Molecular alterations in genes involved in DNA mismatch repair (MMR) promote cancer initiation and foster tumour progression1. Cancers deficient in MMR frequently show favourable prognosis and indolent progression2. The functional basis of the clinical outcome of patients with tumours that are deficient in MMR is not clear. Here we genetically inactivate MutL homologue 1 (MLH1) in colorectal, breast and pancreatic mouse cancer cells. The growth of MMR-deficient cells was comparable to their proficient counterparts in vitro and on transplantation in immunocompromised mice. By contrast, MMR-deficient cancer cells grew poorly when transplanted in syngeneic mice. The inactivation of MMR increased the mutational burden and led to dynamic mutational profiles, which resulted in the persistent renewal of neoantigens in vitro and in vivo, whereas MMR-proficient cells exhibited stable mutational load and neoantigen profiles over time. Immune surveillance improved when cancer cells, in which MLH1 had been inactivated, accumulated neoantigens for several generations. When restricted to a clonal population, the dynamic generation of neoantigens driven by MMR further increased immune surveillance. Inactivation of MMR, driven by acquired resistance to the clinical agent temozolomide, increased mutational load, promoted continuous renewal of neoantigens in human colorectal cancers and triggered immune surveillance in mouse models. These results suggest that targeting DNA repair processes can increase the burden of neoantigens in tumour cells; this has the potential to be exploited in therapeutic approaches.

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We thank B. Van Emburgh, A. Sogari, C. Falcomatà, V. Amodio and C. Cancelliere for assistance with in vitro experiments; F. Sassi and S. Giove for help with the immunohistochemistry; M. Buscarino for suggestions on MSI analysis; D. Cantarella for performing RNA-seq; G. Corti and L. Novara for bioinformatic analysis and comments; D. Sangiolo and M. Rescigno for discussion; D. Hananhan and K. Shchors for providing the PDAC cell model; and C. Torrance and J. Roix for scientific support, suggestions and critically reading the manuscript. This study was supported by European Community’s Seventh Framework Programme under grant agreement no. 602901 MErCuRIC (A.Bard. and G.G.); no. 635342-2 MoTriColor (A.Bard. and S.S.); IMI contract no. 115749 CANCER-ID (A.Bard.); AIRC 2010 Special Program Molecular Clinical Oncology 5 per mille, Project no. 9970 (A.Bard. and S.S.); AIRC IG no. 16788 (A.Bard.); AIRC IG no. 17707 (F.D.N.); Fondazione Piemontese per la Ricerca sul Cancro-ONLUS 5 per mille 2011 Ministero della Salute (A.Bard. and F.D.N.); AIRC-IG Grant no.15645 and Swiss National Science Foundation (SNSF), Sinergia Grant (CRSII3 160742/1) (E.G.); AIRC and the EU under a Marie Curie COFUND (G.G.); AIRC 3-year fellowship (G.S.); grant Terapia Molecolare Tumori by Fondazione Oncologia Niguarda Onlus (A.S.-B. and S.S.); and Merck grant for Oncology Innovation -GOI- 2016 (A.Bard.).

Author information


  1. Candiolo Cancer Institute – FPO, IRCCS, Candiolo 10060, Turin, Italy

    • Giovanni Germano
    • , Simona Lamba
    • , Giuseppe Rospo
    • , Ludovic Barault
    • , Alessandro Magrì
    • , Federica Maione
    • , Mariangela Russo
    • , Giovanni Crisafulli
    • , Alice Bartolini
    • , Giulia Lerda
    • , Giulia Siravegna
    • , Benedetta Mussolin
    • , Monica Montone
    • , Nabil Amirouchene-Angelozzi
    • , Silvia Marsoni
    • , Enrico Giraudo
    • , Federica Di Nicolantonio
    •  & Alberto Bardelli
  2. University of Turin, Department of Oncology, Candiolo 10060, Turin, Italy

    • Giovanni Germano
    • , Ludovic Barault
    • , Alessandro Magrì
    • , Mariangela Russo
    • , Giovanni Crisafulli
    • , Giulia Lerda
    • , Giulia Siravegna
    • , Federica Di Nicolantonio
    •  & Alberto Bardelli
  3. IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan 20156, Italy

    • Roberta Frapolli
    •  & Maurizio D’Incalci
  4. Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan 20133, Italy

    • Federica Morano
    • , Filippo de Braud
    •  & Filippo Pietrantonio
  5. Department of Oncology and Hemat-Oncology Università degli Studi di Milano, Milan 20122, Italy

    • Filippo de Braud
    •  & Salvatore Siena
  6. FIRC Institute of Molecular Oncology (IFOM), Milan 20139, Italy

    • Nabil Amirouchene-Angelozzi
  7. Policlinico Universitario A. Gemelli, Roma 00168, Italy

    • Armando Orlandi
  8. University of Torino, Department of Science and Drug Technology, Turin 10125, Italy

    • Enrico Giraudo
  9. Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan 20142, Italy.

    • Andrea Sartore-Bianchi
    •  & Salvatore Siena


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G.G. and A.Bard. conceived the study. G.G., A.M., F.Ma, R.F. and M.M. performed animal experiments. A.Bart., B.M., G.S. and M.M. performed DNA analyses and experiments. G.R. and G.C. conducted bioinformatics data analyses. M.R. performed cell-based drug screens. L.B. designed and performed the TMZ experiments on human cells. S.L. performed gene-editing experiments. G.G., G.L., A.M., E.G. and N.A.-A. conducted studies on mouse cells. F.Mo, F.D.N., A.O., F.d.B., F.P., S.S. and A.S.-B. designed and performed clinical studies, obtained tissue samples and performed patient data analysis. S.M. and M.D. were involved in data analysis. G.G. and A.Bard. wrote the manuscript. A.Bard. and F.D.N. supervised the study. All authors read and approved the final submitted manuscript

Competing interests

A.Bard. and G.G. are inventors on patent applications related to this research. The transfer of certain materials to third parties is subject to terms contained within license and intellectual property agreements held between PhoreMost Limited, the University of Turin, A.Bard. and G.G. A.Bard. is a shareholder of PhoreMost Limited. A.Bard. and G.G. are co-founders and shareholders of NeoPhore Limited; A.Bard is a member of the NeoPhore scientific advisory board. A.Bard. receives research funding from Merck related to this project. The other authors declare no competing financial interests.

Corresponding author

Correspondence to Alberto Bardelli.

Reviewer Information Nature thanks J. de Vries and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

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    Life Sciences Reporting Summary

  2. 2.

    Supplementary Figure 1

    This file contains uncropped western blot scans (size marker and protein identified are indicated).

Excel files

  1. 1.

    Supplementary Table 1 - List of predicted neoantigens in murine and human cell lines.

    As described in details in the M&M section, output calculations from exome data and RNAseq expression analyses were combined to generate predicted neoantigens. The lists (in excel format) reported here are based on the output from NetMHC4.0. The excel file incudes: gene, accession number, chromosome coordinates, nucleotide change, amino-acidic change, allele frequency, predicted antigenic peptide and number of different epitopes generated. The amount of generated epitopes (N. family) in each datasheet of the excel file indicates the total number of neoantigens (private, shared and common) present in the indicated time-point. For constitutively and transiently Cas9-expressing CT26 cells, time points correspond to those listed in Figure 3a. For RCM1, SW620 and SCKO1 the time points are described in Fig.4e. Whenever multiple peptides, obtained from the same variation, were identified, the best ranking ones are specified.

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