In vivo screens using a selective CRISPR antigen removal lentiviral vector system reveal immune dependencies in renal cell carcinoma

Dubrot, Juan; Lane-Reticker, Sarah Kate; Kessler, Emily; Ayer, Austin; Mishra, Gargi; Wolfe, Clara; Zimmer, Margaret D.; Du, Peter; Mahapatra, Animesh; Ockerman, Kyle M.; Davis, Thomas; Kohnle, Ian C.; Pope, Hans W.; Allen, Peter M.; Olander, Kira E.; Iracheta-Vellve, Arvin; Doench, John G.; Haining, W. Nicholas; Yates, Kathleen B.; Manguso, Robert T.

doi:10.1016/j.immuni.2021.01.001

Cited by 66 publications

(61 citation statements)

References 78 publications

(125 reference statements)

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“…This included STAT1, which is required to successfully relay IFN-g and IFN-a/b signals 64 (Figure 3A and B). Puzzlingly, the loss of receptors for IFN-g, IFNGR1 and IFNGR2 were found to reduce immune sensitivity of the queried tumors in vivo in the Manguso and Dubrot screens, whereas they enhanced sensitivity in the screen of Lawson and colleagues 3,5,6 (Figure 3C and D). Below, we will discuss for each of the major pathways identified, translational implications of the findings made in the genetic screens, while also offering a biological context (Figure 4).…”

Section: Common Hits Between the Different Screensmentioning

confidence: 99%

“…2 In recent years, we and others have used this powerful functional genetic screening approach to understand the process of tumor cell-intrinsic immune resistance. [3][4][5][6][7][8][9] This tool has proven its merit for the current challenges of immunotherapy, in particular that of immune checkpoint blockade (ICB). By antibody blockade of the inhibitory T-cell checkpoints programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), ICB can cause durable clinical responses for cancer patients with various tumor indications.…”

Section: Introductionmentioning

confidence: 99%

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The (re)discovery of tumor-intrinsic determinants of immune sensitivity by functional genetic screens

Vredevoogd¹,

Apriamashvili²,

Peeper

2021

Immuno-Oncology and Technology

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Functional genetic screens by CRISPR-Cas9 allow for the unbiased discovery of proteins causally involved in complex biological processes. In recent years, this approach has been used by multiple laboratories to uncover a range of tumor cell regulators determining immune sensitivity. In this review, we provide an overview of genetic screens carried out both in vitro and in vivo. By comparative analysis we highlight commonly identified proteins and pathways that are key in establishing tumor-intrinsic immune susceptibility. Together, these screens demonstrated the importance of the antigen presentation, interferon-g, tumor necrosis factor and autophagy pathways in governing sensitivity of tumor cells to immune attack. Moreover, they underline the complex interplay between tumor cells and their microenvironment, providing both fundamental and clinically relevant insights into the mechanisms of tumor immune resistance.

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Section: Common Hits Between the Different Screensmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The (re)discovery of tumor-intrinsic determinants of immune sensitivity by functional genetic screens

Vredevoogd¹,

Apriamashvili²,

Peeper

2021

Immuno-Oncology and Technology

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“…First, we generated all the knockout lines by electroporating the gRNA/Cas9 RNP directly into cells. This non-viral approach leaves no traces behind and does not permanently introduce foreign elements, such as Cas9 protein, antibiotic resistance marker or fluorescent protein, which can artificially enhance the immunogenicity of the cells expressing them 39 . In addition, IFNγ–a pleiotropic cytokine–is a double-edged sword that not only increases the immunogenicity of the tumours and primes them for growth inhibition, but also upregulates several inhibitory immune proteins 54,55 .…”

Section: Discussionmentioning

confidence: 99%

“…To study the molecular role of Stub1 , we used virus-free CRISPR-editing to delete Stub1 from an ICB-resistant and poorly immunogenic 37 murine melanoma line (B16-F10) by electroporating the corresponding crRNA/tracrRNA/Cas9 ribonucleoprotein (RNP) into the cells. This transient approach is favoured as recent reports have highlighted the capability of viral based approaches to artificially increase immunogenicity of syngeneic mouse lines 38,39 . After CRISPR editing, we isolated a total of nine Stub1 -null cells by single-cell subcloning (Supplementary Table 2).…”

Section: Introductionmentioning

confidence: 99%

STUB1 is an intracellular checkpoint for interferon gamma sensing

Ng¹,

Lim²,

Sim³

et al. 2020

Preprint

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Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published mouse model CRISPR screen suggested that targeting an E3 ligase (STUB1) involved with protein homeostasis, may overcome ICB resistance but the molecular basis behind this observation is unclear. Using the ICB-resistant and poorly immunogenic B16-F10 murine melanoma model, we reveal an under-appreciated role of STUB1 to dampen the interferon gamma (IFNγ) response. Deletion of Stub1 in tumour cells increased IFNGR1 abundance on cellular surface, thus lowering the stimulating threshold of IFNγ. These outcomes translated to IFNγ-enhanced antigen presentation and upregulation of the immunoproteasome complexes. Through proteomics and gene expression profiling, we confirmed STUB1 as a negative regulator of the IFNγ signaling pathway. To block the function of STUB1 in tumour cells, we stably expressed a rationally designed inhibitory biologic, which recapitulated the Stub1-null phenotypes in both murine and human tumour cells. Overall, our findings elucidate STUB1 as a barrier for IFNγ sensing and offer a roadmap to pursue STUB1 inhibitors, which may improve tumour response to checkpoint inhibitory therapy.

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“…In order to better understand cell-autonomous mechanisms that protect tumors from immune clearance, genome-wide CRISPR-Cas9 screens have been performed in melanoma, renal-, colorectal-and breast cancer cell lines. Together they identified the interferon-ɣ (IFNɣ) response, TNF-mediated NF B signaling and autophagy as core pathways involved in immune evasion across different cancer types [10][11][12][13][14][15][16][17][18] . However, to our knowledge, a comprehensive genetic analysis of potential target genes to enhance anti-tumor immunity in PDA is still missing.…”

Section: Introductionmentioning

confidence: 99%

Loss of Rnf31 and Vps4b sensitizes pancreatic cancer to T cell-mediated killing

Frey

Tortola

Egli

et al. 2021

Preprint

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Pancreatic ductal adenocarcinoma (PDA) is an inherently immune cell deprived tumor, characterized by desmoplastic stroma and suppressive immune cells. Here we systematically dissected PDA intrinsic mechanisms of immune evasion by in vitro and in vivo CRISPR screening, and identified Rnf31 and Vps4b as essential factors required for escaping CD8+ T cell-killing. Using murine PDA cells and human PDA organoids, we demonstrate that Rnf31 protects from TNF-mediated caspase 8 cleavage and subsequent apoptosis induction. For Vps4b we found that inactivation impairs autophagy, resulting in increased accumulation of CD8+ T cell-derived granzyme B and subsequent tumor cell lysis. Orthotopic transplantation of Rnf31- or Vps4b deficient pancreatic tumors, moreover, revealed increased CD8+ T cell infiltration and effector function, and markedly reduced tumor growth in mice. Our work uncovers vulnerabilities in PDA that might be exploited to render these tumors more susceptible to the immune system.

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In vivo screens using a selective CRISPR antigen removal lentiviral vector system reveal immune dependencies in renal cell carcinoma

Cited by 66 publications

References 78 publications

The (re)discovery of tumor-intrinsic determinants of immune sensitivity by functional genetic screens

The (re)discovery of tumor-intrinsic determinants of immune sensitivity by functional genetic screens

STUB1 is an intracellular checkpoint for interferon gamma sensing

Loss of Rnf31 and Vps4b sensitizes pancreatic cancer to T cell-mediated killing

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