Development Of Kaposi's Sarcoma Research Articles

Abstract 1483: Sugar analogs inhibit KSHV replication by blocking N-glycosylation and inducing the unfolded protein response

Abstract Background: Kaposi´s Sarcoma (KS) herpesvirus (KSHV) is one of the seven known human oncogenic viruses. KSHV infection precedes the development of KS, an AIDS-associated malignancy. While the incidence of AIDS-KS globally has seen a decline, this endothelial cancer is refractory to treatment. The standard of care for AIDS-KS has gone virtually unchanged for over 20 years and effective treatments against the oncovirus KSHV have yet to be developed. 2-DG (2-deoxy-D-glucose), 2-FDG (2-fluoro-deoxy-D-glucose), and2-DFM (2-deoxy-2-fluoro-D-mannose) are sugar analogs. 2-DG and 2-FDG are D-glucose analogs and strong glycolysis inhibitors. 2-DG is also a D-mannose analog so that can inhibit N-glycosylation. In this work we aimed to further characterize and compare the inhibition of the three drug analogs on KSHV replication and pathogenesis. Design/Methods: We determined the ability of each drug to inhibit KSHV reactivation and infection by flow cytometry using the KSHV producer cell line iSLK.219 Virus titers were measured by de novo infection of naïve AdHEK293 cells and quantifying the number of AdHEK293 infected cells. We further characterized sugar analog inhibition assessing viral glycoprotein expression by western blot and quantifying virion production by qPCR. To show that N-glycosylation impairment is the main contributor of virus replication inhibition, we carried out D-mannose rescue experiments and analyzed activation of the unfolded protein response (UPR). When UPR cannot be resolved, it leads to either apoptosis or autophagy. For this reason, we analyzed the cellular fate of reactivated sugar analog-treated cells by western blot using apoptotic and autophagic markers. Additionally, we calculated the cell death index using the IncuCyte® machine. Results: All three sugar analogs negatively affected KSHV replication and virion infectivity, but 2-DFM displayed the strongest inhibition. Viral glycoproteins K8.1 and gB were down-regulated and virion production was diminished especially with 2-FDG and 2-DFM sugar analogs. D-mannose could rescue viral glycoprotein expression and virion infectivity, but could not reverse the number of reactivated cells, indicating that N-glycosylation is the main target of the drugs. In line with this, all analogs triggered UPR and even overcame KSHV-induced suppression of the PERK pathway. Finally, 2-DG, 2-FDG, and 2-DFM played a protective role in reactivated cells, displaying the autophagic marker instead of the apoptotic signature observed in the untreated cells. Conclusion: Collectively, this work identifies the non-toxic inhibition of N-glycosylation by sugar analogs as an important viral target and reinforces the potential of 2-DG as an antiviral for KSHV and other enveloped viruses. We also found that 2-DFM is a more potent and target-oriented compound and a promising therapeutic antiviral agent against an oncogenic virus. Citation Format: Mariana Schlesinger, Christian McDonald, Anuj Ahuja, Carolina Alvez-Canete, Zelmira Nunez Del Prado, Julian Naipauer, Theodore Lampidis, Enrique Mesri, Noula Shembade. Sugar analogs inhibit KSHV replication by blocking N-glycosylation and inducing the unfolded protein response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1483.

Soluble biomarkers of HIV-1-related systemic immune activation are associated with high plasma levels of growth factors implicated in the pathogenesis of Kaposi sarcoma in adults.

Human Herpesvirus-8 (HHV-8) is the etiologic agent of Kaposi's sarcoma (KS), a multicentric angio-proliferative cancer commonly associated with Human Immunodeficiency Virus (HIV) infection. KS pathogenesis is a multifactorial condition hinged on immune dysfunction yet the mechanisms underlying the risk of developing KS in HHV-8 seropositive adults remains unclear. Here we explored whether soluble markers of HIV-1-related systemic immune activation (SIA) and angiogenesis (VEGF and FGF acidic) are involved in the pathogenesis of KS in adults with HHV8. Blood samples from 99 HIV-1 infected and 60 HIV-1 uninfected adults were collected in Yaoundé, Cameroon. CD3+/CD4+ T cell counts and HIV-1 plasma viral load were determined using the Pima Analyzer and the RT-PCR technique, respectively. Plasma levels of SIA biomarkers (sCD163, sCD25/IL-2Rα, and sCD40/TNFRSF5) and biomarkers of progression to KS (VEGF and FGF acidic) were measured using the Luminex assay. Seropositivity (IgG) for HHV-8 was determined using the ELISA method. Overall, 20.2% (20/99) of HIV-1 infected and 20% (12/60) of HIV-1 uninfected participants were seropositive for HHV8. Levels of sCD163, sCD25/IL-2Rα, sCD40/TNFRSF5, and FGF acidic were higher in the HIV-1 and HHV8 co-infection groups compared to the HIV-1 and HHV8 uninfected groups (all P <0.05). In addition, Higher plasma levels of VEGF correlated with sCD163 (rs = 0.58, P =0.0067) and sCD40/TNFRSF5 (rs = 0.59, P = 0.0064), while FGF acidic levels correlated with sCD40/TNFRSF5 (rs = 0.51, P = 0.022) in co-infected. In HIV-1 mono-infected donors, VEGF and FGF acidic levels correlated with sCD163 (rs =0.25, P = 0.03 and rs = 0.30, P = 0.006 respectively), sCD25/IL-2Rα (rs = 0.5, P <0.0001 and rs = 0.55, P <0.0001 respectively) and sCD40/TNFRSF5 (rs = 0.7, P <0.0001 and rs = 0.59, P <0.0001 respectively) and even in patients that were virally suppressed sCD25/IL-2Rα (rs = 0.39, P = 0.012 and rs = 0.53, P = 0.0004 respectively) and sCD40/TNFRSF5 (rs = 0.81, P <0.0001 and rs = 0.44, P = 0.0045 respectively). Our findings suggest that although the development of KS in PLWH is multifactorial, HIV-associated SIA might be among the key drivers in coinfections with HHV8 and is independent of the patients' viremic status.

DP19 Two patients with Kaposi sarcoma from the 2-week-wait clinic

Abstract We present two cases of Kaposi sarcoma (KS) in HIV-negative men who have sex with men (MSM) with no coexisting immunosuppressive treatment or conditions. They both presented with suspicious lesions on lower extremities, with no other mucocutaneous involvement. The initial clinical presentation of both patients was atypical, with uncharacteristic aspects of lesions on clinical examination and dermoscopy, which prompted further investigations. Skin biopsies were performed for both patients. Both cases had similar histological findings in keeping with KS: slit-like vascular spaces within the dermis, proliferation of capillary blood vessels with associated inflammatory infiltrate with lymphocytes and plasma cells and focal aggregates of spindle cells. Immunohistochemistry revealed endothelial cells displaying positive staining for human herpesvirus (HHV)-8. Immunodeficiency screening revealed no abnormalities, and repeat HIV screening was negative in both patients. These cases present an interesting occurrence of an increasingly encountered new subtype of KS seen in HIV-seronegative immunocompetent MSM. In addition to the four established variants of KS, this is an emerging variant that arises mostly in middle-aged MSM. HHV-8 has been identified as the aetiological agent in KS; indeed, the viral infection precedes the development of the disease. The fifth subtype is increasingly recognized: nonepidemic KS in MSM who are HIV-negative and have no other causes for immunodeficiency, as seen in our two cases. This subtype has been reported in the literature since the 1980s in this specific population. The clinical presentation resembles classic KS, with predominantly cutaneous involvement of the lower limbs, running a similar indolent course. There is a strong connection between HHV-8 infection and the development of KS; however, in countries where there is a low prevalence of the virus, in the general population, there is increased detection in the MSM group. Correlating the high detection rate of HHV-8 in MSM with its role in the development in KS, immunocompetent MSM are at risk of developing the condition. However, this subtype carries an overall good prognosis, with an indolent course and good response to similar therapeutic options used in the classic subtype.

Variation within major internal repeats ofKSHV invivo.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), yet the viral genetic factors that lead to the development of KS in KSHV-infected individuals have not been fully elucidated. Nearly, all previous analyses of KSHV genomic evolution and diversity have excluded the three major internal repeat regions: the two origins of lytic replication, internal repeats 1 and 2 (IR1 and IR2), and the latency-associated nuclear antigen (LANA) repeat domain (LANAr). These regions encode protein domains that are essential to the KSHV infection cycle but have been rarely sequenced due to their extended repetitive nature and high guanine and cytosine (GC) content. The limited data available suggest that their sequences and repeat lengths are more heterogeneous across individuals than in the remainder of the KSHV genome. To assess their diversity, the full-length IR1, IR2, and LANAr sequences, tagged with unique molecular identifiers (UMIs), were obtained by Pacific Biosciences' single-molecule real-time sequencing (SMRT-UMI) from twenty-four tumors and six matching oral swabs from sixteen adults in Uganda with advanced KS. Intra-host single-nucleotide variation involved an average of 0.16 per cent of base positions in the repeat regions compared to a nearly identical average of 0.17 per cent of base positions in the remainder of the genome. Tandem repeat unit (TRU) counts varied by only one from the intra-host consensus in a majority of individuals. Including the TRU indels, the average intra-host pairwise identity was 98.3 per cent for IR1, 99.6 per cent for IR2 and 98.9 per cent for LANAr. More individuals had mismatches and variable TRU counts in IR1 (twelve/sixteen) than in IR2 (two/sixteen). There were no open reading frames in the Kaposin coding sequence inside IR2 in at least fifty-five of ninety-six sequences. In summary, the KSHV major internal repeats, like the rest of the genome in individuals with KS, have low diversity. IR1 was the most variable among the repeats, and no intact Kaposin reading frames were present in IR2 of the majority of genomes sampled.

Endemic Kaposi's Sarcoma.

Kaposi's sarcoma (KS) is a common neoplasm in Eastern and central Africa reflecting the spread of human gammaherpesvirus-8 (HHV-8), now considered a necessary causal agent for the development of KS. The endemic KS subtype can follow an aggressive clinical course with ulcerative skin lesions with soft tissue invasion or even bone or visceral involvement. In the latter cases, a thorough imaging work-up and better follow-up schedules are warranted. As KS is a chronic disease, the therapeutic goal is to obtain sustainable remission in cutaneous and visceral lesions and a good quality of life. Watchful monitoring may be sufficient in localized cutaneous forms. Potential therapeutic modalities for symptomatic advanced KS include systemic chemotherapies, immunomodulators, immune checkpoint inhibitors, and antiangiogenic drugs.

Genomic changes in Kaposi Sarcoma-associated Herpesvirus and their clinical correlates.

Kaposi sarcoma (KS), a common HIV-associated malignancy, presents a range of clinicopathological features. Kaposi sarcoma-associated herpesvirus (KSHV) is its etiologic agent, but the contribution of viral genomic variation to KS development is poorly understood. To identify potentially influential viral polymorphisms, we characterized KSHV genetic variation in 67 tumors from 1-4 distinct sites from 29 adults with advanced KS in Kampala, Uganda. Whole KSHV genomes were sequenced from 20 tumors with the highest viral load, whereas only polymorphic genes were screened by PCR and sequenced from 47 other tumors. Nine individuals harbored ≥1 tumors with a median 6-fold over-coverage of a region centering on K5 and K6 genes. K8.1 gene was inactivated in 8 individuals, while 5 had mutations in the miR-K10 microRNA coding sequence. Recurring inter-host polymorphisms were detected in K4.2 and K11.2. The K5-K6 region rearrangement breakpoints and K8.1 mutations were all unique, indicating that they arise frequently de novo. Rearrangement breakpoints were associated with potential G-quadruplex and Z-DNA forming sequences. Exploratory evaluations of viral mutations with clinical and tumor traits were conducted by logistic regression without multiple test corrections. K5-K6 over-coverage and K8.1 inactivation were tentatively correlated (p<0.001 and p = 0.005, respectively) with nodular rather than macular tumors, and with individuals that had lesions in ≤4 anatomic areas (both p≤0.01). Additionally, a trend was noted for miR-K10 point mutations and lower survival rates (HR = 4.11, p = 0.053). Two instances were found of distinct tumors within an individual sharing the same viral mutation, suggesting metastases or transmission of the aberrant viruses within the host. To summarize, KSHV genomes in tumors frequently have over-representation of the K5-K6 region, as well as K8.1 and miR-K10 mutations, and each might be associated with clinical phenotypes. Studying their possible effects may be useful for understanding KS tumorigenesis and disease progression.

Kaposi's sarcoma-associated herpes virus-derived microRNA K12-1 over-activates the PI3K/Akt pathway to facilitate cancer progression in HIV-related gastrointestinal Kaposi's sarcoma.

Kaposi's sarcoma-associated herpes virus (KSHV) initiate and accelerate the development of Kaposi's sarcoma (KS), and KSHV possesses many cancer-associated genes, including KSHV-derived microRNA miR-K12-1, which has been identified to be closely associated with KS progression. However, the detailed mechanisms by which miR-K12-1 facilitates HIV-related gastrointestinal KS development are still not fully delineated. This study strived to evaluate the effect of miR-K12-1 on the progression of HIV-related gastrointestinal KS. The expression levels of miR-K12-1 in HIV-related gastrointestinal KS tissues were determined by RT-qPCR. Proliferation and apoptosis were assessed by colony formation, CCK-8 and flow cytometry, respectively. The expression of all proteins was detected by Western blot. The in vivo effect of miR-K12-1 on the formation of a tumor was explored by using the mouse xenograft model. In this study, we uncovered that KSHV-miR-K12-1 was upregulated in HIV-related gastrointestinal KS tissues and associated with poor outcome in HIV-related gastrointestinal KS patients. Compared with the control group, after miR-K12-1 inhibitor transfection, BCBL-1 cell viability was decreased, and the cell apoptosis was significantly increased, whereas transfection of miR-K12-1 mimics promoted cell proliferation and mitosis. In addition, our rescuing experiments verified that miR-K12-1 promoted cell proliferation via activating the PI3K/Akt pathway, and inhibition of the PI3K/Akt pathway by LY294002 abrogated the tumor-promoting effects of miR-K12-1 in HIV-related gastrointestinal KS. In summary, we concluded that KSHV-derived miR-K12-1 activate the PI3K/Akt pathway to initiate and accelerate the development of KS, which convinces us that miR-K12-1 can be used as potential biomarkers for KS diagnosis, treatment and prognosis.

Kaposi's sarcoma, biologics and small molecules: Navigating the complex interplay between host immunity and viral biology. A case series with focused review of the literature.

The consequences of cytokine-specific immune modulation in the development and course of Kaposi's sarcoma (KS) are poorly understood. A retrospective chart review of patients treated with biologic/small molecule drugs and followed at the dedicated KS outpatient service of our Dermatology Unit was performed. The literature on biologic and small molecule drug use in KS patients was also reviewed. Data concerning 12 KS patients treated with biologic/small molecule drugs were collected. After a median delay of 6months following biologic or small molecule drug introduction, nine patients experienced either KS onset or reactivation. Drugs associated with KS onset or flaring were: rituximab, infliximab, ruxolitinib apremilast (1), mirikizumab, abatacept (1). After a median follow-up of 25 months, all cases achieved persistent complete response through culprit drug discontinuation or drug withdrawal plus treatment. No effect on KS course was recorded with tocilizumab and vedolizumab. Based on our experience with the largest case series reported to date as well as the available literature, tocilizumab and ustekinumab seem to exert an overall neutral effect on KS. On the other hand, rituximab, infliximab, and ruxolitinib have been associated with the development or worsening of pre-existing KS and should be carefully pondered before use. Due to limited and partly controversial evidence, no definitive conclusions can be drawn on vedolizumab, apremilast, mirikizumab, abatacept.

A UNIQUE PRESENTATION OF PULMONARY KAPOSI SARCOMA IN A YOUNG PATIENT WITH CONCOMITANT CMV

TOPIC: Chest Infections TYPE: Medical Student/Resident Case Reports INTRODUCTION: Kaposi sarcoma (KS) is an AIDS-defining illness characterized by low-grade vascular tumors thought to be caused by HHV-8. Commonly described features include raised purplish skin nodules millimeters to centimeters in size on the face, groin, and lower extremities (1). Less discussed are the visceral manifestations and the co-infections which may contribute to the presentation. Our case demonstrates a young male with pulmonary KS confirmed by imaging and bronchoscopy with biopsy found to have co-infection with CMV. CASE PRESENTATION: A 29-year-old male with AIDS-related cutaneous Kaposi sarcoma (diagnosed 11/2020) presented with dyspnea, cough, hemoptysis, and lower extremity edema in 2/2021. On arrival, vitals were stable with SpO2 98% on room air. Admission labs revealed CD4 of 17, HIV viral load <30, WBC 7.4 with 1% atypical lymphocytes, AST/ALT 101/84, and negative testing for SARS-CoV-2 and influenza A/B. Chest X-ray on admission showed patchy bilateral airspace disease. CTA chest showed bilateral peribronchovascular flame-shaped ground glass opacities, bilateral pulmonary nodules, mediastinal and axillary lymphadenopathy, and moderate bilateral pleural effusions. Right thoracentesis was performed with removal of 900mL of serosanguinous fluid exudative by Light's criteria. Subsequent diagnostic bronchoscopy showed areas of thickened erythematous mucosa with predominance in the bilateral upper lobes. Bronchoalveolar lavage (BAL) revealed CMV level of 224 IU/mL without additional infectious agents. Transbronchial and endobronchial biopsy revealed neoplastic cells positive for CD31 and HHV-8, confirming pulmonary parenchymal and endobronchial KS. Patient continued with HAART and was started on liposomal doxorubicin. DISCUSSION: This case describes a young male with AIDS and cutaneous KS who presented with productive cough, dyspnea, and hemoptysis found to have pulmonary KS. An interesting aspect of this case is the possible contribution of CMV co-infection. In addition to BAL findings, imaging was consistent with aspects of both KS and CMV including flame shaped GGOs and pulmonary nodules, respectively (2,3). Also demonstrated were labs consistent with CMV, including atypical lymphocytes and moderately elevated AST/ALT (6). Interestingly, there are publications from as early as 1980 suggesting that CMV may contribute to the development of KS by working synergistically with HHV-8 (4,5). A 2009 study even went on to postulate that a particular CMV locus can activate the full KS-associated herpesvirus lytic replication cycle (7). CONCLUSIONS: Our case presents a patient with KS and CMV co-infection. CMV may directly or indirectly influence the presentation of respiratory illness in co-infected patients. Though some work has been done already, future studies should work to further elucidate the exact association between KS and CMV in AIDS patients. REFERENCE #1: Reference #1: Aboulafia D.M. The epidemiologic, pathologic, and clinical features of AIDS-associated pulmonary Kaposi's sarcoma. Chest. 2000;117(4):1128–1145.Reference #2: Restrepo C et al. Imaging manifestations of Kaposi sarcoma. Radiographics. 2006;99(4).Reference #3: McGuinness G et al. Cytomegalovirus pneumonitis: spectrum of parenchymal CT findings with pathologic correlation in 21 AIDS patients. Radiology. 1994;192(2). REFERENCE #2: Reference #4: Giraldo G, Beth E, Huang E. Kaposi's sarcoma and its relationship to cytomegalovirus (CMNV). III. CMV DNA and CMV early antigens in Kaposi's sarcoma. Int J Cancer. 1980;26(1).Reference #5: Goldman, J.M., Epstein M.A. Vaccine intervention against virus-induced tumors. Palgrave Macmillan, London;c1986. 93-103p.Reference #6: Goncalves C et al. Cytomegalovirus acute infection with pulmonary involvement in an immunocompetent patient. ID Cases. 2018;14. REFERENCE #3: Reference #7: Wells R, Stensland L, Vieira J. The human cytomegalovirus UL112-113 locus can activate the full kaposi's sarcoma-associated herpesvirus lytic replica ion cycle. Journal of Virology. Apr 2009;83(9) 4695-4699p. DISCLOSURES: No relevant relationships by Jacob Patrick, source=Web Response No relevant relationships by Yiseiry Perez-Melendez, source=Web Response

Expression of CD40L on CD4+T cells distinguishes active versus inactive HIV-associated Kaposi's Sarcoma

Kaposi's sarcoma (KS) is a malignancy of vascular origin. It is caused by the Kaposi's sarcoma-associated herpes virus (KSHV). Immune dysregulation is a key feature in the development and progression of KS. The main aim of this study was to determine and compare circulating CD4+ and CD8+T cell subsets including their expression of CD40 ligand (CD40L) and programmed cell death protein 1 (PD1), natural killer (NK) cells, and NK T cells between individuals with active HIV-associated KS versus those in remission. We found that the proportion of CD4+T cells was significantly higher in individuals in remission compared to those with active KS (26.3% vs 13.9%; p = 0.01). We also observed that the proportion of CD4+T cells and central memory CD4+T cells expressing CD40L was significantly higher in individuals with active KS versus those in remission, (10.6% vs 5.4%; p = 0.03) and (14.8% vs 5.9%; p = 0.01) respectively. There was no significant difference in proportion of CD4+ and CD8+ naïve, central memory, effector memory, and terminal effector cells between the two groups. In addition, there was no difference in expression of PD1 on the T cell subsets between the two groups. Furthermore, the proportion of NK cells and NK T cells were not differential between individuals with active disease versus those in remission. CD40L expression is higher in individuals with active HIV-associated KS compared to those in remission. The proportion of CD4+T cells is higher in individuals in remission compared to those with active HIV-associated KS.

Reactivation and Persistence of Human Herpesvirus-8 Infection in B Cells and Monocytes by Th-1 Cytokines Increased in Kaposi's Sarcoma

AbstractPatients with Kaposi's sarcoma (KS) have a human herpesvirus-8 (HHV-8) load higher than patients without KS and present a CD8+ T-cell activation with production of Th1-type cytokines both in tissues and peripheral blood mononuclear cells (PBMC). Because in tissues of KS patients detection of inflammatory cytokines (IC) can precede detection of HHV-8 DNA and because signs of immunoactivation and/or dysregulation can precede KS development, we investigated the effect of IC on HHV-8 infection. To achieve this goal, PBMC and purified cell populations from 45 patients with KS and 45 patients at risk of KS were analyzed for HHV-8 DNA and/or gene expression and for cell survival, growth, and phenotype before or after culture with or without the IC increased in KS. The results indicate that PBMC that are polymerase chain reaction (PCR)-positive at day 0 generally loose the virus upon culture. However, the presence of IC maintains HHV-8 DNA load in cultured cells. In addition, IC increase viral load to detectable levels in PBMC from serologically positive patients that were PCR-negative before culture. γ Interferon is sufficient for these effects, whereas tumor necrosis factor and interleukin-6 have little or no activity. The increase of HHV-8 DNA by IC is observed after short-term (7 days) or long-term (28 days) culture of the cells and occurs in one or both of the two circulating cell types that are infected in vivo: B cells and monocytes. In both cases it is associated with lytic gene expression, suggesting that virus reactivation is one of the most likely mechanisms for the effect of IC on virus load. However, IC have also effects on the cells target of HHV-8 infection, because they increase B-cell survival and induce the growth and differentiation of monocytes into KS-like spindle cells with markers of endothelial macrophages. Because cells with markers of endothelial macrophages are present in blood and lesions from KS patients and are infected by HHV-8, these data may explain the high HHV-8 load associated with KS development and suggest that infected monocytes may carry the virus to tissues, transmit the infection, or differentiate in loco in spindle cells with endothelial macrophage markers.

SOX5 Regulates Cell Proliferation, Apoptosis, Migration and Invasion in KSHV-Infected Cells.

Kaposi's sarcoma (KS) originates from vascular endothelial cells, with KS-associated herpesvirus (KSHV) as the etiological agent. SRY-box transcription factor 5 (SOX5) plays different roles in various types of cancer, although its role in KS remains poorly understood. In this study, we identified the role of SOX5 in KS tissues and KSHV-infected cells and elucidated the molecular mechanism. Thirty-two KS patients were enrolled in this study. Measurement of SOX5 mRNA and protein levels in human KS tissues and adjacent control tissues revealed lower levels in KS tissues, with KS patients having higher SOX5 level in the early stages of the disease compared to the later stages. And SOX5 mRNA and protein was also lower in KSHV-infected cells (iSLK-219 and iSLK-BAC) than normal cells (iSLK-Puro). Additionally, SOX5 overexpression inhibited cell proliferation and promoted apoptosis and decreased KSHV-infected cell migration and invasion. Moreover, we found that SOX5 overexpression suppressed the epithelial-to-mesenchymal transition of KSHV-infected cells. These results suggest SOX5 is a suppressor factor during KS development and a potential target for KS treatment.

The Presence of Antibody-Dependent Cell Cytotoxicity–Mediating Antibodies in Kaposi Sarcoma–Associated Herpesvirus–Seropositive Individuals Does Not Correlate with Disease Pathogenesis or Progression

Although the immune response is likely to play a pivotal role in controlling Kaposi sarcoma (KS)-associated herpesvirus (KSHV) and preventing disease development, the exact factors responsible for that control remain ill defined. T cell responses are weak and variable, and neutralizing Abs are more frequently detected in individuals with KS. This suggests a potential role for nonneutralizing Abs, which to date have been largely uninvestigated. Ab-dependent cell cytotoxicity (ADCC) is a common effector function for nonneutralizing Abs and is known to play a protective role in other herpesvirus infections; yet, ADCC has never been investigated in the context of KSHV infection. In this study, we provide, to our knowledge, the first evidence that anti-KSHV Abs are capable of mediating ADCC responses against infected human cells undergoing lytic reactivation. ADCC activity significantly higher than seronegative controls was detected in 24 of 68 KSHV-seropositive individuals tested. However, ADCC responses were not associated with KS development or progression. ADCC activity was also found to be independent of HIV status, sex, age, KSHV Ab titer, and KSHV-neutralizing activity. Nevertheless, additional investigations into effector cell function between KS and asymptomatic individuals are needed to determine whether ADCC has a role in preventing KS.

Donor-derived human herpesvirus 8 and development of Kaposi sarcoma among 6 recipients of organs from donors with high-risk sexual and substance use behavior.

Kaposi sarcoma (KS) can develop following organ transplantation through reactivation of recipient human herpesvirus 8 (HHV‐8) infection or through donor‐derived HHV‐8 transmission. We describe 6 cases of donor‐derived HHV‐8 infection and KS investigated from July 2018 to January 2020. Organs from 6 donors, retrospectively identified as HHV‐8‐positive, with a history of drug use disorder, were transplanted into 22 recipients. Four of 6 donors had risk factors for HHV‐8 infection reported in donor history questionnaires. Fourteen of 22 organ recipients (64%) had evidence of posttransplant HHV‐8 infection. Lung recipients were particularly susceptible to KS. Four of the 6 recipients who developed KS died from KS or associated complications. The US opioid crisis has resulted in an increasing number and proportion of organ donors with substance use disorder, and particularly injection drug use history, which may increase the risk of HHV‐8 transmission to recipients. Better awareness of the risk of posttransplant KS for recipients of organs from donors with HHV‐8 infection risk could be useful for recipient management. Testing donors and recipients for HHV‐8 is currently challenging with no validated commercial serology kits available. Limited HHV‐8 antibody testing is available through some US reference laboratories and the Centers for Disease Control and Prevention.

KSHV enhances mesenchymal stem cell homing and promotes KS-like pathogenesis

Kaposi's sarcoma (KS) tends to occur in injured or inflamed sites of the body, which is described as the “Koebner phenomenon”. KS is also unique in its extraordinary angio-hyperplastic inflammatory phenotype. Recently, evidence has accrued indicating that KS may derive from KSHV-infected mesenchymal stem cells (MSCs), which possess enhanced migration and homing ability. Inspired by these findings, we hypothesized that KS may arise from KSHV-infected MSCs that chemotactically migrate to preexisting inflammatory or injured sites. Here we report that KSHV infection of human MSCs significantly up-regulated expression of several chemokine receptors and enhanced cell migration ability in vitro. Furthermore, using a wound mouse model, we demonstrated that KSHV infection dramatically promotes MSCs migrating and settling in the wound sites. In addition, two mice in the KSHV-infected group showed purpura and tumors with KS-like features. Taken together, KSHV-enhanced MSC migration ability and inflammatory microenvironment play crucial roles in KS development.

DLX1008 (brolucizumab), a single-chain anti-VEGF-A antibody fragment with low picomolar affinity, leads to tumor involution in an in vivo model of Kaposi Sarcoma.

Kaposi Sarcoma (KS) is among the most angiogenic cancers in humans and an AIDS-defining condition. KS-associated herpesvirus (KSHV) is necessary for KS development, as is vascular endothelial growth factor (VEGF-A). DLX1008 is a novel anti-VEGF-A antibody single-chain variable fragment (scFv) with low picomolar affinity for VEGF-A. In vivo imaging techniques were used to establish the efficacy of DLX1008 and to establish the mechanism of action; this included non-invasive imaging by ultrasound and optical fluorescence, verified by post-mortem histochemistry. The results showed that DLX1008 was efficacious in a KS mouse model. The NSG mouse xenografts suffered massive internal necrosis or involution, consistent with a lack of blood supply. We found that imaging by ultrasound was superior to external caliper measurements in the validation of the angiogenesis inhibitor DLX1008. Further development of DLX1008 against VEGF-dependent sarcomas is warranted.

Reduction of Kaposi's Sarcoma-Associated Herpesvirus Latency Using CRISPR-Cas9 To Edit the Latency-Associated Nuclear Antigen Gene.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), an AIDS-defining cancer in HIV-1-infected individuals or immune-suppressed transplant patients. The prevalence for both KSHV and KS are highest in sub-Saharan Africa where HIV-1 infection is also epidemic. There is no effective treatment for advanced KS; therefore, the survival rate is low. Similar to other herpesviruses, KSHV's ability to establish latent infection in the host presents a major challenge to KS treatment or prevention. Strategies to reduce KSHV episomal persistence in latently infected cells might lead to approaches to prevent KS development. The CRISPR-Cas9 system is a gene editing technique that has been used to specifically manipulate the HIV-1 genome but also Epstein-Barr virus (EBV) which, similar to KSHV, belongs to the Gammaherpesvirus family. Among KSHV gene products, the latency-associated nuclear antigen (LANA) is absolutely required in the maintenance, replication, and segregation of KSHV episomes during mitosis, which makes LANA an ideal target for CRISPR-Cas9 editing. In this study, we designed a replication-incompetent adenovirus type 5 to deliver a LANA-specific Cas9 system (Ad-CC9-LANA) into various KSHV latent target cells. We showed that KSHV latently infected epithelial and endothelial cells transduced with Ad-CC9-LANA underwent significant reductions in the KSHV episome burden, LANA RNA and protein expression over time, but this effect is less profound in BC3 cells due to the low infection efficiency of adenovirus type 5 for B cells. The use of an adenovirus vector might confer potential in vivo applications of LANA-specific Cas9 against KSHV infection and KS.IMPORTANCE The ability for Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma (KS), to establish and maintain latency has been a major challenge to clearing infection and preventing KS development. This is the first study to demonstrate the feasibility of using a KSHV LANA-targeted CRISPR-Cas9 and adenoviral delivery system to disrupt KSHV latency in infected epithelial and endothelial cell lines. Our system significantly reduced the KSHV episomal burden over time. Given the safety record of adenovirus as vaccine or delivery vectors, this approach to limit KSHV latency may also represent a viable strategy against other tumorigenic viruses and may have potential benefits in developing countries where the viral cancer burden is high.

FoxO1 Suppresses Kaposi’s Sarcoma-Associated Herpesvirus Lytic Replication and Controls Viral Latency

Kaposi's sarcoma-associated herpesvirus (KSHV) has latent and lytic replication phases, both of which contribute to the development of KSHV-induced malignancies. Among the numerous factors identified to regulate the KSHV life cycle, oxidative stress, caused by imbalanced clearing and production of reactive oxygen species (ROS), has been shown to robustly disrupt KSHV latency and induce viral lytic replication. In this study, we identified an important role of the antioxidant defense factor forkhead box protein O1 (FoxO1) in the KSHV life cycle. Either chemical inhibition of the FoxO1 function or knockdown of FoxO1 expression led to an increase in the intracellular ROS level that was subsequently sufficient to disrupt KSHV latency and induce viral lytic reactivation. On the other hand, treatment with N-acetyl-l-cysteine (NAC), an oxygen free radical scavenger, led to a reduction in the FoxO1 inhibition-induced ROS level and, ultimately, the attenuation of KSHV lytic reactivation. These findings reveal that FoxO1 plays a critical role in keeping KSHV latency in check by maintaining the intracellular redox balance.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several cancers, including Kaposi's sarcoma (KS). Both the KSHV latent and lytic replication phases are important for the development of KS. Identification of factors regulating the KSHV latent phase-to-lytic phase switch can provide insights into the pathogenesis of KSHV-induced malignancies. In this study, we show that the antioxidant defense factor forkhead box protein O1 (FoxO1) maintains KSHV latency by suppressing viral lytic replication. Inhibition of FoxO1 disrupts KSHV latency and induces viral lytic replication by increasing the intracellular ROS level. Significantly, treatment with an oxygen free radical scavenger, N-acetyl-l-cysteine (NAC), attenuated the FoxO1 inhibition-induced intracellular ROS level and KSHV lytic replication. Our works reveal a critical role of FoxO1 in suppressing KSHV lytic replication, which could be targeted for antiviral therapy.

Quantitative RNAseq analysis of Ugandan KS tumors reveals KSHV gene expression dominated by transcription from the LTd downstream latency promoter.

KSHV is endemic in Uganda and the HIV epidemic has dramatically increased the incidence of Kaposi sarcoma (KS). To investigate the role of KSHV in the development of KS, we obtained KS biopsies from ART-naïve, HIV-positive individuals in Uganda and analyzed the tumors using RNAseq to globally characterize the KSHV transcriptome. Phylogenetic analysis of ORF75 sequences from 23 tumors revealed 6 distinct genetic clusters with KSHV strains exhibiting M, N or P alleles. RNA reads mapping to specific unique coding sequence (UCDS) features were quantitated using a gene feature file previously developed to globally analyze and quantitate KSHV transcription in infected endothelial cells. A pattern of high level expression was detected in the KSHV latency region that was common to all KS tumors. The clear majority of transcription was derived from the downstream latency transcript promoter P3(LTd) flanking ORF72, with little evidence of transcription from the P1(LTc) latency promoter, which is constitutive in KSHV-infected lymphomas and tissue-culture cells. RNAseq data provided evidence of alternate P3(LTd) transcript editing, splicing and termination resulting in multiple gene products, with 90% of the P3(LTd) transcripts spliced to release the intronic source of the microRNAs K1-9 and 11. The spliced transcripts encode a regulatory uORF upstream of Kaposin A with alterations in intervening repeat sequences yielding novel or deleted Kaposin B/C-like sequences. Hierarchical clustering and PCA analysis of KSHV transcripts revealed three clusters of tumors with different latent and lytic gene expression profiles. Paradoxically, tumors with a latent phenotype had high levels of total KSHV transcription, while tumors with a lytic phenotype had low levels of total KSHV transcription. Morphologically distinct KS tumors from the same individual showed similar KSHV gene expression profiles suggesting that the tumor microenvironment and host response play important roles in the activation level of KSHV within the infected tumor cells.

A retrospective review of patients with Kaposi's sarcoma in Botswana.

Despite widespread antiretroviral coverage in Botswana, Kaposi's sarcoma (KS) remains among the most common malignancies. To date, adult KS in Botswana is not well characterized. The diagnosis relies on clinical suspicion that is often confirmed by histopathology given the implications of treatment; however, this poses a significant resource burden. We conducted a retrospective review of the cohort of patients biopsied for possible KS at Princess Marina Hospital, the main dermatology referral site in Botswana, from September 2008 through June 2015 to describe the demographics, human immunodeficiency virus (HIV) characteristics, and clinical presentations of these patients. Histopathologic diagnoses were reviewed, and positive predictive value (PPV) was used to characterize the accuracy of clinical suspicion of KS. A total of 441 patients received 450 biopsies where KS was on the differential diagnosis, and 239 patients (54%) were ultimately diagnosed with KS. The KS cohort was more likely to be male (58% vs. 37%, P<0.001), HIV positive (94% vs. 85%, P<0.05), and have lower CD4 counts at the time of biopsy (274cells/μl vs. 362cells/μl, P<0.05). The PPV of clinical suspicion of KS was 58%. When KS was not histopathologically diagnosed, clinically benign diseases were found in 17%, medically significant conditions requiring alternative therapies in 78%, and life-threatening diseases in 5%. Our study reinforces the risk factors in development of KS. The poor PPV supports the important role of histology in KS diagnosis to both ensure appropriate treatment and prevent overtreatment. Improved accessibility to biopsy and augmentation of local dermatopathologic services would likely improve diagnostic accuracy and treatment.