Synergistic activity of bortezomib and HDACi in preclinical models of B-cell precursor acute lymphoblastic leukemia via modulation of p53, PI3K/AKT, and NF-κB.

PURPOSE: Relapse of disease and subsequent resistance to established therapies remains a major challenge in the treatment of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). New therapeutic options, such as proteasome and histone deacetylase inhibitors (HDACi) with a toxicity profile differing from that of conventional cytotoxic agents, are needed for these extensively pretreated patients. EXPERIMENTAL DESIGN: Antiproliferative and proapoptotic effects of combined HDACi/proteasome inhibitor treatments were analyzed using BCP-ALL monocultures, cocultures with primary mesenchymal stroma cells from patients with ALL, and xenograft mouse models. The underlying molecular mechanisms associated with combined treatment were determined by gene expression profiling and protein validation. RESULTS: We identified the proteasome inhibitor bortezomib as a promising combination partner for HDACi due to the substantial synergistic antileukemic activity in BCP-ALL cells after concomitant application. This effect was maintained or even increased in the presence of chemotherapeutic agents. The synergistic effect of combined HDACi/BTZ treatment was associated with the regulation of genes involved in cell cycle, JUN/MAPK, PI3K/AKT, p53, ubiquitin/proteasome, and NF-κB pathways. We observed an activation of NF-κB after bortezomib treatment and the induction of apoptosis-related NF-κB target genes such as TNFαRs after concomitant treatment, indicating a possible involvement of NF-κB as proapoptotic mediator. In this context, significantly lower NF-κB subunits gene expression was detected in leukemia cells from patients who developed a relapse during frontline chemotherapy, compared with those who relapsed after cessation of frontline therapy. CONCLUSION: These results provide a rationale for the integration of HDACi/BTZ combinations into current childhood BCP-ALL treatment protocols.

High VLA-4 expression is associated with adverse outcome and distinct gene expression changes in childhood B-cell precursor acute lymphoblastic leukemia at first relapse.

BACKGROUND: Resistance to therapy and subsequent relapse remain major challenges in the clinical management of relapsed childhood acute lymphoblastic leukemia. As the bone marrow environment plays an important role in survival and chemotherapy resistance of leukemia cells by activating different signaling pathways, such as the VLA-4 and PI3K/Akt pathways, we studied the prognostic and biological impact of VLA-4 expression in leukemia cells from children with relapsed B-cell precursor acute lymphoblastic leukemia and its influence on the sensitivity of the leukemia cells to drugs. DESIGN AND METHODS: VLA-4 expression was quantified by real-time polymerase chain reaction in leukemia cells from 56 patients with relapsed acute lymphoblastic leukemia enrolled in the ALL-REZ BFM 2002 trial of the Berlin-Frankfurt-Münster study group. Gene expression changes related to VLA-4 expression were investigated by microarray-based mRNA profiling. The effect of VLA-4 signaling on proliferation and drug resistance was studied in co-cultures of leukemia and stromal cells. RESULTS: High expression of VLA-4 at first relapse was associated with adverse prognostic factors, poor molecular response to therapy and significantly worse probabilities of event-free and overall survival. VLA-4 expression was an independent prognostic parameter. Comparing gene expression profiles of leukemia cells with high versus low VLA-4 expression, we identified 27 differentially expressed genes primarily involved in the PI3K/Akt, ephrin and Rho GTPase pathways. Blocking of VLA-4 signaling in combination with cytarabine treatment abolished the growth supportive effect of stromal cells. CONCLUSIONS: Our results show that high VLA-4 expression is a marker of poor prognosis and a potential therapeutic target in children with relapsed acute lymphoblastic leukemia and confirm that cellular interactions and biological effects related to VLA-4 play a decisive role in the survival of leukemia cells and response to therapy. (ClinicalTrials.gov identifier: NCT00114348).

Leukemia-associated genetic aberrations in mesenchymal stem cells of children with acute lymphoblastic leukemia.

Childhood acute lymphoblastic leukemia (ALL) is caused by malignant immature lymphocytes. Even though childhood ALL can be cured in a large number of patients, around 20% of the patients suffer a relapse after chemotherapy. The origin of the relapse is unclear at the present time. Given the high plasticity of cells, we searched for leukemia-associated genetic aberrations and immunoglobulin (IG) gene rearrangements in mesenchymal stem cells (MSC) from childhood B-cell precursor ALL patients. MSC from all ten ALL patients analyzed presented the chromosomal translocations that had been detected in leukemia cells (TEL-AML1, E2A-PBX1, or MLL rearrangement). The proportions of translocation-positive MSC varied between 10% and 54% depending on the patients and the time point of analysis. Leukemia-specific IG gene rearrangements were detected in the MSC from three ALL patients. The detection of leukemia-associated genetic aberrations in MSC indicates a clonal relationship between MSC and leukemia cells and suggests their involvement in the pathogenesis and/or pathophysiology of childhood ALL.

Anti-tumor effect of DNA-based vaccination and dSLIM immunomodulatory molecules in mice with Ph+ acute lymphoblastic leukaemia.

Since the prognosis of patients with Philadelphia chromosome positive acute lymphoblastic leukaemia (Ph+ ALL) still remains poor, new relapse prevention strategies are needed. We evaluated the pre-immunization of mice with DNA-based vaccines subsequently challenged by the syngeneic Ph+ ALL cell line BM185. Ballistic transfer of minimalistic immunogenically defined gene expression (MIDGE) vectors encoding a BCR-ABLp185 fusion specific peptide or GM-CSF were used for in vivo transfection. DNA-based double stem-loop immunomodulators (dSLIM) were used as immune adjuvant. We present survival and functional data that DNA-based vaccination with BCR-ABLp185 fusion specific sequences, GM-CSF and dSLIM leads to an anti-tumor effect in mice challenged with a lethal Ph+ ALL dose and this effect depends on leukaemia-specific sequences.

Insulin-like growth factor-1 increases intracellular calcium concentration in human primary neuroendocrine pancreatic tumor cells and a pancreatic neuroendocrine tumor cell line (BON-1) via R-type Ca2+ channels and regulates chromogranin a secretion in BON-1 cells.

Insulin-like growth factor 1 (IGF-1) is a potent mitogenic and secretory factor that acts on voltage operated Ca(2+) channels (VOCCs). VOCCs are categorized into L-type channels (Ca(V)1.1-1.4), P/Q-type channels (Ca(V)2.1), N-type channels (Ca(V)2.2), R-type channels (Ca(V)2.3), and T-type channels (Ca(V)3.1-3.3). Aside from regulating membrane excitability, VOCCs influence chromogranin A (CgA) secretion in neuroendocrine tumor (NET) cells. It is not known, whether VOCCs play a role in the IGF-1-dependent regulation of CgA secretion in NET cells. We therefore studied the effects of IGF-1 on individual VOCC subtypes and characterized their role in mediating IGF-1-dependent regulation of CgA secretion in NET cells. Using specific modulators of VOCC subtypes, we identified the functional expression of L-, N-, P/Q- and R-type channels in primary as well as permanent models of NET. The IGF-1-induced intracellular Ca(2+) increase in NET cells was mainly due to the activation of R-type channel activity. The effects on intracellular calcium, observed in whole-cell patch-clamp recordings and fluorescence imaging, were partially blocked by the specific R-type channel blocker SNX-482 and antisense oligonucleotides against the alpha(1) subunit of this channel. IGF-1 potently induced CgA secretion. The effect of IGF-1 was reduced by both, inhibition of R-type channel activity and a reduction of R-type channel expression using antisense oligonucleotides. Since R-type channels exist in NET cells and couple to both, IGF-1 receptor signaling as well as CgA secretion, pharmacological interference with R-type channels may represent a new therapeutic option by blocking Ca(2+) signaling thereby abrogating IGF-1-dependent hypersecretion in NET disease.