Targeted reduction of cholesterol uptake in cholesterol-addicted lymphoma cells blocks turnover of oxidized lipids to cause ferroptosis.

Normal human cells can either synthesize cholesterol or take it up from lipoproteins to meet their metabolic requirements. In some malignant cells, de novo cholesterol synthesis genes are transcriptionally silent or mutated, meaning that cholesterol uptake from lipoproteins is required for survival. Recent data suggest that lymphoma cells dependent upon lipoprotein-mediated cholesterol uptake are also subject to ferroptosis, an oxygen- and iron-dependent cell death mechanism triggered by accumulation of oxidized lipids in cell membranes unless the lipid hydroperoxidase, glutathione peroxidase 4 (GPX4), reduces these toxic lipid species. To study mechanisms linking cholesterol uptake with ferroptosis and determine the potential role of the high-density lipoprotein (HDL) receptor as a target for cholesterol depleting therapy, we treated lymphoma cell lines known to be sensitive to the reduction of cholesterol uptake with HDL-like nanoparticles (HDL NPs). HDL NPs are a cholesterol-poor ligand that binds to the receptor for cholesterol-rich HDLs, scavenger receptor type B1 (SCARB1). Our data reveal that HDL NP treatment activates a compensatory metabolic response in treated cells toward increased de novo cholesterol synthesis, which is accompanied by nearly complete reduction in expression of GPX4. As a result, oxidized membrane lipids accumulate, leading to cell death through a mechanism consistent with ferroptosis. We obtained similar results in vivo after systemic administration of HDL NPs in mouse lymphoma xenografts and in primary samples obtained from patients with lymphoma. In summary, targeting SCARB1 with HDL NPs in cholesterol uptake-addicted lymphoma cells abolishes GPX4, resulting in cancer cell death by a mechanism consistent with ferroptosis.

Tri-ethylene glycol modified class B and class C CpG conjugated gold nanoparticles for the treatment of lymphoma.

CpG oligodeoxynucleotides (CpGs) can induce an anti-tumor immune response, but also uniquely cause direct lymphoma cytotoxicity. To improve the delivery and efficacy of CpGs, we utilized a tri-ethylene modified CpG conjugated gold nanoparticle (tmCpG NP) platform that is compatible with both class B and class C CpGs, to treat various types of lymphoma, including diffuse large B cell lymphoma, high-grade lymphoma, Burkitt's lymphoma, and mantle cell lymphoma. Both classes of tmCpG NPs reduced viability of human and murine lymphoma cells via apoptosis compared with free CpGs, while having no toxic effects on dendritic cells. TmCpG NPs increased CD19, CD20, and OX40 expression on the lymphoma cells. Overall, we introduced a stable tmCpG NP design that has significant anti-lymphoma effects.

In vivo gold nanoparticle delivery of peptide vaccine induces anti-tumor immune response in prophylactic and therapeutic tumor models.

Gold nanoparticles (AuNPs) are promising vehicles for cancer immunotherapy, with demonstrated efficacy in immune delivery and innate cell stimulation. Nevertheless, their potential has yet to be assessed in the in vivo application of peptide cancer vaccines. In this study, it is hypothesized that the immune distribution and adjuvant qualities of AuNPs could be leveraged to facilitate delivery of the ovalbumin (OVA) peptide antigen and the CpG adjuvant and enhance their therapeutic effect in a B16-OVA tumor model. AuNP delivery of OVA (AuNP-OVA) and of CpG (AuNP-CpG) enhanced the efficacy of both agents and induced strong antigen-specific responses. In addition, it is found that AuNP-OVA delivery alone, without CpG, is sufficient to promote significant antigen-specific responses, leading to subsequent anti-tumor activity and prolonged survival in both prophylactic and therapeutic in vivo tumor models. This enhanced therapeutic efficacy is likely due to the adjuvant effect of peptide coated AuNPs, as they induce inflammatory cytokine release when cultured with bone marrow dendritic cells. Overall, AuNP-mediated OVA peptide delivery can produce significant therapeutic benefits without the need of adjuvant, indicating that AuNPs are effective peptide vaccine carriers with the potential to permit the use of lower and safer adjuvant doses during vaccination.

In vivo immune cell distribution of gold nanoparticles in naïve and tumor bearing mice.

Gold nanoparticles (AuNP) have been widely used for drug delivery and have recently been explored for applications in cancer immunotherapy. Although AuNPs are known to accumulate heavily in the spleen, the particle distribution within immune cells has not been thoroughly studied. Here, cellular distribution of Cy5 labeled 50 nm AuNPs is characterized within the immune populations of the spleen from naïve and tumor bearing mice using flow cytometry. Surprisingly, approximately 30% of the detected AuNPs are taken up by B cells at 24 h, with about 10% in granulocytes, 18% in dendritic cells, and 8% in T cells. In addition, 3% of the particles are detected within myeloid derived suppressor cells, an immune suppressive population that could be targeted for cancer immunotherapy. Furthermore, it is observed that, over time, the particles traveled from the red pulp and marginal zone to the follicles of the spleen. Taking into consideration that the particle cellular distribution does not change at 1, 6 and 24 h, it is highly suggestive that the immune populations carry the particles and migrate through the spleen instead of the particles migrating through the tissue by cell-cell transfer. Finally, no difference is observed in particle distribution between naïve and tumor bearing mice in the spleen, and nanoparticles are detected within 0.7% of dendritic cells of the tumor microenvironment. Overall, these results can help inform and influence future AuNP delivery design criteria including future applications for nanoparticle-mediated immunotherapy.

Discovering the fibroblastic reticular cell in the immune tumor microenvironment in lymphoma.

The study of the cellular and molecular microenvironment in B cell lymphoma, especially diffuse large B cell lymphoma (DLBCL), has led to prognostic and therapeutic algorithms that may improve patient outcomes. Emerging gene signature panels provide a granular understanding of DLBCL based on the immune tumor microenvironment (iTME). In addition, some gene signatures identify lymphomas that are more responsive to immune-based treatment, indicating that the iTME has a biological signature that could affect outcomes when targeted. In this issue of the JCI, Apollonio et al. report on fibroblastic reticular cells (FRCs) as potential targets in aggressive lymphoma. FRCs interacted with lymphoma cells and induced a state of chronic inflammation that suppressed immune function by impeding optimal T cell migration and inhibiting CD8+ T cell lytic function. These findings suggest that manipulating the iTME by directly targeting FRCs may enhance responses to immunotherapy in DLBCL.

Immune Checkpoint Blockade for the Treatment of Hodgkin Lymphoma.

Classical Hodgkin lymphoma is biologically different than other lymphomas. The cancer cells only occupy a small amount of the lymph node and evade the immune system by amplification of PD-L1 and PD-L2. Therefore, checkpoint inhibitors are a logical treatment option for Hodgkin lymphoma patients to unlock the immune system. Checkpoint inhibitors have shown high response rates in clinical trials in advanced-stage Hodgkin lymphoma. The two most commonly used checkpoint inhibitors are pembrolizumab and nivolumab, both FDA approved as third-line therapy. There is increasing interest in the use of checkpoint inhibitors with combination chemotherapy or with other targeted agents in the second-line or even frontline setting. In this review, we will highlight the clinical trials that led to approvals of checkpoint inhibitors for Hodgkin lymphoma.

Enhanced systemic antilymphoma immune response by photothermal therapy with CpG deoxynucleotide-coated nanoparticles.

In preclinical studies, we investigated a novel mechanism of in situ vaccination in lymphoma. Radiation therapy (RT) can induce abscopal responses in lymphoma models, but this has not translated into clinical efficacy. We hypothesized that immune stimulation with cytosine guanine dinucleotide (CpG) deoxynucleotides could enhance abscopal effects induced by RT or photothermal therapy (PTT), which has been shown to have an immune stimulatory effect in solid tumors but has not been studied in lymphoma. We designed a branched gold nanoparticle (NP) platform to carry CpG deoxynucleotides while maintaining PTT function and compared the immunologic profile of the tumor microenvironment after PTT or RT in a dual-flank lymphoma model. One flank was treated with CpG deoxynucleotides with RT or PTT, and the other tumor was left untreated. We found that the CpG deoxynucleotide/PTT group had significant reduction in growth in both treated (primary) and untreated (secondary) tumors, suggesting an improved abscopal response, with a concomitant increase in CD8/CD4 and cytotoxic T-cell/regulatory T-cell ratios in both primary and secondary tumors compared with CpG deoxynucleotides/RT. Dendritic cells in primary and secondary draining lymph nodes had increased maturation markers in the CpG deoxynucleotide/PTT group, and the effector memory T cells (both CD4 and CD8) in the secondary tumor and spleen were increased, suggesting a systemic vaccination effect. These data suggest that in a lymphoma model, PTT using a CpG deoxynucleotide NP platform resulted in enhanced in situ vaccination and abscopal response compared with RT.

Ibrutinib for chronic lymphocytic leukemia in the setting of respiratory failure from severe COVID-19 infection: Case report and literature review.

Ibrutinib, a known Burton's tyrosine kinase (BTK) and interleukin-2 inducible T-cell kinase (ITK) inhibitor, is used for the treatment of B-cell disorders (chronic lymphocytic leukemia [CLL] and various other lymphomas) and chronic graft versus host disease following allogeneic hematopoietic cell transplantation. Because it is considered an immunosuppressant, continuation of ibrutinib is often debated when patients have an active infection, and this becomes an especially difficult decision in the setting of coronavirus disease 2019 (COVID-19). Here, we describe a patient with CLL who was on ibrutinib then developed severe COVID-19 infection requiring mechanical ventilation. We elected to continue ibrutinib the same day he was intubated, reasoning that BTK inhibition in myeloid immune cells has been shown to reduce or even reverse influenza-mediated acute lung injury and that ITK inhibition in T cells has correlated with reduction in viral replication, and therefore may have an advantage in this setting. Ibrutinib also has been shown to block Src family kinases, which potentially could result in reduction of viral entry and the inflammatory cytokine response in the lungs. The patient was extubated after 9 days with a complex hospital course and eventually discharged on room air. The only way to rationally inform these decisions and explore similar potentially promising leads in this pandemic is to conduct carefully done clinical trials.

Moxetumomab pasudotox for hairy cell leukemia: preclinical development to FDA approval.

Moxetumomab pasudotox (MP) is an immunotoxin that recently received US Food and Drug Administration (FDA) approval for the treatment of hairy cell leukemia (HCL) that has failed at least 2 prior lines of therapy, including a purine analog. MP is a recombinant immunotoxin that consists of an anti-CD22 immunoglobulin variable domain genetically joined to Pseudomonas exotoxin (PE38). Unlike most antibody-drug conjugates, which use a chemical linker, recombinant DNA techniques are used to produce MP. MP and its predecessor, BL22, were initially developed to treat non-Hodgkin lymphoma, acute lymphoblastic leukemia, and HCL. However, MP was found to be particularly effective in HCL due to the high level of CD22 cell-surface expression. The recent pivotal phase 3 trial of MP in relapsed/refractory HCL demonstrated a durable complete remission rate of 30%, and 85% of complete responders achieved minimal residual disease negativity, which is associated with improved disease-free survival outcomes in HCL. In addition to an exceptional depth of response, MP appears to be less immunosuppressive than purine analogs. MP is generally well tolerated but has unique toxicities, including capillary leak syndrome and hemolytic uremic syndrome, which are poorly understood. This review will encompass the preclinical and clinical development of MP, with particular attention to its current indication in HCL.

Laminin Peptide-Immobilized Hydrogels Modulate Valve Endothelial Cell Hemostatic Regulation.

Valve endothelial cells (VEC) have unique phenotypic responses relative to other types of vascular endothelial cells and have highly sensitive hemostatic functions affected by changes in valve tissues. Furthermore, effects of environmental factors on VEC hemostatic function has not been characterized. This work used a poly(ethylene glycol) diacrylate (PEGDA) hydrogel platform to evaluate the effects of substrate stiffness and cell adhesive ligands on VEC phenotype and expression of hemostatic genes. Hydrogels of molecular weights (MWs) 3.4, 8, and 20 kDa were polymerized into platforms of different rigidities and thiol-modified cell adhesive peptides were covalently bound to acrylate groups on the hydrogel surfaces. The peptide RKRLQVQLSIRT (RKR) is a syndecan-1 binding ligand derived from laminin, a trimeric protein and a basement membrane matrix component. Conversely, RGDS is an integrin binding peptide found in many extracellular matrix (ECM) proteins including fibronectin, fibrinogen, and von Willebrand factor (VWF). VECs adhered to and formed a stable monolayer on all RKR-coated hydrogel-MW combinations. RGDS-coated platforms supported VEC adhesion and growth on RGDS-3.4 kDa and RGDS-8 kDa hydrogels. VECs cultured on the softer RKR-8 kDa and RKR-20 kDa hydrogel platforms had significantly higher gene expression for all anti-thrombotic (ADAMTS-13, tissue factor pathway inhibitor, and tissue plasminogen activator) and thrombotic (VWF, tissue factor, and P-selectin) proteins than VECs cultured on RGDS-coated hydrogels and tissue culture polystyrene controls. Stimulated VECs promoted greater platelet adhesion than non-stimulated VECs on their respective culture condition; yet stimulated VECs on RGDS-3.4 kDa gels were not as responsive to stimulation relative to the RKR-gel groups. Thus, the syndecan binding, laminin-derived peptide promoted stable VEC adhesion on the softer hydrogels and maintained VEC phenotype and natural hemostatic function. In conclusion, utilization of non-integrin adhesive peptide sequences derived from basement membrane ECM may recapitulate balanced VEC function and may benefit endothelialization of valve implants.

High-density sub-100-nm peptide-gold nanoparticle complexes improve vaccine presentation by dendritic cells in vitro.

Nanocarriers have been explored to improve the delivery of tumor antigens to dendritic cells (DCs). Gold nanoparticles are attractive nanocarriers because they are inert, non-toxic, and can be readily endocytosed by DCs. Here, we designed novel gold-based nanovaccines (AuNVs) using a simple self-assembling bottom-up conjugation method to generate high-peptide density delivery and effective immune responses with limited toxicity. AuNVs were synthesized using a self-assembling conjugation method and optimized using DC-to-splenocyte interferon-γ enzyme-linked immunosorbent spot assays. The AuNV design has shown successful peptide conjugation with approximately 90% yield while remaining smaller than 80 nm in diameter. DCs uptake AuNVs with minimal toxicity and are able to process the vaccine peptides on the particles to stimulate cytotoxic T lymphocytes (CTLs). These high-peptide density AuNVs can stimulate CTLs better than free peptides and have great potential as carriers for various vaccine types.

Gold nanoparticle delivery of modified CpG stimulates macrophages and inhibits tumor growth for enhanced immunotherapy.

Gold nanoparticle accumulation in immune cells has commonly been viewed as a side effect for cancer therapeutic delivery; however, this phenomenon can be utilized for developing gold nanoparticle mediated immunotherapy. Here, we conjugated a modified CpG oligodeoxynucleotide immune stimulant to gold nanoparticles using a simple and scalable self-assembled monolayer scheme that enhanced the functionality of CpG in vitro and in vivo. Nanoparticles can attenuate systemic side effects by enhancing CpG delivery passively to innate effector cells. The use of a triethylene glycol (TEG) spacer on top of the traditional poly-thymidine spacer increased CpG macrophage stimulatory effects without sacrificing DNA content on the nanoparticle, which directly correlates to particle uptake. In addition, the immune effects of modified CpG-AuNPs were altered by the core particle size, with smaller 15 nm AuNPs generating maximum immune response. These TEG modified CpG-AuNP complexes induced macrophage and dendritic cell tumor infiltration, significantly inhibited tumor growth, and promoted survival in mice when compared to treatments with free CpG.