Protocol for in vivo CRISPR screening using selective CRISPR antigen removal lentiviral vectors.

Recognition of Cas9 and other proteins encoded in delivery vectors has limited CRISPR technology in vivo. Here, we present a protocol for genome engineering using selective CRISPR antigen removal (SCAR) lentiviral vectors in Renca mouse model. This protocol describes how to conduct an in vivo genetic screen with a sgRNA library and SCAR vectors that can be applied to different cell lines and contexts. For complete details on the use and execution of this protocol, please refer to Dubrot et al. (2021).1.

Targeting TBK1 to overcome resistance to cancer immunotherapy.

Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking1,2. Here we identify the innate immune kinase TANK-binding kinase 1 (TBK1)3 as a candidate immune-evasion gene in a pooled genetic screen4. Using a suite of genetic and pharmacological tools across multiple experimental model systems, we confirm a role for TBK1 as an immune-evasion gene. Targeting TBK1 enhances responses to PD-1 blockade by decreasing the cytotoxicity threshold to effector cytokines (TNF and IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumour models, with concordant findings in matched patient-derived organotypic tumour spheroids and matched patient-derived organoids. Tumour cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNF and IFNγ in a JAK-STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is an effective strategy to overcome resistance to cancer immunotherapy.

In vivo CRISPR screens reveal the landscape of immune evasion pathways across cancer.

The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1b/HLA-E. Surprisingly, loss of tumor interferon-γ (IFNγ) signaling sensitizes many models to immunity. The immune inhibitory effects of tumor IFN sensing are mediated through two mechanisms. First, tumor upregulation of classical MHC class I inhibits natural killer cells. Second, IFN-induced expression of Qa-1b inhibits CD8+ T cells via the NKG2A/CD94 receptor, which is induced by ICB. Finally, we show that strong IFN signatures are associated with poor response to ICB in individuals with renal cell carcinoma or melanoma. This study reveals that IFN-mediated upregulation of classical and non-classical MHC class I inhibitory checkpoints can facilitate immune escape.

Mutant IDH Inhibits IFNγ-TET2 Signaling to Promote Immunoevasion and Tumor Maintenance in Cholangiocarcinoma.

Isocitrate dehydrogenase 1 mutations (mIDH1) are common in cholangiocarcinoma. (R)-2-hydroxyglutarate generated by the mIDH1 enzyme inhibits multiple α-ketoglutarate-dependent enzymes, altering epigenetics and metabolism. Here, by developing mIDH1-driven genetically engineered mouse models, we show that mIDH1 supports cholangiocarcinoma tumor maintenance through an immunoevasion program centered on dual (R)-2-hydroxyglutarate-mediated mechanisms: suppression of CD8+ T-cell activity and tumor cell-autonomous inactivation of TET2 DNA demethylase. Pharmacologic mIDH1 inhibition stimulates CD8+ T-cell recruitment and interferon γ (IFNγ) expression and promotes TET2-dependent induction of IFNγ response genes in tumor cells. CD8+ T-cell depletion or tumor cell-specific ablation of TET2 or IFNγ receptor 1 causes treatment resistance. Whereas immune-checkpoint activation limits mIDH1 inhibitor efficacy, CTLA4 blockade overcomes immunosuppression, providing therapeutic synergy. The findings in this mouse model of cholangiocarcinoma demonstrate that immune function and the IFNγ-TET2 axis are essential for response to mIDH1 inhibition and suggest a novel strategy for potentiating efficacy. SIGNIFICANCE: Mutant IDH1 inhibition stimulates cytotoxic T-cell function and derepression of the DNA demethylating enzyme TET2, which is required for tumor cells to respond to IFNγ. The discovery of mechanisms of treatment efficacy and the identification of synergy by combined CTLA4 blockade provide the foundation for new therapeutic strategies. See related commentary by Zhu and Kwong, p. 604. This article is highlighted in the In This Issue feature, p. 587.

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

CRISPR-Cas9 genome engineering has increased the pace of discovery for immunology and cancer biology, revealing potential therapeutic targets and providing insight into mechanisms underlying resistance to immunotherapy. However, endogenous immune recognition of Cas9 has limited the applicability of CRISPR technologies in vivo. Here, we characterized immune responses against Cas9 and other expressed CRISPR vector components that cause antigen-specific tumor rejection in several mouse cancer models. To avoid unwanted immune recognition, we designed a lentiviral vector system that allowed selective CRISPR antigen removal (SCAR) from tumor cells. The SCAR system reversed immune-mediated rejection of CRISPR-modified tumor cells in vivo and enabled high-throughput genetic screens in previously intractable models. A pooled in vivo screen using SCAR in a CRISPR-antigen-sensitive renal cell carcinoma revealed resistance pathways associated with autophagy and major histocompatibility complex class I (MHC class I) expression. Thus, SCAR presents a resource that enables CRISPR-based studies of tumor-immune interactions and prevents unwanted immune recognition of genetically engineered cells, with implications for clinical applications.

Understanding and treatment of chronic abdominal pain in pediatric primary care.

This study examined the practices used by primary care pediatricians to assess and treat chronic abdominal pain (CAP), as an initial step in guiding clinical practice guideline (CPG) development. A survey was mailed to a random sample of office-based pediatrician members (primary care pediatricians [PCPs]) of the American Medical Association. PCPs (n = 470) provided information about the typical presentation of CAP, assessment/treatment approaches used in their own practice, their definition of a functional gastrointestinal disorder (FGID), and their familiarity with the Rome Criteria for diagnosing FGIDs. Substantial variability among PCPs was noted across all these areas. Results suggest that perceptions and practices of pediatric CAP vary widely among PCPs; no single standard of care emerged to guide development of a CPG for this population. Future research should evaluate the efficacy of specific strategies currently in use to identify potential opportunities for improving assessment and treatment of CAP in pediatric primary care.

Six degrees of separation: an exploratory network analysis of mentoring relationships in pediatric psychology.

OBJECTIVE: The present study is an exploratory social network analysis of mentee-mentor relationships in the field of pediatric psychology. METHOD: An online survey was distributed to members of the Division 54 Society of Pediatric Psychology (SPP) listserv asking them to name up to 10 psychologists from whom they had "received mentoring" and who influenced their careers. Directed network analyses were conducted to examine features of the resulting mentoring network. RESULTS: Participants reported receiving mentoring in a wide variety of relationships and settings. The average "degrees of separation" between individuals in the network was 5.30. CONCLUSION: : The field of pediatric psychology is interconnected with professionals learning from multiple mentors in multiple settings, extending beyond just graduate student advisors. Overall, many different mentors were listed, and there does not appear to be only one or two individuals providing the majority of mentoring within the field.

Perceived benefits of mentoring in pediatric psychology: a qualitative approach.

OBJECTIVE: To identify tangible and intangible benefits of mentoring cited by a select group of identified mentors. METHODS: Twenty frequently named mentors within pediatric psychology provided responses to open-ended questions regarding benefits they have experienced through the mentoring process. RESULTS: Mentors identified many personal and professional benefits of the mentoring relationship, although they did not clearly distinguish between tangible and intangible advantages to the relationship. The most commonly reported benefits included career development of the mentee, mentor's career enhancement, and a sense of giving back to the field of pediatric psychology. CONCLUSIONS: A bidirectional definition of mentoring more accurately describes the relationship than a more traditional unidirectional definition. These results suggest that mentors experience a wide variety of benefits that could be examined more closely within the field.