Oral Presentation 35th Lorne Cancer Conference 2023

METTL3 inhibition enhances anti-tumour immunity by way of a cell-intrinsic interferon response   (#27)

Andrew A Guirguis 1 2 3 , Yaara Ofir-Rosenfeld 4 , Kathy Knezevic 1 , Wesley Blackaby 4 , David Hardick 4 , Chih Chan 1 3 , Ali Motazedian 1 3 , Andrea Gillespie 1 , Dane Vassiliadis 1 3 , Enid Lam 1 3 , Kevin Tran 1 , Byron Andrews 4 , Michael Harbour 4 , Lina Vasiliauskaite 4 , Claire Saunders 4 , Georgia Tsagkogeorga 4 5 , Ewa Pilka 6 , Marie Carkill 7 , Laura MacPherson 1 3 , Elanor Wainwright 1 3 , Brian Liddicoat 1 3 , Benjamin Blyth 1 3 , Mark Albertella 4 , Oliver Rausch 4 , Mark A Dawson 1 2 3 8
  1. Peter MacCallum Cancer Institute, St Kilda, VICTORIA, Australia
  2. Clinical Haematology, Peter MacCallum Cancer Institute & Royal Melbourne Hospital, Melbourne, Victoria, Australia
  3. Sir Peter MacCallum Department of Oncology, Melbourne University, Parkville, VIC, Australia
  4. Storm Therapeutics, Cambridge, UK
  5. Milner Therapeutics Institute, University of Cambridge, Cambridge, UK
  6. Evotec UK Ltd, Abingdon, UK
  7. Charles River Laboratories, Portishead, UK
  8. Centre for Cancer Research, University of Melbourne, Melbourne, Vic, Australia
  • While immunotherapeutic approaches which enhance anti-tumour immunity have altered the natural course of a number of cancers – a significant proportion of cases exhibit primary resistance or partial responses1–3. As such, leveraging alternate non-overlapping mechanisms to increase the immunogenicity of cancer cells remains a priority.
  • N6-methyladenosine (m6A) is one of the most frequent modifications known to exist on RNA4. In this context, genetic modulation of METTL3, the methyltransferase responsible for laying down this modification has highlighted the role of m6A in regulating normal and malignant haematopoiesis5,6. Genetic ablation of METTL3 has also been associated with activation of endogenous retroviral elements7, viral immune evasion8–10 and formation of dsRNA11.
  • Using a novel class of RNA methyltransferase inhibitors targeting METTL312, we demonstrate that a global decrease in N6-methyladenosine (m6A) results in the formation of dsRNA and an associated cell-intrinsic interferon response by way of viral mimicry.
  • In association with loss of m6A, we show that as part of an interferon-stimulated response, genes associated with antigen presentation via MHC-I are increased and concurrently stabilised.
  • Using unbiased CRISPR screening approaches – we demonstrate the importance of dsRNA sensing and interferon signalling in the potentiation of T-cell killing of cancer cells following METTL3 inhibition.
  • Given PDL1’s known function as an interferon-stimulated gene13, we combine METTL3 inhibition with anti-PD1 therapy in vivo and demonstrate that the combination of therapies is more efficacious than either therapy alone.
  • To investigate the basis of the varied responses to therapy, we apply SPLINTR14, an expressed molecular barcoding strategy to our models and proceed to interrogate clonal diversity in the face of therapeutic pressure and identify transcriptional programs of resistant cells.
  • We demonstrate at single clone resolution that anti-PD1 therapy and METTL3 inhibition target distinct malignant clones and that the combination of therapies is capable of eradicating clones insensitive to the individual therapies alone.
  • Our data provides a molecular and pre-clinical rationale for using METTL3 inhibitors in the clinic to promote anti-tumour killing.
  1. Zaretsky, J. M. et al. Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma. New Engl J Medicine 375, 819–829 (2016).
  2. Gao, J. et al. Loss of IFN-γ Pathway Genes in Tumor Cells as a Mechanism of Resistance to Anti-CTLA-4 Therapy. Cell 167, 397-404.e9 (2016).
  3. Shin, D. S. et al. Primary Resistance to PD-1 Blockade Mediated by JAK1/2 Mutations. Cancer Discov 7, 188–201 (2017).
  4. Cantara, W. A. et al. The RNA modification database, RNAMDB: 2011 update. Nucleic Acids Res 39, D195–D201 (2011).
  5. Barbieri, I. et al. Promoter-bound METTL3 maintains myeloid leukaemia by m6A-dependent translation control. Nature 552, 126–131 (2017).
  6. Vu, L. P. et al. The N(6)-methyladenosine (m(6)A)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells. Nat Med 23, 1369–1376 (2017).
  7. Chelmicki, T. et al. m6A RNA methylation regulates the fate of endogenous retroviruses. Nature 1–5 (2021) doi:10.1038/s41586-020-03135-1.
  8. Lu, M. et al. N6-methyladeonisine modification enables viral RNA to escape recognition by RNA sensor. Nat Microbiol 5, 584–598 (2020).
  9. Lu, M. et al. Nonsegmented Negative-Sense RNA Viruses Utilize N 6 -Methyladenosine (m 6 A) as a Common Strategy To Evade Host Innate Immunity. J Virol 95, (2021).
  10. Winkler, R. et al. m6A modification controls the innate immune response to infection by targeting type I interferons. Nat Immunol 20, 173–182 (2018).
  11. Gao, Y. et al. m6A Modification Prevents Formation of Endogenous Double-Stranded RNAs and Deleterious Innate Immune Responses during Hematopoietic Development. Immunity (2020) doi:10.1016/j.immuni.2020.05.003.
  12. Yankova, E. et al. Small molecule inhibition of METTL3 as a strategy against myeloid leukaemia. Nature 1–8 (2021) doi:10.1038/s41586-021-03536-w.
  13. Garcia-Diaz, A. et al. Interferon Receptor Signaling Pathways Regulating PD-L1 and PD-L2 Expression. Cell Reports 19, 1189–1201 (2017).
  14. Fennell, K. A. et al. Non-genetic determinants of malignant clonal fitness at single-cell resolution. Nature 601, 125–131 (2022).