Supplementary Figures 5-7 from Genome-Wide siRNA Screen for Modulators of Cell Death Induced by Proteasome Inhibitor Bortezomib

Supplementary Figures 1-4 from Genome-Wide siRNA Screen for Modulators of Cell Death Induced by Proteasome Inhibitor Bortezomib

Supplementary Figure 8 from Genome-Wide siRNA Screen for Modulators of Cell Death Induced by Proteasome Inhibitor Bortezomib

Acyl derivatives of 4-(aminomethyl)-N-hydroxybenzamide are potent sub-type selective HDAC6 inhibitors. Constrained heterocyclic analogs based on 1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine show further enhanced HDAC6 selectivity and inhibitory activity in cells. Homology models suggest that the heterocyclic spacer can more effectively access the wider catalytic channel of HDAC6 compared to other HDAC sub-types.

A screen for HDAC6 inhibitors identified acyl derivatives of 4-(aminomethyl)-N-hydroxybenzamide as potent leads with unexpected selectivity over the other subtypes. We designed and synthesized constrained heterocyclic analogues such as tetrahydroisoquinolines that show further enhanced HDAC6 selectivity and inhibitory activity in cellular assays. Selectivity may be attributed to the benzylic spacer more effectively accessing the wider channel of HDAC6 compared to other HDAC subtypes as well as hydrophobic capping groups interacting with the protein surface near the rim of the active site.

Abstract Multiple pathways have been proposed to explain how proteasome inhibition induces cell death, but mechanisms remain unclear. To approach this issue, we performed a genome-wide siRNA screen to evaluate the genetic determinants that confer sensitivity to bortezomib (Velcade (R); PS-341). This screen identified 100 genes whose knockdown affected lethality to bortezomib and to a structurally diverse set of other proteasome inhibitors. A comparison of three cell lines revealed that 39 of 100 genes were commonly linked to cell death. We causally linked bortezomib-induced cell death to the accumulation of ASF1B, Myc, ODC1, Noxa, BNIP3, Gadd45α, p-SMC1A, SREBF1, and p53. Our results suggest that proteasome inhibition promotes cell death primarily by dysregulating Myc and polyamines, interfering with protein translation, and disrupting essential DNA damage repair pathways, leading to programmed cell death. Cancer Res; 70(11); 4318–26. ©2010 AACR.

Multiple pathways have been proposed as the mechanism by which proteasome inhibition induces cell death. To clarify their relative importance, we performed a genome‐wide siRNA screen to evaluate the genetic determinants that confer sensitivity of the HCT‐116 colon cancer cell line to bortezomib (VELCADE®, PS‐341). The screen identified 100 genes whose knock‐down affects the lethality of bortezomib. From this list, the accumulation of the proteins ASF1B, Myc, ODC1, PMAIP1 (Noxa), BNIP3, Gadd45α, p‐SMC1A, SREBF1, and p53 by proteasome inhibition was linked to the induction of cell death. Fifty‐nine genes in the A375 melanoma cell line and 56 genes in the HeLa cervical cancer cell line showed similar interactions with bortezomib to those seen in HCT‐116 and a subset of 39 genes were common to all three cell lines. Finally, knockdown of these 100 genes in HCT‐116 cells similarly affected their responsiveness to a structurally diverse set of proteasome inhibitors. Our results suggest that proteasome inhibition promotes cell death primarily by dysregulating Myc and polyamines, interfering with protein translation, and disrupting essential DNA damage repair pathways, leading to programmed cell death. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A70.