‘Seed’ analysis of off-target siRNAs reveals an essential role of Mcl-1 in resistance to the small-molecule Bcl-2/Bcl-XL inhibitor ABT-737

Abstract

ABT-737 is a subnanomolar inhibitor of the antiapoptotic proteins Bcl-2, Bcl-X L and Bcl-w. Although ABT-737 triggers extensive cell death in many small-cell lung carcinoma (SCLC) cell lines, some of the SCLC cell lines and the majority of the cancer cell lines derived from other solid tumors were found to be resistant to ABT-737. To better understand the mechanism of resistance to ABT-737, we screened a short interfering RNA library consisting of short interfering RNA against 4000 'druggable' targets in an SCLC-derived cell line, NCI-H196. By comparing the knockdowns with phenotypes, all of the three top 'hits' from the screen were found to result from off-target gene silencing. Interestingly, the three offtarget siRNAs were found to knock down an antiapoptotic Bcl-2 family protein Mcl-1 owing to the complementation between their seed regions with the 3 0 untranslated region (3 0 UTR) of Mcl-1. Furthermore, reducing the level of Mcl-1 using siRNAs or the small-molecule compounds Bay43-9006 and Seliciclib was sufficient to overcome the resistance to ABT-737 in the resistant SCLC cell line and cancer cell lines derived from other solid tumors. These results provide further evidence that Mcl-1 is the major factor that causes resistance to ABT-737 in cancer cells derived from diverse solid tumors, and the combination of Mcl-1 downregulating agents with ABT-737 could be potent therapeutic regimens for patient with ABT-737-resistant SCLC and many other types of solid tumors.

ABT-737 Induces Apoptosis in Mantle Cell Lymphoma Cells with a Bcl-2high/Mcl-1low Profile and Synergizes with Other Antineoplastic Agents

“…Our investigation confirms that flavopiridol downregulates Mcl-1 at both the mRNA and protein levels in MCL cells and shows a highly synergistic apoptotic effect when combined with ABT-737 in ABT-737-resistant cell lines. The effectiveness of combining ABT-737 with compounds that inactivate Mcl-1 firmly establishes the validity of such a therapeutic approach in the treatment of MCL (12,(35)(36)(37). A similar synergistic effect is measured when ABT-737 is combined with ara-C, which is a heavily prescribed drug in the treatment of MCL (24,38,39).…”

“…Nontreated cells were used as a control. biomarker regardless of both the level of Bcl-2 (12)(13)(14) and the nature of the tumor cell. Indeed, several studies and ours have identified a uniform profile (Bcl-2 high /Mcl-1 low ) for ABT-737-sensitive cells.…”

“…ABT-737 displaces proapoptotic BH3-only from Bcl-2 or Bcl-xL, leading to activation of Bax and Bak and downstream caspases (9). Because of the low affinity of ABT-737 for Mcl-1, high basal levels of Mcl-1 have been associated with ABT-737 resistance (11)(12)(13)(14). Previous studies have also shown that ABT-737 is effective as a single agent against some leukemia/ lymphoma cell lines both in vitro and in vivo (11,(15)(16)(17).…”

Alterations in the Noxa/Mcl-1 axis determine sensitivity of small cell lung cancer to the BH3 mimetic ABT-737

“…However, there is considerable variation in the sensitivities of different SCLC cell lines to this drug (18,19). Studies in a variety of cell types have suggested that indicators of sensitivity include low levels of Mcl-1 and high levels of Bcl-2, Bcl-X L , Bim, and Noxa expression (19)(20)(21)(22)(23)(24)(25). In particular, studies in SCLC have documented that decreased Mcl-1 expression (19,20) and amplification of a region of chromosome 18 containing both the BCL-2 and NOXA genes correlates with increased sensitivity to ABT-737 (24).…”

In vitro modulation of Bcl-2 levels in small cell lung cancer cells: effects on cell viability

“…In fact, the Bcl-2 family inhibitor ABT-737 is effective against some SCLC lines by simultaneously inhibiting three antiapoptotic proteins (Bcl-2, Bcl-xL, and Bcl-w). In addition, in the case of the expression of a fourth anti-apoptotic protein (Mcl-1), its knockdown is further required to achieve increased tumor cell death, thus reinforcing the importance of a multitargeted approach (40).…”