CD209+ monocyte-derived myeloid dendritic cells were increased in patients with leukemic cutaneous T-cell lymphoma undergoing extracorporeal photopheresis via the CELLEXTM system.

BACKGROUND/PURPOSE: We previously reported that myeloid dendritic cells (mDC) were increased in patients with leukemic cutaneous T-cell lymphoma (L-CTCL) following extracorporeal photopheresis (ECP) using the Therakos UVAR XTS™ system. We now assessed monocyte-derived mDCs (Mo-DCs) in L-CTCL patients treated with the CELLEXTM photopheresis system. CD209, a transmembrane receptor, was used to define Mo-DCs. METHODS: Peripheral blood samples from baseline pre-ECP and at Day 2, 1 month, 3 months, and 6 months post-ECP were analyzed by flow cytometry for Lin- HLA-DR+ CD123+ plasmacytoid dendritic cells (pDCs), Lin- HLA-DR+ CD11c+ mDCs, and CD209+ mDCs. The expression of CD209 mRNA was assessed by real-time PCR. RESULTS: At baseline, 7 of 19 patients had lower than normal mDCs, and all patients had lower than normal CD209+ mDCs in peripheral blood mononuclear cells (0.005% in patients, n = 19, vs 0.50% in healthy donors, n = 7, P < .0001). The CD209+ mDC numbers only accounted for 3.28% out of total mDCs in patients compared with 66.51% in healthy donors. After treatment, the CD209+ mDC numbers showed increasing trends in patients. The average absolute numbers of CD209+ mDCs went up by 4.8-fold at 3 months (n = 10, P = .103) and by 6.4-fold at 6 months (n = 9, P = .100). CD209 mRNA expression went up in two patients responsive to therapy, parallel to CD209+ mDC numbers. L-CTCL patients achieved 70% overall clinical response rate (7/10) following ECP therapy with the CELLEXTM system. CONCLUSIONS: Our results suggest that the CELLEXTM photopheresis system is effective for treating L-CTCL patients like the UVAR XTS™ system, and in vivo-generated Mo-DCs increase following ECP.

Temperature effects on the hydrodynamic radius of the intrinsically disordered N-terminal region of the p53 protein.

Intrinsically disordered proteins (IDPs) are often characterized in terms of the hydrodynamic radius, Rh . The Rh of IDPs are known to depend on fractional proline content and net charge, where increased numbers of proline residues and increased net charge cause larger Rh . Though sequence and charge effects on the Rh of IDPs have been studied, the temperature sensitivity has been noted only briefly. Reported here are Rh measurements in the temperature range of 5-75°C for the intrinsically disordered N-terminal region of the p53 protein, p53(1-93). Of note, the Rh of this protein fragment was highly sensitive to temperature, decreasing from 35 Å at 5°C to 26 Å at 75°C. Computer generated simulations of conformationally dynamic and disordered polypeptide chains were performed to provide a hypothesis for the heat-induced compaction of p53(1-93) structure, which was opposite to the heat-induced increase in Rh observed for a model folded protein. The simulations demonstrated that heat caused Rh to trend toward statistical coil values for both proteins, indicating that the effects of heat on p53(1-93) structure could be interpreted as thermal denaturation. The simulation data also predicted that proline content contributed minimally to the native Rh of p53(1-93), which was confirmed by measuring Rh for a substitution variant that had all 22 proline residues changed for glycine.

Toward understanding polymer micelle stability: Density ultracentrifugation offers insight into polymer micelle stability in human fluids.

Micelles, as a class of drug delivery systems, are underrepresented among United States Food and Drug Administration approved drugs. A lack of clinical translation of these systems may be due to, in part, to a lack of understanding of micelle interactions with biologic fluids following injection. Despite the limited clinical translation, micelles remain an active area of research focus and pre-clinical development. The goal of the present study was to examine the stability of amphiphilic block copolymer micelles in biologic fluids to identify the properties and components of biologic fluids that influence micelle stability. Micelle stability, measured via Förster resonance energy transfer-based fluorescent spectrometry, was complemented with density ultracentrifugation to reveal the colocalized, or dissociated, state of the dye cargo after exposure to human biologic fluids. Polymeric micelles composed of poly(ethylene glycol-block-caprolactone) (mPEG-CL) and poly(ethylene glycol-block-lactide) (mPEG-LA) were unstable in fetal bovine serum, human serum and synovial fluid, with varying levels of instability observed in ascites and pleural fluid. All polymeric micelles exhibited stability in cerebrospinal fluid, highlighting the potential for local cerebro-spinal administration of micelles. Interestingly, mPEG2.2k-CL3.1k and mPEG2k-LA2.7k micelles favored dissolution whereas mPEG5.4k-LA28.5k micelles favored stability. Taken together, our data offers both quantitative and qualitative evidence for micelle stability within human biologic fluids and offers evidence of polymer micelle instability in biologic fluids that is not explained by either total protein content or total unsaturated lipid content. The results help to identify potential sites for local delivery where stability is maintained.

ONC201 selectively induces apoptosis in cutaneous T-cell lymphoma cells via activating pro-apoptotic integrated stress response and inactivating JAK/STAT and NF-κB pathways.

Cutaneous T-cell lymphomas (CTCLs) are extremely symptomatic and still incurable, and more effective and less toxic therapies are urgently needed. ONC201, an imipridone compound, has shown efficacy in pre-clinical studies in multiple advanced cancers. This study was to evaluate the anti-tumor activity of ONC201 on CTCL cells. The effect of ONC201 on the cell growth and apoptosis were evaluated in CTCL cell lines (n=8) and primary CD4+ malignant T cells isolated from CTCL patients (n=5). ONC201 showed a time-dependent cell growth inhibition in all treated cell lines with a concentration range of 1.25-10.0 µM. ONC201 also induced apoptosis in tested cells with a narrow concentration range of 2.5-10.0 µM, evidenced by increased Annexin V+ cells, accompanied by accumulated sub-G1 portions. ONC201 only induced apoptosis in CD4+ malignant T cells, not in normal CD4+ T cells. The activating transcription factor 4 (ATF4), a hallmark of integrated stress response, was upregulated in response to ONC201 whereas Akt was downregulated. In addition, molecules in JAK/STAT and NF-κB pathways, as well as IL-32ß, were downregulated following ONC201 treatment. Thus, ONC201 exerts a potent and selective anti-tumor effect on CTCL cells. Its efficacy may involve activating integrated stress response through ATF4 and inactivating JAK/STAT and NF-κB pathways.

Depletion of regulatory T cells by targeting CC chemokine receptor type 4 with mogamulizumab.

The CC chemokine receptor 4 (CCR4) is highly expressed on type 2 helper T cells and regulatory T (Treg) cells. Mogamulizumab, an anti-CCR4 antibody, reduces the numbers of CCR4+ malignant T cells and CCR4+ Treg cells in cutaneous T-cell lymphoma. Depleting Treg cells by targeting CCR4 has great potential in cancer immunotherapies.

Genomic profiling of Sézary syndrome identifies alterations of key T cell signaling and differentiation genes.

Sézary syndrome is a rare leukemic form of cutaneous T cell lymphoma characterized by generalized redness, scaling, itching and increased numbers of circulating atypical T lymphocytes. It is rarely curable, with poor prognosis. Here we present a multiplatform genomic analysis of 37 patients with Sézary syndrome that implicates dysregulation of cell cycle checkpoint and T cell signaling. Frequent somatic alterations were identified in TP53, CARD11, CCR4, PLCG1, CDKN2A, ARID1A, RPS6KA1 and ZEB1. Activating CCR4 and CARD11 mutations were detected in nearly one-third of patients. ZEB1, encoding a transcription repressor essential for T cell differentiation, was deleted in over one-half of patients. IL32 and IL2RG were overexpressed in nearly all cases. Our results demonstrate profound disruption of key signaling pathways in Sézary syndrome and suggest potential targets for new therapies.