Abstract

Heat shock proteins (Hsp) are increasingly employed as therapeutic targets in various solid tumors and leukemias. We have recently shown that Hsp32 is expressed in leukemic cells and serves as a survival-factor and molecular target in Ph+ chronic myeloid leukemia. In the present study, we examined the expression and functional role of Hsp32 in acute lymphoblastic leukemia (ALL). Leukemic cells were obtained from patients with Ph+ ALL (n=5) and Ph− ALL (n=5). In addition, Ph+ ALL cell lines (Z-119, BV-173, TOM-1, NALM-1) and Ph− ALL cell lines (RAJI, RAMOS, REH, BL-41) were used. As assessed by immunocytochemistry and qRT-PCR, leukemic cells were found to express the Hsp32 protein as well as Hsp32 mRNA in all patients and in all cell lines examined. The Hsp32-inductor hemin was found to promote the expression of Hsp32 in leukemic cells. To determine the functional role of Hsp32 in lymphoblasts, an siRNA against Hsp32 was applied. The siRNA-induced knock down of Hsp32 in RAJI cells was found to be associated with reduced growth and with an increase in apoptotic cells compared to a control siRNA against luciferase (p<0.05). In a next step, two pharmacologic inhibitors of Hsp32, pegylated zinc protoporphyrine (PEG-ZnPP) and styrene maleic acid-micelle-encapsulated ZnPP (SMA-ZnPP), were applied. As assessed by 3H-thymidine uptake experiments, both drugs were found to inhibit proliferation in the BCR/ABL+ cell lines Z-119, BV-173, and TOM-1, and in the BCR/ABL-negative ALL cell lines RAJI, RAMOS, REH, and BL-41. The effects of PEG-ZnPP and SMA-ZnPP were dose-dependent with IC50 values ranging between 1 and 10 μM, and were found to be associated with apoptosis as determined by microscopy as well as by flow cytometry and AnnexinV-staining. In NALM-1 cells, PEG-ZnPP and SMA-ZnPP also produced apoptosis and growth arrest, but the IC50 for SMA-ZnPP was slightly higher compared to other cell lines (20 μM). Effects of Hsp32-targeting drugs were also observed in primary leukemic cells obtained from patients with Ph+ ALL and Ph− ALL, with IC50 values ranging between 1 and 10 μM. No major differences were found when comparing results in imatinib-sensitive and imatinib-resistant patients. In drug combination experiments, Hsp32-targeting drugs were found to cooperate with imatinib and with AMN107 (nilotinib) in producing growth-inhibition and apoptosis in all Ph+ ALL cell lines tested. Furthermore, we were able to demonstrate strong cooperative antileukemic effects when applying Hsp32-targeting drugs in combination with bendamustine. Overall, these results suggest that Hsp32 may be a novel molecular target in ALL.

Disclosures: Valent:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding.

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