Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis.

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.

Vascular amounts and dispersion of caliber-classified vessels as key parameters to quantitate 3D micro-angioarchitectures in multiple myeloma experimental tumors.

Blood vessel micro-angioarchitecture plays a pivotal role in tumor progression, metastatic dissemination and response to therapy. Thus, methods able to quantify microvascular trees and their anomalies may allow a better comprehension of the neovascularization process and evaluation of vascular-targeted therapies in cancer. To this aim, the development of a restricted set of indexes able to describe the arrangement of a microvascular tree is eagerly required. We addressed this goal through 3D analysis of the functional microvascular network in sulfo-biotin-stained human multiple myeloma KMS-11 xenografts in NOD/SCID mice. Using image analysis, we show that amounts, spatial dispersion and spatial relationships of adjacent classes of caliber-filtered microvessels provide a near-linear graphical "fingerprint" of tumor micro-angioarchitecture. Position, slope and axial projections of this graphical outcome reflect biological features and summarize the properties of tumor micro-angioarchitecture. Notably, treatment of KMS-11 xenografts with anti-angiogenic drugs affected position and slope of the specific curves without degrading their near-linear properties. The possibility offered by this procedure to describe and quantify the 3D features of the tumor micro-angioarchitecture paves the way to the analysis of the microvascular tree in human tumor specimens at different stages of tumor progression and after pharmacologic interventions, with possible diagnostic and prognostic implications.

Circulating tumor DNA reveals genetics, clonal evolution, and residual disease in classical Hodgkin lymphoma.

The rarity of neoplastic cells in the biopsy imposes major technical hurdles that have so far limited genomic studies in classical Hodgkin lymphoma (cHL). By using a highly sensitive and robust deep next-generation sequencing approach for circulating tumor DNA (ctDNA), we aimed to identify the genetics of cHL in different clinical phases, as well as its modifications on treatment. The analysis was based on specimens collected from 80 newly diagnosed and 32 refractory patients with cHL, including longitudinal samples collected under ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) chemotherapy and longitudinal samples from relapsing patients treated with chemotherapy and immunotherapy. ctDNA mirrored Hodgkin and Reed-Sternberg cell genetics, thus establishing ctDNA as an easily accessible source of tumor DNA for cHL genotyping. By identifying STAT6 as the most frequently mutated gene in ∼40% of cases, we refined the current knowledge of cHL genetics. Longitudinal ctDNA profiling identified treatment-dependent patterns of clonal evolution in patients relapsing after chemotherapy and patients maintained in partial remission under immunotherapy. By measuring ctDNA changes during therapy, we propose ctDNA as a radiation-free tool to track residual disease that may integrate positron emission tomography imaging for the early identification of chemorefractory patients with cHL. Collectively, our results provide the proof of concept that ctDNA may serve as a novel precision medicine biomarker in cHL.

Dual PI3K/ERK inhibition induces necroptotic cell death of Hodgkin Lymphoma cells through IER3 downregulation.

PI3K/AKT and RAF/MEK/ERK pathways are constitutively activated in Hodgkin lymphoma (HL) patients, thus representing attractive therapeutic targets. Here we report that the PI3K/ERK dual inhibitor AEZS-136 induced significant cell proliferation inhibition in L-540, SUP-HD1, KM-H2 and L-428 HL cell lines, but a significant increase in necroptotic cell death was observed only in two out of four cell lines (L-540 and SUP-HD1). In these cells, AEZS-136-induced necroptosis was associated with mitochondrial dysfunction and reactive oxygen species (ROS) production. JNK was activated by AEZS-136, and AEZS-136-induced necroptosis was blocked by the necroptosis inhibitor necrostatin-1 or the JNK inhibitor SP600125, suggesting that JNK activation is required to trigger necroptosis following dual PI3K/ERK inhibition. Gene expression analysis indicated that the effects of AEZS-136 were associated with the modulation of cell cycle and cell death pathways. In the cell death-resistant cell lines, AEZS-136 induced the expression of immediate early response 3 (IER3) both in vitro and in vivo. Silencing of IER3 restored sensitivity to AEZS-136-induced necroptosis. Furthermore, xenograft studies demonstrated a 70% inhibition of tumor growth and a 10-fold increase in tumor necrosis in AEZS-136-treated animals. Together, these data suggest that dual PI3K/ERK inhibition might be an effective approach for improving therapeutic outcomes in HL.