Triplet Combos Targeting ER, CDK4/6, and PIK3CA Inhibit Tumor Growth in ER+ Breast Cancer

April 14, 2021
Courtney Marabella
Courtney Marabella

Senior Editor, OncLive®
Courtney Marabella joined the MJH Life Sciences team in 2021 and is Senior Editor for OncLive®. Prior to joining the company she worked as the Audience Development Editor for the Asbury Park Press, part of the USA Today Network. Email: cmarabella@onclive.com

A triplet combination regimen that targets PIK3CA mutations, ER, and CDK4/6 can reduce and inhibit tumor growth in preclinical models of estrogen receptor–positive breast cancer that is resistant to fulvestrant plus a CDK4/6 inhibitor or PI3K inhibitor.

A triplet combination regimen that targets PIK3CA mutations, ER, and CDK4/6 can reduce and inhibit tumor growth in preclinical models of estrogen receptor (ER)–positive breast cancer that is resistant to fulvestrant (Faslodex) plus a CDK4/6 inhibitor or PI3K inhibitor, according to data that were virtually presented during AACR Annual Meeting 2021.1

Findings showed that the triple inhibition of the ER, CDK4/6, and PI3Kpathwaysreduced protein expression in ER, PI3K/AKT/mTOR, and in the cyclin D-CDK4/6-Rb pathway; it also induced apoptosis and cell cycle arrest.

Additionally, the triple inhibition delayed or prevented acquisition of cells that are resistant to fulvestrant plus a CDK4/6 or PI3K inhibitor. Notably, switching between fulvestrant/CDK4/6 inhibitor and fulvestrant/PI3K inhibitor after acquired resistance did not prevent disease progression, which indicates that the simultaneous blocking of all 3 mutational pathways is required for this patient population.

Additionally, in cells specifically resistant to fulvestrant plus a PI3K inhibitor, an increase in CDK2 levels was observed.

“Together, our data support the use of combined therapy with either PI3K or CDK4/6 inhibitors and fulvestrant following progression on or after an endocrine-based regimen,” lead study author and coinvestigators noted in their presentation of the data. “However, following disease progression on combined therapy with fulvestrant and either CDK4/6 or PI3K inhibitors, triple combination targeting ER, PI3K, and either CDK4/6 or CDK2 is needed to inhibit tumor progression.”

While outcomes have improved for patients with ER-positive advanced breast cancer, those treated with a combination of a CDK4/6 inhibitor and endocrine therapy often experience disease progression. Additionally, while the approved combination of the a-specific PI3K inhibitor alpelisib (Piqray) and fulvestrant in patients with PIK3CA-mutant ER-positive metastatic breast cancer has shown clinical benefit, patients often develop resistance.

As such, an unmet need remains for more novel and effective treatment regimens, as well as a defined treatment sequence following disease progression on combination treatment with endocrine therapy and either a PI3K or CDK4/6 inhibitor.

In the study presented at the meeting, investigators developed 4 cell models of PIK3CA-mutant, ER-positive breast cancer resistant to fulvestrant plus a CDK4/6 or PI3K inhibitor. These cell models were derived from MCF-7- (MPFR and MPiFR) and T47D- (TPFR and TPiFR) derived cells, as well as 2 orthotopic cell line xenograft models (MPFR and MPiFR).

Investigators then used these cells models to determine whether ER–positive breast cancer cells that are resistant to fulvestrant and either PI3K or CDK4/6 inhibitors would benefit from switching between combination regimens, or if a triple combination would be more effective.

The cell models derived from MCF-7 and T47D cells that were resistant to the combination of palbociclib (Ibrance) and fulvestrant were exposed to either vehicle, fulvestrant alone, palbociclib alone, alpelisib alone, as well as alpelisib/fulvestrant, palbociclib/fulvestrant, alpelisib/palbociclib, and alpelisib/palbociclib plus fulvestrant. Exposure lasted for 2, 4, or 6 days, or 8 weeks, and cell growth was measured by crystal violet colorimetric assay.

The palbociclib- and fulvestrant-resistant MPFR and TPFR cells were also exposed to vehicle, single-agent treatment with fulvestrant, palbociclib, alpelisib, as well as the combinations for 6 days. Cell lysate were then prepared and analyzed using Western blotting with various antibodies, such as ER, p-AKT S473, Pan AKT, p-PRAS40 T246, PRAS40, p-Rb S780, Rb, p-S6 S234/s236c, S6, and glyceraldehyde 3-phosphate dehydrogenase, which was utilized as a loading control.

Palbociclib and fulvestrant-resistant MPFR cells were then orthotopically transplanted into the mammary fat pad of NOD/Shi-scid IL2rγnull Central Institute for Experimental Animals (CIEA) mice, which were divided into groups and treated with vehicle (n=10), fulvestrant plus alpelisib (n=9), fulvestrant and palbociclib (n=10), and the triplet combination of alpelisib, palbociclib, and fulvestrant (n=8). Fulvestrant was administered once weekly subcutaneously, while alpelisib and palbociclib were administered orally 5 times per week, daily.

Results showed that tumor growth was significantly less with the triplet regimen compared with the vehicle and doublet combinations.

MPiF-R and TPiF-R cells that were resistant to alpelisib plus fulvestrant were also treated with vehicle; single-agent treatment with fulvestrant, palbociclib, or alpelisib; alpelisib/fulvestrant, palbociclib/fulvestrant, alpelisib/palbociclib, and alpelisib/palbociclib plus fulvestrant. The effect on cell growth was assessed over 6 to 8 days, while the outgrowth of resistant colonies was determined weekly via evaluation of the confluency of 48 wells over 12 weeks. Any positive wells were defined as 50% or greater confluency.

The fold change of upregulated CDKs and cyclins in the MPiF-R compared with the MCF-7/S0.5 cells was 15.25 for CDK6, 6.84 for CDK2, 3.63 for CCNE1, and 7.31 for CCNE2. Additionally, MPiF-R and MCF-7/S0.5 cell lines were exposed to vehicle, fulvestrant alone, alpelisib alone, the CDK2 inhibitor dinaciclib alone, as well as combinations of dinaciclib/fulvestrant, dinaciclib/alpelisib, and dinaciclib/alpelisib plus fulvestrant. Cell growth was again assessed by crystal violet colorimetric assay after 2, 4, and 6 days of exposure, and the outgrowth of resistant colonies was evaluated in the MPiF-R and MCF-7/S0.5 cells by weekly evaluation of the confluency of 48 wells over 12 weeks.

Reference

Karimi L, Alves C, Terp M, et al. Triple combination targeting ER, CDK4/6, and PI3K inhibits tumor growth in ER+ breast cancer resistant to combined fulvestrant and CDK4/6 or PI3K inhibitor. Presented at: AACR Annual Meeting 2021; April 10-15, 2021; virtual. Abstract 1415.