11 research outputs found
Disrupting BCR-ABL in Combination with Secondary Leukemia-Specific Pathways in CML Cells Leads to Enhanced Apoptosis and Decreased Proliferation
Chronic myeloid leukemia (CML) is a myeloproliferative
disorder
caused by expression of the fusion gene BCR-ABL following a chromosomal
translocation in the hematopoietic stem cell. Therapeutic management
of CML uses tyrosine kinase inhibitors (TKIs), which block ABL-signaling
and effectively kill peripheral cells with BCR-ABL. However, TKIs
are not curative, and chronic use is required in order to treat CML.
The primary failure for TKIs is through the development of a resistant
population due to mutations in the TKI binding regions. This led us
to develop the mutant coiled-coil, CC<sup>mut2</sup>, an alternative
method for BCR-ABL signaling inhibition by targeting the N-terminal
oligomerization domain of BCR, necessary for ABL activation. In this
article, we explore additional pathways that are important for leukemic
stem cell survival in K562 cells. Using a candidate-based approach,
we test the combination of CC<sup>mut2</sup> and inhibitors of unique
secondary pathways in leukemic cells. Transformative potential was
reduced following silencing of the leukemic stem cell factor Alox5
by RNA interference. Furthermore, blockade of the oncogenic protein
MUC-1 by the novel peptide GO-201 yielded reductions in proliferation
and increased cell death. Finally, we found that inhibiting macroautophagy
using chloroquine in addition to blocking BCR-ABL signaling with the
CC<sup>mut2</sup> was most effective in limiting cell survival and
proliferation. This study has elucidated possible combination therapies
for CML using novel blockade of BCR-ABL and secondary leukemia-specific
pathways
A Single Mutant, A276S of p53, Turns the Switch to Apoptosis
The tumor suppressor protein p53
induces apoptosis, cell cycle
arrest, and DNA repair along with other functions in a transcription-dependent
manner [Vousden, K. H. <i>Cell</i> <b>2000</b>, <i>103</i>(5), 691–694]. The selection of these functions
depends on sequence-specific recognition of p53 to a target decameric
sequence of gene promoters [Kitayner, M.; et al. <i>Mol. Cell</i> <b>2006</b>, <i>22</i>(6), 741–753]. Amino
acid residues in p53 that directly bind to DNA were analyzed, and
the replacement of A276 in p53 with selected amino acids elucidated
its importance in promoter transcription. For most apoptotic and cell
cycle gene promoters, position 9 of the target decameric sequence
is a cytosine, while for DNA repair gene promoters, thymine is found
instead. Therefore, selective binding to the cytosine at the ninth
position may transcribe apoptotic gene promoters and thus can induce
apoptosis and cell cycle arrest. Molecular modeling with PyMOL indicated
that substitution of a hydrophilic residue, A276S, would prefer binding
to cytosine at the ninth position of the target decameric sequence,
whereas substitution of a hydrophobic residue (A276F) would fail to
do so. Correspondingly, A276S demonstrated higher transcription of
PUMA, PERP, and p21<sup>WAF1/CIP1</sup>gene promoters containing a
cytosine at the ninth position and lower transcription of GADD45 gene
promoter containing a thymine at the ninth position compared to wild-type
p53. Cell cycle analysis showed that A276S maintained similar G1/G0
phase arrest as wild-type p53. Additionally, A276S induced higher
apoptosis than wild-type p53 as measured by DNA segmentation and 7-AAD
assay. Since the status of endogenous p53 can influence the activity
of the exogenous p53, we examined the activity of A276S in HeLa cells
(wild-type endogenous p53) in addition to T47D cells (mutated and
mislocalized endogenous p53). The same apoptotic trend in both cell
lines suggested A276S can induce cell death regardless of endogenous
p53 status. Cell proliferation assay depicted that A276S efficiently
reduced the viability of T47D cells more than wild-type p53 over time.
We conclude that the predicted preferred binding of A276S to cytosine
at the ninth position better transactivates a number of apoptotic
gene promoters. Higher induction apoptosis than wild-type p53 makes
A276S an attractive candidate for therapy to eradicate cancer
Solid Phase Synthesis of Mitochondrial Triphenylphosphonium-Vitamin E Metabolite Using a Lysine Linker for Reversal of Oxidative Stress
<div><p>Mitochondrial targeting of antioxidants has been an area of interest due to the mitochondria's role in producing and metabolizing reactive oxygen species. Antioxidants, especially vitamin E (α-tocopherol), have been conjugated to lipophilic cations to increase their mitochondrial targeting. Synthetic vitamin E analogues have also been produced as an alternative to α-tocopherol. In this paper, we investigated the mitochondrial targeting of a vitamin E metabolite, 2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman (α-CEHC), which is similar in structure to vitamin E analogues. We report a fast and efficient method to conjugate the water-soluble metabolite, α-CEHC, to triphenylphosphonium cation via a lysine linker using solid phase synthesis. The efficacy of the final product (MitoCEHC) to lower oxidative stress was tested in bovine aortic endothelial cells. In addition the ability of MitoCEHC to target the mitochondria was examined in type 2 diabetes db/db mice. The results showed mitochondrial accumulation <em>in vivo</em> and oxidative stress decrease <em>in vitro</em>.</p> </div
Effect of MitoCEHC on lowering ROS.
<p>ROS was measured via FACSCAN. The effect of 2 µM α-CEHC and 2 µM MitoCEHC on lowering ROS induced by high glucose in endothelial cells was tested 36 hours after treatment. MitoCEHC displays a higher significant effect on decreasing ROS than α-CEHC alone. Data are expressed as the percent of basal (5 mM glucose). Mean values were analyzed using one-way ANOVA with Tukey's posttest (*p<0.05, ***p<0.001).</p
The DNA Binding Domain of p53 Is Sufficient To Trigger a Potent Apoptotic Response at the Mitochondria
The
tumor suppressor p53 is one of the most studied proteins in
human cancer.− While nuclear p53 has been utilized for cancer gene
therapy, mitochondrial targeting of p53 has not been fully exploited
to date., In response to cellular stress, p53 translocates
to the mitochondria and directly interacts with Bcl-2 family proteins
including antiapoptotic Bcl-XL and Bcl-2 and proapoptotic Bak and
Bax. Antiapoptotic Bcl-XL forms inhibitory
complexes with proapoptotic Bak and Bax preventing their homo-oligomerization. Upon translocation to the mitochondria, p53 binds
to Bcl-XL, releases Bak and Bax from the inhibitory complex and enhances
their homo-oligomerization. Bak and Bax
homotetramer formation disrupts the mitochondrial outer membrane,
releases antiapoptotic factors such as cytochrome <i>c</i> and triggers a rapid apoptotic response mediated by caspase induction. It is still unclear if the MDM2 binding domain
(MBD), the proline-rich domain (PRD) and/or DNA binding domain (DBD)
of p53 are the domains responsible for interaction with Bcl-XL.− The purpose of this work is to determine if a smaller functional
domain of p53 is capable of inducing apoptosis similarly to full length
p53. To explore this question, different domains of p53 (MBD, PRD,
DBD) were fused to the mitochondrial targeting signal (MTS) from Bcl-XL
to ensure Bcl-XL specific targeting. The
designed constructs were tested for apoptotic activity (TUNEL, Annexin-V,
and 7-AAD) in 3 different breast cancer cell lines (T47D, MCF-7, MDA-MB-231),
in a cervical cancer cell line (HeLa) and in non-small cell lung adenocarcinoma
cells H1373. Our results indicate that DBD-XL (p53 DBD fused to the
Bcl-XL MTS) reproduces (in T47D cells) or demonstrates increased apoptotic
activity (in MCF-7, MDA-MB-231, and HeLa cells) compared to p53-XL
(full length p53 fused to Bcl-XL MTS). Additionally, mitochondrial
dependent apoptosis assays (TMRE, caspase-9), co-IP and overexpression
of Bcl-XL in T47D cells suggest that DBD fused to XL MTS may bind
to and inhibit Bcl-XL. Taken together, our data demonstrates for the
first time that the DBD of p53 may be the minimally necessary domain
for achieving apoptosis at the mitochondria in multiple cell lines.
This work highlights the role of small functional domains of p53 as
a novel cancer biologic therapy
Computational Modeling of Stapled Peptides toward a Treatment Strategy for CML and Broader Implications in the Design of Lengthy Peptide Therapeutics
The
oncogenic gene product Bcr-Abl is the principal cause of chronic
myeloid leukemia, and although several therapies exist to curb the
aberrant kinase activity of Bcr-Abl through targeting of the Abl kinase
domain, these therapies are rendered ineffective by frequent mutations
in the corresponding gene. It has been demonstrated that a designed
protein, known as CCmut3, is able to produce a dominant negative inactivating
effect on Bcr-Abl kinase by preferentially oligomerizing with the
N-terminal coiled-coil oligomerization domain of Bcr-Abl (Bcr-CC)
to effectively reduce the oncogenic potential of Bcr-Abl. However,
the sheer length of the CCmut3 peptide introduces a high degree of
conformational variability and opportunity for targeting by intracellular
proteolytic mechanisms. Here, we have examined the effects of introducing
one or two molecular staples, or cross-links, spanning <i>i</i>, <i>i</i> + 7 backbone residues of the CCmut3 construct,
which have been suggested to reinforce α-helical conformation,
enhance cellular internalization, and increase resistance to proteolytic
degradation, leading to enhanced pharmacokinetic properties. The importance
of optimizing staple location along a highly tuned biological construct
such as CCmut3 has been widely emphasized and, as such, we have employed
in silico techniques to swiftly build, relax, and characterize a large
number of candidates. This approach effectively allowed exploring
each and every possible staple location along the peptide backbone
so that every possible candidate is considered. Although many of the
stapled candidate peptides displayed enhanced binding characteristics
for Bcr-CC and improved conformational stability in the (Bcr-CC) bound
form, simulations of the stapled peptides in the unbound form revealed
widespread conformational variability among stapled candidates dependent
on staple type and location, implicating the molecular replacement
of helix-stabilizing residues with staple-containing residues in disrupting
the native α-helical conformation of CCmut3, further highlighting
a need for careful optimization of the CCmut3 construct. A candidate
set has been assembled, which retains the native backbone α-helical
integrity in both the bound and unbound forms while providing enhanced
binding affinity for the Bcr-CC target, as research disseminated in
this manuscript is intended to guide the development of a next-generation
CCmut3 inhibitor peptide in an experimental setting
Multidomain Targeting of Bcr-Abl by Disruption of Oligomerization and Tyrosine Kinase Inhibition: Toward Eradication of CML
The oncoprotein Bcr-Abl, the causative
agent of chronic myeloid
leukemia (CML), requires homo-oligomerization via a coiled-coil domain
to function [Bartram, C. R.; et al. <i>Nature</i> <b>1983</b>, <i>306</i> (5940), 277–280; and Zhao,
X.; et al. <i>Nat. Struct. Biol.</i> <b>2002</b>, <i>9</i>(2), 117–120]. While tyrosine kinase inhibitors
(TKIs) have shown great efficacy as treatment options for CML, their
use may cause an acquisition of mutations in the tyrosine kinase domain,
which prevent TKI binding and lead to a loss in activity [Woessner,
D. W.; et al. <i>Cancer J.</i> <b>2011</b>, <i>17</i>(6), 477–486]. Previously, we have shown that a
rationally modified coiled-coil domain (CC<sup>mut3</sup>) can disrupt
this oligomerization, inhibit proliferation, and induce apoptosis
in CML cells [Dixon, A. S.; et al. <i>Mol. Pharmaceutics</i> <b>2012</b>, <i>9</i>(1), 187–195]. Here,
we show that using the most recently approved TKI, ponatinib (Iclusig),
in combination with CC<sup>mut3</sup> allows a dose reduction of ponatinib
and increased therapeutic efficacy in vitro measured by reduction
in kinase activity, induction of apoptosis via caspase-3/7 and 7-AAD/Annexin
V assays, and reduced transformative ability measured by a colony
forming assay. The combination was effective not only in cells containing
wild-type Bcr-Abl (K562, Ba/F3-p210) but also cells with Bcr-Abl containing
the T315I mutation (Ba/F3-p210-T315I). In addition, we report for
the first time the ability of CC<sup>mut3</sup> alone to inhibit the
T315I mutant form of Bcr-Abl. This novel combination may prove to
be more potent than single agent therapies and should be further explored
for clinical use
Enhanced and Selective Killing of Chronic Myelogenous Leukemia Cells with an Engineered BCR-ABL Binding Protein and Imatinib
The oncoprotein Bcr-Abl stimulates prosurvival pathways
and suppresses apoptosis from its exclusively cytoplasmic locale,
but when targeted to the mitochondrial compartment of leukemia cells,
Bcr-Abl was potently cytotoxic. Therefore, we designed a protein construct
to act as a mitochondrial chaperone to move Bcr-Abl to the mitochondria.
The chaperone (i.e., the 43.6 kDa intracellular cryptic escort (iCE))
contains an EGFP tag and two previously characterized motifs: (1)
an optimized Bcr-Abl binding motif that interacts with the coiled-coil
domain of Bcr (ccmut3; 72 residues), and (2) a cryptic mitochondrial
targeting signal (cMTS; 51 residues) that selectively targets the
mitochondria in oxidatively stressed cells (i.e., Bcr-Abl positive
leukemic cells) via phosphorylation at a key residue (T193) by protein
kinase C. While the iCE colocalized with Bcr-Abl, it did not relocalize
to the mitochondria. However, the iCE was selectively toxic to Bcr-Abl
positive K562 cells as compared to Bcr-Abl negative Cos-7 fibroblasts
and 1471.1 murine breast cancer cells. The toxicity of the iCE to
leukemic cells was equivalent to 10 μM imatinib at 48 h and
the iCE combined with imatinib potentiated cell death beyond imatinib
or the iCE alone. Substitution of either the ccmut3 or the cMTS with
another Bcr-Abl binding domain (derived from Ras/Rab interaction protein
1 (RIN1; 295 residues)) or MTS (i.e., the canonical IMS derived from
Smac/Diablo; 49 residues) did not match the cytotoxicity of the iCE.
Additionally, a phosphorylation null mutant of the iCE also abolished
the killing effect. The mitochondrial toxicity of Bcr-Abl and the
iCE in Bcr-Abl positive K562 leukemia cells was confirmed by flow
cytometric analysis of 7-AAD, TUNEL, and annexin-V staining. DNA segmentation
and cell viability were assessed by microscopy. Subcellular localization
of constructs was determined using confocal microscopy (including
statistical colocalization analysis). Overall, the iCE was highly
active against K562 leukemia cells and the killing effect was dependent
upon both the ccmut3 and functional cMTS domains
A Chimeric p53 Evades Mutant p53 Transdominant Inhibition in Cancer Cells
Because
of the dominant negative effect of mutant p53, there has
been limited success with wild-type (wt) p53 cancer gene therapy.
Therefore, an alternative oligomerization domain for p53 was investigated
to enhance the utility of p53 for gene therapy. The tetramerization
domain of p53 was substituted with the coiled-coil (CC) domain from
Bcr (breakpoint cluster region). Our p53 variant (p53-CC) maintains
proper nuclear localization in breast cancer cells detected via fluorescence
microscopy and shows a similar expression profile of p53 target genes
as wt-p53. Additionally, similar tumor suppressor activities of p53-CC
and wt-p53 were detected by terminal deoxynucleotidyl transferase
dUTP nick end labeling (TUNEL), annexin-V, 7-aminoactinomycin D (7-AAD),
and colony-forming assays. Furthermore, p53-CC was found to cause
apoptosis in four different cancer cell lines, regardless of endogenous
p53 status. Interestingly, the transcriptional activity of p53-CC
was higher than wt-p53 in 3 different reporter gene assays. We hypothesized
that the higher transcriptional activity of p53-CC over wt-p53 was
due to the sequestration of wt-p53 by endogenous mutant p53 found
in cancer cells. Co-immunoprecipitation revealed that wt-p53 does
indeed interact with endogenous mutant p53 via its tetramerization
domain, while p53-CC escapes this interaction. Therefore, we investigated
the impact of the presence of a transdominant mutant p53 on tumor
suppressor activities of wt-p53 and p53-CC. Overexpression of a potent
mutant p53 along with wt-p53 or p53-CC revealed that, unlike wt-p53,
p53-CC retains the same level of tumor suppressor activity. Finally,
viral transduction of wt-p53 and p53-CC into a breast cancer cell
line that harbors a tumor derived transdominant mutant p53 validated
that p53-CC indeed evades sequestration and consequent transdominant
inhibition by endogenous mutant p53
Re-Engineered p53 Chimera with Enhanced Homo-Oligomerization That Maintains Tumor Suppressor Activity
The use of the tumor suppressor p53 for gene therapy of cancer
is limited by the dominant negative inactivating effect of mutant
endogenous p53 in cancer cells. We have shown previously that swapping
the tetramerization domain (TD) of p53 with the coiled-coil (CC) from
Bcr allows for our chimeric p53 (p53-CC) to evade hetero-oligomerization
with endogenous mutant p53. This enhances the utility of this construct,
p53-CC, for cancer gene therapy. Because domain swapping to create
p53-CC could result in p53-CC interacting with endogenous Bcr, which
is ubiquitous in cells, modifications on the CC domain are necessary
to minimize potential interactions with Bcr. Hence, we investigated
the possible design of mutations that will improve homodimerization
of CC mutants and disfavor hetero-oligomerization with wild-type CC
(CCwt), with the goal of minimizing potential interactions with endogenous
Bcr in cells. This involved integrated computational and experimental
approaches to rationally design an enhanced version of our chimeric
p53-CC tumor suppressor. Indeed, the resulting lead candidate p53-CCmutE34K-R55E
avoids binding to endogenous Bcr and retains p53 tumor suppressor
activity. Specifically, p53-CCmutE34K-R55E exhibits potent apoptotic
activity in a variety of cancer cell lines, regardless of p53 status
(in cells with mutant p53, wild-type p53, or p53-null cells). This
construct overcomes the dominant negative effect limitation of wt
p53 and has high significance for future gene therapy for treatment
of cancers characterized by p53 dysfunction, which represent over
half of all human cancers