Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

Cancer cells acquire genetic heterogeneity to escape from immune surveillance during tumor evolution, but a systematic approach to distinguish driver from passenger mutations is lacking. Here we investigate the impact of different immune pressure on tumor clonal dynamics and immune evasion mechanism, by combining massive parallel sequencing of immune edited tumors and CRISPR library screens in syngeneic mouse tumor model and co-culture system. We find that the core microRNA (miRNA) biogenesis and targeting machinery maintains the sensitivity of cancer cells to PD-1-independent T cell-mediated cytotoxicity. Genetic inactivation of the machinery or re-introduction of ANKRD52 frequent patient mutations dampens the JAK-STAT-interferon-γ signaling and antigen presentation in cancer cells, largely by abolishing miR-155-targeted silencing of suppressor of cytokine signaling 1 (SOCS1). Expression of each miRNA machinery component strongly correlates with intratumoral T cell infiltration in nearly all human cancer types. Our data indicate that the evolutionarily conserved miRNA pathway can be exploited by cancer cells to escape from T cell-mediated elimination and immunotherapy.


nature research | reporting summary
April 2020 For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
The raw FASTQ files and the source datasets generated and analyzed in this study for the sequencing data are available in the Genome Sequence Archive (GSA) database upon accession number (CRA004140, CRA004141, CRA004145, CRA004146). Descriptions of the analyses, tools and algorithms are provided in the Methods or GSA. Source data are provided with this paper.
For CRISPR screens, the mutagenized cell pool was guaranteed with at least 1000x coverage of the sgRNA library volume in every step of. Using these sample sizes allowed for the application of robust statistical assessments for each experiment or analysis. The sample sizes were chosen according to the convention in the field. Group sizes for in vivo validation experiments were selected empirically based upon prior knowledge of the intragroup variation of tumor challenges and immunotherapy treatment. For small-scale experiments, the number of replicates exceeds at least 3 technical replicates repeated measurements of the same original sample).
No data were excluded from any experiments and figures shown.
Replicates were used in all experiments as noted in text, figure legends and methods. For CRISPR screens, 10 sgRNAs per gene, timepoints, at least 5 tumors, high library coverage and sample correlations for each group were used for reproducibility improvement. All in vivo experiments were repeated at least twice with consonant results, with the exception of those that were supporting/confirmatory in nature and appear ONLY in Extended Data (e.g. SizeMatch experiment). All experiments presented for which replication was attempted were successfully replicated.
Mice were age-matched and randomized where appropriate (e.g. prior to initiating treatment for matched conditions). For in vitro experiments, cells are randomized planted for treatment and randomized collected.
No blinding was performed in the in vivo experiments due to requirements for cage labeling and staffing needs.
No blinding was performed in the in vitro experiments due to requirements for distinguishing gene-edited cells from control cells.

nature research | reporting summary
April 2020

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research 293FT (R70007) and 293T (/17) (CRL-11268) cell lines were originally purchased from Thermo Fisher and ATCC respectively and cultured according to the manufacturers' manual specifically. MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS) respectively. MC38-OVA cell lines were constructed by introducing cDNA of OVA peptide into plenti6.3 vector (Thermo Fisher, K533000). The cDNA sequences for OVA peptide (p.257-264, SIINFEKL) were synthesized at GENEWIZ. Blasticidin (Gibco, A1113903) selection was used for lentivirus transduced cells (MOI # 0.3) to generate OVA stably expressing cells.
None of the cell lines were independently authenticated.
All cell lines used in this study were tested as mycoplasma-negative using the Universal Mycoplasma Detection Kit (ATCC, 30-1012K). None.
C57BL/6J and Foxn1nu (nude) mice (female, aged at 6-8 weeks and weighing approximately 18-22g) were used for tumor implantation experiments and purchased from Shanghai SLAC Laboratory Animal. OT-I transgenic mouse was a gift from Bing Sun's Lab at SIBCB. OT-I transgenic mouse (male or female, aged at 6-10 weeks) were used to provide fresh CD8+ T cells.