Tumor cell-released kynurenine biases MEP differentiation into megakaryocytes in individuals with cancer by activating AhR–RUNX1

Tumor-derived factors are thought to regulate thrombocytosis and erythrocytopenia in individuals with cancer; however, such factors have not yet been identified. Here we show that tumor cell-released kynurenine (Kyn) biases megakaryocytic–erythroid progenitor cell (MEP) differentiation into megakaryocytes in individuals with cancer by activating the aryl hydrocarbon receptor–Runt-related transcription factor 1 (AhR–RUNX1) axis. During tumor growth, large amounts of Kyn from tumor cells are released into the periphery, where they are taken up by MEPs via the transporter SLC7A8. In the cytosol, Kyn binds to and activates AhR, leading to its translocation into the nucleus where AhR transactivates RUNX1, thus regulating MEP differentiation into megakaryocytes. In addition, activated AhR upregulates SLC7A8 in MEPs to induce positive feedback. Importantly, Kyn–AhR–RUNX1-regulated MEP differentiation was demonstrated in both humanized mice and individuals with cancer, providing potential strategies for the prevention of thrombocytosis and erythrocytopenia.


Ethics oversight
Human protocol was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University(2019-KY-256), Women and Children's Hospital of Xiamen University(KY-2019-073) and Peking University People's Hospital (NKRDP2021005-EC-2).
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
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Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
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Sample size
Sample sizes were chosen based on the previous experience of the investigators with similar experimental models.No statistical methods were used to predetermine sample sizes but our sample sizes are similar to those reported in previous publications including those from our group (cancer cell. 2018;33(3):480-94;Blood. 2019;134 (18):1547-57).
Data exclusions No data were excluded from analysis.

Replication
Replicates were used in all experiments as noted in text, figure legends and methods.All experiments presented for which replication was attempted were successfully replicated.
Randomization For in vitro experiments, MEPs were divided equally to each group, then treated with drug agents or transduced with siRNA or lentivirus.

Wild animals
No Wild animals were used in this study

Reporting on sex
For colon cancer model,we using both male and female mice; For breast cancer model,we using female mice.
Field-collected samples No Field-collected samples were used in this study

Ethics oversight
These animals were maintained in the Animal Facilities of Chinese Academy of Medical Science under pathogen-free conditions.All studies involving mice were approved by the Animal Care and Use Committee of Chinese Academy of Medical Science.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry Plots
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Methodology Sample preparation
Preparation of single cell suspension from mouse bone marrow: bone marrow of hind limbs were harvested into a tissue culture dish with 5 mL PBS containing 0.5% FBS, then single-cell suspension were obtained by gently aspirating several times using the 1 mL syringe.Pass cell suspension through the cell strainer to eliminate clumps and debris, then centrifuge at 500 g for 5 minutes at 4°C to harvest the cell pellet, and repeat the wash step one time.Finally, re-suspend the cell pellet in PBS to the final concentration of 1 x 10^7 cells/mL and used for cell surface staining.Isolation of PBMC from human bone marrow or umbilical cord blood: Dilute the blood at 1:1 with PBS, underlay the diluted blood with the same volume of Ficoll.Then centrifuge at 400 g for 20 minutes at room temperature.

Instrument
Invitrogen Attune NxT and Sony MA900

Software
FlowJo software

Cell population abundance
The single cell suspension from bone marrow or umbilical cord blood were staining with mouse or human MEP surface mark, then cells were sorted in Sony MA900 with purity of priority.

Gating strategy
For all experiments, debris was first excluded by a morphology gate based on FSC-A and SSC-A.Then, non-singlets were eliminated from analysis by a single cell gate based on FSC-H and FSC-A.All gates were set based on FMO (full-minus one) stains and isotype control antibodies after appropriate compensation using single-stained compensation controls.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.
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.April 2023weeks tdTomato transgene mice were purchased from Shanghai Model Organisms Center (Shanghai, China).Ahr-/-mice were presented by Dr. Jun Yan (Third Military Medical University) and 6-8 weeks Ahr-/-mice were used for experiments.