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Cyclin-Dependent Kinases and
CDC7 as Therapeutic Targets
Geoffrey Shapiro, M.D., Ph.D.
Dana-Farber Cancer Institute
Gary K. Schwartz, M.D.
Columbia University
Disclosures
Geoffrey Shapiro, MD, PhD
Advisory Board: G1 Therapeutics, Syros, Vertex, Millenium
Research Funding: Pfizer
Funding to Dana-Farber Cancer Institute:
• Pfizer
• Lilly
• Novartis
• Merck
• Cyclacel
• Bayer
Investigator: palbociclib, abemaciclib, ribociclib,
dinaciclib, seliciclib, roniciclib
Disclosures
Gary K. Schwartz, MD
Consultant for and honoraria received from:
• Novartis
• Astra-Zeneca
• Boehringer
Cell Cycle and Transcriptional CDKs
Malumbres. Genome Biology 2014 15:122
Model of CDK Activation
Malumbres. Genome Biology 2014 15:122
Overview of CDK Function
Malumbres. Genome Biology 2014 15:122
CDK Inhibitor Compounds
CDK4/6
Selective
CDK 1/2/9 or
pan-CDK
(+CDK 4/5/7)
CDK7
Palbociclib
(PD0332991)
Flavopiridol
THZ1 (covalent)
Abemaciclib
(LY2835219)
Dinaciclib
(SCH727965)
Ribociclib
(LEE011)
Seliciclib
(CYC202)
CYC065
Roniciclib
(BAY1000394)
Key Issues for CDK4/6 Inhibitors
• What governs a G1 arrest response vs. cytotoxic response?
Solid tumor models tend to undergo G1 arrest, whereas abrupt
apoptosis has been observed in models of T Cell Leukemia and
Burkitt Lymphoma.
CDK6 is a chromatin-bound NF-κB co-factor.
• Among cell types that undergo initial G1 arrest, what
governs a senescent response?
HER2-amplified BC, ER+ BC and KRAS-mutant NSCLC likely
undergo senescence following CDK4/6 inhibition, possibly with
associated tumor regression that can occur late. CDK4/6
inhibition destabilizes FOXM1, which suppresses senescence.
In KRAS-mutant NSCLC, does the tumor suppressive background
matter (KRAS alone, KRAS/TP53, KRAS/LKB1, KRAS/CDKN2A/B)?
Key Issues for CDK4/6 Inhibitors
• What are mechanisms of resistance to CDK4/6 inhibitors?
De novo resistance linked to Rb-negativity
Acquired resistance linked to CDK2 activation
(Cyclin E overexpression or p27Kip1 loss).
• What are the most compelling combinations for future
development?
Hormonal Therapy in ER+ breast cancer
(Basis of initial palbociclib US FDA approval in first-line
metastatic ER+ breast cancer)
MEK inhibition in RAS-driven cancers
PI3K inhibition in tumors with PI3K pathway activation
Other signal transduction inhibitors (IGF-1R)
Chemotherapy (e.g. Taxane) when appropriately sequenced
Palbociclib Induces G1 Cell Cycle
Arrest in all Liposarcoma Cell Lines
Kovatcheva M et al, Oncotarget, 2015
Palbociclib induces senescence in some
but not all RB+ liposarcoma cell lines
Senescence
Non-Senescence
senescence-associated β- galactosidase
Senescence
Non-Senescence
HPγ1 staining for senescence-associated
heterochromatic foci formation
Kovatcheva M et al, Oncotarget, 2015
shCDK4 Replicates the Effect of
Palbociclib
Senescence
Non-Senescence
Kovatcheva M et al, Oncotarget, 2015
Palbociclib or shCDK4 Suppresses MDM2
Expression in Senescent Liposarcoma Cells
B
Δ by densitometry
Senescence
Non-Senescence
0.47 0.24 1.03
0.74
Kovatcheva M et al, Oncotarget, 2015
MDM2 Knockdown Induces Cellular
Senescence in All Liposaracoma Cells
Senescence
Non-Senescence
Kovatcheva M et al, Oncotarget, 2015
Palbociclib Induces MDM2 Knockdown and Senescence in
GBM and Breast Cancer Cells Independent of p53 Status
GBM
Breast Cancer
Kovaatcheva et al, Oncotarget, 2015
MDM2 Loss is Associated with Prolonged
Clinical Benefit in Liposarcoma Patients
Treated with Palbociclib
Kovatcheva M et al, Oncotarget, 2015
ATRX is Required for Palbocilcib-mediated
MDM2 loss and Induction of Senescence
C.
c
D
Senescence
Non-Senescence
CC
Model of MDM2 Regulation by ATRX
to Induce Senescence
CDK4 inhibition causes dissociation of HAUSP/USP7 from MDM2, priming it for
autoubiquitination; (2) Degradation of MDM2 also requires ATRX; (3) MDM2 likely
ubiquinates a SAP other than p53; removal of MDM2 promotes senescence.
Kovatcheva M et al, Oncotarget, 2015
CDK2 as a target
• Replacement of
p27Kip1
• Dominant negative
mutants
• CDK2 inhibitory
peptides
• Targeted cyclin A
degradation
• CDK2 anti-sense
and siRNA
experiments
• cdk2-/- knockout
cells
Combined CDK1/2 depletion is required
for antiproliferative effects
Cai et al. Cancer Research, 2006
Putative CDK2-dependent tumors:
HGSOC
Ovarian and TNBCs
BRCA-deficient/
HR-deficient
• Cisplatin sensitive
• PARP inhibitor sensitive
based on synthetic
lethality of PARP
inhibition and HR
deficiency
CCNE1-amplified/
HR-dependent
• High cyclin E expression
• Replicative stress
upregulates HR genes
and HR dependency
• Often rapid recurrence
and platinum-insensitive
tumors
• Likely CDK2 dependent
• CDK1/CDK2 required for
initiation of HR
CDK2 and 1 targeting agents also inhibit transcriptional CDKs to varying degrees
Cell Cycle Kinases
Malumbres Physiol Rev 2011; 91: 973-1007
CDC7 and CDK in DNA Replication
Labib. Genes Dev 2010; 24: 1208-19
CDC7 in Translesion Synthesis
Yamada et al. Cell Cycle 2014; 13: 1859-66
RNA Pol II CTD Phosphorylation
Heidemann et al. BBA 2013; 1829: 55-62
CDK-dependent regulation of initiation to
elongation switch of RNA polymerase II
Larochelle et al. Nat Struct Mol Biol 2012; 19: 1108-1115
Concept of Super-enhancers
Typical enhancers
• composed of transcription factor binding sites located at a distance
from the transcriptional start site that act through chromosomal
looping events to enhance transcription.
Super-enhancers
• consist of very large clusters of enhancers that are densely occupied
by transcription factors, co-factors and chromatin regulators (e.g.
BRD4)
• arise via gene amplification, translocation or transcription factor
overexpression
• facilitate high level of expression of genes involved in cell identity,
growth and proliferation; often genes and encoded proteins have
short half-life, so high-level transcription is critical to maintenance of
their expression
• highly sensitive to perturbation
Whyte et al. Cell 2013; 153: 307-19; Lovén et al. Cell 2013; 153: 320-34;
Chapuy et al. Cancer Cell 2013; 24: 777-90.
Example of MYC
Super-enhancer
THZ1 is a covalent CDK7
inhibitor-drug bind with a unique
cysteine residue outside of the
kinase domain results in
prolonged and irreversible
CDK7 inactivation
BRD4 inhibition (bromodomain
Inhibitor) expected to produce
similar effects
CDK9 inhibitor may also
produce similar effects; known
CDK9 inhibitors (flavopiridol,
Dinaciclib) are ATPcompetitive and reversible
Kwiatkowski et al. Nature 2014; 511: 616-20;
Chipumuro et al. Cell 2014; 159: 1126-39;
Christensen et al. Cancer Cell 2014; 26: 909-22
Concept of Super-enhancers
Lovén et al. Cell 2013; 153: 320-34
Transcriptional CDK depletion increases
cytotoxicity following CDK1/2 depletion
Cai et al. Cancer Research, 2006
CDK12 is a CTD Kinase that regulates
transcriptional elongation of a small subset of genes
Pathway analysis indicates preferential loss of genes involved in
DNA replication, recombination and repair
(e.g. BRCA1, ATR, FANCI, FANCD2)
Blazec et al. Genes Dev 2011: 25: 2158-72
CDK12
causes defects in
homologous recombination repair
Bajrami et al. Cancer Res 2014: 74: 287-97
Loss of CDK12 Activity in a Subset of
Ovarian Tumors
TCGA, Nature 2011
CDKs in the DNA Damage Response
CDK Family Member
CDK1
CDK2
CDK9
CDK12
CDK5
CDK4/6
Target
BRCA1, CtIP
ATRIP, CtIP, {BRCA2}
RNA Polymerase II
(RAD51)
RNA Polymerase II (DDR
cassette)
Sensitization to PARP
inhibition in siRNA screen
G1 arrest and reliance on
NHEJ
Cell Cycle and Transcriptional CDK Inhibitors:
Summary
• CDK4/6 inhibitors have anti-tumor activity; additional work needed
on biological outcomes in a variety of tumor backgrounds,
mechanisms of resistance and development of optimal combinations
• CDK2 is easily compensated by CDK1; CDK2-dependent tumors
may exist (e.g. subset of HGSOCs), requiring clinical validation
• CDK7, BRD4 and MYC all can overcome transcriptional pausing
and activate or recruit CDK9 or 12 for productive transcriptional
elongation
• Targeting CDK7 or BRD4 has greatest effect on super-enhancerdirected transcription and so may be relatively selective for
transformed cells
• CDK12 is involved in the transcription of DDR genes; mutations
confer homologous recombination repair deficiency in ovarian
cancer cells