Dagmar Wider

Potent in vitro and in vivo activity of sorafenib in multiple myeloma: induction of cell death, CD138-downregulation and inhibition of migration through actin depolymerization.

Despite considerable advances, multiple myeloma (MM) remains incurable and the development of novel therapies targeting the interplay between plasma cells (PCs) and their bone marrow (BM) microenvironment remains essential. We investigated the effect of various agents in vitro on the proliferation, phenotype, morphology, actin polymerization and migration of MM cells and, in vivo, the tumour growth of L363‐bearing non‐obese diabetic severe combined immunodeficient mice with a deficient interleukin‐2 receptor gamma chain (NSG). In vitro, we observed a dose‐dependent cytotoxicity with bortezomib and sorafenib. Using RPMI8226 cells co‐expressing histone 2B‐mCherry and cytochrome c‐GFP, bortezomib‐ and sorafenib‐induced apoptosis was confirmed, and both agents combined showed synergism. Sorafenib induced CD138‐downregulation and abolished CXCL12‐induced actin polymerization. L363 cells expressed CCR4 and CCR5 and migrated to their common ligand CCL5. Chemotaxis to BM stroma cells was notable and significantly reduced by sorafenib. Downregulation of phospho‐ERK appeared relevant for the inhibition of actin polymerization and chemotaxis. Sorafenib alone, and combined with bortezomib, showed substantial antitumour activity in L363‐bearing NSG. Correspondingly, sorafenib induced clinical responses in MM‐/AL‐amyloidosis patients. We conclude that, in addition to the cytotoxic and anti‐angiogenic effects of sorafenib, blocking of MM cell migration and homing represent promising mechanisms to interrupt the interplay between PCs and their supportive microenvironment.

Effective Eradication of Glioblastoma Stem Cells by Local Application of an AC133/CD133-Specific T-cell–Engaging Antibody and CD8 T Cells

Cancer stem cells (CSC) drive tumorigenesis and contribute to genotoxic therapy resistance, diffuse infiltrative invasion, and immunosuppression, which are key factors for the incurability of glioblastoma multiforme (GBM). The AC133 epitope of CD133 is an important CSC marker for GBM and other tumor entities. Here, we report the development and preclinical evaluation of a recombinant AC133×CD3 bispecific antibody (bsAb) that redirects human polyclonal T cells to AC133(+) GBM stem cells (GBM-SC), inducing their strong targeted lysis. This novel bsAb prevented the outgrowth of AC133-positive subcutaneous GBM xenografts. Moreover, upon intracerebral infusion along with the local application of human CD8(+) T cells, it exhibited potent activity in prophylactic and treatment models of orthotopic GBM-SC-derived invasive brain tumors. In contrast, normal hematopoietic stem cells, some of which are AC133-positive, were virtually unaffected at bsAb concentrations effective against GBM-SCs and retained their colony-forming abilities. In conclusion, our data demonstrate the high activity of this new bsAb against patient-derived AC133-positive GBM-SCs in models of local therapy of highly invasive GBM.

Monitoring bortezomib therapy in multiple myeloma: screening of cyclin D1, D2, and D3 via reliable real-time polymerase chain reaction and association with clinico-pathological features and outcome

Cyclins D1, D2, and D3 (CCND1, 2, 3) are regulated by proteasomal degradation. Their overexpression in multiple myeloma (MM) has prognostic value. We performed this pilot study to analyze a possible association between CCND1–3 overexpression and response to treatment with the proteasome inhibitor bortezomib, since a specific prognostic marker for bortezomib response has not been reported, but would be ideal to predict who benefits most from bortezomib in times of several potentially efficient therapeutic options. Bone marrow (BM) specimens of 20/47 consecutive patients were available for reliable CCND1–3 analyses by real-time PCR. With CCND1 overexpression in 9/20 patients, the risk for progression after bortezomib treatment was significantly decreased (HR 0.102, 95% CI 0.021–0.498, p = 0.0048) and progression-free survival substantially prolonged (p = 0.0011). Our study is the first to suggest that overexpressed CCND1 in MM is an independent prognostic marker associated with a more durable response to bortezomib. These preliminary results warrant a larger study.

Early and mature endothelial progenitors and VEGFR2+-cells in multiple myeloma: Association with disease characteristics and variation in different cell compartments

We analyzed (1) early endothelial progenitors (EPCs; CD34(+)/AC133(+)/VEGFR2(+)), mature EPCs (CD34(+)/VEGFR2(+)) and VEGFR2(+)-cells in bone marrow (BM)-specimens of multiple myeloma (MM)- vs. monoclonal gammopathy (MGUS)-patients and healthy controls; (2) differences of BM-, peripheral blood (PB)- and leukapheresis (LP)-samples; and (3) the association of EPCs and VEGFR2(+)-cells with MM-parameters. MM patients demonstrated highest early and mature EPCs and VEGFR2(+)-cells in the BM, particularly with advanced and active disease. Endothelial cells differed in BM-, PB- and LP-specimens, albeit seemed less associated with unfavorable prognostic MM-parameters. Our data suggest that especially VEGFR2(+)-cells and mature EPCs in MM are of value to explore further.

Proteasome inhibition enhances the efficacy of volasertib-induced mitotic arrest in AML in vitro and prolongs survival in vivo

Elderly and frail patients, diagnosed with acute myeloid leukemia (AML) and ineligible to undergo intensive treatment, have a dismal prognosis. The small molecule inhibitor volasertib induces a mitotic block via inhibition of polo-like kinase 1 and has shown remarkable anti-leukemic activity when combined with low-dose cytarabine. We have demonstrated that AML cells are highly vulnerable to cell death in mitosis yet manage to escape a mitotic block through mitotic slippage by sustained proteasome-dependent slow degradation of cyclin B. Therefore, we tested whether interfering with mitotic slippage through proteasome inhibition arrests and kills AML cells more efficiently during mitosis. We show that therapeutic doses of bortezomib block the slow degradation of cyclin B during a volasertib-induced mitotic arrest in AML cell lines and patient-derived primary AML cells. In a xenotransplant mouse model of human AML, mice receiving volasertib in combination with bortezomib showed superior disease control compared to mice receiving volasertib alone, highlighting the potential therapeutic impact of this drug combination.

From CLL to Multiple Myeloma - Spleen Tyrosine Kinase (SYK) influences multiple myeloma cell survival and migration

Arnaud Petit Andr e Baruchel Jacqueline Clavel Benôıt Brethon Yves Bertrand Patrick Lutz Fabienne Nacka St ephane Ducassou Unit of Paediatric Haematology and Oncology, Bordeaux University Hospital, Bordeaux-Cedex, Unit of Paediatric Haematology and Oncology, Armand Trousseau Hospital, Unit of Paediatric Haematology and Immunology, Robert Debr e Hospital, Paris, Institut National de la Sant e et de la recherche M edicale (INSERM), Unit754, Registry of Malignant Hemopathies in childhood, Villejuif, Institute of Paediatric Haematology and Oncology, 1, place du Professeur Joseph Renault, Lyon, Unit of Paediatric Haematology and Oncology, Hautepierre Hospital, Strasbourg, and Clinical Investigation Centre in Paediatrics CIC 1401, Bordeaux University Hospital, Bordeaux-Cedex, France E-mail: stephane.ducassou@chu-bordeaux.fr

Safety and efficacy of vorinostat, bortezomib, doxorubicin and dexamethasone in a phase I/II study for relapsed or refractory multiple myeloma (VERUMM study: vorinostat in elderly, relapsed and unfit multiple myeloma).

This phase I/II trial was conducted to investigate the safety, efficacy, and pharmacodynamics of the pan-HDAC-inhibitor (HDACi) vorinostat combined with bortezomib, doxorubicin, and dexamethasone (VBDD) in patients suffering from relapsed/refractory multiple myeloma (RRMM). In the phase I part of this study, 9/33 patients received dose-escalated vorinostat (100, 200, 300mg), using a 4-day-on and 4-day-off schedule, and a standard 3+3 design. In the phase II part of the study, 24/33 patients were included to further assess VBDDs safety and efficacy. Complementary analyses were performed throughout the trial to correlate clinical outcome with patient fitness, pharmacodynamics and potential biomarkers. The number of prior therapy lines was substantial with a median of 3 (1-9; prior bortezomib treatment: 88%). VBDD was well tolerated with no dose-limiting toxicity: 15 adverse events occurred in 9/33 (27%) patients. The best overall response rate was 67% and clinical benefit rate 94%. With a median follow-up of 30.8 months, median progression-free- and overall-survival were 9.6 and 33.8 months, respectively. VBDD-responders showed early pan-HDAC activity decreases in peripheral blood mononuclear cell samples (median 45% of pre-treatment levels), of bone marrow (BM) infiltration rates and of BM-HDAC6 expression. VBDD proved to be an effective, well-tolerated outpatient quadruplet regimen with promising responses in RRMM. Our intermittent HDACi schedule provides a novel treatment option as compared to previous maximum-tolerated-dose-driven HDACi approaches and may serve as a useful example for other HDACi combinations, demonstrating that a continuous epigenetic treatment, with proven synergy to antimyeloma agents, is relevant before HDACi are dismissed as antimyeloma agents.

APC/CCdh1 regulates the balance between maintenance and differentiation of hematopoietic stem and progenitor cells

Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously regenerate the hematopoietic system through differentiation and self-renewal. The process of differentiation is initiated in the G1 phase of the cell cycle, when stem cells leave their quiescent state. During G1, the anaphase-promoting complex or cyclosome associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate cell proliferation. Following Cdh1 knockdown in HSPCs, we analyzed human and mouse hematopoiesis in vitro and in vivo in competitive transplantation assays. We found that Cdh1 is highly expressed in human CD34+ HSPCs and downregulated in differentiated subsets; whereas, loss of Cdh1 restricts myeloid differentiation, supports B cell development and preserves immature short-term HSPCs without affecting proliferation or viability. Our data highlight a role of Cdh1 as a regulator of balancing the maintenance of HSPCs and differentiation into mature blood cells.