Ligand-dependent hedgehog signaling maintains an undifferentiated, malignant osteosarcoma phenotype.

TP53 and RB1 loss-of-function mutations are common in osteosarcoma. During development, combined loss of TP53 and RB1 function leads to downregulation of autophagy and the aberrant formation of primary cilia, cellular organelles essential for the transmission of canonical Hedgehog (Hh) signaling. Excess cilia formation then leads to hypersensitivity to Hedgehog (Hh) ligand signaling. In mouse and human models, we now show that osteosarcomas with mutations in TP53 and RB1 exhibit enhanced ligand-dependent Hh pathway activation through Smoothened (SMO), a transmembrane signaling molecule required for activation of the canonical Hh pathway. This dependence is mediated by hypersensitivity to Hh ligand and is accompanied by impaired autophagy and increased primary cilia formation and expression of Hh ligand in vivo. Using a conditional genetic mouse model of Trp53 and Rb1 inactivation in osteoblast progenitors, we further show that deletion of Smo converts the highly malignant osteosarcoma phenotype to benign, well differentiated bone tumors. Conversely, conditional overexpression of SHH ligand, or a gain-of-function SMO mutant in committed osteoblast progenitors during development blocks terminal bone differentiation. Finally, we demonstrate that the SMO antagonist sonidegib (LDE225) induces growth arrest and terminal differentiation in vivo in osteosarcomas that express primary cilia and Hh ligand combined with mutations in TP53. These results provide a mechanistic framework for aberrant Hh signaling in osteosarcoma based on defining mutations in the tumor suppressor, TP53.

Adaptation of the intact proviral DNA assay to a nanowell-based digital PCR platform.

Quantification of intact proviruses is a critical measurement in HIV cure studies both in vitro and in vivo. The widely adopted 'intact proviral DNA assay' (IPDA), designed to discriminate and quantify genetically intact HIV proviruses based on detection of two HIV sequence-specific targets, was originally validated using Bio-Rad's droplet digital PCR technology (ddPCR). Despite its advantages, ddPCR is limited in multiplexing capability (two-channel) and is both labor- and time intensive. To overcome some of these limitations, we utilized a nanowell-based digital PCR platform (dPCR, QIAcuity from Qiagen) which is a fully automated system that partitions samples into nanowells rather than droplets. In this study we adapted the IPDA assay to the QIAcuity platform and assessed its performance relative to ddPCR. The dPCR could differentiate between intact, 5' defective and 3' defective proviruses and was sensitive to single HIV copy input. We found the intra-assay and inter-assay variability was within acceptable ranges (with coefficient of variation at or below 10%). When comparing the performance of the IPDA in ex vivo CD4+ T cells from people with HIV on antiretroviral therapy, there was a strong correlation in the quantification of intact (rs = 0.93; p < 0.001) and 3' defective proviruses (rs = 0.96; p < 0.001) with a significant but less strong correlation for 5' defective proviruses (rs = 0.7; p = 0.04). We demonstrate that the dPCR platform enables sensitive and accurate quantification of genetically intact and defective proviruses similar to the ddPCR system but with greater speed and efficiency. This flexible system can be further optimized in the future, to detect up to 5 targets, enabling a more precise detection of intact and potentially replication-competent proviruses.

Regional Analysis of Intact and Defective HIV Proviruses in the Brain of Viremic and Virally Suppressed People with HIV.

Here, we provide the first regional analysis of intact and defective HIV reservoirs within the brain. Brain tissue from both viremic and virally suppressed people with HIV (PWH) harbored HIV pol DNA in all regions tested, with lower levels present in basal ganglia and cerebellum relative to frontal white matter. Intact proviruses were primarily found in the frontal white matter but also detected in other brain regions of PWH, demonstrating frontal white matter as a major brain reservoir of intact, potentially replication competent HIV DNA that persists despite antiretroviral therapy. ANN NEUROL 2023;94:798-802.

Chronic immune activation and gut barrier dysfunction is associated with neuroinflammation in ART-suppressed SIV+ rhesus macaques.

HIV-associated neurocognitive disorders (HAND) affect ~40% of virally suppressed people with HIV (PWH), however, the precise viral dependent and independent changes to the brain are unclear. Here we characterized the CNS reservoir and immune environment of SIV-infected (SIV+) rhesus macaques during acute (n = 4), chronic (n = 12) or ART-suppressed SIV infection (n = 11). Multiplex immunofluorescence for markers of SIV infection (vRNA/vDNA) and immune activation was performed on frontal cortex and matched colon tissue. SIV+ animals contained detectable viral DNA+ cells that were not reduced in the frontal cortex or the gut by ART, supporting the presence of a stable viral reservoir in these compartments. SIV+ animals had impaired blood brain barrier (BBB) integrity and heightened levels of astrocytes or myeloid cells expressing antiviral, anti-inflammatory or oxidative stress markers which were not abrogated by ART. Neuroinflammation and BBB dysfunction correlated with measures of viremia and immune activation in the gut. Furthermore, SIV-uninfected animals with experimentally induced gut damage and colitis showed a similar immune activation profile in the frontal cortex to those of SIV-infected animals, supporting the role of chronic gut damage as an independent source of neuroinflammation. Together, these findings implicate gut-associated immune activation/damage as a significant contributor to neuroinflammation in ART-suppressed HIV/SIV infection which may drive HAND pathogenesis.

Non-Human Primate Models of HIV Brain Infection and Cognitive Disorders.

Human Immunodeficiency virus (HIV)-associated neurocognitive disorders are a major burden for people living with HIV whose viremia is stably suppressed with antiretroviral therapy. The pathogenesis of disease is likely multifaceted, with contributions from viral reservoirs including the brain, chronic and systemic inflammation, and traditional risk factors including drug use. Elucidating the effects of each element on disease pathogenesis is near impossible in human clinical or ex vivo studies, facilitating the need for robust and accurate non-human primate models. In this review, we describe the major non-human primate models of neuroHIV infection, their use to study the acute, chronic, and virally suppressed infection of the brain, and novel therapies targeting brain reservoirs and inflammation.

Intact HIV Proviruses Persist in the Brain Despite Viral Suppression with ART.

OBJECTIVE: Human immunodeficiency virus (HIV) persistence in blood and tissue reservoirs, including the brain, is a major barrier to HIV cure and possible cause of comorbid disease. However, the size and replication competent nature of the central nervous system (CNS) reservoir is unclear. Here, we used the intact proviral DNA assay (IPDA) to provide the first quantitative assessment of the intact and defective HIV reservoir in the brain of people with HIV (PWH). METHODS: Total, intact, and defective HIV proviruses were measured in autopsy frontal lobe tissue from viremic (n = 18) or virologically suppressed (n = 12) PWH. Total or intact/defective proviruses were measured by detection of HIV pol or the IPDA, respectively, through use of droplet digital polymerase chain reaction (ddPCR). HIV-seronegative individuals were included as controls (n = 6). RESULTS: Total HIV DNA was present at similar levels in brain tissues from untreated viremic and antiretroviral (ART)-suppressed individuals (median = 22.3 vs 26.2 HIV pol copies/106 cells), reflecting a stable CNS reservoir of HIV that persists despite therapy. Furthermore, 8 of 10 viremic and 6 of 9 virally suppressed PWH also harbored intact proviruses in the CNS (4.63 vs 12.7 intact copies/106 cells). Viral reservoirs in CNS and matched lymphoid tissue were similar in the composition of intact and/or defective proviruses, albeit at lower levels in the brain. Importantly, CNS resident CD68+ myeloid cells in virally suppressed individuals harbored HIV DNA, directly showing the presence of a CNS resident HIV reservoir. INTERPRETATION: Our results demonstrate the first evidence for an intact, potentially replication competent HIV reservoir in the CNS of virally suppressed PWH. ANN NEUROL 2022;92:532-544.

The role of oxidative stress in HIV-associated neurocognitive disorders.

HIV-associated neurocognitive disorders (HAND) are a leading cause of morbidity in up to 50% of individuals living with HIV, despite effective treatment with antiretroviral therapy (ART). Current evidence suggests that chronic inflammation associated with HIV is especially attributed to the dysregulated production of reactive oxygen species (ROS) that contribute to neurodegeneration and poor clinical outcomes. While ROS have beneficial effects in eliciting immune responses to infection, chronic ROS production causes damage to macromolecules such as DNA and lipids that has been linked to altered redox homeostasis associated with antioxidant dysregulation. As a result, this disruption in the balance between antioxidant-dependent mechanisms of ROS inactivation and ROS production by enzymes such as the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family, as well as from the electron transport chain of the mitochondria can result in oxidative stress. This is particularly relevant to the brain, which is exquisitely susceptible to oxidative stress due to its inherently high lipid concentration and ROS levels that have been linked to many neurodegenerative diseases that have similar stages of pathogenesis to HAND. In this review, we discuss the possible role and mechanisms of ROS production leading to oxidative stress that underpin HAND pathogenesis even when HIV is suppressed by current gold-standard antiretroviral therapies. Furthermore, we highlight that pathological ROS can serve as biomarkers for HIV-dependent HAND, and how manipulation of oxidative stress and antioxidant-dependent pathways may facilitate novel strategies for HIV cure.

Trp53 and Rb1 regulate autophagy and ligand-dependent Hedgehog signaling.

Ligand-dependent activation of Hedgehog (Hh) signaling in cancer occurs without mutations in canonical pathway genes. Consequently, the genetic basis of Hh pathway activation in adult solid tumors, such as small-cell lung cancer (SCLC), is unknown. Here we show that combined inactivation of Trp53 and Rb1, a defining genetic feature of SCLC, leads to hypersensitivity to Hh ligand in vitro, and during neural tube development in vivo. This response is associated with the aberrant formation of primary cilia, an organelle essential for canonical Hh signaling through smoothened, a transmembrane protein targeted by small-molecule Hh inhibitors. We further show that loss of both Trp53 and Rb1 disables transcription of genes in the autophagic machinery necessary for the degradation of primary cilia. In turn, we also demonstrate a requirement for Kif3a, a gene essential for the formation of primary cilia, in a mouse model of SCLC induced by conditional deletion of both Trp53 and Rb1 in the adult airway. Our results provide a mechanistic framework for therapeutic targeting of ligand-dependent Hh signaling in human cancers with somatic mutations in both TP53 and RB1.

Hedgehog Signaling in the Maintenance of Cancer Stem Cells.

Cancer stem cells (CSCs) represent a rare population of cells with the capacity to self-renew and give rise to heterogeneous cell lineages within a tumour. Whilst the mechanisms underlying the regulation of CSCs are poorly defined, key developmental signaling pathways required for normal stem and progenitor functions have been strongly implicated. Hedgehog (Hh) signaling is an evolutionarily-conserved pathway essential for self-renewal and cell fate determination. Aberrant Hh signaling is associated with the development and progression of various types of cancer and is implicated in multiple aspects of tumourigenesis, including the maintenance of CSCs. Here, we discuss the mounting evidence suggestive of Hh-driven CSCs in the context of haematological malignancies and solid tumours and the novel strategies that hold the potential to block many aspects of the transformation attributed to the CSC phenotype, including chemotherapeutic resistance, relapse and metastasis.