Hsp90 Inhibitor SNX-7081 dysregulates proteins involved with DNA repair and replication and the cell cycle in human chronic lymphocytic leukemia (CLL) cells.

The proteomic effects of the Hsp90 inhibitor, SNX-7081, have been determined on the p53-mutated B-cell chronic lymphocytic leukemia (CLL) cell line, MEC1. Following SNX-7081 treatment (500 nM, 24 h), 51 proteins changed abundance by more than 2-fold (p < 0.05); 7 proteins increased while 44 proteins decreased. Proteins identified as differentially abundant by LC-MS/MS were validated by Western blotting (DDB1, PCNA, MCM2, Hsp90, Hsp70, GRP78, PDIA6, HLA-DR). RT-PCR showed that SNX-7081 unexpectedly modulates a number of these proteins in MEC1 cells at the mRNA level (PCNA, MCM2, Nup155, Hsp70, GRP78, PDIA6, and HLA-DR). Pathway analysis determined that 3 of the differentially abundant proteins (cyclin D1, c-Myc and pRb) were functionally related. p53 levels did not change upon SNX-7081 treatment of p53 wild-type Raji cells or p53-mutated MEC1 and U266 cells, indicating that SNX-7081 has a p53-independent mechanism. The decreases in DDB1, MCM2, c-Myc, and PCNA and increases of pRb and cyclin D1 were confirmed in MEC1, U266, Raji, and p53 null HL60 cells by Western blotting. These data suggest that SNX-7081 arrests the cell cycle and inhibits DNA replication and r epair and provides evidence for the mechanism of the observed synergy between Hsp90 inhibitors and drugs that induce DNA strand breaks.

Comprehensive proteomic analysis of the effects of purine analogs on human Raji B-cell lymphoma.

Cladribine (CdA) and fludarabine (FdAMP) are purine analogs that induce apoptosis in chronic lymphocytic leukemia and non-Hodgkin's lymphoma, but the mechanisms are undefined. The effects of CdA and fludarabine nucleoside (FdA) on the cytosolic, mitochondrial, and nuclear proteomes in human Raji lymphoma cells have been determined using two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry. Differentially abundant proteins have provided new insights into CdA- and FdA-induced apoptosis. Treatment with these purine analogs induced changes in proteins involved with intermediary metabolism, cell growth, signal transduction, protein metabolism, and regulation of nucleic acids. Differentially abundant mitochondrial 39S ribosomal protein L50, mTERF domain-containing protein 1, Chitinase-3 like 2 protein, and ubiquinone biosynthesis protein COQ9 have been identified in cells undergoing apoptosis. Up-regulation of several stress-associated proteins found in the endoplasmic reticulum (ER) including GRP78, ERp57, and ORP150 suggests that purine analog-induced apoptosis may result from ER stress and unfolded protein response. While mitochondria-dependent apoptosis has been associated with purine analog cytotoxicity, the likely involvement of the ER stress pathway in CdA- and FdA-induced apoptosis has been shown here for the first time.

The Hsp90 inhibitor SNX-7081 synergizes with and restores sensitivity to fludarabine in chronic lymphocytic leukemia cells with lesions in the TP53 pathway: a potential treatment strategy for fludarabine refractory disease.

Drug resistance in chronic lymphocytic leukemia (CLL) associated with lesions in the ATM/TP53 pathway represents a major challenge in clinical management. Evidence suggests that heat shock protein-90 (Hsp90) inhibitors may represent a therapeutic option in combination with more conventional therapies. We explored the effects of combining the Hsp90 inhibitor, SNX-7081, with fludarabine in vitro against CLL cells and hematological cell lines. In seven cell lines and 23 patient samples synergy between SNX-7081 and fludarabine (2-FaraA) was apparent in the three TP53 mutated cell lines and at significantly lower concentrations in TP53 or ATM dysfunctional patient cells. In 11/13 2-FaraA-resistant patient samples, SNX-7081 reduced the 50% inhibitory concentration to within a clinically achievable range. Synergy between SNX-7081 and 2-FaraA was evident in both the cell lines and patient samples as a significant decrease in cell viability. Our data suggest that combining SNX-7081 and fludarabine may be effective in the treatment of fludarabine-refractory CLL.