PVRL2 Suppresses Antitumor Immunity through PVRIG- and TIGIT-independent Pathways.

Poliovirus receptor-related 2 (PVRL2, also known as nectin-2 or CD112) is believed to act as an immune checkpoint protein in cancer; however, most insight into its role is inferred from studies on its known receptor, poliovirus receptor (PVR)-related immunoglobulin domain protein (PVRIG, also known as CD112R). Here, we study PVRL2 itself. PVRL2 levels were found to be high in tumor cells and tumor-derived exosomes. Deletion of PVRL2 in multiple syngeneic mouse models of cancer showed a dramatic reduction in tumor growth that was immune dependent. This effect was even greater than that seen with deletion of PD-L1. PVRL2 was shown to function by suppressing CD8+ T and natural killer cells in the tumor microenvironment. The loss of PVRL2 suppressed tumor growth even in the absence of PVRIG. In contrast, PVRIG loss showed no additive effect in the absence of PVRL2. T-cell immunoreceptor with Ig and ITIM domains (TIGIT) blockade combined with PVRL2 deletion resulted in a near complete block in tumor growth. This effect was not recapitulated by the combined deletion of PVRL2 with its paralog, PVR, which is the ligand for TIGIT. These data uncover PVRL2 as a distinct inhibitor of the antitumor immune response with functions beyond that of its known receptor PVRIG. Moreover, the data provide a strong rationale for combinatorial targeting of PVRL2 and TIGIT for cancer immunotherapy.

Mechanosensitive miR-100 coordinates TGFß and Wnt signaling in osteocytes during fluid shear stress.

Organism scale mechanical forces elicit cellular scale changes through coordinated regulation of multiple signaling pathways. The mechanisms by which cells integrate signaling to generate a unified biological response remains a major question in mechanobiology. For example, the mechanosensitive response of bone and other tissues requires coordinated signaling by the transforming growth factor beta (TGFß) and Wnt pathways through mechanisms that are not well-defined. Here we report a new microRNA-dependent mechanism that mediates mechanosensitive crosstalk between TGFß and Wnt signaling in osteocytes exposed to fluid shear stress (FSS). From 60 mechanosensitive microRNA (miRs) identified by small-RNAseq, miR100 expression is suppressed by in vivo hindlimb loading in the murine tibia and by cellular scale FSS in OCY454 cells. Though FSS activates both TGFß and Wnt signaling in osteocytes, only TGFß represses miR-100 expression. miR-100, in turn, antagonizes Wnt signaling by targeting and inhibiting expression of Frizzled receptors (FZD5/FZD8). Accordingly, miR-100 inhibition blunts FSS- and TGFß-inducible Wnt signaling. Therefore, our results identify FSS-responsive miRNAs in osteocytes, including one that integrates the mechanosensitive function of two essential signaling pathways in the osteoanabolic response of bone to mechanical load.

Fluid shear stress generates a unique signaling response by activating multiple TGFß family type I receptors in osteocytes.

Bone is a dynamic tissue that constantly adapts to changing mechanical demands. The transforming growth factor beta (TGFß) signaling pathway plays several important roles in maintaining skeletal homeostasis by both coupling the bone-forming and bone-resorbing activities of osteoblasts and osteoclasts and by playing a causal role in the anabolic response of bone to applied loads. However, the extent to which the TGFß signaling pathway in osteocytes is directly regulated by fluid shear stress (FSS) is unknown, despite work suggesting that fluid flow along canaliculi is a dominant physical cue sensed by osteocytes following bone compression. To investigate the effects of FSS on TGFß signaling in osteocytes, we stimulated osteocytic OCY454 cells cultured within a microfluidic platform with FSS. We find that FSS rapidly upregulates Smad2/3 phosphorylation and TGFß target gene expression, even in the absence of added TGFß. Indeed, relative to treatment with TGFß, FSS induced a larger increase in levels of pSmad2/3 and Serpine1 that persisted even in the presence of a TGFß receptor type I inhibitor. Our results show that FSS stimulation rapidly induces phosphorylation of multiple TGFß family R-Smads by stimulating multimerization and concurrently activating several TGFß and BMP type I receptors, in a manner that requires the activity of the corresponding ligand. While the individual roles of the TGFß and BMP signaling pathways in bone mechanotransduction remain unclear, these results implicate that FSS activates both pathways to generate a downstream response that differs from that achieved by either ligand alone.

LIN28B Impairs the Transition of hESC-Derived ß Cells from the Juvenile to Adult State.

Differentiation of human embryonic stem cells into pancreatic ß cells holds great promise for the treatment of diabetes. Recent advances have led to the production of glucose-responsive insulin-secreting cells in vitro, but resulting cells remain less mature than their adult primary ß cell counterparts. The barrier(s) to in vitro ß cell maturation are unclear. Here, we evaluated a potential role for microRNAs. MicroRNA profiling showed high expression of let-7 family microRNAs in vivo, but not in in vitro differentiated ß cells. Reduced levels of let-7 in vitro were associated with increased levels of the RNA binding protein LIN28B, a negative regulator of let-7 biogenesis. Ablation of LIN28B during human embryonic stem cell (hESC) differentiation toward ß cells led to a more mature glucose-stimulated insulin secretion profile and the suppression of juvenile-specific genes. However, let-7 overexpression had little effect. These results uncover LIN28B as a modulator of ß cell maturation in vitro.

Suppression of Exosomal PD-L1 Induces Systemic Anti-tumor Immunity and Memory.

PD-L1 on the surface of tumor cells binds its receptor PD-1 on effector T cells, thereby suppressing their activity. Antibody blockade of PD-L1 can activate an anti-tumor immune response leading to durable remissions in a subset of cancer patients. Here, we describe an alternative mechanism of PD-L1 activity involving its secretion in tumor-derived exosomes. Removal of exosomal PD-L1 inhibits tumor growth, even in models resistant to anti-PD-L1 antibodies. Exosomal PD-L1 from the tumor suppresses T cell activation in the draining lymph node. Systemically introduced exosomal PD-L1 rescues growth of tumors unable to secrete their own. Exposure to exosomal PD-L1-deficient tumor cells suppresses growth of wild-type tumor cells injected at a distant site, simultaneously or months later. Anti-PD-L1 antibodies work additively, not redundantly, with exosomal PD-L1 blockade to suppress tumor growth. Together, these findings show that exosomal PD-L1 represents an unexplored therapeutic target, which could overcome resistance to current antibody approaches.

High-throughput, Efficient, and Unbiased Capture of Small RNAs from Low-input Samples for Sequencing.

MicroRNAs hold great promise as biomarkers of disease. However, there are few efficient and robust methods for measuring microRNAs from low input samples. Here, we develop a high-throughput sequencing protocol that efficiently captures small RNAs while minimizing inherent biases associated with library production. The protocol is based on early barcoding such that all downstream manipulations can be performed on a pool of many samples thereby reducing reagent usage and workload. We show that the optimization of adapter concentrations along with the addition of nucleotide modifications and random nucleotides increases the efficiency of small RNA capture. We further show, using unique molecular identifiers, that stochastic capture of low input RNA rather than PCR amplification influences the biased quantitation of intermediately and lowly expressed microRNAs. Our improved method allows the processing of tens to hundreds of samples simultaneously while retaining high efficiency quantitation of microRNAs in low input samples from tissues or bodily fluids.

DGCR8 is essential for tumor progression following PTEN loss in the prostate.

In human prostate cancer, the microRNA biogenesis machinery increases with prostate cancer progression. Here, we show that deletion of the Dgcr8 gene, a critical component of this complex, inhibits tumor progression in a Pten-knockout mouse model of prostate cancer. Early stages of tumor development were unaffected, but progression to advanced prostatic intraepithelial neoplasia was severely inhibited. Dgcr8 loss blocked Pten null-induced expansion of the basal-like, but not luminal, cellular compartment. Furthermore, while late-stage Pten knockout tumors exhibit decreased senescence-associated beta-galactosidase activity and increased proliferation, the simultaneous deletion of Dgcr8 blocked these changes resulting in levels similar to wild type. Sequencing of small RNAs in isolated epithelial cells uncovered numerous miRNA changes associated with PTEN loss. Consistent with a Pten-Dgcr8 association, analysis of a large cohort of human prostate tumors shows a strong correlation between Akt activation and increased Dgcr8 mRNA levels. Together, these findings uncover a critical role for microRNAs in enhancing proliferation and enabling the expansion of the basal cell compartment associated with tumor progression following Pten loss.

miR-19, miR-345, miR-519c-5p serum levels predict adverse pathology in prostate cancer patients eligible for active surveillance.

Serum microRNAs hold great promise as easily accessible and measurable biomarkers of disease. In prostate cancer, serum miRNA signatures have been associated with the presence of disease as well as correlated with previously validated risk models. However, it is unclear whether miRNAs can provide independent prognostic information beyond current risk models. Here, we focus on a group of low-risk prostate cancer patients who were eligible for active surveillance, but chose surgery. A major criteria for the low risk category is a Gleason score of 6 or lower based on pre-surgical biopsy. However, a third of these patients are upgraded to Gleason 7 on post surgical pathological analysis. Both in a discovery and a validation cohort, we find that pre-surgical serum levels of miR-19, miR-345 and miR-519c-5p can help identify these patients independent of their pre-surgical age, PSA, stage, and percent biopsy involvement. A combination of the three miRNAs increased the area under a receiver operator characteristics curve from 0.77 to 0.94 (p<0.01). Also, when combined with the CAPRA risk model the miRNA signature significantly enhanced prediction of patients with Gleason 7 disease. In-situ hybridizations of matching tumors showed miR-19 upregulation in transformed versus normal-appearing tumor epithelial, but independent of tumor grade suggesting an alternative source for the increase in serum miR-19a/b levels or the release of pre-existing intracellular miR-19a/b upon progression. Together, these data show that serum miRNAs can predict relatively small steps in tumor progression improving the capacity to predict disease risk and, therefore, potentially drive clinical decisions in prostate cancer patients. It will be important to validate these findings in a larger multi-institutional study as well as with independent methodologies.

PML-RAR{alpha} and Dnmt3a1 cooperate in vivo to promote acute promyelocytic leukemia.

The PML-RARα oncogene is the central effector of acute promyelocytic leukemia (APL). PML-RARα physically interacts with epigenetic-modifying enzymes including DNA methyltransferases (Dnmt) to suppress critical downstream targets. Here, we show that increased expression of Dnmt3a1 cooperates with PML-RARα in vivo to promote early lethality secondary to myeloid expansion and dysfunction in primary mice. Bone marrow cells from these mice cause leukemogenesis with a shortened latency and a higher penetrance on transplantation into irradiated recipients. Furthermore, leukemic cells overexpressing PML-RARα and Dnmt3a1 display increased methylation at a target promoter compared with PML-RARα or Dnmt3a1 controls. Our findings show a cooperation between the PML-RARα oncogene and the Dnmt3a1 enzyme in vivo and that Dnmt levels can be rate limiting in APL progression.

Posttranscriptional crossregulation between Drosha and DGCR8.

The Drosha-DGCR8 complex, also known as Microprocessor, is essential for microRNA (miRNA) maturation. Drosha functions as the catalytic subunit, while DGCR8 (also known as Pasha) recognizes the RNA substrate. Although the action mechanism of this complex has been intensively studied, it remains unclear how Drosha and DGCR8 are regulated and if these proteins have any additional role(s) apart from miRNA processing. Here, we report that Drosha and DGCR8 regulate each other posttranscriptionally. The Drosha-DGCR8 complex cleaves the hairpin structures embedded in the DGCR8 mRNA and thereby destabilizes the mRNA. We further find that DGCR8 stabilizes the Drosha protein via protein-protein interaction. This crossregulation between Drosha and DGCR8 may contribute to the homeostatic control of miRNA biogenesis. Furthermore, microarray analyses suggest that a number of mRNAs may be downregulated in a Microprocessor-dependent, miRNA-independent manner. Our study reveals a previously unsuspected function of Microprocessor in mRNA stability control.