Cancer Therapy: Preclinical Synergistic Activity of Bortezomib and HDACi in Preclinical Models of B-cell Precursor Acute Lymphoblastic Leukemia via Modulation of p53, PI3K/AKT, and NF-kB Lorenz Bastian 1 , Jana Hof 1,2 , Madlen Pfau 1 , Iduna Fichtner 3 , Cornelia Eckert 1 ,G€ unter Henze 1 , Javier Prada 1 , Arend von Stackelberg 1 , Karl Seeger 1 , and Shabnam Shalapour 1 Abstract 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 mesenchy- mal 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-kB pathways. We observed an activation of NF-kB after bortezomib treatment and the induction of apoptosis-related NF- kB target genes such as TNFaRs after concomitant treatment, indicating a possible involvement of NF-kB as proapoptotic mediator. In this context, significantly lower NF-kB 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. Clin Cancer Res; 19(6); 1445–57. Ó2013 AACR. Introduction Despite continuous improvement of therapeutic options for children with acute lymphoblastic leukemia (ALL), the prognosis for patients who suffer a relapse remains poor with a probability of even-free survival of 30% at 10 years (1). New treatment approaches with a different toxicity profile in comparison to conventional cytotoxic agents are required for these extensively pretreated patients. As a promising alternative, histone deacetylase inhibitors (HDACi) have been suggested (2, 3). We have previously shown that HDACi are able to limit the expansion of B-cell precursor (BCP)-ALL cells in vivo (4). The inclusion of HDACi into current ALL chemotherapy concepts requires the identification of suitable combination partners, as first clinical trials achieved only limited therapeutic responses (5, 6) while establishing a good tolerance of HDACi in pediatric patients with malignant diseases. So far, only additive or even antagonistic interactions have been observed after combined treatment with HDACi and dif- ferent conventional cytostatic drugs in myeloid leukemia and T-ALL cells (7). In BCP-ALL leukemia cells, we have recently observed that the sequence of drug application determined synergistic or antagonistic responses to com- bined treatment with HDACi and methotrexate (8). Authors' Affiliations: 1 Department of Pediatric Oncology/Hematology, 2 Department of Pediatrics, Division of General Pediatrics, Charit e-Univer- sit€ atsmedizin Berlin; and 3 Max-Delbr€ uck-Center for Molecular Medicine, Experimental Pharmacology & Oncology, Berlin, Germany Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). K. Seeger and S. Shalapour are co-last authors. Corresponding Author: Shabnam Shalapour, Charit e-Universit€ atsmedi- zin Berlin, Berlin 13353, Germany. Phone: 49-30-450-559568; Fax: 49-30- 450-566946; E-mail: shabnam.shalapour@charite.de doi: 10.1158/1078-0432.CCR-12-1511 Ó2013 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 1445 on August 18, 2017. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst January 28, 2013; DOI: 10.1158/1078-0432.CCR-12-1511