Wilms' tumor 1 (WT1) antigen is overexpressed in Kaposi Sarcoma and is regulated by KSHV vFLIP.

In people living with HIV, Kaposi Sarcoma (KS), a vascular neoplasm caused by KS herpesvirus (KSHV/HHV-8), remains one of the most common malignancies worldwide. Individuals living with HIV, receiving otherwise effective antiretroviral therapy, may present with extensive disease requiring chemotherapy. Hence, new therapeutic approaches are needed. The Wilms' tumor 1 (WT1) protein is overexpressed and associated with poor prognosis in several hematologic and solid malignancies and has shown promise as an immunotherapeutic target. We found that WT1 was overexpressed in >90% of a total 333 KS biopsies, as determined by immunohistochemistry and image analysis. Our largest cohort from ACTG, consisting of 294 cases was further analyzed demonstrating higher WT1 expression was associated with more advanced histopathologic subtypes. There was a positive correlation between the proportion of infected cells within KS tissues, assessed by expression of the KSHV-encoded latency-associated nuclear antigen (LANA), and WT1 positivity. Areas with high WT1 expression showed sparse T-cell infiltrates, consistent with an immune evasive tumor microenvironment. We show that major oncogenic isoforms of WT1 are overexpressed in primary KS tissue and observed WT1 upregulation upon de novo infection of endothelial cells with KSHV. KSHV latent viral FLICE-inhibitory protein (vFLIP) upregulated total and major isoforms of WT1, but upregulation was not seen after expression of mutant vFLIP that is unable to bind IKKÆ´ and induce NFκB. siRNA targeting of WT1 in latent KSHV infection resulted in decreased total cell number and pAKT, BCL2 and LANA protein expression. Finally, we show that ESK-1, a T cell receptor-like monoclonal antibody that recognizes WT1 peptides presented on MHC HLA-A0201, demonstrates increased binding to endothelial cells after KSHV infection or induction of vFLIP expression. We propose that oncogenic isoforms of WT1 are upregulated by KSHV to promote tumorigenesis and immunotherapy directed against WT1 may be an approach for KS treatment.

Generation and Characterization of CYP2E1-Overexpressing HepG2 Cells to Study the Role of CYP2E1 in Hepatic Hypoxia-Reoxygenation Injury.

The mechanisms of hepatic ischemia/reperfusion (I/R) injury, which occurs during liver transplantation or surgery, are poorly understood. The purpose of the current study was to generate and characterize a HepG2 cell line with a stable overexpression of CYP2E1 to investigate the role of the enzyme in hypoxia/reperfusion (H/R) injury in an ex vivo setting. GFP-tagged CYP2E1 and control clones were developed, and their gene expression and protein levels of GFP and CYP2E1 were determined using RT-PCR and ELISA/Western blot analysis, respectively. Additionally, the CYP2E1 catalytic activity was determined by UPLC-MS/MS analysis of 6-hydroxychlorzoxazone formed from the chlorzoxazone substrate. The CYP2E1 and control clones were subjected to hypoxia (10 h) and reoxygenation (0.5 h), and cell death and reactive oxygen species (ROS) generation were quantitated using LDH and flow cytometry, respectively. Compared with the control clone, the selected CYP2E1 clone showed a 720-fold increase in CYP2E1 expression and a prominent band in the western blot analysis, which was associated with a 150-fold increase in CYP2E1 catalytic activity. The CYP2E1 clone produced 2.3-fold more ROS and 1.9-fold more cell death in the H/R model. It is concluded that the constitutive CYP2E1 in the liver may play a detrimental role in hepatic I/R injury.

IL-21 signaling promotes the establishment of KSHV infection in human tonsil lymphocytes by increasing differentiation and targeting of plasma cells.

Introduction: Factors influencing Kaposi's sarcoma-associated herpesvirus (KSHV) transmission and the early stages of KSHV infection in the human immune system remain poorly characterized. KSHV is known to extensively manipulate the host immune system and the cytokine milieu, and cytokines are known to influence the progression of KSHV-associated diseases. Our previous work identified the early targeting of plasma cells for KSHV infection. In this study, we examine whether IL-21, a cytokine known to profoundly influence plasma cell fate, influences the early stages of KSHV infection in B lymphocytes. Methods: Using our unique model of ex vivo KSHV infection in tonsil lymphocytes, we investigate the influence of IL-21 supplementation, IL-21 neutralization, the distribution of IL-21 receptor on B cell subsets and IL-21 secreting T cell subsets on the establishment of KSHV infection in human B cells. Results: We show that IL-21 signaling promotes KSHV infection by promoting both total plasma cell numbers and increasing KSHV infection in plasma cells as early as 3 days post-infection. We further demonstrate that the synergistic effect of KSHV infection and IL-21 treatment on plasma cell frequencies is due to differentiation of new plasma cells from naïve B cell precursors. We examine T cells secreting IL-21 in our tonsil specimens and determine that IL-21 producing CD8+ central memory T cells are correlated with plasma cell frequencies and KSHV targeting of plasma cells. Discussion: These results demonstrate the novel finding that differentiation of new plasma cells is involved in the early stages of KSHV infection in B cells, and that IL-21 signaling can potentiate this effect thereby increasing the overall magnitude of KSHV infection at early timepoints. These results suggest that IL-21 signaling represents a host-level susceptibility factor for the establishment of KSHV infection.

Host-Level Susceptibility and IRF1 Expression Influence the Ability of IFN-γ to Inhibit KSHV Infection in B Lymphocytes.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with vascular endothelial cell tumor, Kaposi's sarcoma (KS) and lymphoproliferative disorder, multicentric Castleman's disease (MCD), primary effusion lymphoma (PEL) and KSHV inflammatory cytokine syndrome (KICS). Dysregulation of proinflammatory cytokines is found in most KSHV associated diseases. However, little is known about the role of host microenvironment in the regulation of KSHV establishment in B cells. In the present study, we demonstrated that IFN-γ has a strong inhibitory effect on KSHV infection but only in a subset of tonsil-derived lymphocyte samples that are intrinsically more susceptible to infection, contain higher proportions of naïve B cells, and display increased levels of IRF1 and STAT1-pY701. The effect of IFN-γ in responsive samples was associated with increased frequencies of germinal center B cells (GCB) and decreased infection of plasma cells, suggesting that IFN-γ-mediated modulation of viral dynamics in GC can inhibit the establishment of KSHV infection.

Local Renin-Angiotensin System Activation and Myofibroblast Formation in Graft Versus Host Disease-Associated Conjunctival Fibrosis.

Purpose: The present study was designed to investigate the role of myofibroblast transdifferentiation and the conjunctival renin-angiotensin system (RAS) in the pathogenesis of graft-versus-host disease (GVHD)-associated conjunctival fibrosis. Methods: A mouse model of major histocompatibility-matched allogeneic transplantation was used to induce GVHD, with male B10.D2 mice as donors and female BALB/c mice as recipients. Male BALB/c to female BALB/c syngeneic transplantation was used as control. Y chromosome staining in the spleen cells obtained from female recipient mice was used to confirm engraftment. The phenol red thread test and fluorescein staining were used to quantify tears and corneal keratopathy. Eyes were harvested at 4 and 8 weeks after the transplant for alpha-smooth muscle actin (α-SMA), angiotensinogen, and angiotensin-converting enzyme (ACE) immunostaining. Conjunctiva was harvested for gene expression quantification of α-SMA, angiotensinogen, and ACE. Results: More than 80% of the spleen cells in the recipient mice were chromosome Y positive, thus conforming successful engraftment. A significant decrease in tear secretion and a marked increase in corneal keratopathy score after allogeneic transplantation indicated the onset of ocular GVHD in these mice. A significant increase in α-SMA gene expression and the presence of a large number of α-SMA-positive cells was noted in the bulbar orbital conjunctiva of mice after allogeneic transplantation. Allogenic transplantation also caused a significant increase in the gene expression and protein expression of angiotensinogen and ACE in the subconjunctival eyelid area. Conclusions: Results of the present study demonstrate that GVHD-associated conjunctival fibrosis is accompanied by myofibroblast formation and activation of the local conjunctival RAS.

Proteoglycan-4 is an essential regulator of synovial macrophage polarization and inflammatory macrophage joint infiltration.

BACKGROUND: Synovial macrophages perform a multitude of functions that include clearance of cell debris and foreign bodies, tissue immune surveillance, and resolution of inflammation. The functional diversity of macrophages is enabled by distinct subpopulations that express unique surface markers. Proteoglycan-4 (PRG4) is an important regulator of synovial hyperplasia and fibrotic remodeling, and the involvement of macrophages in PRG4's synovial role is yet to be defined. Our objectives were to study the PRG4's importance to macrophage homeostatic regulation in the synovium and infiltration of pro-inflammatory macrophages in acute synovitis and investigate whether macrophages mediated synovial fibrosis in Prg4 gene-trap (Prg4GT/GT) murine knee joints. METHODS: Macrophage phenotyping in Prg4GT/GT and Prg4+/+ joints was performed by flow cytometry using pan-macrophage markers, e.g., CD11b, F4/80, and surface markers of M1 macrophages (CD86) and M2 macrophages (CD206). Characterizations of the various macrophage subpopulations were performed in 2- and 6-month-old animals. The expression of inflammatory markers, IL-6, and iNOS in macrophages that are CD86+ and/or CD206+ was studied. The impact of Prg4 recombination on synovial macrophage populations of 2- and 6-month-old animals and infiltration of pro-inflammatory macrophages in response to a TLR2 agonist challenge was determined. Macrophages were depleted using liposomal clodronate and synovial membrane thickness, and the expression of fibrotic markers α-SMA, PLOD2, and collagen type I (COL-I) was assessed using immunohistochemistry. RESULTS: Total macrophages in Prg4GT/GT joints were higher than Prg4+/+ joints (p<0.0001) at 2 and 6 months, and the percentages of CD86+/CD206- and CD86+/CD206+ macrophages increased in Prg4GT/GT joints at 6 months (p<0.0001), whereas the percentage of CD86-/CD206+ macrophages decreased (p<0.001). CD86+/CD206- and CD86+/CD206+ macrophages expressed iNOS and IL-6 compared to CD86-/CD206+ macrophages (p<0.0001). Prg4 re-expression limited the accumulation of CD86+ macrophages (p<0.05) and increased CD86-/CD206+ macrophages (p<0.001) at 6 months. Prg4 recombination attenuated synovial recruitment of pro-inflammatory macrophages in 2-month-old animals (p<0.001). Clodronate-mediated macrophage depletion reduced synovial hyperplasia, α-SMA, PLOD2, and COL-I expressions in the synovium (p<0.0001). CONCLUSIONS: PRG4 regulates the accumulation and homeostatic balance of macrophages in the synovium. In its absence, the synovium becomes populated with M1 macrophages. Furthermore, macrophages exert an effector role in synovial fibrosis in Prg4GT/GT animals.

Synthesis and antiviral activity of fatty acyl conjugates of remdesivir against severe acute respiratory syndrome coronavirus 2 and Ebola virus.

We report here the synthesis, purification, and characterization of mono- and di-fatty acyl conjugates of remdesivir (RDV) and their in vitro antiviral activity against SAR-CoV-2, an Ebola virus transcription- and replication-competent virus-like particle (trVLP) system, and infectious Ebola virus. The most potent monofatty acyl conjugate was 4b, containing a 4-oxatetradecanolyl at the 3' position. Monofatty acyl conjugates, 3'-O-tetradecanoyl (4a) (IC50(VeroE6) = 2.3 µM; IC50(Calu3) = 0.24 µM), 3'-O-4-oxatetradodecanoyl (4b) (IC50(VeroE6) = 2.0 µM; IC50(Calu3) = 0.18 µM), and 3'-O-(12-ethylthiododecanoyl) (4e) (IC50(VeroE6) = 2.4 µM; IC50(Calu3) = 0.25 µM) derivatives exhibited less activity than RDV (IC50(VeroE6) = 0.85 µM; IC50(Calu3) = 0.06 µM) in both VeroE6 and Calu3 cells. Difatty acylation led to a significant reduction in the antiviral activity of RDV (as shown in conjugates 5a and 5b) against SARS-CoV-2 when compared with monofatty acylation (3a-e and 4a-e). About 77.9% of 4c remained intact after 4 h incubation with human plasma while only 47% of parent RDV was observed at the 2 h time point. The results clearly indicate the effectiveness of fatty acylation to improve the half-life of RDV. The antiviral activities of a number of monofatty acyl conjugates of RDV, such as 3b, 3e, and 4b, were comparable with RDV against the Ebola trVLP system. Meanwhile, the corresponding physical mixtures of RDV and fatty acids 6a and 6b showed 1.6 to 2.2 times less antiviral activity than the corresponding conjugates, 4a and 4c, respectively, against SARS-CoV-2 in VeroE6 cells. A significant reduction in viral RNA synthesis was observed for selected compounds 3a and 4b consistent with the IC50 results. These studies indicate the potential of these compounds as long-acting antiviral agents or prodrugs of RDV.

Suppression of DC-SIGN and gH Reveals Complex, Subset-Specific Mechanisms for KSHV Entry in Primary B Lymphocytes.

Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of multiple cancers in immunocompromised patients including two lymphoproliferative disorders associated with KSHV infection of B lymphocytes. Despite many years of research into the pathogenesis of KSHV associated diseases, basic questions related to KSHV molecular virology remain unresolved. One such unresolved question is the cellular receptors and viral glycoproteins needed for KSHV entry into primary B lymphocytes. In this study, we assess the contributions of KSHV glycoprotein H (gH) and the cellular receptor DC-SIGN to KSHV infection in tonsil-derived B lymphocytes. Our results show that (1) neither KSHV-gH nor DC-SIGN are essential for entry into any B cell subset, (2) DC-SIGN does play a role in KSHV entry into tonsil-derived B cells, but in all B cell subtypes alternative entry mechanisms exist, (3) KSHV-gH can participate in KSHV entry into centrocytes via a DC-SIGN independent entry mechanism, and (4) in the absence of KSHV-gH, DC-SIGN is required for KSHV entry into centrocytes. Our results provide a first glimpse into the complexity of KSHV entry in the lymphocyte compartment and highlight that multiple subset-dependent entry mechanisms are employed by KSHV which depend upon multiple cellular receptors and multiple KSHV glycoproteins.

Molecular Virology of KSHV in the Lymphocyte Compartment-Insights From Patient Samples and De Novo Infection Models.

The incidence of Kaposi's sarcoma-associated herpesvirus (KSHV)-associated Kaposi Sarcoma has declined precipitously in the present era of effective HIV treatment. However, KSHV-associated lymphoproliferative disorders although rare, have not seen a similar decline. Lymphoma is now a leading cause of death in people living with HIV (PLWH), indicating that the immune reconstitution provided by antiretroviral therapy is not sufficient to fully correct the lymphomagenic immune dysregulation perpetrated by HIV infection. As such, novel insights into the mechanisms of KSHV-mediated pathogenesis in the immune compartment are urgently needed in order to develop novel therapeutics aimed at prevention and treatment of KSHV-associated lymphoproliferations. In this review, we will discuss our current understanding of KSHV molecular virology in the lymphocyte compartment, concentrating on studies which explore mechanisms unique to infection in B lymphocytes.

An Update of the Virion Proteome of Kaposi Sarcoma-Associated Herpesvirus.

The virion proteins of Kaposi sarcoma-associated herpesvirus (KSHV) were initially characterized in 2005 in two separate studies that combined the detection of 24 viral proteins and a few cellular components via LC-MS/MS or MALDI-TOF. Despite considerable advances in the sensitivity and specificity of mass spectrometry instrumentation in recent years, leading to significantly higher yields in detections, the KSHV virion proteome has not been revisited. In this study, we have re-examined the protein composition of purified KSHV virions via ultra-high resolution Qq time-of-flight mass spectrometry (UHR-QqTOF). Our results confirm the detection of all previously reported virion proteins, in addition to 17 other viral proteins, some of which have been characterized as virion-associated using other methods, and 10 novel proteins identified as virion-associated for the first time in this study. These results add KSHV ORF9, ORF23, ORF35, ORF48, ORF58, ORF72/vCyclin, K3, K9/vIRF1, K10/vIRF4, and K10.5/vIRF3 to the list of KSHV proteins that can be incorporated into virions. The addition of these proteins to the KSHV virion proteome provides novel and important insight into early events in KSHV infection mediated by virion-associated proteins. Data are available via ProteomeXchange with identifier PXD022626.

Cytokine-Targeted Therapeutics for KSHV-Associated Disease.

Kaposi's sarcoma-associated herpesvirus (KSHV) also known as human herpesvirus 8 (HHV-8), is linked to several human malignancies including Kaposi sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman's disease (MCD) and recently KSHV inflammatory cytokine syndrome (KICS). As with other diseases that have a significant inflammatory component, current therapy for KSHV-associated disease is associated with significant off-target effects. However, recent advances in our understanding of the pathogenesis of KSHV have produced new insight into the use of cytokines as potential therapeutic targets. Better understanding of the role of cytokines during KSHV infection and tumorigenesis may lead to new preventive or therapeutic strategies to limit KSHV spread and improve clinical outcomes. The cytokines that appear to be promising candidates as KSHV antiviral therapies include interleukins 6, 10, and 12 as well as interferons and tumor necrosis factor-family cytokines. This review explores our current understanding of the roles that cytokines play in promoting KSHV infection and tumorigenesis, and summarizes the current use of cytokines as therapeutic targets in KSHV-associated diseases.

Analysis of KSHV B lymphocyte lineage tropism in human tonsil reveals efficient infection of CD138+ plasma cells.

Despite 25 years of research, the basic virology of Kaposi Sarcoma Herpesviruses (KSHV) in B lymphocytes remains poorly understood. This study seeks to fill critical gaps in our understanding by characterizing the B lymphocyte lineage-specific tropism of KSHV. Here, we use lymphocytes derived from 40 human tonsil specimens to determine the B lymphocyte lineages targeted by KSHV early during de novo infection in our ex vivo model system. We characterize the immunological diversity of our tonsil specimens and determine that overall susceptibility of tonsil lymphocytes to KSHV infection varies substantially between donors. We demonstrate that a variety of B lymphocyte subtypes are susceptible to KSHV infection and identify CD138+ plasma cells as a highly targeted cell type for de novo KSHV infection. We determine that infection of tonsil B cell lineages is primarily latent with few lineages contributing to lytic replication. We explore the use of CD138 and heparin sulfate proteoglycans as attachment factors for the infection of B lymphocytes and conclude that they do not play a substantial role. Finally, we determine that the host T cell microenvironment influences the course of de novo infection in B lymphocytes. These results improve our understanding of KSHV transmission and the biology of early KSHV infection in a naïve human host, and lay a foundation for further characterization of KSHV molecular virology in B lymphocyte lineages.

A multivalent Kaposi sarcoma-associated herpesvirus-like particle vaccine capable of eliciting high titers of neutralizing antibodies in immunized rabbits.

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and the causative agent of multiple cancers in immunocompromised patients. To date, there is no licensed prophylactic KSHV vaccine. In this study, we generated a novel subunit vaccine that incorporates four key KSHV envelope glycoproteins required for viral entry in diverse cell types (gpK8.1, gB, and gH/gL) into a single multivalent KSHV-like particle (KSHV-LP). Purified KSHV-LPs were similar in size, shape, and morphology to KSHV virions. Vaccination of rabbits with adjuvanted KSHV-LPs generated strong glycoprotein-specific antibody responses, and purified immunoglobulins from KSHV-LP-immunized rabbits neutralized KSHV infection in epithelial, endothelial, fibroblast, and B cell lines (60-90% at the highest concentration tested). These findings suggest that KSHV-LPs may be an ideal platform for developing a safe and effective prophylactic KSHV vaccine. We envision performing future studies in animal models that are susceptible to KSHV infection, to determine correlates of immune protection in vivo.

Kaposi Sarcoma-Associated Herpesvirus Glycoprotein H Is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types.

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and is the causative infectious agent of Kaposi sarcoma and two malignancies of B cell origin. To date, there is no licensed KSHV vaccine. Development of an effective vaccine against KSHV continues to be limited by a poor understanding of how the virus initiates acute primary infection in vivo in diverse human cell types. The role of glycoprotein H (gH) in herpesvirus entry mechanisms remains largely unresolved. To characterize the requirement for KSHV gH in the viral life cycle and in determination of cell tropism, we generated and characterized a mutant KSHV in which expression of gH was abrogated. Using a bacterial artificial chromosome containing a complete recombinant KSHV genome and recombinant DNA technology, we inserted stop codons into the gH coding region. We used electron microscopy to reveal that the gH-null mutant virus assembled and exited from cells normally, compared to wild-type virus. Using purified virions, we assessed infectivity of the gH-null mutant in diverse mammalian cell types in vitro Unlike wild-type virus or a gH-containing revertant, the gH-null mutant was unable to infect any of the epithelial, endothelial, or fibroblast cell types tested. However, its ability to infect B cells was equivocal and remains to be investigated in vivo due to generally poor infectivity in vitro Together, these results suggest that gH is critical for KSHV infection of highly permissive cell types, including epithelial, endothelial, and fibroblast cells.IMPORTANCE All homologues of herpesvirus gH studied to date have been implicated in playing an essential role in viral infection of diverse permissive cell types. However, the role of gH in the mechanism of KSHV infection remains largely unresolved. In this study, we generated a gH-null mutant KSHV and provided evidence that deficiency of gH expression did not affect viral particle assembly or egress. Using the gH-null mutant, we showed that gH was indispensable for KSHV infection of epithelial, endothelial, and fibroblast cells in vitro This suggests that gH is an important target for the development of a KSHV prophylactic vaccine to prevent initial viral infection.

KSHV induces immunoglobulin rearrangements in mature B lymphocytes.

Kaposi sarcoma herpesvirus (KSHV/HHV-8) is a B cell tropic human pathogen, which is present in vivo in monotypic immunoglobulin λ (Igλ) light chain but polyclonal B cells. In the current study, we use cell sorting to infect specific B cell lineages from human tonsil specimens in order to examine the immunophenotypic alterations associated with KSHV infection. We describe IL-6 dependent maturation of naïve B lymphocytes in response to KSHV infection and determine that the Igλ monotypic bias of KSHV infection in vivo is due to viral induction of BCR revision. Infection of immunoglobulin κ (Igκ) naïve B cells induces expression of Igλ and isotypic inclusion, with eventual loss of Igκ. We show that this phenotypic shift occurs via re-induction of Rag-mediated V(D)J recombination. These data explain the selective presence of KSHV in Igλ B cells in vivo and provide the first evidence that a human pathogen can manipulate the molecular mechanisms responsible for immunoglobulin diversity.

Extrafollicular activities: perspectives on HIV infection, germinal center-independent maturation pathways, and KSHV-mediated lymphoproliferation.

Early events in the pathogenesis of KSHV-associated lymphoproliferations in the context of HIV disease remain poorly understood. Recent research indicates that latent HIV infection causes persistent immune dysfunction in B cell follicles. Simultaneously, lack of T cell immune surveillance in the lymph nodes dysregulates the biology of EBV. In sum, these defects bias B lymphocyte maturation away from traditional T cell-dependent germinal center-mediated pathways and towards extrafollicular pathways. Recent advances in B lymphocyte immunology suggest that extrafollicular maturation pathways for antibody secreting cells are more flexible and robust than previously believed. These responses are now understood to be both durable and antigen-specific, and even canonically germinal center-restricted events such as class switch recombination and somatic hypermutation have now been demonstrated in an extrafollicular context. As a lymphotrophic pathogen which causes disease primarily in the context of HIV and EBV co-infection, future studies examining the interactions of KSHV biology with extrafollicular B cell maturation pathways will be critical to uncovering key aspects of KSHV-mediated immune pathology.

Inhibition of Hsp90 Suppresses PI3K/AKT/mTOR Signaling and Has Antitumor Activity in Burkitt Lymphoma.

Hsp90 is a molecular chaperone that protects proteins, including oncogenic signaling complexes, from proteolytic degradation. PU-H71 is a next-generation Hsp90 inhibitor that preferentially targets the functionally distinct pool of Hsp90 present in tumor cells. Tumors that are driven by the MYC oncoprotein may be particularly sensitive to PU-H71 due to the essential role of Hsp90 in the epichaperome, which maintains the malignant phenotype in the setting of MYC. Burkitt lymphoma (BL) is an aggressive B-cell lymphoma characterized by MYC dysregulation. In this study, we evaluated Hsp90 as a potential therapeutic target in BL. We found that primary BL tumors overexpress Hsp90 and that Hsp90 inhibition has antitumor activity in vitro and in vivo, including potent activity in a patient-derived xenograft model of BL. To evaluate the targets of PU-H71 in BL, we performed high-affinity capture followed by proteomic analysis using mass spectrometry. We found that Hsp90 inhibition targets multiple components of PI3K/AKT/mTOR signaling, highlighting the importance of this pathway in BL. Finally, we found that the anti-lymphoma activity of PU-H71 is synergistic with dual PI3K/mTOR inhibition in vitro and in vivo Overall, this work provides support for Hsp90 as a therapeutic target in BL and suggests the potential for combination therapy with PU-H71 and inhibitors of PI3K/mTOR. Mol Cancer Ther; 16(9); 1779-90. ©2017 AACR.

Identification of a nucleoside analog active against adenosine kinase-expressing plasma cell malignancies.

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase-inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI-sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers.

Does persistent HIV replication explain continued lymphoma incidence in the era of effective antiretroviral therapy?

Non-Hodgkin lymphomas are highly increased in incidence in individuals infected with HIV, and this continues to be the case in spite of highly effective combined antiretroviral therapy (cART). New evidence has demonstrated that while successful virtual recovery of CD4 counts and elimination of HIV from peripheral blood can be achieved with cART, viral replication can still occur in lymphoid tissues. In addition, recent studies have suggested that adipose tissue provides an additional reservoir for HIV-infected macrophages and T lymphocytes even in the context of successful cART therapy. In this review article, we discuss possible mechanisms leading to the development of lymphoma in the cART era.