Triple‐negative breast cancers (TNBCs) represent 15% to 20% of all breast cancers and are often associated with poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Aberrations in the phosphoinositide‐3‐kinase (PI3K) and mitogen‐activated protein kinase pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. While the crosstalk between PI3K and extracellular signal‐regulated kinase 1/2 (ERK1/2) pathways has been characterized in several systems, new evidence suggests that MEK5/ERK5 signaling is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/protein kinase B (Akt) and MEK5/ERK5 in the MDA‐MB‐231, BT‐549, and MDA‐MB‐468 TNBC cell lines. We used the Akt inhibitor ipatasertib, ERK5 inhibitors XMD8‐92 and AX15836, and the novel MEK5 inhibitor SC‐1‐181 to investigate the effects of dual inhibition. Our results indicated that dual inhibition of PI3K/Akt and MEK5/ERK5 signaling was more effective at reducing the proliferation and survival of TNBCs than single inhibition of either pathway alone. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. In addition, the dual inhibition strategy was determined to be synergistic in MDA‐MB‐231 and BT‐549 cells and was relatively nontoxic in the nonneoplastic MCF‐10 cell line. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.

Triple negative breast cancers (TNBCs) represent 15-20% of all breast cancers and are often associated with a poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Therefore, there is a need to identify survival pathways that may be targeted in TNBCs. Aberrations in the Phosphoinositide-3-kinase (PI3K) and Mitogen Activated Protein Kinase (MAPK) pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. The crosstalk between PI3K/Akt and MEK1/2/ERK1/2 has been characterized in several systems. However, new evidence suggests that MEK5/ERK5, a member of the MAPK family, is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/Akt and MEK5/ERK5 in three TNBC cell lines: MDA-MB-231, BT549, and MDA-MB-468. We achieved Akt inhibition by using the clinically relevant inhibitors MK-2206 and Ipatasertib. We used a novel compound synthesized in our lab, SC-1-181, and the research tool, XMD8-92, to inhibit MEK5 and ERK5, respectively. Our results indicate that the dual inhibition strategy was more effective than single inhibition due to the loss of crosstalk between the two pathways. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Interestingly, this signaling pattern was observed without disturbing the ERK1/2 pathway. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. Additionally, the dual inhibition strategy was determined to be synergistic in TNBCs and was nontoxic in non-neoplastic cell lines. Lastly, we evaluated the contributions of Bromodomain 4 (BDR4), an off-target effect of XMD8-92, to the synergy mechanism. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs. Citation Format: Thomas D. Wright, Christopher Raybuck, Nathan Gartland, Katy Wendekier, Darlene Monlish, Suravi Chakrabarty, Patrick T. Flaherty, Matthew E. Burow, Jane E. Cavanaugh. Evaluation of synergistic PI3K/Akt and MEK5/ERK5 inhibition in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2392.

Aberrations in the MAPK/extracellular signal-regulated kinase (MEK/ERK) and phosphoinositide-3-kinase (PI3K) pathways have been linked to increased proliferation and survival in triple negative breast cancer (TNBC) cells. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. Promising combinations of MEK and PI3K inhibition have been evaluated in phase I clinical trials for various cancer types. However, these clinical trials have had limited efficacy and have yet to encompass the MEK5/ERK5 pathway, which has been shown to promote cell survival. The goal of this study was to examine the crosstalk between the MEK1/2, MEK5, and PI3K pathways and determine the most promising combination of the MEK1/2, ERK5, and PI3K inhibitors, U0126, XMD8-92, and LY294002, respectively, in a diverse panel of triple negative breast cancer cell lines: BT549, MDA-MB-231, and MDA-MB-468. Our results indicate that dual inhibition of the MEK5 and PI3K pathways significantly reduced both proliferation (45.53%) and migration (39.37%) in MDA-MB-231 TNBC cells. In contrast, inhibition of ERK1/2 alone or in combination with PI3K or ERK5 inhibition yielded mixed responses. Interestingly, dual inhibition of the MEK5 and PI3K pathways increased the activity of the MEK1/2 pathway. These data suggest that crosstalk between these kinases occurs and dual inhibition of PI3K and ERK5 may be a novel therapeutic approach for treating TNBC. Citation Format: Thomas Wright, Christopher Raybuck, Katheryn Wendekier, Jane E. Cavanaugh. Dual inhibition of the MEK5 and PI3K pathways most effectively reduces proliferation and migration in triple negative breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 182.

Aberrations in the MAPK/extracellular signal-regulated kinase (MEK/ERK) and phosphoinositide-3-kinase (PI3K) pathways have been linked to increased proliferation and survival in triple negative breast cancer (TNBC) cells. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. Promising combinations of MEK and PI3K inhibition have been evaluated in phase I clinical trials for various cancer types. However, these clinical trials have had limited efficacy and have yet to encompass the MEK5/ERK5 pathway, which has been shown to promote cell survival. The goal of this study was to examine the crosstalk between the MEK1/2, MEK5, and PI3K pathways and determine the most promising combination of the MEK1/2, ERK5, and PI3K inhibitors, U0126, XMD8-92, and LY294002, respectively, in a diverse panel of triple negative breast cancer cell lines: BT549, MDA-MB-231, and MDA-MB-468. Our results indicate that dual inhibition of the MEK5 and PI3K pathways significantly reduced proliferation (45.53%) in MDA-MB-231 TNBC cells. Also, the combination of MEK5 and PI3K inhibition was shown to be synergistic. In contrast, inhibition of ERK1/2 alone or in combination with PI3K or ERK5 inhibition yielded mixed responses. Additionally, treatment with LY294004 in MDA-MB-231 (ERK 5 KO) was more potent (IC50= 2.5 uM) than treatment in the native MDA-MB-231 cell line (IC50= 13.7 uM). These data suggest that crosstalk between these kinases occurs and dual inhibition of PI3K and ERK5 may be a novel therapeutic approach for treating TNBC. Citation Format: Wright TD, Raybuck C, Wendekier K, Cavanaugh JE. Dual inhibition of the MEK5 and PI3K pathways synergistically reduces proliferation and viability in triple negative breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-19.