BackgroundA significant challenge to overcome in pancreatic ductal adenocarcinoma (PDAC) is the profound systemic immunosuppression that renders this disease non-responsive to immunotherapy. Our supporting data provide evidence that CD200, a regulator of myeloid cell activity, is expressed in the PDAC microenvironment. Additionally, myeloid-derived suppressor cells (MDSC) isolated from patients with PDAC express elevated levels of the CD200 receptor (CD200R). Thus, we hypothesize that CD200 expression in the PDAC microenvironment limits responses to immunotherapy by promoting expansion and activity of MDSC.MethodsImmunofluorescent staining was used to determine expression of CD200 in murine and human PDAC tissue. Flow cytometry was utilized to test for CD200R expression by immune populations in patient blood samples. In vivo antibody blocking of CD200 was conducted in subcutaneous MT-5 tumor-bearing mice and in a genetically engineered PDAC model (KPC-Brca2 mice). Peripheral blood mononuclear cells (PBMC) from patients with PDAC were analyzed by single-cell RNA sequencing. MDSC expansion assays were completed using healthy donor PBMC stimulated with IL-6/GM-CSF in the presence of recombinant CD200 protein.ResultsWe found expression of CD200 by human pancreatic cell lines (BxPC3, MiaPaca2, and PANC-1) as well as on primary epithelial pancreatic tumor cells and smooth muscle actin+ stromal cells. CD200R expression was found to be elevated on CD11b+CD33+HLA-DRlo/− MDSC immune populations from patients with PDAC (p=0.0106). Higher expression levels of CD200R were observed in CD15+ MDSC compared with CD14+ MDSC (p<0.001). In vivo studies demonstrated that CD200 antibody blockade limited tumor progression in MT-5 subcutaneous tumor-bearing and in KPC-Brca2 mice (p<0.05). The percentage of intratumoral MDSC was significantly reduced in anti-CD200 treated mice compared with controls. Additionally, in vivo blockade of CD200 can also significantly enhance the efficacy of PD-1 checkpoint antibodies compared with single antibody therapies (p<0.05). Single-cell RNA sequencing of PBMC from patients revealed that CD200R+ MDSC expressed genes involved in cytokine signaling and MDSC expansion. Further, in vitro cytokine-driven expansion and the suppressive activity of human MDSC was enhanced when cocultured with recombinant CD200 protein.ConclusionsThese results indicate that CD200 expression in the PDAC microenvironment may regulate MDSC expansion and that targeting CD200 may enhance activity of checkpoint immunotherapy.

Richter’s Transformation (RT) is a poorly understood and fatal progression of chronic lymphocytic leukemia (CLL) manifesting histologically as diffuse large B-cell lymphoma. Protein arginine methyltransferase 5 (PRMT5) is implicated in lymphomagenesis, but its role in CLL or RT progression is unknown. We demonstrate herein that tumors uniformly overexpress PRMT5 in patients with progression to RT. Furthermore, mice with B-specific overexpression of hPRMT5 develop a B-lymphoid expansion with increased risk of death, and Eµ-PRMT5/TCL1 double transgenic mice develop a highly aggressive disease with transformation that histologically resembles RT; where large-scale transcriptional profiling identifies oncogenic pathways mediating PRMT5-driven disease progression. Lastly, we report the development of a SAM-competitive PRMT5 inhibitor, PRT382, with exclusive selectivity and optimal in vitro and in vivo activity compared to available PRMT5 inhibitors. Taken together, the discovery that PRMT5 drives oncogenic pathways promoting RT provides a compelling rationale for clinical investigation of PRMT5 inhibitors such as PRT382 in aggressive CLL/RT cases.

Hematopoietic stem cells (HSCs) are functionally and genetically diverse and this diversity decreases with age and disease. Numerous systems have been developed to quantify HSC diversity by genetic barcoding, but no framework has been established to empirically validate barcode sequences. Here we have developed an analytical framework, Selection of informative Amplicon Barcodes from Experimental Replicates (SABER), that identifies barcodes that are unique among a large set of experimental replicates. Amplicon barcodes were sequenced from the blood of 56 adult zebrafish divided into training and validation sets. Informative barcodes were identified and samples with a high fraction of informative barcodes were chosen by bootstrapping. There were 4.2 ± 1.8 barcoded HSC clones per sample in the training set and 3.5 ± 2.1 in the validation set (p = 0.3). SABER reproducibly quantifies functional HSCs and can accommodate a wide range of experimental group sizes. Future large-scale studies aiming to understand the mechanisms of HSC clonal evolution will benefit from this new approach to identifying informative amplicon barcodes.

We currently have little understanding of the mechanisms by which hematopoietic stem and progenitor cells (HSPCs) gain a selective advantage in patients with clonal hematopoiesis and other myeloid neoplasms. The chemokine CXCL8 is elevated in a subset of patients with myeloid neoplasms. Our previous work in zebrafish has discovered a novel role for cxcl8 and its receptor, cxcr1, in supporting colonization of HSPCs within the sinusoidal endothelial cell niche of the embryonic zebrafish known as the caudal hematopoietic tissue (CHT). We hypothesized that mosaic overexpression of cxcl8 in a population of HSPCs during development would alter HSPC-niche interactions, selectively favor HSPCs expressing cxcl8 and lead to their expansion in adults. To test this hypothesis, we microinjected DNA constructs encoding cxcl8-2A-GFP or GFP alone under the control of the HSPC-specific Runx1+23 enhancer into zebrafish embryos at the single-cell stage. Time lapse fluorescence video microscopy and single-cell tracking was performed on HSPCs within the CHT. Overexpression of cxcl8 nearly doubled the amount of time HSPCs resided within the CHT when compared to expression of GFP alone as a control (cxcl8: 4.94 ± 0.86 h vs GFP: 2.54 ± 0.18 h, p=0.01, N=142 tracked cells). Substitution of WT cxcl8 with a mutant cxcl8 construct lacking the ELRCXC motif required for receptor binding reduced these effects (WT cxcl8: 6.6 ± 0.48 h vs ELRCXC-cxcl8: 5.3 ± 0.33 h, p=0.02, N=355 tracked cells). To observe HSPC-niche interactions, kdrl:mCherry endothelial cell reporter zebrafish were microinjected with Runx1+23:cxcl8-2A-GFP or Runx1+23:GFP DNA constructs. The percent of time individual HSPCs spent closely interacting with a single group of CHT endothelial cells (endothelial cell cuddling) was quantified over the period from 52 to 72 hours post-fertilization. Overexpression of cxcl8 by HSPCs increased HSPC-endothelial cell cuddling time by 30% (cxcl8: 87% vs GFP: 57%, p=0.001). To directly test competition between wild type and cxcl8 overexpressing HSPCs, zebrafish embryos were microinjected with a 1:1 molar ratio of Runx1+23:cxcl8-2A-mCherry and Runx1+23:clover DNA. Single cxcl8-2A-mCherry+ and clover+ competitor cells were tracked by time-lapse fluorescence confocal microscopy. HSPCs expressing cxcl8 resided longer within the CHT than competitor HSPCs when quantified over the period from 72 to 96 hours post-fertilization (cxcl8: 4.0 ± 0.20 h vs competitor: 2.5 ± 0.25 h, p=2.0 x 10-6, n=426 tracked cells). Single cell RNA-sequencing (scRNA-seq) of zebrafish embryos with mosaic expression of cxcl8 in HSPCs showed upregulation of cxcl12a in endothelial cells compared to endothelial cells from control embryos (p=5.19 x 10-3), suggesting a possible mechanism to explain the increased CHT residency time. Zebrafish with mosaic expression of Runx1+23:cxcl8 were raised to adulthood and the kidney marrow cells were analyzed by flow cytometry. Compared to clutchmate controls, Runx1+23:cxcl8 mosaic transgenics had a higher hematopoietic progenitor/precursor to lymphocyte ratio, suggesting a mild differentiation block and possible lineage skewing (cxcl8: 2.0 ± 0.15 vs control: 1.6 ± 0.10, p=0.048, N=25 animals). Taken together, these data support a model in which pre-malignant HSPC clones aberrantly express cxcl8 and acquire a selective advantage over normal clones through enhanced interactions with the endothelial cell niche. Disclosures Zon: Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership; CAMP4: Equity Ownership.

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