Linear Chain PEGylated Recombinant Bacillus Thiaminolyticus Thiaminase I Enzyme Has Growth Inhibitory Activity against Lymphoid Leukemia Cell Lines

Abstract

Cancer cells acquire abnormalities in energy metabolism, collectively known as the Warburg effect, affecting substrate availability of thiamine-dependent enzymes. To investigate a strategy to exploit abnormal cancer-associated metabolism related to thiamine, we tested the cytotoxicity of native Bacillus thiaminolyticus thiaminase I enzyme, which digests thiamine, in the NCI60 cell line drug cytotoxicity screening program and found that leukemia cell lines were among the most sensitive to thiaminase I. We obtained additional lymphoid leukemia cell lines and confirmed that native thiaminase I and linear chain PEGylated thiaminase I enzyme (LCPTE) have cytotoxic activity in these cell lines. In addition, the IC 50 of 3 of the 5 leukemia cell lines (Reh, RS4, and Jurkat) were at least 1,000-fold more sensitive than Molt-4 cells, which in turn, were among the most sensitive in the NCI60 panel. The 3 LCPTE-sensitive leukemia cell lines were also sensitive to removal of thiamine from the medium, thus suggesting the mechanism of action of LCPTE involves extracellular thiamine starvation. Surprisingly, rapamycin showed a protective effect against LCPTE toxicity in the 3 LCPTE-sensitive cell lines but not in the other 2 cell lines, suggesting involvement of an mTOR-dependent pathway. Immunoblot analysis of the LCPTE-sensitive cell lines after LCPTE exposure revealed changes in mTOR pathway phosphorylation. Nude mice bearing RS4 leukemia xenografts showed both tumor growth delay and prolonged survival after a single dose of LCPTE. Therefore, disruption of thiamine-dependent metabolism may be a novel therapeutic approach to target altered energy metabolism in leukemia and other cancers. Mol Cancer Ther; 10(9); 1563-70. Ó2011 AACR.

Pharmacologic Properties of Polyethylene Glycol-ModifiedBacillus thiaminolyticusThiaminase I Enzyme

“…We have shown previously that 1k-PEGylated thiaminase has activity in this model (Daily et al, 2011). As shown in Fig.…”

“…9, 5k-PEG thiaminase had no activity compared with the control group, whereas native thiaminase increased the event-free survival (EFS) from 15 to 70 days (p Ͻ 0.001) in an experiment that was terminated at day 100 from the initiation of treatment. In comparison, we have shown previously that 1k-PEG thiaminase increases the EFS to 31 days (Daily et al, 2011). Thus, at their respective MTDs, PEGylated thiaminases are not as effective as native thiaminase in this tumor model.…”

“…Third, in leukemia cell lines growth inhibition occurred at the enzyme concentrations that correspond with thiamine depletion for both native and 5k-PEGylated enzyme, but further cytotoxicity occurred only at the concentrations that were required for cytotoxicity in the solid tumor cell lines, and, again, 5k-PEG thiaminase did not achieve further cytotoxicity. We reported previously the growth inhibition of thiaminase in leukemia cells at low thiaminase concentrations (Daily et al, 2011).…”

“…In the native thiaminase cohort, one of the events was unexplained death. The control group of mice was injected with RS4 cells and left untreated, and results were combined with a previous control group of the same xenograft (Daily et al, 2011). Kaplan- …”

“…We have focused on the disruption of thiamine metabolism in cancer, a strategy that was derived from initial observations that tumors down-regulate thiamine uptake transporters (Liu et al, 2003(Liu et al, , 2004. We have found that recombinant thiaminase enzyme is cytotoxic in breast cancer cells (Liu et al, 2010) and a chemically modified 1k-polyethylene glycol (PEG)ylated form of thiaminase has growth inhibitory effects at very low concentrations in leukemia cell lines (Daily et al, 2011).…”