Photodynamic therapy (PDT) eradicates tumors by the local activation of a photosensitizer with near-infrared light. One of the aspects hampering the clinical use of PDT is the poor selectivity of the photosensitizer. To improve this, we have recently introduced a new approach for targeted PDT by conjugating photosensitizers to nanobodies. Diverse G protein-coupled receptors (GPCRs) show aberrant overexpression in tumors and are therefore interesting targets in cancer therapy. Here we show that GPCR-targeting nanobodies can be used in targeted PDT. We have developed a nanobody binding the extracellular side of the viral GPCR US28, which is detected in tumors like glioblastoma. The nanobody was site-directionally conjugated to the water-soluble photosensitizer IRDye700DX. This nanobody-photosensitizer conjugate selectively killed US28-expressing glioblastoma cells both in 2D and 3D cultures upon illumination with near-infrared light. This is the first example employing a GPCR as target for nanobody-directed PDT. With the emerging role of GPCRs in cancer, this data provides a new angle for exploiting this large family of receptors for targeted therapies.

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein–coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gα_q, Gα_i, and Gα_s proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies.

The G protein–coupled receptor (GPCR) US28 encoded by the human cytomegalovirus (HCMV) is associated with accelerated progression of glioblastomas, aggressive brain tumors with a generally poor prognosis. Here, we showed that US28 increased the malignancy of U251 glioblastoma cells by enhancing signaling mediated by sphingosine-1-phosphate (S1P), a bioactive lipid that stimulates oncogenic pathways in glioblastoma. US28 expression increased the abundance of the key components of the S1P signaling axis, including an enzyme that generates S1P [sphingosine kinase 1 (SK1)], an S1P receptor [S1P receptor 1 (S1P ₁ )], and S1P itself. Enhanced S1P signaling promoted glioblastoma cell proliferation and survival by activating the kinases AKT and CHK1 and the transcriptional regulators cMYC and STAT3 and by increasing the abundance of cancerous inhibitor of PP2A (CIP2A), driving several feed-forward signaling loops. Inhibition of S1P signaling abrogated the proliferative and anti-apoptotic effects of US28. US28 also activated the S1P signaling axis in HCMV-infected cells. This study uncovers central roles for S1P and CIP2A in feed-forward signaling that contributes to the US28-mediated exacerbation of glioblastoma.

Human cytomegalovirus (HCMV) encodes four G protein-coupled receptor (GPCR) homologs. Three of these receptors, UL78, US27 and US28, are known for their roles in HCMV dissemination and latency. Despite importance of its rodent orthologs for viral replication and pathogenesis, such a function is not reported for the HCMV-encoded GPCR UL33. Using the clinical HCMV strain Merlin, we show that UL33 facilitates both cell-associated and cell-free virus transmission. A UL33-deficient virus derivative revealed retarded virus spread, formation of less and smaller plaques, and reduced extracellular progeny during multi-cycle growth analysis in fibroblast cultures compared to parental virus. The growth of UL33-revertant, US28-deficient, and US28-revertant viruses were similar to parental virus under multistep growth conditions. UL33- and US28-deficient Merlin viruses impaired cell-associated virus spread to a similar degree. Thus, the growth defect displayed by the UL33-deficient virus but not the US28-deficient virus reflects UL33’s contribution to extracellular transmission. In conclusion, UL33 facilitates cell-associated and cell-free spread of the clinical HCMV strain Merlin in fibroblast cultures.

The chemokine receptor CXCR4 is overexpressed in many cancers and contributes to pathogenesis, disease progression, and resistance to therapies. CXCR4 is known to form oligomers, but the potential functional relevance in malignancies remains elusive. Using a nanobody-based BRET method, we demonstrate that oligomerization of endogenous CXCR4 on lymphoid cancer cell lines correlates with enhanced expression levels. Specific disruption of CXCR4 oligomers reduced basal cell migration and prosurvival signaling via changes in the phosphoproteome, indicating the existence of constitutive CXCR4 oligomer-mediated signaling. Oligomer disruption also inhibited growth of primary CLL 3D spheroids and sensitized primary malignant cells to clinically used Bcl-2 inhibitor venetoclax. Given its limited efficacy in some patients and the ability to develop resistance, sensitizing malignant B cells to venetoclax is of clinical relevance. Taken together, we established a noncanonical and critical role for CXCR4 oligomers in lymphoid neoplasms and demonstrated that their selective targeting has clinical potential.

Objectives In childhood-onset SLE (cSLE), patients have an increased risk of premature atherosclerosis. The pathophysiological mechanisms for this premature atherosclerosis are not yet completely understood, but besides traditional risk factors, the endothelium plays a major role. The first aim of this study was to measure levels of SLE-associated markers involved in endothelial cell (EC) function and lipids in a cSLE cohort longitudinally in comparison with healthy controls (HC). Next aim was to correlate these levels with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) and nailfold capillaroscopic patterns. Methods Blood serum samples, videocapillaroscopy images and patient characteristics were collected in a multicentre longitudinal cSLE cohort and from age and sex comparable HC. Disease activity was evaluated by SLEDAI. A total of 15 EC markers and six lipids were measured in two longitudinal cSLE samples (minimum interval of 6 months) and in HC. Nailfold videocapillaroscopy images were scored according to the guidelines from the EULAR Study Group on Microcirculation in Rheumatic Diseases. Results In total, 47 patients with cSLE and 42 HCs were analysed. Median age at diagnosis was 15 years (IQR 12–16 years). Median time between t=1 and t=2 was 14.5 months (IQR 9–24 months). Median SLEDAI was 12 (IQR 6–18) at t=1 and 2 (IQR 1–4) at t=2. Serum levels of angiopoietin-2, CCL2, CXCL10, GAS6, pentraxin-3, thrombomodulin, VCAM-1 and vWF-A2 were elevated in cSLE compared with HC at t=1. While many elevated EC markers at t=1 normalised over time after treatment, several markers remained significantly increased compared with HC (angiopoietin-2, CCL2, CXCL10, GAS6, thrombomodulin and VCAM-1). Conclusion In serum from patients with cSLE different markers of endothelial activation were dysregulated. While most markers normalised during treatment, others remained elevated in a subset of patients, even during low disease activity. These results suggest a role for the dysregulated endothelium in early and later phases of cSLE, possibly also during lower disease activity. Trial registration number NL60885.018.17.