Search

BackgroundAntitumor antibody, or targeted immunotherapy, has revolutionized cancer treatment and markedly improved patient outcomes. A prime example is the monoclonal antibody (mAb) trastuzumab, which targets human epidermal growth factor receptor 2 (HER2). However, like many targeted immunotherapies, only a subset of patients benefit from trastuzumab long-term. In addition to tumor-intrinsic factors, we hypothesize that host genetics may influence subsequent immune activation.MethodsTo model the human population, we produced F1 crosses of genetically heterogeneous Diversity Outbred (DO) mice with BALB/c mice (DOCF1). Distinct DOCF1 mice were orthotopically implanted with the BALB/c-syngeneic TUBO mammary tumor line, which expresses the HER2 ortholog rat neu. Treatment with anti-neu mAb clone 7.16.4 began once tumors reached ∼200 mm3. Genetic linkage and quantitative trait locus (QTL) effects analyses in R/qtl2 identified loci associated with tumor growth rates. Locus validation was performed with BALB/c F1 crosses with recombinant-inbred Collaborative Cross (CC) strains selected for therapy-associated driver genetics (CCxCF1). The respective roles of natural killer (NK) cells and macrophages were investigated by selective depletion in vivo. Ex vivo macrophage antibody-dependent phagocytosis (ADCP) assays were evaluated by confocal microscopy using 7.16.4-opsonized E2Crimson-expressing TUBO tumor cells.ResultsWe observed a divergent response to anti-tumor antibody therapy in DOCF1 mice. Genetic linkage analysis detected a locus on chromosome 10 that correlates to a robust response to therapy, which was validated in CCxCF1 models. Single-cell RNA sequencing of tumors from responder and non-responder models identified key differences in tumor immune infiltrate composition, particularly within macrophage (Mφ) subsets. This is further supported byex vivoanalysis showing Mφ ADCP capacity correlates toin vivotreatment outcomes in both DOCF1 and CCxCF1 models.ConclusionsHost genetics play a key regulatory role in targeted immunotherapy outcomes, and putative causal genes are identified in murine chromosome 10 which may govern Mφ function during ADCP.

Immune checkpoint inhibitors (ICI) for cancer therapy have improved outcomes for a variety of malignancies, however many patients fail to benefit. To date, most pre-clinical studies investigating the tumor microenvironment (TME) have utilized different spontaneous or implanted tumor lines to separately investigate immunologically cold or hot TMEs. These models are often in different mouse strains, leading to confounding variables and a disconnect as to whether the tumor or the host is contributing to the observed immunological phenotype. To account for host diversity and reduce variation on the part of the tumor, we have developed immunotherapy models using genetically heterogeneous Diversity Outbred (DO) and recombinant inbred Collaborative Cross (CC) mice. We crossed DO mice with C57BL/6 (B6) to generate DOB6F1 mice that reliably accept B6-syngeneic B16F0 tumors after subcutaneous inoculation. DOB6F1 mice (n=142) treated with combined anti-PD1/anti-CTLA-4 ICI on days 3, 6, and 10 after inoculation exhibited a wide variation in tumor latency, up to a maximum of 65 days, with 19 mice never developing tumor. Quantitative Trait Locus analysis revealed multiple loci influencing response to ICI. We utilized this data to challenge 12 CCB6F1 strains selected based on predicted response where ICI outcomes range from non-responsive to near complete response. In addition, we show evidence that the DOB6F1 model recreates acquired resistance to ICI, with 9 mice having an extremely delayed tumor latency (>40 days). Melanin-free regions were observed in 3/9 of these tumors, suggesting tumor editing. Acquired resistance was also noted 3/10 ICI-treated CC051B6F1 mice. Whole transcriptomic analysis compared tumors from non-responder versus delayed latency DOB6F1 mice. Despite implantation within genetically heterogeneous mice, transcriptomic profiles from late-onset tumors cluster together. Gene Set Enrichment Analysis identified immune processes, with antigen processing and presentation as the most significantly dysregulated gene set. Many of the upregulated genes in late-onset tumors are driven by IFNγ, suggesting IFNγ signaling may contribute to immune escape. We tested this directly by culturing B16 cells with IFNγ prior to inoculation. Short exposure of B16 to IFNγ results in aggressive growth regardless of treatment, but long exposure increases tumor immunogenicity and responsiveness to ICI in inbred B6 mice. Experiments in ICI-responsive CT26-bearing BALB/c mice indicate IFNγ signaling contributes to ICI response, as demonstrated by increased tumor burden after a single 250 µg dose of neutralizing anti-IFNγ antibody concurrent with ICI treatment. Collectively, our DO and CC F1 models allow for reduced tumor variation with a focus on the host and associated TME, and we show differential roles of IFNγ in response to ICI based on the timing of IFNγ exposure. Citation Format: Justin B. Hackett, James Glassbrook, Nasrin Movahhedin, Madeline Bross, Alicia Kevelin, Maria Múniz, Heather Gibson. Genetically heterogeneous mouse models identify IFNg signaling as a shared signature of acquired resistance to immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB065.

In land plants, plastid and mitochondrial RNAs are subject to post-transcriptional C-to-U RNA editing. T-DNA insertions in the ORGANELLE RNA RECOGNITION MOTIF PROTEIN6 gene resulted in reduced photosystem II (PSII) activity and smaller plant and leaf sizes. Exon coverage analysis of the ORRM6 gene showed that orrm6–1 and orrm6–2 are loss-of-function mutants. Compared to other ORRM proteins, ORRM6 affects a relative small number of RNA editing sites. Sanger sequencing of reverse transcription-PCR products of plastid transcripts revealed 2 plastid RNA editing sites that are substantially affected in the orrm6 mutants: psbF-C77 and accD-C794. The psbF gene encodes the β subunit of cytochrome b559, an essential component of PSII. The accD gene encodes the β subunit of acetyl-CoA carboxylase, a protein required in plastid fatty acid biosynthesis. Whole-transcriptome RNA-seq demonstrated that editing at psbF-C77 is nearly absent and the editing extent at accD-C794 was significantly reduced. Gene set enrichment pathway analysis showed that expression of multiple gene sets involved in photosynthesis, especially photosynthetic electron transport, is significantly upregulated in both orrm6 mutants. The upregulation could be a mechanism to compensate for the reduced PSII electron transport rate in the orrm6 mutants. These results further demonstrated that Organelle RNA Recognition Motif protein ORRM6 is required in editing of specific RNAs in the Arabidopsis (Arabidopsis thaliana) plastid.

Loss-of-function mutations in ORGANELLE RNA RECOGNITION MOTIF PROTEIN6 (ORRM6) result in the near absence of RNA editing of psbF-C77 and the reduction in accD-C794 editing in Arabidopsis (Arabidopsis thaliana). The orrm6 mutants have decreased levels of photosystem II (PSII) proteins, especially PsbF, lower PSII activity, pale green pigmentation, smaller leaf and plant sizes, and retarded growth. Stable expression of ORRM6 rescues the orrm6 editing defects and mutant phenotype. Unlike ORRM1, the other known ORRM plastid editing factor, ORRM6, does not contain RNA editing interacting protein/multiple organellar RNA editing factor (RIP/MORF) boxes, which are required for ORRM1 to interact with site-specific pentatricopeptide repeat protein editing factors. ORRM6 interacts with RIP1/MORF8, RIP2/MORF2, and RIP9/MORF9, known components of RNA editosomes. While some plastid RRM proteins are involved in other forms of RNA processing and translation, the primary function of ORRM6 is evidently to mediate psbF-C77 editing, like the essential site-specific pentatricopeptide repeat protein LOW PSII ACCUMULATION66. Stable expression in the orrm6 mutants of a nucleus-encoded, plastid-targeted PsbF protein from a psbF gene carrying a T at nucleotide 77 significantly increases leaf and plant sizes, chlorophyll content, and PSII activity. These transformants demonstrate that plastid RNA editing can be bypassed through the expression of nucleus-encoded, edited forms of plastid genes.