Most cancers preserve functional retinoblastoma (Rb) and may, therefore, respond to inhibition of D-cyclin-dependent Rb kinases, CDK4 and CDK6. To date, CDK4/6 inhibitors have shown promising ...clinical activity in breast cancer and lymphomas, but it is not clear which additional Rb-positive cancers might benefit from these agents. No systematic survey to compare relative sensitivities across tumor types and define molecular determinants of response has been described. We report a subset of cancers highly sensitive to CDK4/6 inhibition and characterized by various genomic aberrations known to elevate D-cyclin levels and describe a recurrent CCND1 3′UTR mutation associated with increased expression in endometrial cancer. The results suggest multiple additional classes of cancer that may benefit from CDK4/6-inhibiting drugs such as abemaciclib.
•A wide range of sensitivity to abemaciclib is observed among Rb+ tumor cells•CDKN2A mutant cancers show only intermediate sensitivity to CDK4/6 inhibition•D-cyclin activating features are associated with highly sensitive cells•About 5% of endometrial cancers bear a stabilizing mutation in the CCND1 3′UTR
Gong et al. identify a subset of cancers highly sensitive to CDK4/6 inhibition, which are characterized by various genomic aberrations known to elevate D-cyclin levels but not by CDKN2A mutations. They also identify a recurrent CCND1 3′UTR mutation associated with increased CCND1 expression in endometrial cancer.
To investigate the role of insulin receptor substrate-1 (IRS-1) and its downstream signaling in insulin-induced thermogenic
differentiation of brown adipocytes, we have reconstituted IRS-1-deficient ...fetal brown adipocytes (IRS-1 â/â ) with wild-type IRS-1 (IRS-1 wt ). The lack of IRS-1 resulted in the inability of insulin to induce IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase)
activity and Akt phosphorylation in IRS-1 â/â brown adipocytes. In addition, these cells showed an impairment in activating α-Akt, β-Akt, and γ-Akt isoforms upon insulin
stimulation. Reconstitution of IRS-1 â/â brown adipocytes with IRS-1 wt restored the IRS-1/PI 3-kinase/Akt signaling pathway. Treatment of wild-type brown adipocytes with insulin for 24 h up-regulated
uncoupling protein-1 (UCP-1) expression and transactivated the UCP-1 promoter; this effect was abolished in the absence of
IRS-1 or in the presence of an Akt inhibitor and further recovered after IRS-1 wt reconstitution. Neither UCP-2 nor UCP-3 was up-regulated by insulin in wild-type and IRS-1-deficient brown adipocytes. Insulin
stimulated the expression of CCAAT/enhancer-binding protein α (C/EBPα) and its DNA binding activity in wild-type brown adipocytes
but not in IRS-1 â/â cells. However, insulin stimulation of both C/EBPα expression and binding activity was restored after IRS-1 wt reconstitution of deficient cells. Retrovirus-mediated expression of C/EBPα and peroxisome proliferator-activated receptor
γ in IRS-1 â/â brown adipocytes up-regulated UCP-1 protein content and transactivated UCP-1 promoter regardless of insulin stimulation.
Both C/EBPα and peroxisome proliferator-activated receptor γ reconstituted FAS mRNA expression, but only C/EBPα restored insulin
sensitivity in the absence of IRS-1. Finally, reconstitution of IRS-1 â/â brown adipocytes with the IRS-1 mutants IRS-1 Phe-895 , which lacks IRS-1/growth factor receptor binding protein 2 binding but not IRS-1/p85-PI 3-kinase binding, or with IRS-1 Tyr-608/Tyr-628/Tyr-658 , which only binds p85-PI 3-kinase, induced UCP-1 expression and transactivated the UCP-1 promoter. These data provide strong
evidence for an essential role of IRS-1 through the PI 3-kinase/Akt signaling pathway inducing UCP-1 gene expression by insulin.
Fetal brown adipocytes are insulin-like growth factor-I (IGF-I) target cells. To assess the importance of the IGF-I receptor (IGF-IR) in brown adipocytes during fetal life, we have generated ...immortalized brown adipocyte cell lines from the IGF-IR(-/-) mice. Using this experimental model, we demonstrate that the lack of IGF-IR in fetal brown adipocytes increased the susceptibility to apoptosis induced by serum withdrawal. Culture of cells in the absence of serum and growth factors produced rapid DNA fragmentation (4 h) in IGF-IR(-/-) brown adipocytes, compared with the wild type (16 h). Consequently, cell viability was decreased more rapidly in fetal brown adipocytes in the absence of IGF-IR. Furthermore, caspase-3 activity was induced much earlier in cells lacking IGF-IR. At the molecular level, IGF-IR deficiency in fetal brown adipocytes altered the balance of the expression of several proapoptotic (Bcl-xS and Bim) and antiapoptotic (Bcl-2 and Bcl-xL) members of the Bcl-2 family. This imbalance was irreversible even though in IGF-IR-reconstituted cells. Likewise, cytosolic cytochrome c levels increased rapidly in IGF-IR-deficient cells compared with the wild type. A rapid entry of Foxo1 into the nucleus accompanied by a rapid exit from the cytosol and an earlier activation of caspase-8 were observed in brown adipocytes lacking IGF-IR upon serum deprivation. Activation of caspase-8 was inhibited by 50% in both cell types by neutralizing anti-Fas-ligand antibody. Adenoviral infection of wild-type brown adipocytes with constitutively active Foxol (ADA) increased the expression of antiapoptotic genes, decreased Bcl-xL and induced caspase-8 and -3 activities, with the final outcome of DNA fragmentation. Up-regulation of uncoupling protein-1 (UCP-1) expression in IGF-IR-deficient cells by transduction with PGC-1alpha or UCP-1 ameliorated caspase-3 activation, thereby retarding apoptosis. Finally, insulin treatment prevented apoptosis in both cell types. However, the survival effect of insulin on IGF-IR(-/-) brown adipocytes was elicited even in the absence of phosphatidylinositol 3-kinase/Akt signaling. Thus, our results demonstrate for the first time the unique role of IGF-IR in maintaining the balance of death and survival in fetal brown adipocytes.
Abemaciclib is an oral, selective cyclin-dependent kinase 4 & 6 inhibitor (CDK4 & 6i), approved for hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2–) advanced ...breast cancer (ABC) as monotherapy for endocrine refractory disease, and with endocrine therapy (ET) for initial treatment and after progression on ET. Abemaciclib has also shown clinical activity in combination with ET in patients with high risk early BC (EBC). Here, we examined the preclinical attributes of abemaciclib and other CDK4 & 6i using biochemical and cell-based assays.
In vitro
, abemaciclib preferentially inhibited CDK4 kinase activity versus CDK6, resulting in inhibition of cell proliferation in a panel of BC cell lines with higher average potency than palbociclib or ribociclib. Abemaciclib showed activity regardless of
HER2
amplification and phosphatidylinositol 3-kinase (
PI3KCA
) gene mutation status. In human bone marrow progenitor cells, abemaciclib showed lower impact on myeloid maturation than other CDK4 & 6i when tested at unbound concentrations similar to those observed in clinical trials. Continuous abemaciclib treatment provided profound inhibition of cell proliferation, and triggered senescence and apoptosis. These preclinical results support the unique efficacy and safety profile of abemaciclib observed in clinical trials.
Abstract
Nearly 70% of newly diagnosed breast cancers are estrogen receptor alpha (ERα) positive, for which endocrine therapy is a primary treatment. However, approximately 40% of those patients on ...endocrine therapy develop resistance, which includes mutations in ERα (ESR1) that drive constitutive activation of the receptor. One approach to overcome this resistance is to develop a potent degrader and pure antagonist of the estrogen receptor, which can effectively suppress estrogen receptor signaling. Fulvestrant, a pure antagonist and selective estrogen receptor degrader (SERD) is approved for the treatment of HR+/HER2- metastatic breast cancer. However, fulvestrant is limited by poor pharmacokinetics properties, limited exposure and sub-optimal in vivo estrogen receptor degradation. Here we describe the preclinical profile of LY3484356, an oral SERD and pure estrogen receptor antagonist, with potent activity against the wild type and mutant estrogen receptor. LY3484356 has Ki values of 0.64 nM and 2.8 nM against wild type ERα and Y537S mutant ERα proteins, respectively. It is a potent and highly efficient degrader of wild type ERα and Y537N mutant ERα proteins in cells, with IC50 values 3.0 nM and 9.6 nM, respectively. LY3484356 is also a potent inhibitor of ERα-mediated transcription in vitro and in vivo. It inhibits cell proliferation in wild type ERα and ESR1 Y537N mutant breast cancer cell lines, with average IC50 values of 3 nM and 17 nM, respectively. In a panel of breast cancer cell lines, 11 out of 12 ER+ breast cancer cell lines were sensitive to LY3484356 (IC50 less than 100 nM), whereas all ER- cell lines tested were insensitive. LY3484356 has demonstrated sustained and prolonged target inhibition (>75% inhibition of PGR transcription up to 96h after last dose) in ESR1 wild type (MCF7) and ESR1 Y537S mutant (ST941/C) xenograft tumors. Consistent with its profile as a pure antagonist, immature rat studies demonstrated no significant effect on uterine wet weight. LY3484356 demonstrated significant tumor growth inhibition and tumor regressions in wild type ESR1 breast cancer xenograft models such as MCF7, T47D and ZR-75-1, as well as ESR1 mutant breast cancer PDX models. LY3484356 has shown synergy or additivity in combination with CDK4/6 inhibitor abemaciclib, mTOR inhibitor everolimus and PIK3CA inhibitor alpelisib in inhibiting cell proliferation in ER+ breast cancer cell lines in vitro and tumor growth inhibition in relevant xenograft or PDX models in vivo. The first-in-human Phase 1/2 clinical trial of LY3484356 (EMBER, ClinicalTrials.gov NCT04188548) is currently ongoing and a window-of-opportunity study evaluating the pharmacodynamic effects of LY3484356 in early stage breast cancer is expected to begin early 2021.
Citation Format: Shripad V. Bhagwat, Baohui Zhao, Weihua Shen, Cecilia Mur, Robert Barr, Lisa J. Kindler, Almudena Rubio, Jolie A. Bastian, Jeffrey D. Cohen, Brian E. Mattioni, Eunice Yuen, Thomas K. Baker, Mark A. Castanares, Dongling Fei, Jason R. Manro, Maria Jose Lallena, Sheng-Bin Peng, Alfonso de Dios. Preclinical characterization of LY3484356, a novel, potent and orally bioavailable selective estrogen receptor degrader (SERD) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1236.
Increased Insulin Sensitivity in IGF-I Receptor–Deficient Brown Adipocytes
Cecilia Mur 1 ,
Angela M. Valverde 1 ,
C. Ronald Kahn 2 and
Manuel Benito 1
1 Departamento de Bioquímica y Biología ...Molecular, Centro Mixto CSIC/UCM, Facultad de Farmacia, Universidad Complutense, Madrid,
Spain
2 Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts
Abstract
Immortalized brown adipocyte cell lines have been generated from fetuses of mice deficient in the insulin-like growth factor
I receptor gene (IGF-IR −/− ), as well as from fetuses of wild-type mice (IGF-IR +/+ ). These cell lines maintained the expression of adipogenic- and thermogenic-differentiation markers and show a multilocular
fat droplets phenotype. IGF-IR −/− brown adipocytes lacked IGF-IR protein expression; insulin receptor (IR) expression remained unchanged as compared with wild-type
cells. Insulin-induced tyrosine autophosphorylation of the IR β-chain was augmented in IGF-IR–deficient cells. Upon insulin
stimulation, tyrosine phosphorylation of (insulin receptor substrate-1) IRS-1 was much higher in IGF-IR −/− brown adipocytes, although IRS-1 protein content was reduced. In contrast, tyrosine phosphorylation of IRS-2 decreased in
IGF-IR–deficient cells; its protein content was unchanged as compared with wild-type cells. Downstream, the association IRS-1/growth
factor receptor binding protein-2 (Grb-2) was augmented in the IGF-IR −/− brown adipocyte cell line. However, SHC expression and SHC tyrosine phosphorylation and its association with Grb-2 were unaltered
in response to insulin in IGF-IR–deficient brown adipocytes. These cells also showed an enhanced activation of mitogen-activated
protein kinase (MAPK) kinase (MEK1/2) and p42/p44 mitogen-activated protein kinase (MAPK) upon insulin stimulation. In addition,
the lack of IGF-IR in brown adipocytes resulted in a higher mitogenic response (DNA synthesis, cell number, and proliferating
cell nuclear antigen expression) to insulin than wild-type cells. Finally, cells lacking IGF-IR showed a much lower association
between IR or IRS-1 and phosphotyrosine phosphatase 1B (PTP1B) and also a decreased PTP1B activity upon insulin stimulation.
However, PTP1B/Grb-2 association remained unchanged in both cell types, regardless of insulin stimulation. Data presented
here provide strong evidence that IGF-IR–deficient brown adipocytes show an increased insulin sensitivity via IRS-1/Grb-2/MAPK,
resulting in an increased mitogenesis in response to insulin.
Footnotes
Address correspondence and reprint requests to Manuel Benito, Departamento de Bioquímica y Biología Molecular, Centro Mixto
CSIC/UCM, Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain. E-mail: benito{at}eucmax.sim.ucm.es .
Received for publication 6 December 2000 and accepted in revised form 19 November 2001.
C.M. and A.M.V. contributed equally to this work. C.R.K. is a member of an advisory panel for Abbott Millenium.
BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; FAS, fatty acid synthase; FS, fetal serum; Grb-2, growth
factor receptor–binding protein-2; IGF, insulin-like growth factor; IR, insulin receptor; IRS, insulin receptor substrate;
MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; PTP1B, phosphotyrosine phosphatase 1B; UCP-1, uncoupling
protein-1.
DIABETES
Aberrant activation of mitogenic signaling pathways in cancer promotes growth and proliferation of cells by activating mTOR and S6 phosphorylation, and D-cyclin kinases and Rb phosphorylation, ...respectively. Correspondingly, inhibition of phosphorylation of both Rb and S6 is required for robust anti-tumor efficacy of drugs that inhibit cell signaling. The best-established mechanism of mTOR activation in cancer is via PI3K/Akt signaling, but mTOR activity can also be stimulated by CDK4 and PIM kinases. In this study, we show that the CDK4/6 inhibitor abemaciclib inhibits PIM kinase and S6 phosphorylation in cancer cells and concurrent inhibition of PIM, CDK4, and CDK6 suppresses both S6 and Rb phosphorylation.
or
mutations obviate the requirement for PIM kinase and circumvent the inhibition of S6 phosphorylation by abemaciclib. Combination with a PI3K inhibitor restored suppression of S6 phosphorylation and synergized to curtail cell growth. By combining abemaciclib with a PI3K inhibitor, three pathways (Akt, PIM, and CDK4) to mTOR activation are neutralized, suggesting a potential combination strategy for the treatment of
-mutant ER+ breast cancer.
Abstract
Breast cancer is the second most common cancer worldwide after lung cancer. About 70% of breast cancers express estrogen receptor α (ER+) and/or progesterone receptor (PR+), and these ...biomarkers are indicative of hormone dependence. However up to 50% acquire resistance to hormone therapy 1, 2. Estrogen independent ER+ breast cancer depends on CDK4 for tumor growth and CDK4 inhibitors have emerged as a promising approach to treat this type of tumors 3. Abemaciclib is a cell cycle inhibitor with selective activity against CDK4 and CDK6 and it is being evaluated in advanced clinical trials for its potential to reduce metastatic ER+ breast cancer growth. We have evaluated combination of abemaciclib with an anti-estrogen therapy in an in vitro breast cancer panel. Phenotypic characterization of sensitive cell lines was carried out by monitoring cell proliferation, senescence, and apoptosis markers using flow cytometry and high content imaging approaches. Using an in vitro panel with a diversity of breast cancer cell lines, a synergistic effect of abemaciclib in combination with the ER down-regulating drug fulvestrant was observed based on Bliss score. This combination treatment demonstrated effective growth inhibition in ER+ cells and exhibited synergism in MCF-7, T47D and ZR-75-1. The mechanistic analyses revealed that the combination of abemaciclib with fulvestrant promoted a decrease in cancer cell proliferation due to G1 phase arrest at doses tested. This growth inhibition was accompanied by increased hallmarks for cell senescence as observed by markers such as SA-β-galactosidase staining or morphological changes. Subsequently, an increase in biomarkers for apoptosis was also observed. These changes occurred in a time dependent manner and were significantly greater with the combination than fulvestrant single agent treatment. We conclude the combination of abemaciclib with fulvestrant better prevented proliferation of breast cancer cell lines by blocking cell proliferation and lead to induction of senescence and apoptosis as compared to fulvestrant treatment alone in ER+ cells.
Bibliography
1 American Cancer Society, Cancer Facts & Figures 2014.
2 Dixon J.M. (2014) New Journal of Science. Volume 2014, Article ID 390618.
3 Miller TW et al. (2011) Cancer Discov. Volume 1 (4): 338-51.
Citation Format: Raquel Torres, Bruna Calsina, Ana Hermoso, Carmen Baquero, Cecilia Mur, Karsten Boehnke, Joaquín Amat, Alfonso De Dios, Xueqian Gong, Sean Buchanan, Richard Paul Beckmann, Maria Jose Lallena. Characterization of the mechanism of action for abemaciclib with antiestrogen combined therapy in human breast cancer cell lines. 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 2836.
Abstract
CDK4/6 inhibitors combined with endocrine therapy (ET) have shown clinical benefit in HR+, HER2- breast cancer. However, development of resistance highlights the need for treatment ...strategies to improve outcomes. To study resistance mechanisms, breast cancer cell lines were treated with CDK4/6i (abemaciclib or palbociclib) in combination with 4-OH-tamoxifen (tamoxifen) for 120-144h and sorted for resistant cells defined as geminin positive (GEM+), a marker of S/G2/M cell cycle accumulation. To confirm the resistant phenotype, cell lines were treated with tamoxifen plus the CDK4/6 inhibitor used to drive resistance. To understand if sequential CDK4/6i treatment is effective in controlling cell proliferation, resistant cells generated through treatment with palbociclib + tamoxifen or abemaciclib + tamoxifen were treated with abemaciclib + ET (fulvestrant or tamoxifen) or palbociclib + ET, respectively. Geminin/Ki67, annexin V and colony formation assays were performed to evaluate cell proliferation and viability. Molecular characterization by western blot and RNAseq analysis were utilized to provide insights into mechanisms of resistance and the effects of sequential treatment with CDK4/6i + ET. Cell lines resistant to palbociclib + tamoxifen subsequently treated with abemaciclib + ET showed decreased %GEM+ and colony formation ability, decreased Ki67 levels, and increased apoptosis. These effects were not observed in cell lines resistant to abemaciclib + tamoxifen following subsequent treatment with palbociclib + ET. Western blot analysis showed that palbociclib and abemaciclib-resistant cells had increased CDK6 and pERK levels, compared to control. Importantly, treatment of palbociclib-resistant cells with abemaciclib + ET decreased FOXM1, a key regulator of senescence and apoptosis. Cyclin A, a marker of mitosis, was also decreased, consistent with decreased %GEM+ subpopulation in palbociclib + ET resistant cells. These effects were not observed in abemaciclib-resistant cells treated with palbociclib + ET.In summary, our in vitro data provides mechanistic insights into resistance to combination CDK4/6i + tamoxifen and suggests the potential benefit of sequential treatment with abemaciclib + ET to overcome resistance in breast cancer.
Citation Format: Elisabet Zapatero-Solana, Maria P. Ganado, Maria J. Ortiz-Ruiz, Cecilia Mur, Lacey Litchfield, Farhana Merzoug, Oscar Puig, Maria Jsoe Lallena. Sequential treatment with abemaciclib plus endocrine therapy inhibits cell proliferation and triggers apoptosis in cell lines resistant to CDK4/6i abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2307.
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