Defining mechanisms of immunotherapy resistance in DNA mismatch-repair deficient colorectal cancer mouse models

Primary supervisor: Sarah Martin, Queen Mary University of London

Secondary supervisor: Anthony Kong, King’s College London

Tertiary supervisor: Marco Gerlinger, Queen Mary University of London

Project

Mismatch repair deficient (MMRd) colorectal cancers (CRCs) have high mutation/neoantigen loads. Many respond to immunotherapy with PD1 or PD1+CTLA4 checkpoint inhibitors (CPIs) but primary or acquired resistance occurs in ~40-60% of cases (1,2). Genetic immune evasion (IE) mechanisms, either through loss of antigen presentation by class I MHC or defective interferon gamma (IFNγ) signalling, are common in these tumours (3). Impaired antigen presentation through B2M inactivation confers CPI resistance in other tumour types (4). Surprisingly, B2M loss neither correlated with CPI resistance in MMRd CRC patients nor in a CT26 CRC mouse model with engineered MMR deficiency (5). How resistance to CPI occurs in MMRd CRCs indeed remains unknown and predictive biomarkers for CPI treatment are lacking. Inactivating mutations in IFNγ-signalling pathway components (e.g. JAK1), are the second commonest genetic mechanism of IE in MMRd CRC3. How these influence CPI sensitivity has not been assessed. Prof Gerlinger’s team furthermore uses circulating tumour DNA (ctDNA) sequencing to reveal novel IE mechanisms that evolve at acquired CPI resistance in patients. This project will use innovative MMRd CRC mouse models to compare how mutations in B2M, the IFNγ-pathway, or in ctDNA-identified IE genes impact tumour immune landscapes and CPI responses. These mechanistic insights should inform the development of better biomarkers and immunotherapies.

Aim 1: Generation and characterization of MMRd cells with defective IFNγ-signalling and B2M mutations

We recently generated murine CRC CT26 MMR-deficient (MLH1-K/O) cell lines using CRISPR-Cas9 gene editing as described (Germano 2021). This project will use CRISPR-Cas9 to next inactivate JAK1 or B2M in MLH1-K/O cells. Knockouts will be extensively characterised with a range of assays (e.g. IFN-responsiveness, MHC and immunoregulatory molecule expression) to systematically assess the differential impact of JAK1 vs. B2M loss on immunological characteristics of MMRd cells.

Aim 2: Elucidating the effect of defective INFγ-signalling vs B2M loss on CPI treatment

The engineered CT26 cells (Aim 1) will be grown in syngeneic immuno-competent Balb/c mice to investigate in vivo how defective IFNγ-signalling influences immune contexture and CPI responses compared to models with B2M-loss or controls. Animals will be treated with isotype-control, PD1 or PD1+CTLA4 CPIs. Tumour volume and survival analyses will determine whether defective IFNγ-signalling and B2M differ in their response to specific ICBs (collaboration Dr Kong). The Vectra3 multiplex-immunofluorescence system and RNA-sequencing will be used to dissect differences in immune responses with/without treatment. This should also inform the development of rational immunotherapy combinations for testing in models that are resistant PD1/PD1+CTLA4 CPI.

Aim 3: Identification and mechanistic validation of drivers of acquired CPI resistance in CRC patient ctDNA

We are currently collecting ctDNA from MMRd CRC patients (clinicaltrials.gov: NCT03572192) to identify novel IE driver mutations that evolve at acquired CPI resistance. Identified mutations will be engineered into our MMRd CT26 model as described (AIM 1) to assess their mechanistic role in CPI resistance. This will reveal hitherto unknown resistance drivers.

Candidate background

The project is particularly suitable for candidates with a strong interest in immunology and translational cancer research who would like to pursue an academic career.

Potential Research Placements

1. Anthony Kong, King’s College London
2. Teresa Marafioti, UCL
3. Jane Sosabowski, Barts Cancer Institute, Queen Mary University of London

References

1. Andre et al. Pembrolizumab in Microsatellite-Instability-High Advanced Colorectal Cancer. The New England journal of medicine 2020; 383:2207 – 2218
2. Lenz et al. Nivolumab (NIVO) + low-dose ipilimumab (IPI) as first-line (1L) therapy in microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) metastatic colorectal cancer (mCRC): Two-year clinical update. Journal of Clinical Oncology 2020; 38:4040 – 4040.
3. Challoner et al. Genetic and immune landscape evolution defines subtypes of MMR deficient colorectal cancer. bioRxiv 2022; https://doi.org/10.1101/2022.02.16.479224
4. Sade-Feldman et al. Resistance to checkpoint blockade therapy through inactivation of antigen presentation. Nature Communications 2017; 8: 1136
5. Germano et al. CD4 T Cell-Dependent Rejection of Beta-2 Microglobulin Null Mismatch Repair-Deficient Tumors. Cancer Discovery 2021; 11:1844-1859