting (Table 1). Most cell lines expressed detectable MLH1 levels, which is in accordance with the recent literature (Table 1). HMLH1 loss was observed in CCRF-CEM and REH cells. Contrary to that, MSH2 protein expression could only be confirmed in CCRF-CEM, MOLT-3 and REH cells. T2 cells were not considered as cell line since they derived from a
Discussion
Genetic instability is one of the main pathogenic mechanisms underlying human neoplasia.1, 25 MSI is one phenotype of genetic instability leading to variability in the length of repetitive nucleotide sequences.26 First described in the Lynch snydrome,1 MSI is widespread among sporadic human malignancies,27, 28, 29, 30 but appeared to be rare in primary leukaemia.23, 31 However, other studies disclosed higher incidences of MSI+ in leukaemic patients, especially as an early genetic event.32 In
Conflict of interest statement
None declared.
Acknowledgement
The authors want to thank Miss Christina Mullins for critical language-proofreading.
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These authors contributed equally to this work.