The pure progesterone receptor (PR) antagonist onapristone enhances the anti-proliferative effects of CDK4/6 inhibitors and fulvestrant, a SERD, in preclinical in-vitro breast cancer models Deepak Lala1, PhD; Tasir Haque1, PhD; Hannah Feinman2; Jianghong Wu2, PhD; Yuren Wang2, PhD; Amy Dwyer3, PhD; Thu Truong3, PhD and Carol Lange3, PhD, Institutions: 1Context Therapeutics, Philadelphia, PA, United States, 19104; 2Reaction Biology Corporation, Malvern, PA, United States, 19355 and 3University of Minnesota Masonic Cancer Center, Minneapolis, MN, United States, 55455. San Antonio Breast Cancer Symposium® - December 4-8, 2018 ABSTRACT METHODS A
Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer related T47D cells were treated with various concentrations of onapristone or palbociclib in media death in women. Around 5–10% of cases are metastatic at diagnosis, and close to 30% of patients with containing 10%FBS.10 days after treatment cell viability was determined using Cell Titer Glo. early stage disease will relapse with metastatic disease. Anti-estrogen therapy is an important treatment 1x agarose, IMEM, 10% DCC, 1% P/S 1x agarose, 2.4x103 cells, Fulv or Palbo Cells were also treated with increasing concentrations of palbociclib in the absence or presence Solidify 5 min at 4°C Solidify 5 min at 4°C modality for hormone receptor-positive (HR+) metastatic breast cancer (mBCa) as mono- or combination of onapristone and analyzed for cell proliferation after 10 days and for gene expression after 16 (e.g. with CDK4/6 inhibitors) first-line (1L) therapy. Unfortunately, despite the high rate of clinical benefit hours of treatment. The effects of onapristone were also tested in soft agar anchorage-independent B veh Ona 3 weeks at 37°C in 1L therapy, disease progression still generally occurs and there remains a need for an efficacious 2L growth assays as well as 3D tumorsphere assays using ER+/PR+ MCF7 cells -/+ onapristone for 21-28 therapy. Although anti-estrogens continue to play a role in 2L treatment there is a critical need for days. Formed colonies or spheres were analyzed morphologically using cell stain and by quantifying the Count colonies targeting additional mechanisms in combination with anti-estrogens. Progesterone is a major mitogen in total number and size of colonies or spheres formed. the adult human mammary epithelium and, in addition to the estrogen receptor (ER), is a key driver of breast cancer cell proliferation. Thus, blocking both ER and PR would likely be an effective approach for Figure 6: Onapristone inhibits colony formation in a T47D soft agar model inhibition of all hormone driven breast cancer cell proliferation. RESULTS A: Brief description of the soft agar protocol B: Typical example of colony growth and size in the presence of onapristone (100nM) Onapristone is a unique PR full antagonist that is efficacious as an antitumor agent in multiple preclinical A B breast cancer models and exhibits additive/synergistic effects with antiestrogens. Here, we examine the T47D T47D 125 125 A B effects of onapristone in in-vitro model systems in combination with CDK4/6 inhibitors and the SERD DMSO DMSO
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0 0 DMSO 1000 300 100 CoA Activation of mammary stem cells DMSO 1000 300 100 Onapristone Concentration (nM) Palbociclib Concentration (nM) Migration of cancer cells from early lesions Figure 3: Onapristone (A) and Palbociclib (B) inhibit T47D cell proliferation in a dose-dependent manner PR PR Proliferation in advanced primary tumor cells 1000 Oncogenic progression Palbociclib 800
Contributes to tumor relapse ) Palbociclib + 100 nM Onapristone Figure 7: Onapristone (100nM) has an additive effect on the inhibition of colony growth with
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Figure 1: Progesterone receptor is a key driver of breast cancer1,2,3,4 l 200 CONCLUSIONS and FUTURE DIRECTIONS
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0 Onapristone inhibits T47D proliferation by regulating key cell cycle genes. Additionally, onapristone
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- - - -200 - enhances the anti-proliferative effects of palbociclib in-vitro. Previous studies have provided a clear Onapristone CDK4/6 Palbociclib rationale for combining onapristone and anti-estrogens in the clinic for the treatment of breast cancer. Cyclin D1 Log[Treatment] (M) Our data extend these studies to provide additional rationale for the combination of onapristone with + + CDK4/6 inhibitors and SERDs as an effective new approach for the treatment of breast cancer. A phase 2 P Figure 4: Onapristone enhances the ability of Palbociclib to inhibit T47D cell proliferation ER ER P clinical study is planned for onapristone plus fulvestrant in patients with metastatic breast cancer. P RB
P E2F Genes downregulated by Genes upregulated by Fulvestrant P onapristone in T47D cells ≥ 50% onapristone in T47D cells ≥ 2x + AURKB6 CDK16 MDM27 REFERENCES Progression of cell cycle CCND16 CDC25C6 CDKN2B9 MCM28 CDK610 1. Lee et al. Cancer Letters (2016) 376:310–317 6. Clare et al. BMC Cancer (2016) 16:32 Cell proliferation 2. Hosseini H et al. Nature (2016) 540:552-558 7. LE Giono et al. Oncogene (2017) 36:6762-6773 Figure 5: Onapristone downregulates genes that promote cell proliferation 3. Joshi PA Nature (2010) 465:803-807 8. Yousef et al. Modern Pathology (2017) 30, 682–697 5 4. Knutson et al. Journal of Hematology & Oncology (2017) 10:89 9. Ansems et al. Breast Cancer Res Treat (2015) 149:693–703 Figure 2: PR cross-talk with ER and CDK4/6 offers combination opportunities for treatment of breast cancer and upregulates genes that inhibit cell cycle proliferation in T47D cells 5. Adapted from Morikawa A Clin Cancer Res (2015) 21:3592-3596 10. Yang C et al. Oncogene (2017) 36, 2255–2264
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