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PurposeKaposi sarcoma (KS), an endothelial cell tumor associated with KS herpesvirus (KSHV), remains among the most common malignancies occurring with HIV infection (HIV-KS). As an oral anti-inflammatory, antiangiogenic, and immunomodulatory agent, lenalidomide is potentially an attractive alternative to standard chemotherapy for KS.Experimental designThe primary objectives of this phase I/II trial were to determine the maximum tolerated dose (MTD) and response rates for lenalidomide in HIV-KS. Secondary objectives included correlating response with natural killer (NK) and T-cell subsets, plasma cytokines, viral copy number, and KSHV gene expression in biopsies. Four dose levels of oral lenalidomide taken 21 consecutive days of 28-day cycles were evaluated in adults with HIV-KS on antiretroviral therapy with controlled viremia.ResultsFifteen and 23 participants enrolled in phases I and II, respectively, 76% of whom had received prior KS therapy. The MTD was not reached, declaring 25 mg as the recommended phase II dose (RP2D). The most frequent adverse events were neutropenia, fatigue, leukopenia, and diarrhea. Of the 25 evaluable participants receiving RP2D, 60% responded. Correlative studies performed in a subset of participants demonstrated a significant increase in proportions of blood T cells with T-regulatory phenotype, and plasma cytokines trended toward a less inflammatory pattern. Clinical response was associated with loss of KSHV transcription.ConclusionsLenalidomide is active in HIV-KS. The most common adverse events were manageable. With 60% of participants receiving RP2D obtaining a partial response and

Coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and can affect multiple organs, among which is the circulatory system. Inflammation and mortality risk markers were previously detected in COVID-19 plasma and red blood cells (RBCs) metabolic and proteomic profiles. Additionally, biophysical properties, such as deformability, were found to be changed during the infection. Based on such data, we aim to better characterize RBC functions in COVID-19. We evaluate the flow properties of RBCs in severe COVID-19 patients admitted to the intensive care unit by usingin vitromicrofluidic techniques and automated methods, including artificial neural networks, for an unbiased RBC analysis. We find strong flow and RBC shape impairment in COVID-19 samples and demonstrate that such changes are reversible upon suspension of COVID-19 RBCs in healthy plasma. Vice versa, healthy RBCs immediately resemble COVID-19 RBCs when suspended in COVID-19 plasma. Proteomics and metabolomics analyses allow us to detect the effect of plasma exchanges on both plasma and RBCs and demonstrate a new role of RBCs in maintaining plasma equilibria at the expense of their flow properties. Our findings provide a framework for further investigations of clinical relevance for therapies against COVID-19 and possibly other infectious diseases.

Supplemental material, sj-docx-1-tah-10.1177_20406207221112900 for Outcomes of patients with diffuse large B-cell and high-grade B-cell lymphomas with synchronous CNS and systemic involvement at diagnosis treated with high-dose methotrexate and R-CHOP: a single-center retrospective study by Megan Fleming, Ying Huang, Emily Dotson, David A. Bond, John Reneau, Narendranath Epperla, Lapo Alinari, Jonathan Brammer, Beth Christian, Robert A. Baiocchi, Kami Maddocks and Yazeed Sawalha in Therapeutic Advances in Hematology

Supplemental material, sj-docx-1-tah-10.1177_20406207221112900 for Outcomes of patients with diffuse large B-cell and high-grade B-cell lymphomas with synchronous CNS and systemic involvement at diagnosis treated with high-dose methotrexate and R-CHOP: a single-center retrospective study by Megan Fleming, Ying Huang, Emily Dotson, David A. Bond, John Reneau, Narendranath Epperla, Lapo Alinari, Jonathan Brammer, Beth Christian, Robert A. Baiocchi, Kami Maddocks and Yazeed Sawalha in Therapeutic Advances in Hematology

Additional file 1: Fig. S1. (Left) B-cell were isolated from the peripheral blood from healthy donors and cultured in the absence (left) or presence (right) of cytokines and a CD40L expressing fibroblasts as previously described((1)). Flow cytometry was performed for the indicated markers. (Top right) Resting and activated B cells were fixed and stained with PI for cell cycle analysis. (Bottom right) Lysates were collected from resting and activated B-cells and blotted for p-Btk, total Btk, c-myc, and GAPDH. Fig. S2. (top) UMAP plots from 4 different patients with leukemic MCL showing lineage specific clustering of cells. (bottom) Dot plots verifying lineage specific marker enrichment among the individual clusters. Fig. S3. UMAP plots compiled from 4 different patients with leukemic MCL (left) or a reference PMBC B-cell data set (right) showing their distinct transcriptional states with an overall small amount of normal B-cells in leukemic MCL samples. Fig. S4. Jeko cells were retrovirally transduced with lentiviruses encoding either a non-targeting sh or an sh directed against SAE1. Cells were GFP sorted and the total viable cells in each was enumerated 3 days after sorting (n=3 biological replicates). Fig. S5. B cells and MCL cell lines were treated with TAK-981 (100 nM, 24 hours). Lysates were prepared and blotted for an antibody recognizing the SUMO-TAK-981 adduct (above) and GAPDH (below). Fig. S6. Representative cell cycle profiles of primary MCL patients samples taken at the time of beginning TAK-981 treatment in Fig.3B. Fig. S7. Jeko cells were synchronized with palbociclib (500 nM) and treated with either DMSO or TAK-981 (100nM) and lysates. Cell were fixed at the indicated time points and cell cycle distribution was determined of PI stained cells. Fig. S8. Z-138 cells were synchronized with palbociclib (500 nM) and treated with either DMSO or TAK-981 (100nM) and lysates. Cell were fixed at the indicated time points and cell cycle distribution was determined of PI stained cells. Fig. S9. (Top) Z-138 cells were synchronized with Palbociclib (500 nM, 24 hours) followed by drug washout and treatment with nocodazole (50 ng/mL) either in the presence of DMSO or TAK-981 (100 nM) for 24 hours. Both drugs were then washed out and cells were collected every hour for cell cycle analysis and lysates were prepared for protein for SUMOylation levels. (Bottom) Z-138 cells were synchronized with Palbociclib (500 nM, 24 hours) followed by drug washout and treatment with nocodazole (50 ng/mL) for 24 hours. Cells were then treated with either DMSO or TAK-981 for 3 hours. Cells were then washed from drugs and treated with either DMSO or TAK-981 and collected every hour for cell cycle analysis and lysates were prepared for protein for SUMOylation levels. Fig. S10. Results of DAVID functional annotation analysis performed on proteins enriched for sumoylation for SUMO1 (top) and SUMO2/3 (bottom). The percentage of SUMOylated proteins within individual functional annotations are shown along with the false discovery rate (FDR). Fig. S11. (Top). Jeko cells were synchronized with Palbociclib (500 nM, 24 hours) followed by drug washout and treatment with nocodazole (50 ng/mL) either in the presence of DMSO or TAK-981 (100 nM) for 24 hours. Both drugs were then washed out and cells were fixed and stained for CENP-A. Representative confocal images of cells or chromosomes are shown. (Bottom) Enumeration of CENP-A foci from DMSO or TAK-981 treated cells was performed using cellSens software. Results are representative of two independent experiments.

Additional file 1. Eμ-BRD2 in vivo model. A Kaplan–Meier estimates of overall survival in mice engrafted with ibrutinib resistant BRD2 splenocytes and treated with vehicle, ibrutinib (30 mg/kg daily in drinking water), SNX-5422 (50 mg/kg 3 days/week) or the combination. B Mice spleens were weighed at early removal criteria and compared between treatment groups. C Histopathology performed on spleen, lymph node, liver, lung and bone marrow reveals reduced leukemic infiltration in the liver, lungs, and marrow of SNX-5422 and SNX-5422 + ibrutinib treated groups. (C, lymph node cortex; M, lymph node medulla; W, spleen white pulp; R, spleen red pulp).

Additional file 2. SNX-5422 related toxicity. Histopathology of the murine non-glandular stomach reveals gastric ulcers in SNX-5422 treated groups in both the Eμ-TCL1 and the Eμ-BRD2 mouse models. The black arrows indicate the damage to the gastric mucosal layer in the SNX-5422 and combo treated mice, red arrows indicate immune cell infiltration, and green arrows indicate mucosal hyperplasia.

Figure S1. Tumor-conditioned media contains soluble factors that promote the recruitment and generation of tumor-associated macrophages. Tumor-conditioned media contains inflammatory cytokines. Culture supernatants were harvested from 3 human cancer cell lines following 24 h of incubation in 0.2% FBS medium. A panel of cytokines and chemokines were quantified after being read on the Meso QuickPlex SQ 120. Data are presented as a heat map for expression of soluble factors in supernatants from each cell line. Table S1. List of PCR primers. Comprehensive list of PCR primers used in this manuscript. Figure S2. Additional functional characterization of in vitro generated TAM. M2-like macrophages and in vitro generated TAM were analyzed by real-time PCR for transcript expression of TNF-a (inflammatory mediator), VEGF (angiogenic factor), CSF-1R (colony- stimulating factor 1 receptor; overexpressed on TAM), and arginase (ARG1; a key factor in the suppressive function of TAM). (PPTX 140 kb)

Vascular endothelial growth factor (VEGF) plays an important role in Kaposi’s sarcoma (KS). We administered PTC299, a post-transcriptional inhibitor of pathogenic VEGF, to persons with HIV-related KS. Seventeen participants received three different doses of PTC299. Adverse events typically observed with VEGF-inhibition were absent. Three participants had partial tumor responses and 11 had stable disease. There were no differences in exposure to PTC299 by antiretroviral regimen. Serum VEGF, but not KSHV DNA, decreased on treatment. Given redundancies in the VEGF feedback loop, future trials should consider combining PTC299 with agents that inhibit different pathways implicated in KS and KSHV proliferation.

The Epstein-Barr virus (EBV) is an oncogenic, γ-herpesvirus associated with a broad spectrum of disease. Although most immune-competent individuals can effectivley develop efficient adaptive immune responses to EBV, immunocompromised individuals are at serious risk for developing life-threatening diseases, such as Hodgkin lymphoma and posttransplant lymphoproliferative disorder (PTLD). Given the significant morbidity associated with EBV infection in high-risk populations, there is a need to develop vaccine strategies that restore or enhance EBV-specific immune responses. Here, we identify the EBV immediate-early protein BZLF1 as a potential target antigen for vaccine development. Primary tumors from patients with PTLD and a chimeric human-murine model of EBV-driven lymphoproliferative disorder (EBV-LPD) express BZLF1 protein. Pulsing human dendritic cells (DC) with recombinant BZLF1 followed by incubation with autologous mononuclear cells led to expansion of BZLF1-specific CD8(+) T cells in vitro and primed BZLF1-specific T-cell responses in vivo. In addition, vaccination of hu-PBL-SCID mice with BZLF1-transduced DCs induced specific cellular immunity and significantly prolonged survival from fatal EBV-LPD. These findings identify BZLF1 as a candidate target protein in the immunosurveillance of EBV and provide a rationale for considering BZLF1 in vaccine strategies to enhance primary and recall immune responses and potentially prevent EBV-associated diseases.

The synthesis and initial biological evaluation of 3-carboranylthymidine analogues (3CTAs) that are (radio)halogenated at the closo-carborane cluster are described. Radiohalogenated 3CTAs have the potential to be used in the radiotherapy and imaging of cancer because they may be selectively entrapped in tumor cells through monophosphorylation by human thymidine kinase 1 (hTK1). Two strategies for the synthesis of a (127)I-labeled form of a specific 3CTA, previously designated as N5, are described: (1) direct iodination of N5 with iodine monochloride and aluminum chloride to obtain N5-(127)I and (2) initial monoiodination of o-carborane to 9-iodo-o-carborane followed by its functionalization to N5-(127)I. The former strategy produced N5-(127)I in low yields along with di-, tri-, and tetraiodinated N5 as well as decomposition products, whereas the latter method produced only N5-(127)I in high yields. N5-(127)I was subjected to nucleophilic halogen- and isotope-exchange reactions using Na(79/81)Br and Na(125)I, respectively, in the presence of Herrmann's catalyst to obtain N5-(79/81)Br and N5-(125)I, respectively. Two intermediate products formed using the second strategy, 1-(tert-butyldimethylsilyl)-9-iodo-o-carborane and 1-(tert-butyldimethylsilyl)-12-iodo-o-carborane, were subjected to X-ray diffraction studies to confirm that substitution at a single carbon atom of 9-iodo-o-carborane resulted in the formation of two structural isomers. To the best of our knowledge, this is the first report of halogen- and isotope-exchange reactions of B-halocarboranes that have been conjugated to a complex biomolecule. Human TK1 phosphorylation rates of N5, N5-(127)I, and N5-(79/81)Br ranged from 38.0% to 29.6% relative to that of thymidine, the endogenous hTK1 substrate. The in vitro uptake of N5, N5-(127)I, and N5-(79/81)Br in L929 TK1(+) cells was 2.0, 1.8, and 1.4 times greater than that in L929 TK1(-) cells.

In vitro studies in mantle cell lymphoma (MCL) cell lines and patient-derived cells have demonstrated synergistic apoptosis with combined rituximab and bortezomib (R-bortezomib) compared with single-agent bortezomib. Therefore, the authors of this report evaluated R-bortezomib in a preclinical model and in a phase 2 clinical trial.A Hu-MCL-severe combined immunodeficiency (SCID) model engrafted with the Jeko cell line was treated with R-bortezomib, bortezomib, or rituximab. Twenty-five patients with relapsed follicular lymphoma (n = 11) and MCL (n = 14) received 375 mg/m(2) rituximab on Days 1 and 8 and 1.3 to 1.5 mg/m(2) bortezomib on Days 1, 4, 8, and 11 every 21 days for a median of 3 cycles (range, 1-5 cycles).R-bortezomib resulted in a statistically significant improvement in overall survival in Hu-MCL-SCID mice. In the clinical trial, the overall response rate was 40% in all 25 patients, 55% in patients with follicular lymphoma, and 29% in patients with MCL. The estimated 2-year progression-free survival (PFS) rate was 24% (95% confidence interval [CI], 10%-53%) in all patients and 60% (95% CI, 20%-85%) in responding patients. Thirteen patients (52%) developed grade 3 neurotoxicity, which consisted of constipation/ileus, sensory or motor neuropathy, or orthostatic hypotension. Patients who were heterozygous for the CD32a (Fcγ receptor IIa) 131 histidine (H) to arginine (R) polymorphism had a significantly decreased PFS (P = .009) after R-bortezomib compared with HH and RR homozygotes.R-bortezomib had significant activity in patients with relapsed or refractory follicular lymphoma and MCL, although an unexpectedly high incidence of grade 3 neurologic toxicity was a potential limiting factor with this combination.

Background. T-cell lymphomas are often associated with abnormal T-cell responses. A viral etiology for certain subsets of cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphoma (PTCL) has been proposed, but viral isolates from fresh malignant T-cells are rare. Recently, a significant association between cytomegalovirus (CMV) seropositivity and CTCL was observed (Herne et.al. Blood101(6):2132), suggesting a pathogenic role for CMV. However, no study to date has examined the incidence of CMV viremia in untreated CTCL and PTCL. Over the last 12 months we analyzed CMV viral loads in patients with CTCL or PTCL prior to initiation of systemic therapy to determine the frequency of CMV viremia and study the relationship between CMV viremia and cellular immune status, as defined by the absolute PB CD8+ T-cell count. Methods. A total of 56 patients with either CTCL (n=41) or PTCL (n=15) were assayed for whole blood quantitative CMV viral loads using a hybrid capture assay for CMV DNA (Digene). PB immunophenotyping by flow cytometry was used to determine absolute CD8+ T-cell counts. In-situ PCR was used to detect CMV DNA in tumor samples. Results. Only 2/56 patients had symptoms consistent with CMV reactivation at the time of the assay. Elevated CMV viral loads were seen in 15/56 (27%) of patients with a patient/cutoff ratio range of 1.0–107.6 and a median of 1.52 for CTCL and 2.14 for PTCL (>1.0 = positive). While all 15 patients with CMV viremia had positive serology for CMV, only 16/41 patients without elevated CMV viral loads were seropositive for CMV. Thus, although CMV viremia paralleled CMV seropositive status, the overall seroprevalence of CMV in this cohort 31/56 (55.3%) was not significantly different from that of the general population. The absolute CD8+ T-cell count was Conclusions. We found that CMV seroprevalence in this cohort of PTCL and CTCL was not greater than that reported in the general population (55.3%). However, we report a previously undescribed high frequency (27%) of CMV viremia, before systemic therapy, and a significant correlation between higher CMV viral loads and degree of CD8+ T-cell lymphopenia. Whether this association reflects a yet to be defined pathogenic role of CMV in T-cell lymphoma or is simply the result of decreased T-cell immunosurveillance remains to be established. The absence of CMV DNA in a limited number of tumor samples from patients with elevated viral load does not support a direct pathogenic role of CMV, although a larger study is needed to confirm this hypothesis. The correlation between lower CD8+ T-cells and CMV viral load suggests defective T-cell immunosurveillance against CMV. A prospective quantitative monitoring study of CMV-specific T-cell responses, CMV DNA viral loads and expression in tumor cells from patients with T-cell lymphoma and appropriate controls (normal volunteers and patients with other hematological malignancies) is in development.

Introduction T cell mediated acute graft-versus-host disease (aGVHD) is the main cause of non-relapse mortality in allogeneic hematopoietic cell transplant recipients. Protein arginine methyltransferase 5 (PRMT5) is a post-translational modifier and promotes activation of memory TH cells. We investigate mechanisms by which PRMT5 regulates T cell function and propose PRMT5 inhibition as a therapeutic strategy for aGVHD. Materials and Methods PRMT5 expression and function was evaluated in T cells of healthy and aGVHD mice and humans. We assessed T cell proliferation and effector function, using C220, a novel PRMT5 inhibitor. We tested effects of PRMT5 inhibition using in vivo mouse models of aGVHD, where mice received T cell depleted bone marrow + allogeneic splenocytes. In graft-versus-leukemia (GVL) experiments, P815 tumor cells were administered. Mice were treated with C220 or vehicle and monitored for survival and clinical aGVHD scores. Results PRMT5 expression and function is upregulated in T cells of mice and humans with aGVHD. Inhibition of PRMT5 reduces T cell proliferation, perturbs cell cycle and downregulates STAT-1 mediated interferon response. Administration of C220 improved survival in aGVHD mouse models and maintained tumor specific CTL response contributing to retention of Graft versus leukemia (GVL) effect. Conclusions Inhibition of PRMT5 using C220, down-regulates T cell proliferative and effector response, induces cell-cycle arrest, reduces interferon response and perturbs signaling pathways. C220 shows potent biological activity in vivo by reducing aGVHD clinical severity and prolonging survival in mouse models without compromising GVL. Therefore, PRMT5 is a novel and druggable target for aGVHD.

BackgroundCentral nervous system Langerhans cell histiocytosis (CNS-LCH) brain involvement may include mass lesions and/or a neurodegenerative disease (LCH-ND) of unknown etiology. The goal of this study was to define the mechanisms of pathogenesis that drive CNS-LCH.MethodsCerebrospinal fluid (CSF) biomarkers including CSF proteins and extracellular BRAFV600E DNA were analyzed in CSF from patients with CNS-LCH lesions compared with patients with brain tumors and other neurodegenerative conditions. Additionally, the presence of BRAFV600E was tested in peripheral mononuclear blood cells (PBMCs) as well as brain biopsies from LCH-ND patients, and the response to BRAF-V600E inhibitor was evaluated in 4 patients with progressive disease.ResultsOsteopontin was the only consistently elevated CSF protein in patients with CNS-LCH compared with patients with other brain pathologies. BRAFV600E DNA was detected in CSF of only 2/20 (10%) cases, both with LCH-ND and active lesions outside the CNS. However, BRAFV600E+ PBMCs were detected with significantly higher frequency at all stages of therapy in LCH patients who developed LCH-ND. Brain biopsies of patients with LCH-ND demonstrated diffuse perivascular infiltration by BRAFV600E+ cells with monocyte phenotype (CD14+ CD33+ CD163+ P2RY12- ) and associated osteopontin expression. Three of 4 patients with LCH-ND treated with BRAF-V600E inhibitor experienced significant clinical and radiologic improvement.ConclusionIn LCH-ND patients, BRAFV600E+ cells in PBMCs and infiltrating myeloid/monocytic cells in the brain is consistent with LCH-ND as an active demyelinating process arising from a mutated hematopoietic precursor from which LCH lesion CD207+ cells are also derived. Therapy directed against myeloid precursors with activated MAPK signaling may be effective for LCH-ND. Cancer 2018;124:2607-20. © 2018 American Cancer Society.

Richter’s Transformation (RT) is a poorly understood and fatal progression of chronic lymphocytic leukemia (CLL) manifesting histologically as diffuse large B-cell lymphoma. Protein arginine methyltransferase 5 (PRMT5) is implicated in lymphomagenesis, but its role in CLL or RT progression is unknown. We demonstrate herein that tumors uniformly overexpress PRMT5 in patients with progression to RT. Furthermore, mice with B-specific overexpression of hPRMT5 develop a B-lymphoid expansion with increased risk of death, and Eµ-PRMT5/TCL1 double transgenic mice develop a highly aggressive disease with transformation that histologically resembles RT; where large-scale transcriptional profiling identifies oncogenic pathways mediating PRMT5-driven disease progression. Lastly, we report the development of a SAM-competitive PRMT5 inhibitor, PRT382, with exclusive selectivity and optimal in vitro and in vivo activity compared to available PRMT5 inhibitors. Taken together, the discovery that PRMT5 drives oncogenic pathways promoting RT provides a compelling rationale for clinical investigation of PRMT5 inhibitors such as PRT382 in aggressive CLL/RT cases.

Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)–induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV+ lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas. This work was supported by grants from the American Society of Hematology/European Hematology Association (L.Alinari), the Leukemia & Lymphoma Society Translational Research Project (LLS TRP) (R.A.B.), Friends of Jason Gould Foundation (R.A.B., P.L.S., J.T.P.), The Ohio State University Drug Development Institute (R.A.B., C.L.), National Institutes of Health, National Institute of Neurological Disorders and Stroke grant R21NS071346 (R.A.B., C.L.) and National Cancer Institute grant R01CA116093 (S.S., R.A.B.).