The Mutant KRAS Gene Up-regulates BCL-XL Protein via STAT3 to Confer Apoptosis Resistance That Is Reversed by BIM Protein Induction and BCL-XL Antagonism.

In colorectal cancers with oncogenic GTPase Kras (KRAS) mutations, inhibition of downstream MEK/ERK signaling has shown limited efficacy, in part because of failure to induce a robust apoptotic response. We studied the mechanism of apoptosis resistance in mutant KRAS cells and sought to enhance the efficacy of a KRAS-specific MEK/ERK inhibitor, GDC-0623. GDC-0623 was shown to potently up-regulate BIM expression to a greater extent versus other MEK inhibitors in isogenic KRAS HCT116 and mutant KRAS SW620 colon cancer cells. ERK silencing enhanced BIM up-regulation by GDC-0623 that was due to its loss of phosphorylation at Ser(69), confirmed by a BIM-EL phosphorylation-defective mutant (S69G) that increased protein stability and blocked BIM induction. Despite BIM and BIK induction, the isogenic KRAS mutant versus wild-type cells remained resistant to GDC-0623-induced apoptosis, in part because of up-regulation of BCL-XL. KRAS knockdown by a doxycycline-inducible shRNA attenuated BCL-XL expression. BCL-XL knockdown sensitized KRAS mutant cells to GDC-0623-mediated apoptosis, as did the BH3 mimetic ABT-263. GDC-0623 plus ABT-263 induced a synergistic apoptosis by a mechanism that includes release of BIM from its sequestration by BCL-XL. Furthermore, mutant KRAS activated p-STAT3 (Tyr(705)) in the absence of IL-6 secretion, and STAT3 knockdown reduced BCL-XL mRNA and protein expression. These data suggest that BCL-XL up-regulation by STAT3 contributes to mutant KRAS-mediated apoptosis resistance. Such resistance can be overcome by potent BIM induction and concurrent BCL-XL antagonism to enable a synergistic apoptotic response.

Zaanan A ，Okamoto K ，Kawakami H ，Khazaie K ，Huang S ，Sinicrope FA ... -  《-》

Reversal of Mutant KRAS-Mediated Apoptosis Resistance by Concurrent Noxa/Bik Induction and Bcl-2/Bcl-xL Antagonism in Colon Cancer Cells.

KRAS mutations are frequently detected in human colorectal cancer and contribute to de novo apoptosis resistance and ultimately therapeutic failure. To overcome KRAS-mediated apoptosis resistance, the irreversible proteasome inhibitor, carfilzomib, was evaluated and found to potently induce Noxa, which was dependent upon c-Myc, and Bik. Isogenic mutant versus wild-type KRAS carcinoma cells showed elevated Bcl-xL, confirmed by KRAS siRNA or ectopic expression. Upregulated Bcl-xL by mutant KRAS was mediated by ERK as indicated by ERK knockdown. Bcl-xL expression was regulated at the level of mRNA and protein as shown using actinomycin D and cyclohexamide, respectively. Suppression of Bcl-xL by shRNA sensitized mutant KRAS cells to carfilzomib. Concurrent Bcl-xL antagonism by the BH3 mimetic ABT-263 combined with carfilzomib synergistically enhanced apoptosis that was dependent on Bax or p53, and was attenuated by Noxa or Bik shRNA. In support of this strategy, ectopically expressed Noxa enhanced apoptosis by ABT-263. Carfilzomib-induced Noxa and Bik sequestered Mcl-1 and ABT-263 released Bik and Bak from Bcl-xL, suggesting a mechanism for drug synergy. These preclinical findings establish mutant KRAS-mediated Bcl-xL upregulation as a key mechanism of apoptosis resistance in KRAS-mutant colorectal cancer. Furthermore, antagonizing Bcl-xL enabled carfilzomib-induced Noxa and Bik to induce synergistic apoptosis that reversed KRAS-mediated resistance. This novel study reveals a promising treatment strategy to overcome apoptosis resistance in KRAS-mutant colorectal cancer by concurrent upregulation of Noxa/Bik and antagonism of Bcl-xL.

Okamoto K ，Zaanan A ，Kawakami H ，Huang S ，Sinicrope FA ... -  《-》

Dual inhibition of Bcl-2 and Bcl-xL strikingly enhances PI3K inhibition-induced apoptosis in human myeloid leukemia cells through a GSK3- and Bim-dependent mechanism.

Rahmani M ，Aust MM ，Attkisson E ，Williams DC Jr ，Ferreira-Gonzalez A ，Grant S ... -  《-》

Failure to induce apoptosis via BCL-2 family proteins underlies lack of efficacy of combined MEK and PI3K inhibitors for KRAS-mutant lung cancers.

Although several groups have demonstrated that concomitant use of MEK and phosphoinositide 3-kinase (PI3K) inhibitors (MEKi/PI3Ki) can induce dramatic tumor regressions in mouse models of KRAS-mutant non-small cell lung cancer (NSCLC), ongoing clinical trials investigating this strategy have been underwhelming to date. While efficacy may be hampered by a narrow therapeutic index, the contribution of biologic heterogeneity in the response of KRAS-mutant NSCLCs to MEKi/PI3Ki has been largely unexplored. In this study, we find that most human KRAS-mutant NSCLC cell lines fail to undergo marked apoptosis in response to MEKi/PI3Ki, which is key for tumor responsiveness in vivo. This heterogeneity of apoptotic response occurs despite relatively uniform induction of growth arrest. Using a targeted short hairpin RNA screen of BCL-2 family members, we identify BIM, PUMA, and BCL-XL as key regulators of the apoptotic response induced by MEKi/PI3Ki, with decreased expression of BIM and PUMA relative to BCL-XL in cell lines with intrinsic resistance. In addition, by modeling adaptive resistance to MEKi/PI3Ki both in vitro and in vivo, we find that, upon the development of resistance, tumors have a diminished apoptotic response due to downregulation of BIM and PUMA. These results suggest that the inability to induce apoptosis may limit the effectiveness of MEKi/PI3Ki for KRAS-mutant NSCLCs by contributing to intrinsic and adaptive resistance to this therapy.

Hata AN ，Yeo A ，Faber AC ，Lifshits E ，Chen Z ，Cheng KA ，Walton Z ，Sarosiek KA ，Letai A ，Heist RS ，Mino-Kenudson M ，Wong KK ，Engelman JA ... -  《-》

Inhibition of HSP90 by AUY922 Preferentially Kills Mutant KRAS Colon Cancer Cells by Activating Bim through ER Stress.

Oncogenic mutations of KRAS pose a great challenge in the treatment of colorectal cancer. Here we report that mutant KRAS colon cancer cells are nevertheless more susceptible to apoptosis induced by the HSP90 inhibitor AUY922 than those carrying wild-type KRAS. Although AUY922 inhibited HSP90 activity with comparable potency in colon cancer cells irrespective of their KRAS mutational statuses, those with mutant KRAS were markedly more sensitive to AUY922-induced apoptosis. This was associated with upregulation of the BH3-only proteins Bim, Bik, and PUMA. However, only Bim appeared essential, in that knockdown of Bim abolished, whereas knockdown of Bik or PUMA only moderately attenuated apoptosis induced by AUY922. Mechanistic investigations revealed that endoplasmic reticulum (ER) stress was responsible for AUY922-induced upregulation of Bim, which was inhibited by a chemical chaperone or overexpression of GRP78. Conversely, siRNA knockdown of GRP78 or XBP-1 enhanced AUY922-induced apoptosis. Remarkably, AUY922 inhibited the growth of mutant KRAS colon cancer xenografts through activation of Bim that was similarly associated with ER stress. Taken together, these results suggest that AUY922 is a promising drug in the treatment of mutant KRAS colon cancers, and the agents that enhance the apoptosis-inducing potential of Bim may be useful to improve the therapeutic efficacy.

Wang CY ，Guo ST ，Wang JY ，Liu F ，Zhang YY ，Yari H ，Yan XG ，Jin L ，Zhang XD ，Jiang CC ... -  《-》