. 2021 May;23(5):526-537.

doi: 10.1038/s41556-021-00672-3. Epub 2021 May 6.

LIMIT is an immunogenic lncRNA in cancer immunity and immunotherapy

Gaopeng Li^{1

2}, Ilona Kryczek^{1

2}, Jutaek Nam³, Xiong Li^{1

2}, Shasha Li^{1

2}, Jing Li^{1

2}, Shuang Wei^{1

2}, Sara Grove^{1

2}, Linda Vatan^{1

2}, Jiajia Zhou^{1

2}, Wan Du^{1

2}, Heng Lin^{1

2}, Ton Wang¹, Chitra Subramanian¹, James J Moon^{3

4}, Marcin Cieslik^{5

6}, Mark Cohen^{1

4

7}, Weiping Zou^{8

9

10

11

12}

Affiliations

¹ Department of Surgery, University of Michigan, Ann Arbor, MI, USA.
² Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA.
³ Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI, USA.
⁴ Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA.
⁵ Department of Computational Medicine & Bioinformatics, University of Michigan, Ann Arbor, MI, USA.
⁶ Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
⁷ Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA.
⁸ Department of Surgery, University of Michigan, Ann Arbor, MI, USA. wzou@med.umich.edu.
⁹ Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA. wzou@med.umich.edu.
¹⁰ Department of Pathology, University of Michigan, Ann Arbor, MI, USA. wzou@med.umich.edu.
¹¹ Graduate Programs in Immunology, University of Michigan, Ann Arbor, MI, USA. wzou@med.umich.edu.
¹² Tumor Biology, University of Michigan, Ann Arbor, MI, USA. wzou@med.umich.edu.

PMID: 33958760
PMCID: PMC8122078
DOI: 10.1038/s41556-021-00672-3

LIMIT is an immunogenic lncRNA in cancer immunity and immunotherapy

Gaopeng Li et al. Nat Cell Biol. 2021 May.

. 2021 May;23(5):526-537.

doi: 10.1038/s41556-021-00672-3. Epub 2021 May 6.

Authors

Affiliations

¹ Department of Surgery, University of Michigan, Ann Arbor, MI, USA.
² Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA.
³ Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI, USA.
⁴ Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA.
⁵ Department of Computational Medicine & Bioinformatics, University of Michigan, Ann Arbor, MI, USA.
⁶ Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
⁷ Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA.
⁸ Department of Surgery, University of Michigan, Ann Arbor, MI, USA. wzou@med.umich.edu.
⁹ Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA. wzou@med.umich.edu.
¹⁰ Department of Pathology, University of Michigan, Ann Arbor, MI, USA. wzou@med.umich.edu.
¹¹ Graduate Programs in Immunology, University of Michigan, Ann Arbor, MI, USA. wzou@med.umich.edu.
¹² Tumor Biology, University of Michigan, Ann Arbor, MI, USA. wzou@med.umich.edu.

PMID: 33958760
PMCID: PMC8122078
DOI: 10.1038/s41556-021-00672-3

Abstract

Major histocompatibility complex-I (MHC-I) presents tumour antigens to CD8⁺ T cells and triggers anti-tumour immunity. Humans may have 30,000-60,000 long noncoding RNAs (lncRNAs). However, it remains poorly understood whether lncRNAs affect tumour immunity. Here, we identify a lncRNA, lncRNA inducing MHC-I and immunogenicity of tumour (LIMIT), in humans and mice. We found that IFNγ stimulated LIMIT, LIMIT cis-activated the guanylate-binding protein (GBP) gene cluster and GBPs disrupted the association between HSP90 and heat shock factor-1 (HSF1), thereby resulting in HSF1 activation and transcription of MHC-I machinery, but not PD-L1. RNA-guided CRISPR activation of LIMIT boosted GBPs and MHC-I, and potentiated tumour immunogenicity and checkpoint therapy. Silencing LIMIT, GBPs and/or HSF1 diminished MHC-I, impaired antitumour immunity and blunted immunotherapy efficacy. Clinically, LIMIT, GBP- and HSF1-signalling transcripts and proteins correlated with MHC-I, tumour-infiltrating T cells and checkpoint blockade response in patients with cancer. Together, we demonstrate that LIMIT is a cancer immunogenic lncRNA and the LIMIT-GBP-HSF1 axis may be targetable for cancer immunotherapy.

PubMed Disclaimer

Conflict of interest statement

Competing Interest Declaration

W.Z. has served as a scientific advisor or consultant for NGM, Cstone, Oncopia, and Hengenix. All other authors declare no competing interests.

Figures

**Extended Data Fig. 1:**
LIMIT correlates to effector immune genes across multiple cancer types.

**Extended Data Fig. 2:**
Genetic loci and sequences of human LIMIT and mouse Limit.

**Extended Data Fig. 3:**
LIMIT augments MHC-1 expression

**Extended Data Fig. 4:**
LIMIT augments MHC-1 expression.

**Extended Data Fig. 5:**
LIMIT augments antigen-loaded MHC-1 expression *in vivo*.

**Extended Data Fig. 6:**
LIMIT cis-activates GBPs to boost MHC-1 and tumor immunity.

**Extended Data Fig. 7:**
GBPs activate HSF1 to stimulate MHC-I expression.

**Extended Data Fig. 8:**
HSF1 drives MHC-I expression and tumor immunity.

**Extended Data Fig. 9:**
HSF1 signaling genes correlated with MHC-I and tumor immunity.

**Extended Data Fig. 10:**
Scheme showing how LIMIT-GBP-HSF1 axis affects MHC-I and tumor immunity.

**Fig. 1:. LIMIT is an immunogenic lncRNA.**
a. Human melanoma samples (TCGA data set, SKCM, n = 472 patients) were divided into hot and cold tumors on the basis of CD8A transcripts. The volcano plots showed the fold change and p-value of 3926 lncRNA candidates in hot tumor (CD8A, top 10%) vs. cold tumor (CD8A, bottom 10%). P value by 2-sided t-test. **b-d**. Correlation of LIMIT with IFNG (b), MHC-I (c), or CD8 (d) in human melanoma patients (TCGA, SKCM). P value by 2-sided linear regression. **e-h**. Human melanoma samples (TCGA, SKCM) were divided into high (n = 236 patients) and low (n = 236 patients) LIMIT tumors. Gene set enrichment analysis showed the indicated gene signatures. P value by GSEA analysis. i. Cancer patients having received immune checkpoint blockade (ICB) therapy were divided into low and high LIMIT groups (bottom 15% vs top 15%). The response rates to ICB were calculated as the percentages of partial response (PR) plus complete response (CR). P value by Chi-square test. Patients were from 4 cohorts. j. Survival plot of melanoma patients (TCGA, SKCM). Patients were divided into high (n = 236 patients) and low (n = 236 patients) LIMIT groups. P value by 2-sided log-rank test. k. A375 cells were treated with indicated cytokines (5 ng/ml) for 24 hours. LIMIT was detected by Northern blotting. The 28S rRNA, 18S rRNA, and 5S rRNA are shown as loading controls.1 of 2 experiments is shown. **l-m**. 5’RACE and 3’RACE of human LIMIT (l) or murine Limit (m). 1 of 2 experiments is shown. n. A375 cells were treated with IFNγ for 24 hours. LIMIT was detected by RT-PCR in nuclear or cytoplasmic RNAs. The unspliced and mature ACTB were served as controls for nuclear and cytoplasmic RNAs, respectively. 1 of 2 experiments is shown. o. RPKM of LIMIT in different cancer cells in response to IFNγ. p. Wild type (WT) or STAT1 knockout (KO) A375 cells were treated with 5 ng/ml IFNγ for 24 hours. RNA levels of LIMIT were quantified by qRT-PCR. All Data are mean ± SD. n = 3 biological independent samples in (**o, p**). Source data are provided in Soure_data_Fig1.xlsx and Unmodified_blots_Fig1.pdf.

**Fig. 2:. LIMIT augments MHC-I expression.**
a. A375 shFluc or shLIMIT cells were treated with IFNγ for 24 hours. RNA levels of LIMIT were determined by qRT-PCR. P value by 2-sided t-test. b. A375 shFluc or shLIMIT cells were treated with IFNγ for the indicated time. Protein levels of phospho-STAT1 (p-Y701), STAT1, and GAPDH were determined by Western blotting. 1 of 2 experiments is shown. c. A375 shFluc or shLIMIT cells were treated with IFNγ for 48 hours. Surface expression of HLA-ABC were determined by flow cytometry (FACS). P value by 2-sided t-test. **d-g**. YUMM1.7 (**d, e**) or CT26 (**f, g**) cells carrying shFluc or shLimit were treated with IFNγ. RNA levels of Limit were determined 24 hours after treatment (**d, f**). Surface staining of MHC-I (H2-D^b) was detected 48 hours after treatment (**e, g**). P value by 2-sided t-test. **h-i**. A375 WT or LIMIT promoter deletion cells were treated with IFNγ. RNA levels of LIMIT were determined 24 hours after treatment (h). Surface expression of HLA-ABC were determined 48 hours after treatment (i). P value by 2-sided t-test. **j-k**. B16 cells were transfected with dCas9-VPR, together with non-targeting sgRNA (sgNT) or sgRNA targeting the promoter of Limit (sgLimit). RNA levels of Limit were determined 24 hours after treatment (j). Surface expression of MHC-I (H2-D^b) or PD-L1 were determined 48 hours after treatment (k). P value by 2-sided t-test. l. B16-OVA cells carrying shFluc or shB2m were manipulated with Limit CRISPRa (sgLimit) for 48 hours. Surface expression of OVA-H2K^b were determined by FACS. P value by 2-sided t-test. **m-n**. B16-OVA cells were manipulated with B2m knocking down (shB2m) or Limit CRISPRa (sgLimit), and co-cultured with OT-I cell at the ratio of 1:4. Cell death was measured by PI staining in CD45⁻ tumor cells. Dot plots (m) and statistical results (n) are shown. P value by 2-sided t-test. All data are mean ± SD. n = 3 biological independent samples in (**a, c, d, e, f, g, h, i, j, k, l, n**). Source data are provided in Soure_data_Fig2.xlsx and Unmodified_blots_Fig2.pdf.

**Fig. 3:. LIMIT enhances anti-tumor immunity.**
a. Tumor growth curve of YUMM1.7 shFluc or YUMM1.7 shLimit cells in NSG mice. n = 5 animals, P value by 2-sided t-test for tumor volume at end point. b. Tumor growth curve and tumor size of YUMM1.7 shFluc or YUMM1.7 shLimit cells inoculated in wild type C57BL/6 mice. n = 6 (shFluc) or 7 (shLimit) animals, P value by 2-sided t-test for tumor volume at end point. c. Tumor growth curve of CT26 shFluc or CT26 shLimit cells in wild type BALB/c mice. n = 7 animals, P value by 2-sided t-test for tumor volume at end point. d. Dot plots of intratumoral CD3⁺ T cells, IFNγ⁺ T cells, and TNFα⁺ T cells in YUMM1.7 shFluc or YUMM1.7 shLimit tumors. e. The percentages of intra-tumoral CD3⁺ T cells (of CD45⁺), IFNγ⁺ T cells, and TNFα⁺ T cells in YUMM1.7 shFluc or YUMM1.7 shLimit tumors. P value by 2-sided t-test. f. Tumor growth curve of B16 sgNT or B16 sgLimit (CRISPRa) cells in wild type C57BL/6 mice. n = 5 animals. P value by 2-sided t-test. g. Dot plots of intral-tumoral T cell population and activation in B16 sgNT or B16 sgLimit tumors. h. Intral-tumoral T cell population or activation in B16 sgNT or B16 sgLimit tumors. P value by 2-sided t-test. i. Tumor growth curve of B16 sgNT cells and B16 sgLimit cells in response to IgG or anti-PD-L1 antibody. n = 5 animals. All data are mean ± SD. n = 5 biological independent samples in (**e, h**). Source data are provided in Soure_data_Fig3.xlsx.

**Fig. 4:. LIMIT cis-activates GBPs to boost MHC-I and tumor immunity.**
a. A375 shFluc or shLIMIT cells were treated with IFNγ for 24 hours. RNA (a) or protein (b) levels of GBPs were determined. P value by 2-sided t-test. 1 of 3 blots is shown. c. B16 cells were manipulated with Limit CRISPRa for 24 hours. Gbp2 precursor and mature RNAs were determined. P value by 2-sided t-test. **d-e**. YUMM1.7 shFluc, shLimit, shGbp2 or shLimit+shGbp2 cells were treated with IFNγ. Gbp2 protein was detected 24 hours after treatment (d). Surface expression of MHC-I (H2-D^b) was measured 48 hours after treatment (e). 1 of 2 blots is shown. P value by 2-sided t-test. f. Tumor growth curves of YUMM1.7 shFluc, shLimit, shGbp2, or shLimit+shGbp2 cells inoculated in C57BL/6 mice. n = 5 animals, P value by 2-sided t-test for tumor volume at end point. g. Percentages of intral-tumoral CD8⁺ T cells or IFNγ⁺CD8⁺ T cells in YUMM1.7 tumors carrying shFluc, shLimit, shGbp2, or shLimit+shGbp2. n = 5 animals, P value by 2-sided t-test. h. MC38 shFluc or shGbp2 cells were treated with IFNγ . Surface staining of H2-D^b were determined 48 hours after treatment. P value by 2-sided t-test. i. Tumor growth curves of MC38 shFluc and MC38 shGbp2 cells. Tumor bearing mice were treated with IgG or anti-PD-L1 antibody. n = 6 (shFluc) or 7 (shGbp2) animals, P value by t-test for end point tumor volume. j. A375 cells were treated with indicated cytokines for 24 hours. Protein levels of GBP1-5 were determined. 1 of 2 experiments is shown. **k-m**. A375 WT or GBP1-5 KO cells were treated with IFNγ. GBP1-5 protein (k) and MHC-I-related gene transcripts (l) were determined 24 hours after treatment. MHC-I surface expression (m) were determined 48 hours after treatment. 1 of 3 blots is shown. P value by 2-sided t-test. All data are mean ± SD. n = 3 biological independent samples in (**a, c, e, h, l, m**). Source data are provided in Soure_data_Fig4.xlsx and Unmodified_blots_Fig4.pdf.

**Fig. 5:. GBPs activate HSF1 to stimulate MHC-I expression and tumor immunity.**
**a-c**. A375 cells were forced expression of GBPs. MHC-I RNA (a), surface expression (b) or total protein (c) levels were determined 24 hours (a) or 48 hours (b, c) afterwards. P value by 2-sided t-test. 1 of 2 blots is shown. d. MHC-I surface expression in YUMM1.7 or B16 cells upon Gbp2 overexpression. P value by 2-sided t-test. e. HSF1 chromatin IP for indicated gene promoters were performed in IFNγ-pretreated A375 cells. P value by 2-sided t-test. f. A375 cells were treated with 17-AAG to activate HSF1. Protein levels of HLA-ABC were determined 48 hours after treatment. 1 of 2 experiments is shown. g. A375 HSE-luc cells were forced expression of GBPs. Luciferase activity were detected 48 hours afterwards. P value by 2-sided t-test. h. A375 cells were forced expression of GBP1. Indicated proteins were determined 12 hours afterwards. 1 of 2 experiments is shown. i. A375 WT or GBPs KO cells were treated with IFNγ. Indicated proteins were determined 24 hours afterwards. 1 of 2 experiments is shown. j. A375 cells were forced expression of GBP1, and treated with KRIBB11. MHC-I surface expression was determined 48 hours afterwards. P value by 2-sided t-test. **k-l**. MC38 shFluc, shGbp2, shHsf1, or shGbp2+shHsf1 cells were treated with IFNγ. Indicated proteins (k) or MHC-I surface expression (l) were determined 48 hours afterwards. 1 of 2 experiments is shown. P value by 2-sided t-test. m. Tumor growth curves of MC38 shFluc, shGbp2, shHsf1, and shGbp2+shHsf1 cells in C57BL/6 mice. n = 5 animals, P value by 2-sided t-test for end point tumor volume. n. Percentages of intral-tumoral IFNγ⁺CD8⁺ T cells or TNFα⁺CD8⁺ T cells in MC38 tumors carrying shFluc, shGbp2, shHsf1, or shGbp2+shHsf1. n = 5 biological independent samples, P value by 2-sided t-test. All data are mean ± SD. n = 3 biological independent samples in (**a, b, d, e, g, j, l**). Source data are provided in Soure_data_Fig5.xlsx and Unmodified_blots_Fig5.pdf.

**Fig. 6:. LIMIT-GBP-HSF1 axis drives MHC-I and tumor immunity.**
a. Co-IP of GBP1-5 with HSP90 antibody in IFNγ-pretreated A375 cells. 1 of 3 experiments is shown. b. Co-IP of HSP90 with Flag antibody in Flag-GBP1-overexpressed A375 cells. * indicated the band shift of HSP90 upon GBP1 overexpression. 1 of 2 experiments is shown. c. Immunofluorescence staining of GBP1 and HSP90 in IFNγ-pretreated A375 cells. 1 of 4 images is shown. d. 293T cells were forced expression of Flag-HSF1 and increased doses of Flag-GBP1. Co-IP of HSF1 or GBP1 with HSP90 antibody were performed 24 hours afterwards. 1 of 2 experiments is shown. e. A375 cells were treated with HSP90 inhibitor, or forced expression of GBP1. Indicated proteins were detected 48 hours afterwards. 1 of 2 experiments is shown. **f-g**. A375 cells were treated with IFNγ and KRIBB11. RNA (f) or surface staining (g) levels of indicated genes were determined 48 hours afterwards. P value by 2-sided t-test. h. YUMM1.7 shFluc or shHsf1 cells were treated with IFNγ. Total protein (h) or surface expression (i) levels of indicated genes were determined 48 hours afterwards. 1 of 2 experiments is shown. P value by 2-sided t-test. **j-k**. Tumor growth curves of YUMM1.7 shFluc or shHsf1 cells in NSG mice (j) or wild type C57BL/6 mice (k). n = 5 (j) or 6 (k) animals, P value by 2-sided t-test for end point tumor volume. l. Percentages of CD3⁺, Ki67⁺, IFNγ⁺, and TNFα⁺ T cells in YUMM1.7 shFluc or shHsf1 tumors. n = 5 biological independent samples, P value by 2-sided t-test. m. A375 shFluc or shLIMIT cells were transfected with HSE-luc and PRL-SV40 overnight, and then treated with IFNγ for additional 48 hours. HSF1 transcriptional activity is depicted as the relative luciferase activity. P value by 2-sided t-test. n. B16 cells were manipulated with Limit CRISPRa and treated with KRIBB11. Surface expression of MHC-I (H2-D^b) was determined 48 hours afterwards. P value by 2-sided t-test. All data are mean ± SD. n = 3 biological independent samples in (**f, g, i, m, n**). Source data are provided in Soure_data_Fig6.xlsx and Unmodified_blots_Fig6.pdf.

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Comment in

LIMITing tumours with an immunogenic lncRNA.
Hou J, Peng W. Hou J, et al. Nat Cell Biol. 2021 May;23(5):443-445. doi: 10.1038/s41556-021-00682-1. Nat Cell Biol. 2021. PMID: 33958759 No abstract available.

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LIMIT is an immunogenic lncRNA in cancer immunity and immunotherapy

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LIMIT is an immunogenic lncRNA in cancer immunity and immunotherapy

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