Figure 5 from The UBA1–STUB1 Axis Mediates Cancer Immune Escape and Resistance to Checkpoint Blockade
Authors: Bao Yi , Cruz Gabriel , Zhang Yuping , Qiao Yuanyuan , Mannan Rahul , Hu Jing , Yang Fan , Gondal Mahnoor , Shahine Miriam , Kang Sarah , Mahapatra Somnath , Chu Alec , Choi Jae Eun , Yu Jiali , Lin Heng , Miner Stephanie J. , Robinson Dan R. , Wu Yi-Mi , Zheng Yang , Cao Xuhong , Su Fengyun , Wang Rui , Hosseini Noshad , Cieslik Marcin , Kryczek Ilona , Vaishampayan Ulka , Zou Weiping , Chinnaiyan Arul M. 
Published: February 2025

<p>UBA1 inactivation upregulates interferon signaling via stabilization of JAK1. <b>A,</b> Hallmark pathways enriched by bulk RNA-seq of tumors with <i>Uba1</i> depletion (sg<i>Uba1</i>) versus control from the B16-BL6 (left) or Myc-CaP (right) subcutaneous tumor models. <b>B,</b> Hallmark pathways enriched by bulk RNA-seq of tumors with <i>Uba1</i> overexpression (OE) versus control (empty vector) from the Myc-CaP subcutaneous tumor model (left) or of Myc-CaP subcutaneous tumors in TAK-243–treated vs. control mice (right). <b>C,</b> Left: UMAP of pooled CD45<sup>+</sup> and CD45<sup>−</sup> cells from the indicated Myc-CaP tumors subjected to scRNA-seq. Clusters of malignant cells and leukocytes are shown. Right: hallmark pathways enriched by the scRNA-seq (shown in the left) of tumors with <i>Uba1</i> depletion (sg<i>Uba1</i>) versus control or tumors in TAK-243–treated vs. control mice. TAK-243 was administered via intravenous injection in <b>B</b> (right) and <b>C</b>. <b>D,</b> hallmark pathways enriched by bulk RNA-seq of Myc-CaP cells with <i>Uba1</i> depletion (sg<i>Uba1</i>) vs. control, with or without IFN-γ stimulation, or Myc-CaP cells treated with or without 50 nmol/L TAK-243 for 18 hours, and stimulated with or without IFN-γ. IFN-γ or IFN-α response pathways are highlighted in red in <b>A</b>–<b>D</b>. <b>E,</b> Surface expression of MHC-I measured by flow cytometry in Myc-CaP cells with <i>Uba1</i> depletion (sg<i>Uba1</i>) or UBA1 inhibition by 18 hours of 50 nmol/L TAK-243 treatment, in the presence or absence of IFN-γ stimulation. Nontargeting sgRNA or DMSO were used as controls, respectively. Data were acquired from biological triplicates. <b>F,</b> Surface expression of MHC-I measured by flow cytometry in GFP-labeled Myc-CaP tumor cells that were <i>Uba1</i> depleted or inactivated (<i>n</i> = 4 mice, per group). <b>G,</b> Mass spectrometry measuring protein abundance in Myc-CaP cells treated with 100 nmol/L TAK-243 for 4 hours and subsequently 50 µg/mL of cycloheximide (CHX) for an additional 6 hours. LFC, Log<sub>2</sub> fold change. <b>H,</b> CRISPR knockout screens with sgRNAs targeting genes that were robustly upregulated by TAK-243 in <b>G</b>, in Myc-CaP cells that received TAK-243 and IFN-γ co-treatment (left) or TAK-243 and IFN-β co-treatment (right). <b>I,</b> Immunoblot analysis assessing levels of the indicated proteins in Myc-CaP cells with <i>Uba1</i> depletion (sg<i>Uba1</i>) or 18 hours of 50 nmol/L TAK-243 treatment in the presence of IFN-γ stimulation. Nontargeting sgRNA or DMSO were used as controls, respectively. <b>J,</b> Left: immunoblot analysis assessing JAK1 expression in Myc-CaP cells that received knockout of <i>Jak1</i> (<i>Jak1</i> KO). Cells receiving nontargeting sgRNA were used as control. Right: surface expression of MHC-I measured by flow cytometry in the indicated cells treated with or without 50 nmol/L TAK-243 and stimulated with or without IFN-γ. Data were acquired from technical triplicates, representative of two independent experiments. <b>K,</b> Volumes of tumors derived from Myc-CaP cells established as in <b>J</b>, in mice treated with or without the combination (combo) of anti-PD-1 and TAK-243 (<i>n</i> = 5 mice, per group). Data in <b>J</b> and <b>K</b> are representative of two independent experiments with two distinct sgRNAs. IFN-γ stimulation was performed at 1 ng/mL for 18 hours. All immunoblot analysis was representative of two independent experiments. Data are presented as mean ± SEM. Statistics were acquired by the two-tailed Student <i>t</i> test in <b>E</b>, <b>F</b> (TAK-243 vs. DMSO), and <b>J</b>, or by two-way ANOVA in <b>F</b> (sg<i>Uba1</i> vs. control) and <b>K</b>. **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; n.s., not significant. MFI, mean fluorescent index.</p>