Extended Data Fig. 1: AC484 increases tumour cell sensitivity to IFNγ and immune cell activation in vitro.

a, IFNγ-induced growth inhibition of wildtype (WT) or Ptpn1-null or Ptpn2-null B16 tumour cell (n = 3 for each cell line). b, In vitro growth curve of control and Ptpn1-null (left) and Ptpn2-null (right) B16 cells ± AC484 (0.1 µM) and ± IFNγ (10 ng mL⁻¹) (n = 2). c, PCA of top 500 highly variable genes from in vitro transcriptional profiling of B16 control or Ptpn2/n1-null cells ± IFNγ and ± AC484. d, GSEA of Hallmark TNFα signaling, IFNγ and IFNα response gene signatures in control or Ptpn2/n1-null B16 cells treated with AC484 (10 μM) incubated overnight in the presence of IFNγ (10 ng mL⁻¹). e, Western blot for total and phosphorylated STAT1 protein in B16 cells that were stimulated for 16 h with IFNγ (100 ng mL⁻¹) ± AC484 and then washed out of IFNγ and incubated for 4 h in AC484 only (where applicable). f, CXCL10 (blue) and CXCL9 (orange) production by IFNγ-treated B16 tumour cells (n ≥ 4). g, B16-OVA tumour cells that were pretreated with IFNγ and AC484 overnight and incubated with OT-I T cells. Percent IFNγ (blue) or TNFα (orange) producing OT-I T cells (n = 4). h, Viability at increasing doses of AC484 + IFNγ for the human cell line collection screened with the PRISM pipeline. i, PRISM screen cell lines ranked by their sensitivity to AC484 with and without IFNγ (1 ng mL⁻¹). j, Heatmap showing viability across cancers from different tissue types in the PRISM platform against increasing doses of AC484 + IFNγ. k, PRISM screen cell lines ranked by their sensitivity to AC484 + IFNγ (1 ng mL⁻¹) (top). Tracks show cell lines with mutations in IFNGR1, IFNGR2, JAK, or STAT genes and the enrichment of these mutations in cell lines that are more or less sensitive to AC484 + IFNγ (bottom). FDR for enrichment of mutant cell lines shown to the right. l, Pearson correlation of gene expression in PRISM cell lines with sensitivity to AC484 alone (top) and GSEA of these correlations (bottom). m, Activation (Percent CD25-expressing CD8 T cells, n = 6) and TNFα production (n = 8) of anti-CD3/CD28 stimulated primary splenic pan T cells isolated from C57BL/6N mice. n, TNFα production of anti-CD3/CD28 stimulated primary splenic CD4 T cells (left, n = 4) and CD8 T cells (right, n = 4) isolated from C57BL6J mice. o, Western blot for total LCK and pLCK in naive CD8 T cells stimulated with crosslinked anti-CD3 (2.5 μg mL⁻¹) ± AC484 (20 μM) for 0, 5, 10, or 15 min. Quantification of pLCK expression normalized to total LCK at the right. p, Western blot for total FYN and pFYN in naive CD8 T cells stimulated with crosslinked anti-CD3 (2.5 μg mL⁻¹) ± AC484 (20 μM) for 0, 5, 10, or 15 min. Quantification of pFYN expression normalized to total FYN at the right. q, Representative histogram (left) and quantification (right) for CFSE-labeled naïve CD44⁺CD8⁺ T cells from WT mouse spleen sub-optimally stimulated with plate-bound anti-CD3 (1 μg mL⁻¹), soluble anti-CD28 (2 μg mL⁻¹), IL-2 (100 ng mL⁻¹), with or without AC484 (20 μM) for 96 h. T-cell proliferation (CFSE dilution) determined by flow cytometry. r-v, Representative raw data of AC484 human whole blood pathway engagement and immune activation. r, IFNγ-mediated STAT1 phosphorylation and IL-2-induced STAT5 phosphorylation in AC484-treated whole blood from one healthy donor assessed by flow cytometry. s, IFNγ-induced CXCL10 (IP10) production in whole blood from one healthy donor. t, IFNγ-induced STAT1 phosphorylation (pSTAT1) and CXCL10 (IP10) production in whole blood from healthy donors (left, n = 8) or cancer patients (right, CXCL10 n = 6, pSTAT1 n = 5). u, IFNγ and TNFα production following TCR stimulation with Cytostim^TM of whole blood from one healthy donor. v, Frequency of CD69-expressing T cells following TCR with Cytostim^TM of whole blood from one healthy donor.

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