HSP90 is essential for Jak-STAT signaling in classical Hodgkin lymphoma cells

In classical Hodgkin lymphoma (cHL) chemotherapeutic regimens are associated with stagnant rates of secondary malignancies requiring the development of new therapeutic strategies. We and others have shown that permanently activated Signal Transducer and Activator of Transcription (STAT) molecules are essential for cHL cells. Recently an overexpression of heat-shock protein 90 (HSP90) in cHL cells has been shown and inhibition of HSP90 seems to affect cHL cell survival. Here we analysed the effects of HSP90 inhibition by geldanamycin derivative 17-AAG or RNA interference (RNAi) on aberrant Jak-STAT signaling in cHL cells. Treatment of cHL cell lines with 17-AAG led to reduced cell proliferation and a complete inhibition of STAT1, -3, -5 and -6 tyrosine phosphorylation probably as a result of reduced protein expression of Janus kinases (Jaks). RNAi-mediated inhibition of HSP90 showed similar effects on Jak-STAT signaling in L428 cHL cells. These results suggest a central role of HSP90 in permanently activated Jak-STAT signaling in cHL cells. Therapeutics targeting HSP90 may be a promising strategy in cHL and other cancer entities associated with deregulated Jak-STAT pathway activation

Novel highly potent CD4bs bNAb with restricted pathway to HIV-1 escape

Purpose: Broadly HIV-1 neutralizing antibodies (bNAbs) can suppress viremia in humans and represent a novel approach for effective immunotherapy. However, bNAb monotherapy selects for antibody-resistant viral variants. Thus, we focused on the identification of new antibody combinations and/or novel bNAbs that restrict pathways of HIV-1 escape. Methods: We screened HIV-1 positive patients for their neutralizing capacities. Following, we performed single cell sorting and PCR of HIV-1 Env-reactive mature B cells of identified elite neutralizers. Found antibodies were tested for neutralization and binding capacities in vitro. Further, their antiviral activity was tested in an HIV-1 infected humanized mouse model. Results: Here we report the isolation of antibody 1–18, a VH1–46-encoded CD4 binding site (CD4bs) bNAb identified in an individual ranking among the top 1% neutralizers of 2,274 HIV-1-infected subjects. Tested on a 119-virus panel, 1–18 showed to be exceptionally broad and potent with a coverage of 97% and a mean IC50 of 0.048 lg/mL, exceeding the activity of most potent CD4bs bNAbs described to-date. A 2.4 Å cryo-EM structure of 1–18 bound to a native-like Env trimer revealed that it interacts with HIV-1 env similar to other CD4bs bNAbs, but includes additional contacts to the V3 loop of the adjacent protomer. Notably, in vitro, 1–18 maintained activity against viruses carrying mutations associated with escape from VRC01-class bNAbs. Further, its HIV-1 env wide escape profile differed critically from other CD4bs bNAbs. In humanized mice, monotherapy with 1–18 was sufficient to prevent the development of viral escape variants that rapidly emerged during treatment with other CD4bs bNAbs. Finally, 1–18 overcame classical HIV-1 mutations that are driven by VRC01-like bNAbs in vivo. Conclusion: 1–18 is a highly potent and broad bNAb that restricts escape and overcomes frequent CD4bs escape pathways, providing new options for bNAb combinations to prevent and treat HIV-1 infection

Differentiation of Chronic Lymphocytic Leukemia B Cells into Immunoglobulin Secreting Cells Decreases LEF-1 Expression

Lymphocyte enhancer binding factor 1 (LEF-1) plays a crucial role in B lineage development and is only expressed in B cell precursors as B cell differentiation into mature B and plasma cells silences its expression. Chronic lymphocytic leukemia (CLL) cells aberrantly express LEF-1 and its expression is required for cellular survival. We hypothesized that modification of the differentiation status of CLL cells would result in loss of LEF-1 expression and eliminate the survival advantage provided by its aberrant expression. In this study, we first established a methodology that induces CLL cells to differentiate into immunoglobulin (Ig) secreting cells (ISC) using the TLR9 agonist, CpG, together with cytokines (CpG/c). CpG/c stimulation resulted in dramatic CLL cell phenotypic and morphologic changes, expression of cytoplasmic Ig, and secretion of light chain restricted Ig. CpG/c stimulation also resulted in decreased CLL cell LEF-1 expression and increased Blimp-1 expression, which is crucial for plasma cell differentiation. Further, Wnt pathway activation and cellular survival were impaired in differentiated CLL cells compared to undifferentiated CLL cells. These data support the notion that CLL can differentiate into ISC and that this triggers decreased leukemic cell survival secondary to the down regulation of LEF-1 and decreased Wnt pathway activation

The JAK inhibitor AZD1480 regulates proliferation and immunity in Hodgkin lymphoma

Aberrant activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway has been reported to promote proliferation and survival of Hodgkin and Reed–Sternberg cells of Hodgkin lymphoma (HL). We investigated the activity of the JAK inhibitor AZD1480 in HL-derived cell lines and determined its mechanisms of action. AZD1480 at low doses (0.1–1 μ) potently inhibited STATs phosphorylation, but did not predictably result in antiproliferative effects, as it activated a negative-feedback loop causing phosphorylation of JAK2 and extracellular signal-regulated kinases 1 and 2 (ERK1/2), and increased IP-10, RANTES and interleukin (IL)-8 concentrations in the supernatants. Inhibition of the ERK activity by mitogen-activated extracellular signal regulated kinase (MEK) inhibitors (UO126 and PD98059) enhanced the cytotoxic activity of AZD1480. Interestingly, submicromolar concentrations of AZD1480 demonstrated significant immunoregulatory effects by downregulating T-helper 2 cytokines and chemokines, including IL-13 and thymus- and activation-regulated chemokine, and the surface expression of the immunosuppressive programmed death ligands 1 and 2. Higher concentrations of AZD1480 (5 μ) induced G2/M arrest and cell death by inhibiting Aurora kinases. Our study demonstrates that AZD1480 regulates proliferation and immunity in HL cell lines and provides mechanistic rationale for evaluating AZD1480 alone or in combination with MEK inhibitors in HL

Inferior outcomes of EU versus US patients treated with CD19 CAR-T for relapsed/refractory large B-cell lymphoma: association with differences in tumor burden, systemic inflammation, bridging therapy utilization, and CAR-T product use

Real-world evidence suggests a trend toward inferior survival of patients receiving CD19 chimeric antigen receptor (CAR) T-cell therapy in Europe (EU) and with tisagenlecleucel. The underlying logistic, patient- and disease-related reasons for these discrepancies remain poorly understood. In this multicenter retrospective observational study, we studied the patient-individual journey from CAR-T indication to infusion, baseline features, and survival outcomes in 374 patients treated with tisagenlecleucel (tisa-cel) or axicabtagene-ciloleucel (axi-cel) in EU and the United States (US). Compared with US patients, EU patients had prolonged indication-to-infusion intervals (66 versus 50 d; P < 0.001) and more commonly received intermediary therapies (holding and/or bridging therapy, 94% in EU versus 74% in US; P < 0.001). Baseline lactate dehydrogenase (LDH) (median 321 versus 271 U/L; P = 0.02) and ferritin levels (675 versus 425 ng/mL; P = 0.004) were significantly elevated in the EU cohort. Overall, we observed inferior survival in EU patients (median progression-free survival [PFS] 3.1 versus 9.2 months in US; P < 0.001) and with tisa-cel (3.2 versus 9.2 months with axi-cel; P < 0.001). On multivariate Lasso modeling, nonresponse to bridging, elevated ferritin, and increased C-reactive protein represented independent risks for treatment failure. Weighing these variables into a patient-individual risk balancer (high risk [HR] balancer), we found higher levels in EU versus US and tisa-cel versus axi-cel cohorts. Notably, superior PFS with axi-cel was exclusively evident in patients at low risk for progression (according to the HR balancer), but not in high-risk patients. These data demonstrate that inferior survival outcomes in EU patients are associated with longer time-to-infusion intervals, higher tumor burden/LDH levels, increased systemic inflammatory markers, and CAR-T product use

Apoptotic cell-based therapies against transplant rejection: role of recipient’s dendritic cells

One of the ultimate goals in transplantation is to develop novel therapeutic methods for induction of donor-specific tolerance to reduce the side effects caused by the generalized immunosuppression associated to the currently used pharmacologic regimens. Interaction or phagocytosis of cells in early apoptosis exerts potent anti-inflammatory and immunosuppressive effects on antigen (Ag)-presenting cells (APC) like dendritic cells (DC) and macrophages. This observation led to the idea that apoptotic cell-based therapies could be employed to deliver donor-Ag in combination with regulatory signals to recipient’s APC as therapeutic approach to restrain the anti-donor response. This review describes the multiple mechanisms by which apoptotic cells down-modulate the immuno-stimulatory and pro-inflammatory functions of DC and macrophages, and the role of the interaction between apoptotic cells and APC in self-tolerance and in apoptotic cell-based therapies to prevent/treat allograft rejection and graft-versus-host disease in murine experimental systems and in humans. It also explores the role that in vivo-generated apoptotic cells could have in the beneficial effects of extracorporeal photopheresis, donor-specific transfusion, and tolerogenic DC-based therapies in transplantation