Philip Sutera
Mentors: Ian Pollack, MD; Daniel Premkumar, PhD; Department of Neurological Surgery
Children’s Hospital of Pittsburgh
Elucidating the molecular mechanism of Dinaciclib synergizing with ABT-737 to induce
cell death in malignant human glioma cell Lines
Background:
T98G
LN18
Analysis of genomic alterations
in over 500 glioblastoma tumors
revealed 78.9% of tumors have
alterations affecting pRb function6.
Alterations included mutations and
deletions of pRb, amplification of CDKs,
ABT-737, 0.1µM+Dinaciclib, 1,000nM
ABT-737, 0.1µM+Dinaciclib, 250nM
ABT-737, 0.1µM+Dinaciclib, 100nM
ABT-737, 0.1µM+ Dinaciclib, 25nM
ABT-737, 0.1µM
Dinaciclib, 1,000nM
Dinaciclib, 250nM
Dinaciclib, 100nM
Dinaciclib, 25nM
Control
% Cell Death
Malignant glioma is an aggressive brain tumor that responds poorly to conventional treatment
modalities1. Due to multiple modes of resistance, including pro-survival redundancy, single
agent therapy has proven to be an ineffective treatment2. Many studies have aimed to determine
the molecular mechanisms of glioma resistance and generate targets to induce cell death in these
cell lines. Overactive pathways have
100
been identified in glioma, including
90
PI3K/Akt/mTORC1 signaling, epidermal
80
growth factor receptor (EGFR) signaling,
70
and anti-apoptotic proteins, such as Bcl-2
60
and Bcl-xl2-4. Mutations in the tumor
50
suppressor genes p53 and genomic
40
alterations affecting Retinoblastoma
30
protein (pRb) function are also common
20
in glioma and contribute to enhanced
10
tumor survival and proliferation5.
0
Inhibition of the Bcl-2 anti-apoptotic
family proteins with ABT-737 has
generated only limited cell death in
glioma and sensitivity has been related to
expression levels of Mcl-1, Bcl-2, Bcl-xl,
Bim and Noxa7-8. Understanding these
pathways, the Pollack group has
demonstrated ABT-737 synergizes with
Histone deacetylase, Survivin, and Akt
inhibitors to induce significant apoptosis
at clinically attainable concentrations2-4,6.
Treatment
Figure 1. Annexin V apoptosis assay of T98G and LN18 cell lines with
varying concentrations of Dinaciclib, ABT-737, and combined
treatment. Showing percent of cells in apoptosis.
and p16 deletion6. These findings guided preliminary screening for co-administering a CDK
inhibitor, Dinaciclib, with ABT-737 to induce significant cell death (Figure 1). Dinaciclib is an
inhibitor of CDKs 1,2,5,9 and primarily acts through inhibition of phosphorylation of pRb7.
Hypophosphorylated pRb remains associated with transcription factor E2F which prevents
progression through the cell cycle9-10. Additionally, a new pro-apoptotic function of pRb has
been described wherein pRb associated with E2F can act as a transcription factor for proapoptotic factors8,11. pRb has also been implicated in the ability of ABT-737 to induce
apoptosis. In response to ABT-737, pRb is cleaved to p68Rb which forms a transcription factor
complex with E2F and RNA polymerase II8. This complex increases the expression of the proapoptotic factor Noxa which also inhibits the anti-apoptotic Mcl-1 leading to increased cell
death.
Hypothesis: The goal of this work will be to assess the inhibition of cell growth and induction of
cell death by the combination of Dinaciclib and ABT-737 in a panel of glioma cell lines with
different genomic alterations, which will be a fundamental step in assessing the relevance of this
combination of pathway inhibitors for clinical therapeutics in all, or a subset of, gliomas. Given
the important role of Rb in both glioma and pathways involving apoptosis modulation, we also
hypothesize that this treatment combination will induce cell death by promoting a proapoptotic
mechanism, and have designed a series of studies to address these hypotheses.
Methodology:
Cell lines: This study will be conducted on established human malignant glioma cell lines, grown
in cell culture. These cell lines include LNZ308 (PTEN mutant, p53 mutant), LN229 (PTEN
wild type, p53mutant), LN18 (PTEN wild type, p53 mutant), U87 (PTEN deleted, p53 wild
type), T98G (PTEN mutant, p53 mutant)
Treatment: The cells will be treated with DMSO, ABT-737, Dinaciclib, or both inhibitors at
varying concentrations.
Mechanism of Apoptosis: Western Blot analysis will be used to determine the involvement of
Noxa, Mcl-1, pRB, E2F, and Caspases-3, 7, and 8, and their downstream targets. Apoptosis will
be analyzed with an AnnexinV/PI apoptosis assay as described7-8. Mitochondrial membrane
dysfunction will be determined with flow cytometry, using a DiOC6 mitochondrial membrane
depolarization assay and by visualizing AIF and Cytochrome C appearance in the cytoplasm by
Western blot analysis as described7-8. Finally, light and immunofluorescence microscopy will be
used to assess the morphologic changes such as cell shrinkage, microtubule catastrophe, and
DNA fragmentation that are characteristic of apoptosis in response to treatment.
Analysis: Flow cytometry software will be used to determine the number of cells in a given
population for all flow cytometry experiments. Student’s two tailed t-tests will be used to
compare pairwise treatments and ANOVA will be used for multiple comparisons, with a
significance cutoff of 0.05. All experiments will be run in triplicate.
Significance: Current treatment for glioma is ineffective with a median survival rate of 14.6
months. Finding new and effective treatments for these tumors is imperative and necessary to
understand the mechanisms by which these treatments work. This study aims to understand the
mechanism of a potential therapy for glioma as well as gain further insights to the molecular
pathways involved in glioma and resistance.
Limitations: The greatest limitation in this project will be my time available. With only an 8
week time span, it will be difficult to complete all studies outlined above.
Student Role: My role in this project is to elucidate the molecular mechanism by which ABT737 synergizes with Dinaciclib to induce apoptosis in glioma cell lines. I will be performing the
experiments outlined above as well as analyzing and interpreting the results.
Ethical Approval: The work in the lab is covered by a current Institutional Biosafety Committee
(IBC) protocol.
1. Omuro A, DeAngelis LM. (2013). Glioblastoma and Other Malignant Gliomas: A
Clinical Review. JAMA, 310(17), 1842-1850.
2. Foster K.A. et al (2014) Co-administration of ABT-737 and SAHA induces apoptosis
mediated by Noxa upregulation, Bax activation and mitochondrial dysfunction in PTENintact malignant human glioma cell lines. J Neurooncol. 120(3):459-72
3. Premkumar D.R. et al (2012). ABT-737 Synergizes with Bortezomib to Induce Apoptosis
Mediated by Bid Cleavage, Bax Activation, and Mitochondrial Dysfunction in an AktDependent Context in Malignant Human Glioma Cell Lines. JPET 341:859-872
4. Jane E.P. et al (2013) YM-155 Potentiates the Effect of ABT-737 in Malignant Human
Glioma Cells via Survivin and Cdl-1 Downregulation in an EGFR-Dependent Context.
Mol Cancer Ther 12:326-338.
5. Brennan C.W. et al (2013). The Somatic Genomic Landscape of Glioblastoma. Cell
155(2): 462–477
6. Jane E.P. et al. Inhibition of Phosphatidylinositol 3-Kinase/AKT Signaling by
NVP-BKM120 Promotes ABT-737–Induced Toxicity in a Caspase-Dependent Manner
through Mitochondrial Dysfunction and DNA Damage Response in Established and
Primary Cultured Glioblastoma Cells. J Pharmacol Exp Ther 350:22–35.
7. Parry D. et al (2010) Dinaciclib (SCH 727965), a Novel and Potent Cyclin-Dependent
Kinase Inhibitor. Mol Cancer Ther August 2010 9;2344
8. Bertin-Ciftci J. et al (2013) pRb/E2F-1-mediated caspase-dependent induction of Noxa
amplifies the apoptotic effects of the Bcl-2/Bcl-xL inhibitor ABT-737. Cell Death and
Differentiation. 20(5):755-764.
9. Harbour J.W. et al (2000) The Rb/E2F pathway: expanding roles and emerging
paradigms. Genes & Dev. 2000. 14:2393-2409
10. Giacinti C. et al (2006) RB and cell cycle progression. Oncogene 25, 5220-5227
11. Ianari A. et al (2009) Pro-apoptotic function of the retinoblastoma tumor suppressor
protein. Cancer Cell 15(3): 184-194