Quantitative Systems Pharmacology Modeling of Avadomide-Induced Neutropenia Enables Virtual Clinical Dose and Schedule Finding Studies.

Avadomide is a cereblon E3 ligase modulator and a potent antitumor and immunomodulatory agent. Avadomide trials are challenged by neutropenia as a major adverse event and a dose-limiting toxicity. Intermittent dosing schedules supported by preclinical data provide a strategy to reduce frequency and severity of neutropenia; however, the identification of optimal dosing schedules remains a clinical challenge. Quantitative systems pharmacology (QSP) modeling offers opportunities for virtual screening of efficacy and toxicity levels produced by alternative dose and schedule regimens, thereby supporting decision-making in translational drug development. We formulated a QSP model to capture the mechanism of avadomide-induced neutropenia, which involves cereblon-mediated degradation of transcription factor Ikaros, resulting in a maturation block of the neutrophil lineage. The neutropenia model was integrated with avadomide-specific pharmacokinetic and pharmacodynamic models to capture dose-dependent effects. Additionally, we generated a disease-specific virtual patient population to represent the variability in patient characteristics and response to treatment observed for a diffuse large B-cell lymphoma trial cohort. Model utility was demonstrated by simulating the avadomide effect in the virtual population for various dosing schedules and determining the incidence of high-grade neutropenia, its duration, and the probability of recovery to low-grade neutropenia.

Avadomide monotherapy in relapsed/refractory DLBCL: safety, efficacy, and a predictive gene classifier.

Treatment options for relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) are limited, with no standard of care; prognosis is poor, with 4- to 6-month median survival. Avadomide (CC-122) is a cereblon-modulating agent with immunomodulatory and direct antitumor activities. This phase 1 dose-expansion study assessed safety and clinical activity of avadomide monotherapy in patients with de novo R/R DLBCL and transformed lymphoma. Additionally, a novel gene expression classifier, which identifies tumors with a high immune cell infiltration, was shown to enrich for response to avadomide in R/R DLBCL. Ninety-seven patients with R/R DLBCL, including 12 patients with transformed lymphoma, received 3 to 5 mg avadomide administered on continuous or intermittent schedules until unacceptable toxicity, disease progression, or withdrawal. Eighty-two patients (85%) experienced ≥1 grade 3/4 treatment-emergent adverse events (AEs), most commonly neutropenia (51%), infections (24%), anemia (12%), and febrile neutropenia (10%). Discontinuations because of AEs occurred in 10% of patients. Introduction of an intermittent 5/7-day schedule improved tolerability and reduced frequency and severity of neutropenia, febrile neutropenia, and infections. Among 84 patients with de novo R/R DLBCL, overall response rate (ORR) was 29%, including 11% complete response (CR). Responses were cell-of-origin independent. Classifier-positive DLBCL patients (de novo) had an ORR of 44%, median progression-free survival (mPFS) of 6 months, and 16% CR vs an ORR of 19%, mPFS of 1.5 months, and 5% CR in classifier-negative patients (P = .0096). Avadomide is being evaluated in combination with other antilymphoma agents. This trial was registered at www.clinicaltrials.gov as #NCT01421524.

Combination lenalidomide-rituximab immunotherapy activates anti-tumour immunity and induces tumour cell death by complementary mechanisms of action in follicular lymphoma.

Chemotherapy plus rituximab has been the mainstay of treatment for follicular lymphoma (FL) for two decades but is associated with immunosuppression and relapse. In phase 2 studies, lenalidomide combined with rituximab (R2 ) has shown clinical synergy in front-line and relapsed/refractory FL. Here, we show that lenalidomide reactivated dysfunctional T and Natural Killer (NK) cells ex vivo from FL patients by enhancing proliferative capacity and T-helper cell type 1 (Th1) cytokine release. In combination with rituximab, lenalidomide improved antibody-dependent cellular cytotoxicity in sensitive and chemo-resistant FL cells, via a cereblon-dependent mechanism. While single-agent lenalidomide and rituximab increased formation of lytic NK cell immunological synapses with primary FL tumour cells, the combination was superior and correlated with enhanced cytotoxicity. Immunophenotyping of FL patient samples from a phase 3 trial revealed that R2 treatment increased circulating T- and NK-cell counts, while R-chemotherapy was associated with reduced cell numbers. Finally, using an in vitro model of myeloid differentiation, we demonstrated that lenalidomide caused a reversible arrest in neutrophil maturation that was distinct from a cytotoxic chemotherapeutic agent, which may help explain the lower rates of neutropenia observed with R2 versus R-chemotherapy. Taken together, we believe these data support a paradigm shift in the treatment of FL - moving from combination immunochemotherapy to chemotherapy-free immunotherapy.

Comparison of RNA-seq and microarray platforms for splice event detection using a cross-platform algorithm.

BACKGROUND: RNA-seq is a reference technology for determining alternative splicing at genome-wide level. Exon arrays remain widely used for the analysis of gene expression, but show poor validation rate with regard to splicing events. Commercial arrays that include probes within exon junctions have been developed in order to overcome this problem. We compare the performance of RNA-seq (Illumina HiSeq) and junction arrays (Affymetrix Human Transcriptome array) for the analysis of transcript splicing events. Three different breast cancer cell lines were treated with CX-4945, a drug that severely affects splicing. To enable a direct comparison of the two platforms, we adapted EventPointer, an algorithm that detects and labels alternative splicing events using junction arrays, to work also on RNA-seq data. Common results and discrepancies between the technologies were validated and/or resolved by over 200 PCR experiments. RESULTS: As might be expected, RNA-seq appears superior in cases where the technologies disagree and is able to discover novel splicing events beyond the limitations of physical probe-sets. We observe a high degree of coherence between the two technologies, however, with correlation of EventPointer results over 0.90. Through decimation, the detection power of the junction arrays is equivalent to RNA-seq with up to 60 million reads. CONCLUSIONS: Our results suggest, therefore, that exon-junction arrays are a viable alternative to RNA-seq for detection of alternative splicing events when focusing on well-described transcriptional regions.

Human Bone Marrow Stromal Cells Differentiate Into Corneal Tissue and Prevent Ocular Graft-Versus-Host Disease in Mice.

Clinical trials have assessed the use of human bone marrow stromal cells (hBMSCs) for the treatment of immune-related disorders such as graft-versus-host disease (GVHD). In the current study, we show that GFP(+)-transduced hBMSCs generated from bone marrow migrate and differentiate into corneal tissue after subconjunctival injection in mice. Interestingly, these hBMSCs display morphological features of epithelial, stromal, and endothelial cells and appear at different layers and with different morphologies depending on their position within the epithelium. Furthermore, these cells display ultrastructural properties, such as bundles of intermediate filaments, interdigitations, and desmosomes with GFP(-) cells, which confirms their differentiation into corneal tissues. GFP(+)-transduced hBMSCs were injected at different time points into the right eye of lethally irradiated mice undergoing bone marrow transplantation, which developed ocular GVHD (oGVHD). Remarkably, hBMSCs massively migrate to corneal tissues after subconjunctival injection. Both macroscopic and histopathological examination showed minimal or no evidence of GVHD in the right eye, while the left eye, where no hBMSCs were injected, displayed features of GVHD. Thus, in the current study, we confirm that hBMSCs may induce their therapeutic effect at least in part by differentiation and regeneration of damaged tissues in the host. Our results provide experimental evidence that hBMSCs represent a potential cellular therapy to attenuate oGVHD.

An activin receptor IIA ligand trap promotes erythropoiesis resulting in a rapid induction of red blood cells and haemoglobin.

Sotatercept (ACE-011), a recombinant human fusion protein containing the extracellular domain of the human Activin receptor IIA, binds to and inhibits activin and other members of the transforming growth factor -ß (TGF-ß) superfamily. Administration of sotatercept led to a rapid and sustained increase in red blood cell (RBC) count and haemoglobin (Hb) in healthy volunteers (phase I clinical trials), but the mechanism is not fully understood. Mice treated with RAP-011 (murine ortholog of ACE-011) respond with a rapid (within 24 h) increase in haematocrit, Hb, and RBC count. These effects are accompanied by an equally rapid stimulation of late-stage erythroid precursors in the bone marrow (BM). RAP-011 also induces a significant increase in erythroid burst-forming units and erythropoietin, which could contribute to additional, sustained effects on RBC production. Further in vitro co-culture studies demonstrate that BM accessory cells are required for RAP-011 effects. To better understand which TGF-ß family ligand(s) mediate RAP-011 effects, we evaluated the impact of several of these ligands on erythroid differentiation. Our data suggest that RAP-011 may act to rescue growth differentiation factor 11/Activin A-induced inhibition of late-stage erythropoiesis. These data define the mechanism of action of a novel agent that regulates RBC differentiation and provide the rationale to develop sotatercept for the treatment of anaemia and ineffective erythropoiesis.

The novel combination of sirolimus and bortezomib prevents graft-versus-host disease but maintains the graft-versus-leukemia effect after allogeneic transplantation.

BACKGROUND: We have previously shown that bortezomib induces a depletion of alloreactive T cells and allows the expansion of T cells with suppressive properties. In the current study, we analyzed the potential synergistic effect of bortezomib in conjunction with sirolimus in order to reduce-graft-versus-host disease without hampering graft-versus-leukemia effect in the allogeneic transplant setting. DESIGN AND METHODS: We evaluated the effect of sirolimus, bortezomib or the combination of both in the proliferation and activation of in vitro stimulated T lymphocytes. Pathways involved in this synergy were also analyzed using Western blot assays. Finally, BALB/c mice receiving C57BL/6 allogeneic donor bone marrow with splenocytes were used to measure in vivo the effect of this novel combination on the risk of graft-versus-host disease. RESULTS: The combination of both drugs synergistically inhibited both activation and proliferation of stimulated T cells. Also, the production of Th1 cytokines (IFN γ, IL-2 and TNF) was significantly inhibited. This effect was due, at least in part, to the inhibition of Erk and Akt phosphorylation. In vivo, the combination reduced the risk of graft-versus-host disease without hampering graft-versus-leukemia effect, as shown in mice receiving graft-versus-host disease prophylaxis with sirolimus plus bortezomib being infused with tumor WEHI cells plus C57BL/6 donor BM and splenocytes. CONCLUSIONS: The current study reveals a synergistic effect of the combination sirolimus and bortezomib to prevent graft-versus-host disease while maintaining the graft-versus-leukemia effect.

Impaired expression of DICER, DROSHA, SBDS and some microRNAs in mesenchymal stromal cells from myelodysplastic syndrome patients.

UNLABELLED: Background Recent findings suggest that a specific deletion of Dicer1 in mesenchymal stromal cell-derived osteoprogenitors triggers several features of myelodysplastic syndrome in a murine model. Our aim was to analyze DICER1 and DROSHA gene and protein expression in mesenchymal stromal cells (the osteoblastic progenitors) obtained from bone marrow of myelodysplastic syndrome patients, in addition to microRNA expression profile and other target genes such as SBDS, a DICER1-related gene that promotes bone marrow dysfunction and myelodysplasia when repressed in a murine model. DESIGN AND METHODS: Mesenchymal stromal cells from 33 bone marrow samples were evaluated. DICER, DROSHA and SBDS gene expression levels were assessed by real-time PCR and protein expression by Western blot. MicroRNA expresion profile was analyzed by commercial low-density arrays and some of these results were confirmed by individual real-time PCR. RESULTS: Mesenchymal stromal cells from myelodysplastic syndrome patients showed lower DICER1 (0.65±0.08 vs. 1.91±0.57; P=0.011) and DROSHA (0.62±0.06 vs. 1.38±0.29; P=0.009) gene expression levels, two relevant endonucleases associated to microRNA biogenesis, in comparison to normal myelodysplastic syndrome. These findings were confirmed at protein levels by Western blot. Strikingly, no differences were observed between paired mononuclear cells from myelodysplastic syndrome and controls. In addition, mesenchymal stromal cells from myelodysplastic syndrome patients showed significant lower SBDS (0.63±0.06 vs. 1.15±0.28; P=0.021) gene expression levels than mesenchymal stromal cells from healthy controls. Furthermore, mesenchymal stromal cells from myelodysplastic syndrome patients showed an underlying microRNA repression compared to healthy controls. Real-time PCR approach confirmed that mir-155, miR-181a and miR-222 were down-expressed in mesenchymal stromal cells from myelodysplastic syndrome patients. Conclusions This is the first description of an impaired microRNA biogenesis in human mesenchymal stromal cells from myelodysplastic syndrome patients, where DICER1 and DROSHA gene and protein downregulation correlated to a gene and microRNA abnormal expression profile, validating the animal model results previously described.

Bone marrow mesenchymal stem cells for improving hematopoietic function: an in vitro and in vivo model. Part 2: Effect on bone marrow microenvironment.

The aim of the present study was to determine how mesenchymal stem cells (MSC) could improve bone marrow (BM) stroma function after damage, both in vitro and in vivo. Human MSC from 20 healthy donors were isolated and expanded. Mobilized selected CD34(+) progenitor cells were obtained from 20 HSCT donors. For in vitro study, long-term bone marrow cultures (LTBMC) were performed using a etoposide damaged stromal model to test MSC effect in stromal confluence, capability of MSC to lodge in stromal layer as well as some molecules (SDF1, osteopontin,) involved in hematopoietic niche maintenance were analyzed. For the in vivo model, 64 NOD/SCID recipients were transplanted with CD34+ cells administered either by intravenous (i.v.) or intrabone (i.b.) route, with or without BM derived MSC. MSC lodgement within the BM niche was assessed by FISH analysis and the expression of SDF1 and osteopontin by immunohistochemistry. In vivo study showed that when the stromal damage was severe, TP-MSC could lodge in the etoposide-treated BM stroma, as shown by FISH analysis. Osteopontin and SDF1 were differently expressed in damaged stroma and their expression restored after TP-MSC addition. Human in vivo MSC lodgement was observed within BM niche by FISH, but MSC only were detected and not in the contralateral femurs. Human MSC were located around blood vessels in the subendoestal region of femurs and expressed SDF1 and osteopontin. In summary, our data show that MSC can restore BM stromal function and also engraft when a higher stromal damage was done. Interestingly, MSC were detected locally where they were administered but not in the contralateral femur.

Titanium and tantalum as mesenchymal stem cell scaffolds for spinal fusion: an in vitro comparative study.

INTRODUCTION: In the last few years, great interest has been focused on tissue engineering as a potential therapeutic approach for musculoskeletal diseases. The role of metallic implants for spinal fusion has been tested in preclinical and clinical settings. Titanium and tantalum have excellent biocompatibility and mechanical properties and are being used in this situation. On the other hand, the therapeutic role of mesenchymal stem cells (MSC) is extensively explored for their multilineage differentiation into osteoblasts. OBJECTIVES: In vitro comparison of titanium and tantalum as MSCs scaffolds. MATERIAL AND METHODS: In the present study, we have compared the in vitro expansion capacity, viability, immunophenotype (both explored by flow cytometry) and multi-differentiation ability of MSC cultured in the presence of either titanium or tantalum fragments. The adherence of MSC to either metal was demonstrated by electron microscopy. RESULTS: Both metals were able to carry MSC when transferred to new culture flasks. In addition, our study shows that culture of MSC with titanium or tantalum improves cell viability and maintains all their biological properties, with no significant differences regarding the metal employed. CONCLUSION: This would support the use of these combinations for clinical purposes, especially in the spinal fusion and reconstruction setting.

Limbus damage in ocular graft-versus-host disease.

Graft-versus-host disease (GVHD) is the major cause of morbidity and mortality among patients undergoing allogeneic hematopoietic stem cell transplantation. Although animal models have been clearly established for the study of skin, liver, and gut, currently there is no equivalent experiemental model for analyzing ocular involvement, which is rather common, especially among patients diagnosed with chronic GVHD. In the current study we have developed a murine model of ocular GVHD and, for the first time, we describe the histopathologic features involving cornea and limbus, which could play a role in the physiopathology of the disease at the ocular level. Our results represent a major finding that allows us to define a model for evaluating new therapeutic strategies for treating ocular GVHD prior to their use in clinical setting.

Isolation and characterization of mesenchymal stromal cells from human degenerated nucleus pulposus: comparison with bone marrow mesenchymal stromal cells from the same subjects.

STUDY DESIGN: To identify mesenchymal stromal cells (MSC) from degenerate human nucleus pulposus (NP) and compare them with bone marrow (BM) MSC. OBJECTIVE: To test whether MSC obtained from NP and BM from the same subjects share similar biologic characteristics. SUMMARY OF BACKGROUND DATA: Recent studies have proposed biologic strategies for the treatment of intervertebral disc degeneration, including cell therapy. Bone marrow (BM) MSC could be an attractive approach to restore disc function, and there is evidence that NP may contain MSC-like cells. METHODS: Tissue samples were obtained from degenerate lumbar NP and from iliac crest of the same 16 patients with degenerative disc diseases, undergoing discectomy and fusion procedures. MSC isolated from both sources were compared regarding their expansion time, immunophenotype, differentiation ability, and molecular analysis. RESULTS: In all cases, MSC from NP were isolated and expanded. They fulfil nearly all morphological, inmunophenotypical, and differentiation criteria described by the International Society of Cell Therapy for MSC, with the exception that NP-MSC are not able to differentiate into adipocytes. Slight differences were observed with BM-MSC from the same subjects. CONCLUSION: The NP contains mesenchymal stem cells. These cells were quite similar to mesenchymal stem cells from BM, with the exception of their adipogenic differentiation ability. These findings suggest that we may treat intervertebral disc degeneration by cell therapy (MSC from BM) and by stimulating endogenous MSC from NP.

Both CD133(+) cells and monocytes provide significant improvement for hindlimb ischemia, although they do not transdifferentiate into endothelial cells.

To address a number of questions regarding the experimental use of bone marrow (BM) stem cells in hindlimb ischemia, including which is the best cell type (e.g., purified hematopoietic stem cell or monocytes), the best route of delivery [intramuscular (IM) or intravenous (IV)], and the mechanism of action (transdifferentiation or paracrine effects), we have compared the neovascularization capacities of CD133(+) stem cells and monocytes (CD11b(+)) from the BM of Tie2-GFP mice either via IV or IM in a murine severe hindlimb ischemia model. To test the effect of cytokine administration, an extra group received BM conditioned medium. Peripheral blood flow as well as capillary density and GPF-positivity detection in ischemic muscles was evaluated 7, 14, and 21 days postinjection. In addition, CD133(+) and CD11b(+) cells from transgenic animals were cultured in vitro with angiogenic media for 7, 14, and 21 days to assess GFP expression. In all four cell-treated groups, blood flow and capillary density significantly recovered compared with the mice that received no cells or conditioned medium. There were no differences with respect to cell types or administration routes, with the exception of a faster flow recovery in the CD133(+)-treated cell group. We did not find GFP(+) cells in the ischemic muscles and there was no GFP expression after in vitro proangiogenic culture. Our study shows that both purified CD133(+) stem cells and myeloid mononuclear cells, either IM or IV administered, have similar neoangiogenic ability. Nevertheless, transdifferentiation into endothelial cells is not the mechanism responsible for their beneficial effect.

Immunomodulatory effect of 5-azacytidine (5-azaC): potential role in the transplantation setting.

Cytokine genes are targets of multiple epigenetic mechanisms in T lymphocytes. 5-azacytidine (5-azaC) is a nucleoside-based DNA methyltransferase inhibitor that induces demethylation and gene reactivation. In the current study, we analyzed the effect of 5-azaC in T-cell function and observed that 5-azaC inhibits T-cell proliferation and activation, blocking cell cycle in the G(0) to G(1) phase and decreasing the production of proinflammatory cytokines such as tumor necrosis factor-alpha and interferon-gamma. This effect was not attributable to a proapoptotic effect of the drug but to the down-regulation of genes involved in T-cell cycle progression and activation such as CCNG2, MTCP1, CD58, and ADK and up-regulation of genes that induce cell-growth arrest, such as DCUN1D2, U2AF2, GADD45B, or p53. A longer exposure to the drug leads to demethylation of FOXP3 promoter, overexpression of FOXP3, and expansion of regulatory T cells. Finally, the administration of 5-azaC after transplantation prevented the development of graft-versus-host disease, leading to a significant increase in survival in a fully mismatched bone marrow transplantation mouse model. In conclusion, the current study shows the effect of 5-azaC in T lymphocytes and illustrates its role in the allogeneic transplantation setting as an immunomodulatory drug, describing new pathways that must be explored to prevent graft-versus-host disease.

Multiparametric comparison of mesenchymal stromal cells obtained from trabecular bone by using a novel isolation method with those obtained by iliac crest aspiration from the same subjects.

Trabecular bone fragments from femoral heads are sometimes used as bone grafts and have been described as a source of mesenchymal progenitor cells. Nevertheless, mesenchymal stromal cells (MSC) from trabecular bone have not been directly compared with MSC obtained under standard conditions from iliac crest aspiration of the same patients. This is the ideal control to avoid inter-individual variation. We have obtained MSC by a novel method (grinding bone fragments with a bone mill without enzymatic digestion) from the femoral heads of 11 patients undergoing hip replacement surgery and compared them with MSC obtained by standard iliac crest aspiration of bone marrow from the same patients. We have shown that trabecular bone MSC obtained by mechanically fragmented femoral heads fulfil the immunophenotypic and multilineage (adipogenic, osteogenic and chondrogenic) differentiation criteria used to define MSC. We have also differentially compared cellular yields, growth kinetics, cell cycle assessment, and colony-forming unit-fibroblast content of MSC from both sources and conclude that these parameters do not significantly differ. Nevertheless, the finding of slight differences, such as a higher expression of the immature marker CD90, a lower expansion time through the different passages, and a higher percentage of cycling cells in the trabecular bone MSC, warrants further studies with the isolation method proposed here in order to gain further knowledge of the status of MSC in this setting.

Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification.

OBJECTIVE: Optimization of the mesenchymal stem cells (MSC) isolation and expansion method. MATERIALS AND METHODS: Mononuclear cells (MNC) from bone marrow aspirates were obtained by both density gradient centrifugation (standard method) and gravity sedimentation. Cells were cultured in standard conditions (10% fetal calf serum and normal oxygen tension [21% O(2)]) and expansion results compared to those obtained with the same culture conditions to which platelet lysate (PL) preparations were added; in addition, the 21% O(2) concentration was compared to a lower (5%) concentration (hypoxia) until the fourth cell passage. Time of expansion, number of cells obtained, morphology, cell surface markers, and differentiation potential were evaluated. RESULTS: MSC obtained by any of the different culture conditions expressed comparable immunophenotype and were able to differentiate into osteoblasts, adipocytes, and chondrocytes. When the number of MSC obtained at fourth passage was analyzed, the highest cell numbers were obtained with gravity sedimentation isolation and PL-supplemented culture and the expansion time was the shortest when cells were cultured under hypoxic conditions. CONCLUSION: MSC isolation by MNC gravity sedimentation together with culture medium supplementation with 5% of PL in a hypoxic atmosphere (5% O(2)) significantly improved MSC yield and reduced expansion time compared to the standard accepted protocols.