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A synthetic lethal screen for NF-κB-dependent plasma cell disorders

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A synthetic lethal screen for NF-κB-dependent plasma cell disorders

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A Synthetic Lethal Screen for NF-kB-Dependent Plasma Cell
Disorders

By

Waiel Halabi

A Thesis Presented to FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

December 2012

Copyright 2012 Waiel Halabi
ii

TABLE OF CONTENTS
Acknowledgements……………………………………………………………………… iii
List of Figures……………………………………………………………………………. iv
List of Tables…………………………………………………………………………….. v
Abstract…………………………………………………………………………………... vi
Introduction: Multiple myeloma………………………………………………………...... 1
Materials & Methods…………………………………………………………………….. 7
Results…………………………………………………………………………………….. 11
Discussion………………………………………………………………………………… 24
Future Experiments………………………………………………………………………. 25
References………………………………………………………………………………… 26

iii

ACKNOWLEDGEMENTS
Special thanks to
Dr. Preet M. Chaudhary
Dr. Hittu Matta
Dr. Ramakrishnan Gopalakrishnan

I could not have done this research without their help and support.

iv

LIST OF FIGURES

Figure1. Canonical and non-canonical NF- B pathway……………………………………… 3
Figure2. NIH clinical collection by therapeutic indication…………………………………… 9
Figure3. Evotec library results from the primary screen presented in heat map……………… 12
Figure4. Lopac library results from the primary screen presented in heat map………………. 13
Figure5. NCI library results from the primary screen presented in heat map………………… 14
Figure6. Dose response of fenretinide in T1165 vector, K13, and K13-ER cells…………….. 20
Figure7. Chemical structure of fenretinide……………………………………………………. 20
Figure8. Dose response of fenretinide in K562 vector and K13………………………............. 21
Figure9. Luciferase activity test for fenretinide……………………………………………….. 22
Figure10. Dose response of fenretinide in BC-1, BC-3, BJAB, Namalwa, MM1S, U266
(myeloma and lymphoma cell lines)………………………………………………………....... 23

v

LIST OF TABLES

Table1. Hits from the primary screen of the EVOTEC library………………………........ 15-17
Table2. Hits from the primary screen of the LOPAC library……………………………. 17-19
Table3. Lymphoma and multiple myeloma cell lines……………………………………. 23

vi

ABSTRACT
Nuclear factor- kappa B (NF- B) was first described as a transcription factor in B cells
that binds to the enhancer element controlling immunoglobulin kappa light chain expression.
Since its discovery in 1986, NF- B and its role in inflammatory responses, immune reactions,
and tumorigenesis has been extensively studied. Although the abnormal activation of the NF-κB
pathway plays a key role in the pathogenesis of several cancers, inflammatory and autoimmune
disorders, no specific NF-κB inhibitor is currently in clinical use or in clinical trial. Additionally,
to date, efforts to target NF-κB signaling have been focused mostly on IKK2-based, in vitro
screening. However, NF-κB pathway activates the expression of a large number of genes, and it
is possible that distal downstream components of the NF-κB-signaling network could be targets
for drug discovery. A strategy that could be used for revealing such NF-κB-linked targets is
synthetic lethal screening. To test this hypothesis, we carried out a screen with an isogenic pair
of cell lines derived from IL6-dependent T1165 mouse plasmacytoma. A derivative of T1165
cells stably expressing Kaposi's sarcoma associated herpesvirus encoded viral FLICE inhibitory
protein (vFLIP) K13 possess increased NF-κB activity and is independent of IL6. We screened
T1165-K13 and T1165-vector cells using libraries of small molecule compounds and identified
Fenretinide as a compound with synthetic lethality against plasmacytomas with increased NF-κB
activity.

1

INTRODUCTION
Multiple myeloma (MM) is an incurable cancer of plasma cells. The growth of these
bone marrow malignant cells makes it harder for the bone marrow to make healthy blood cells.
Multiple myeloma mostly affects older adults. The patient usually has low red blood cell (RBC)
count with abnormal bleeding, bone and back pain due to the uncontrolled growth of cancer cells
in the bone marrow, fatigue, shortness in breathing due to anemia, and fever without any other
cause [1]. Plasma cells major function is to make antibodies to help in fighting infections. In
multiple myeloma these plasma cells become abnormal cells (myeloma cells) and they start
multiplying without any control. The numbers of these abnormal plasma cells increase and
become highly abnormal. Since their major function is to make antibodies, they start making
very high amount of antibodies [2]. The disease usually takes three phases: 1) inactive phase,
abnormal plasma cells accumulate in the bone marrow and they interrupt the production of the
normal cells; 2) active phase, proliferation of a small fraction of cells; 3) fulminant phase,
increase in the proliferation of the abnormal plasmablastic cells. When the disease progress the
plasma cells enter the peripheral circulation and it is called plasma cell leukemia. Cytokine IL-6
is one of the best characterized growth factor for the early stage of multiple myeloma; however,
the cells become IL-6 independent with the disease progression. Malignant cells need one of two
mechanisms for its proliferation and survival: 1) a common mechanism (classical) via autocrine
production of growth factors, 2) an alternative mechanism (i.e. in the case of IL-6 independent)
via genetic and epigenetic alterations that result in constitutive activation of various signaling
pathways including NF- B pathway. Multiple myeloma is associated with constitutive activation
of classical and alternative NF- B pathways.

2

Nuclear factor- kappa B (NF- B) was first described as a transcription factor in B cells
that binds to the enhancer element controlling immunoglobulin kappa light chain expression [3].
Since its discovery in 1986, NF- B and its role in inflammatory responses, immune reactions,
and tumorigenesis has been extensively studied. NF- B complex contains five subunits: c-Rel,
NF- B1 (p50 and its precursor p105), NF- B2 (p52 and its precursor p100), p65 (RelA), and
RelB. The combination of any two of these subunits will form a heterodimer (i.e. RelA and p50).
Any of these heterodimers in most cells is associated with a family of inhibitory proteins (the
most common is I B ). NF- B activation is tightly regulated by signals that degrade IkB. In the
classical or canonical NF- B signaling pathway (Fig. 1), IkB proteins are phosphorylated by an
activated IkB kinase (IKK) complex at specific sites equivalent to Ser32 and Ser36 of IkBa. The
IKK complex is composed of the catalytic subunits IKKα and IKKβ and the regulatory subunit
IKKγ, also known as NF- B essential modulator (NEMO). IKKα and IKKβ, which are 52%
identical, form homodimers or heterodimers. Although IKKα and IKKβ cooperate for IkB
phosphorylation, these proteins differ in the signals that they mediate. The IKKb component is
essential for the signaling via the classical NF- B pathway [4-5]. In a recently identified
alternative or non-canonical pathway for NF- B activation (Fig. 1), upstream NF-kB inducing
kinase (NIK) activates an IKKα homodimer; both IKKβ and IKKγ are dispensable in this
signaling [6]. In the alternative pathway, NF- B2/p100 is phosphorylated at two C-terminal sites
by the IKKα homodimer and ubiquitinated. This modification targets the inhibitory C-terminus
for proteasomal degradation, producing p52.

3

A number of viral proteins, such as SV40 Large T and small T antigens, are known to
modulate the activity of critical cellular signaling pathways and have been used over the years as
molecular tools to discern the involvement of these pathways in cancer development and
progression [7]. Dr. Chaudhary's laboratory has previously shown that Human Herpesvirus 8
Fig.1. Schematic diagram showing the Canonical and non-canonical NFkB pathway
4

(HHV8)-encoded K13 protein possesses the unique ability to activate the classical and
alternative (non-canonical) NF-κB pathways by directly interacting with a ~700-kDa IκB kinase
(IKK) complex consisting of IKK1/IKKα, IKK2/IKKβ and Nemo/IKKγ [8]. Furthermore, they
have shown that by bypassing the upstream components of the NF-κB signaling pathway, such
as RIP, NIK and TRAFs, K13 selectively activates the two NF-κB pathways without
concomitant JNK activation [9-10]. In a recent study, they exploited this unique ability of K13
to selectively activate the classical and alternative NF-κB pathways as a molecular tool to
clarify the contribution of these two NF-κB pathways to myeloma pathogenesis and therapeutic
response. They demonstrated that ectopic expression of K13 protected plasmacytomas against
IL6 withdrawal-induced apoptosis and resulted in emergence of IL6-independent clones that
could proliferate long-term in vitro in the absence of IL6 and form abdominal plasmacytomas
with visceral involvement when injected intraperitoneally into syngeneic mice [11]. These IL6-
independent clones of T1165-K13 cells were dependent on NF-κB activity for their survival and
proliferation and were highly sensitive to NF-κB inhibitor Bay-11-7082, but were resistant to
INCB018424, a selective Janus Kinase 1/2 inhibitor [11].
Although the abnormal activation of the NF-κB pathway plays a key role in the
pathogenesis of several cancers, inflammatory and autoimmune disorders, no specific NF-κB
inhibitor is currently in clinical use or in clinical trial. Additionally, to date, efforts to target NF-
κB signaling have been focused mostly on IKK2-based, in vitro screening. However, NF-κB
pathway activates the expression of a large number of genes, and it is possible that distal
downstream components of the NF-κB-signaling network could be targets for drug discovery. A
strategy that could be used for revealing such NF-κB-linked targets is synthetic lethal screening
[12]. Synthetic lethality is a powerful approach for cancer drug discovery that involves
5

identification of genotype-selective anticancer agents that become lethal to cancer cells only in
the presence of specific oncoproteins, absence of tumor suppressor proteins, or activation of
specific signaling pathways. For a given mutation ‘‘A,’’ if there exists a second mutation ‘‘B’’
that is particularly lethal to the organism in the presence of A, mutation B is synthetic lethal with
mutation A because the lethality requires the synthesis, or bringing together, of the two
mutations. In synthetic lethal screening with NF-κB, the second perturbation can be created by
using a small molecule to alter the function of a target protein. Isogenic cell lines differing only
by the presence of the specific genetic alteration provide the ideal platform for carrying out a
synthetic lethal screening. However, although genetic and epigenetic alterations resulting in NF-
κB activation are seen in up to 20% of patients with myeloma and 40% of myeloma cell lines, it
is not possible to carry out synthetic lethal screening for NF-κB using myeloma cell lines due to
the confounding effect of alterations at other loci. For example, a recent whole genome
sequencing study of 23 myeloma patient samples found approximately 7,450 point mutations per
sample across the genome, including an average of 35 amino-acid-changing point mutations plus
21 chromosomal rearrangements disrupting protein-coding regions [13]. Furthermore, the
genetic and epigenetic alterations that lead to NF-κB activation in myeloma cell lines could also
affect other signaling pathways (e.g. JNK, p38, MAPK, etc.), further confounding the
interpretation of results. In contrast, T1165-vector/K13 cell lines are isogenic in nature and NF-
κB activation in these cells is not accompanied by activation of other signaling pathways. As
such, these cells present an ideal platform to identify drugs that show synthetic lethality with NF-
κB in plasma cell neoplasms. To test this hypothesis, we carried out a screen with T1165-
vector/K13 cells using a library of small molecule compounds (Evotec, Lopac, and NCI
6

libraries). The goal of this study to identify drugs that are best suited for the treatment of multiple
myeloma with increased NF-κB activity.
Scope of the Dissertation
Multiple myeloma is associated with constitutive activation of classical and alternative
NF- B pathways and become IL-6 independent with the disease progression. NF- B is
specifically activated in T1165-K13 cell line. We hypothesize that we can identify genotype-
selective anticancer agents that become lethal to plasmacytoma cells with increased NF-κB
activity.
Aim 1: To carry out a synthetic lethal screen of drugs to identify compounds with selective
activity against plasmacytoma cells with increased NF-κB activity.
Aim 2: To check whether the identified compounds from the above screening will be able to kill
the human cancer cells with increased NF- κB activity.

7

MATERIALS AND METHODS
Cell lines, Media and Reagents
T1165 plasmacytoma cells expressing vector and Flag tagged-K13 and K13-ER have
been described earlier [11], K562, BC-1, BC-3, BJAB, Namalwa, MM1S and U266 were
obtained from American Type Culture Collection (were grown in RPMI medium supplemented
with 10% (v/v) FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mm sodium pyruvate, 2
mm glutamine (all from Invitrogen). The T1165 vector cells are supplemented with supernatants
(4% final) from SP1 cell line for mouse IL-6. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] from Promega
(Cat#G111A), PMS (phenazine methosulfate) from ICN biomedicals (cat#299-1-6), Fenretinide
was obtained from Sigma.
Cell Viability and Screening of library compounds
Cells from exponentially growing cultures were plated in an untreated flat-bottom 96 well
plate at a density of 10 X 10
3
cells/ well. Aliquots of chemical library compounds will be diluted
in PBS and directly transferred to cells so as to result in a 10 μM effective final concentration of
library compounds in 0.1% DMSO. Cell viability was measured after indicated time points using
the MTS reagent (3–4,5-dimethylthiazol-2yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium, inner salt) following manufacturer’s instructions (Promega, Madison, WI).
Absorbance of viable cells was measured at 490nmwith 600nm as a reference wavelength.
Percent cell survival was calculated based on the reading of cells grown in the presence of
DMSO control.

8

Dose-Response studies with putative “hits”:
We will re-screen the candidate hit compounds at four different concentrations (two-fold
serial dilutions starting at 10 µM or 4 µM) in the readout system under identical conditions to
that of the primary screen in order to verify that the compounds are true hits. The IC
50
(the
concentration which results in 50% decrease in viability will be calculated based on the dose-
response data).
Evotec library
The NIH Clinical Collection and NIH Clinical Collection 2 are plated arrays of 446 and
281, respectively, small molecules that have a history of use in human clinical trials. The
collection was assembled by the National Institutes of Health (NIH) through the Molecular
Libraries Roadmap Initiative as part of its mission to enable the use of compound screens in
biomedical research. Similar collections of FDA approved drugs have proven to be rich sources
of undiscovered bioactivity and therapeutic potential. The clinically tested compounds in the
NCC are highly drug-like with known safety profiles. These compounds can provide excellent
starting points for medicinal chemistry optimization and, for high-affinity targets, may even be
appropriate for direct human use in new disease areas.

9

LOPAC
1280TM
library
This is a Library Of Pharmacologically Active Compounds arranged in a 96 well format
at 10mM in DMSO and was obtained from Sigma. These includes drug like molecules in the
field of Antibiotics, Apoptosis, Calcium signaling, Gene regulation and expression, Ion channels,
Lipid signaling, Multi-drug resistance, Neurotransmission and phosphorylation.
NCI library
These compounds were obtained from NCI developmental therapeutics branch which
includes NCI diversity set III, 1597 compounds; NCI mechanistic set, 879 compounds; NCI
natural product set II, 120 compounds.
Fig.2. Pie chart showing the diversity of drugs in the Evotec library collection
10

Luciferase assay
K562- NF-κB luc K13-ER cells have been described previously [14]. Briefly, K13-
ER
TAM
fusion protein was generated by fusing the K13 to the ligand-binding domain of a
mutated estrogen receptor. The mutated estrogen receptor does not bind to the physiological
ligand estrogen, but binds with very high affinity to the synthetic ligand 4-OHT (4-
hydroxytamoxifen) and regulates the activity of its fusion partner in a 4-OHTdependent manner.
Then the K13-ER fusion protein was expressed in a clone of K562 cell line that had been
engineered to express a stably integrated copy of an NF-κB-driven luciferase reporter construct
to generate the K562- NF-κB -Luc reporter cells. The reporter cells were treated with increasing
concentrations of selected "hit" compounds in the presence of 4-hydroxy tamoxifen (20 nM) to
activate the K13-induced NF-κB activity. Cells were lysed using luciferase lysis buffer from
promega (E153A) and the supernatants were assayed for luminescence in the presence of assay
buffer containing D-Luciferin in a luminometer.

11

RESULTS
Primary screening
First, we ran cell viability (MTS) screening assay for T1165-vector and T1165-K13 cells
treated with the Evotec (721 compounds), LOPAC (1280 compounds), and NCI library (Total
2596 compounds including NCI diversity set III, 1597 compounds; NCI mechanistic set, 879
compounds; NCI natural product set II, 120 compounds) at 10 µM concentration and then the
heat maps were generated from the cell viability. Heat maps were generated by taking the 100%
viability as 1 using the NCI CIM miner tool. Fig. 1, 2 and 3 represents the heat maps generated
for Evotec, Lopac and NCI library of compounds.

12

Fig.3. Heat map representing the effect of Evotec library of compounds against
T1165 vector and K13. Intense green represents maximal cell death and intense
red represents maximal cell growth.
Color scale
13

Fig.4. Heat map representing the effect of LOPAC library of compounds against
T1165 vector and K13. Intense green represents maximal cell death and intense
red represents maximal cell growth.
Color scale
14

Fig.5. Heat map representing the effect of NCI library of compounds against
T1165 vector and K13. Intense green represents maximal cell death and intense
red represents maximal cell growth.
Color scale
15

From the above results we got the following hits (compounds) which killed T1165-K13
cells specifically (i.e. the compounds which killed at least 50% of the T1165-K13 cells without
any effect on vector cells) and the list of compounds are presented in table 1 and 2
Table 1. EVOTEC Library hits from primary screening
compound name Description and therapeutic function
19-NORETHINDRONE ACETATE Steroidal progestin
TRIAMCINOLONE ACETONIDE Synthetic corticosteroid
SONAZINE Antipsychotic drug.
S(-)-TIMOLOL MALEATE Potent non-selective β-adrenoceptor
antagonist
ALTRETAMINE Antineoplastic agent. It was approved by
the FDA in 1990
CEFOXITIN SODIUM SALT A semi-synthetic antibiotic derived from
Cephamycin C, possessing high
resistance to β-lactamase inactivation.
An antibacterial.
GEMFIBROZIL Lipid regulating agent
FLUDARABINE Antimetabolites. It is used to treat a type
of cancer of the white blood cells called
B-cell chronic lymphocytic leukemia
(CLL).
BECLOMETHASONE DIPROPIONATE Potent glucocorticoid steroid, used for
asthma
METHAZOLAMIDE Potent inhibitor of carbonic anhydrase
BUDESONIDE Corticosteroid or steroid, used to help
prevent the symptoms of asthma
TESTOSTERONE Male hormone
DILANTIN Antiepileptic drug
FLUCONAZOLE Synthetic triazole antifungal agents
CROMOLYN SODIUM Antihistamine
TERBUTALINE SULFATE Bronchodilator, treatment of asthma
RAMIPRIL Angiotensin-converting enzyme
INHIBITOR used in treatment of
HYPERTENSION and congestive HEART
FAILURE
MUPIROCIN Treat bacterial infections
METHIMAZOLE Methimazole inhibits the synthesis of
thyroid hormones and thus is effective in
the treatment of hyperthyroidism
16

FLUOCINOLONE ACETONIDE 21-ACETATE Reduce skin inflammation and relieve
itching
(+/-)-NOREPINEPHRINE HYDROCHLORIDE Modulates human dendritic cell
activation by altering cytokine release
ZONISAMIDE Control partial seizures (convulsions) in
the treatment of epilepsy
ACEBUTOLOL HYDROCHLORIDE Antihypertensive, antiarrhythmic
AMCINONIDE Corticosteroids, used as anti-
inflammatory and antipruritic agents
BRIMONIDINE To treat increased pressure in the eye
COGENTIN MESYLATE Anticholinergic and antihistaminic agent
TIZANIDINE HYDROCHLORIDE Skeletal muscle relaxant
CYTOXAN Antineoplastic AGENT
AMOXICILLIN CRYSTALLINE Penicillin , antibacterial drug
ITRACONAZOLE Antifungal agent
BENPROPERINE PHOSPHATE Cough inhibitor
OLANZAPINE Used to treat nervous and mental
conditions
RISPERIDONE Atypical antipsychotic drug that is used
for treating schizophrenia, bipolar mania
and autism
ZOLPIDEM TARTRATE Nonbenzodiazepine hypnotic drug used
to treat insomnia
LATANOPROST Antiglaucoma agent
CERIVASTATIN NA Reduce blood cholesterol levels
OXYMETHOLONE Anabolic steroid, treatment of
osteoporosis and anaemia
HYPEROSIDE Plant that has antibacterial and
antioxidant properties
PEFLOXACIN MESYLATE Chemotherapeutic agent used to treat
severe and life threatening bacterial
infections
ESOMEPRAZOLE MG Used in the treatment of dyspepsia,
peptic ulcer disease (PUD),
gastroesophageal reflux disease
(GORD/GERD) and Zollinger-Ellison
syndrome
PIOGLITAZONE HCL Antidiabetic medication.
2',3'-DIDEOXYINOSINE Therapy of Patients With the AIDS
Dementia Complex
ICARIIN Important mediator of signal
transduction
IPRIFLAVONE Treating and preventing osteoporosis
RIFABUTIN Bactericidal antibiotic drug primarily
used in the treatment of tuberculosis
17

SERTRALINE Generic antidepressant
METHYLANDROSTENEDIOL Steroid
OXAPROZIN Non-steroidal anti-inflammatory drug
(NSAID),
[

PANTOPRAZOLE SODIUM SALT Proton pump inhibitor drug that inhibits
gastric acid secretion.
TRIPTOLIDE Vine used for treatment of fever, chills,
edema and carbuncle.
ROLIPRAM PDE4-inhibitors, it is an anti-
inflammatory drug
EPIRUBICIN HYDROCHLORIDE Chemotherapy
SIMVASTATIN Hypolipidemic drug
LOVASTATIN Hypolipidemic agent
DIPHENYLCYCLOPROPENONE Treatment for alopecia areata and
alopecia totalis
2(1H)-PYRIMIDINONE, 4-AMINO-1-D-
ARABINOFURANOSYL- [CAS]
Folic acid antagonist.
PRAZOSIN Sympatholytic drug used to treat high
blood pressure
L-GLUTAMIC ACID, N-[4-[[(2,4-DIAMINO-6-
PTERIDINYL)METHYL]METHYLAMINO]BE
NZOYL]- [CAS]
Folic acid antagonist.
VINORELBINE BITATRATE Cancer chemotherapeutic agents
NITAZOXANIDE Antiprotozoal agent
CEFIXIME TRIHYDRATE Antibacterial
HOMOHARRINGTONINE Chemotherapy
SECOISOLARICIRESINOL A plant product
VECURONIUM BROMIDE Muscle relaxation, anesthesia
ROPIVACAINE HCL Local anesthetic
ANASTROZOLE Nonsteroidal aromatase inhibitor. Used
for treatment of advanced breast
carcinoma
ENROFLOXACIN Fluoroquinolone antibiotic, antibacterial
KETOTIFEN FUMARATE Antihistamine that stabilizes mast cells
RIMCAZOLE Antagonist of the sigma receptor

Table 2. LOPAC Library hits from primary screening
Compound name Description and therapeutic function
TROVAFLOXACIN MESYLATE

Antibiotic, used to treat very serious bacterial
infections
(+)-BROMOCRIPTINE D2 dopamine receptor agonist; inhibits
18

METHANESULFONATE prolaction secretion

CAFFEIC ACID PHENETHYL ESTER NFkB inhibitor

CYPROTERONE ACETATE

Androgen antagonist; synthetic steroid

UCL 2077

Slow afterhyperpolarization (sAHP) channel
blocker.

ESOMEPRAZOLE MAGNESIUM
DIHYDRATE

Esomeprazole magnesium dihydrate is a
leading proton pump inhibitor.

EMODIN

p56lck Tyrosine kinase inhibitor

RETINOIC ACID P-HYDROXYANILIDE

Vitamin A acid analog with antiproliferative
activity in cultured human breast cancer cells

M-IODOBENZYLGUANIDINE
HEMISULFATE

Antitumor agent which inhibits ADP
ribosylation; induces changes in the
mitochondrial membrane potential, activation
of caspase-3 and DNA fragmentation

INDATRALINE HYDROCHLORIDE

Potent inhibitor of dopamine, norepinephrine
and serotonin reuptake

BIO

Potent, selective, reversible, and ATP-
competitive glycogen synthase kinase
3alpha/beta (GSK-3alpha/beta) inhibitor.

L-ALPHA-METHYL DOPA

L-aromatic amino acid decarboxylase inhibitor;
antihypertensive

SB 242084 DIHYDROCHLORIDE
HYDRATE

Selective 5-HT2c serotonin receptor
antagonist; crosses the blood-brain barrier

PAPAVERINE HYDROCHLORIDE

Phosphodiesterase inhibitor

6(5H)-PHENANTHRIDINONE

Poly(ADP-ribose) Polymerase (PARP)
inhibitor

N-OLEOYLDOPAMINE

Endogenous vanilloid; weak CB1 cannabinoid
receptor ligand.

RETINOIC ACID

Induces caspase-dependent apoptosis

4-(3,4-DIHYDROXYPHENYL)-4,5,6,7- D1 dopamine receptor agonist
19

TETRAHYDROTHIENO[2,3-
C]PYRIDINE

TYRPHOSTIN 23
TETRAETHYLTHIURAM DISULFIDE

Protein tryrosine kinase EGFR inhibitor.
Alcohol dehydrogenase inhibitor

TREQUINSIN HYDROCHLORIDE

Phosphodiesterase III (PDE III) inhibitor

BAY 61-3606 HYDROCHLORIDE
HYDRATE

Spleen tyrosine kinase (Syk) inhibitor; anti-
inflammatory

PD-166866

PD-166866 is a selective inhibitor of the FGF-
1 receptor tyrosine kinase (FGFR1) with IC50
= 55 nM, and no effect on c-Src, PDGFR-b,
EGFR or insulin receptor tyrosine kinases or
MEK, PKC, and CDK4.

APRINDINE HYDROCHLORIDE

Class Ib antiarrhythmic and hERG channel
blocker.

ARA-G HYDRATE

Ara-G is an inducer of apoptosis; inhibitor of
DNA synthesis; antineoplastic; and
antimetabolite.

TYRPHOSTIN 47

EGFR tyrosine kinase inhibitor

YC-1

NO-independent guanylyl cyclase activator

At the dose tested (i.e. 10 µM) none of the NCI library compounds met the criteria as hits
Secondary screening
We repeated MTS assay for the positive hits from the first run of the EVOTEC and
LOPAC libraries (for EVOTEC compounds we used 4 µM, 2 µM, 1 µM, and 0.5 µM
concentrations) (for LOPAC library compounds we used 10 µM, 5 µM, 2.5 µM, 1.25 µM
concentrations).From the above secondary screening we found that Retinoic acid p-
20

hydroxyanilide (Fenretenide) selectively killed the T1165-K13 and T1165-K13 ER cells grown
in the presence of 4-OHT (Fig. 6).

Retinoic acid p-hydroxyanilide (Fenretinide)
N-(4-hydroxyphenyl) retinamide (4HPR, fenretinide) is a synthetic retinoid with
chemopreventive/cytotoxic activity against various cancers [15-19] 4HPR was shown to induce
apoptosis in solid and hematologic tumor cells through reactive oxygen species (ROS)
generation [20-23] and Bcl-2/Mcl-1 decrease [22, 24]. This agent also inhibits tumor growth by
modulating angiogenesis-associated growth factors and their receptors and exhibits retinoid
receptor-independent apoptotic properties [25]

Fig. 6 shows the effect of Fenretinide on T1165 Vector, K13 and K13 ER
cells at indicated doses for 72h
Fig. 7 Chemical structure of Fenretinide
Fenretinide ( M)
% Viability
0.00 5.00 10.00
0
25
50
75
100
125
Vector
K13
K13-ER
21

To check, Whether the K13 specific effect of Fenretinide can be observed in other cell
lines. We generated K562 cells stably expressing Flag-epitipe tagged-K13 and vector alone and
did the dose response of Fenretinide on these cells. As you can see in Fig. 7 Fenretinide
selectively killed the K562-K13 with a IC
50
of 2.45 µM whereas the IC
50
of vector is 17.5 µM
the vector which confirmed the K13-specific effect of Fenretinide.

Since K13 is a strong activator of NF-κB [26]. We wanted to check whether K13 specific
effect of Fenretinide is associated with inhibition of NF-κB. As such, we treated the K562-NF-
κB luc K13-ER (described in Materials and Methods section) with 10 and 20 µM of Fenretinide
in the presence of 20nM 4-OHT for 15 hrs. As depicted in (Fig. 9), Fenretinide didn’t inhibit
K13-induced NF-κB activity

Fig. 8 shows the effect of Fenretinide on K562 vector and K13
cells at indicated doses for 72h
Fenretinide ( M)
% Viability
0.0 2.5 5.0 7.5 10.0 12.5
0
25
50
75
100
125
Vector
K13
22

The above results suggested that Fenretinide didn’t inhibit NF-κB and uses some other
alternative pathway to selectively kill the K13-expressing cells. Finally, we examined whether
Fenretinide would show selective cytotoxicity towards KSHV-infected primary effusion
lymphoma (PEL) or Multiple Myeloma cell lines. We treated a panel of cell lines (see table 3)
with increasing concentrations of Fenretinide. (Fig.10). The IC50 of Fenretinide was smaller for
myeloma cell lines as compared to KSHV-infected PEL cell lines (BC1 and BC3) or the Burkitt's
lymphoma derived cell lines BJAB and Namalwa (Table 3). These results suggest that
Relative Luciferase activity
Untreated
20 nm (4-OHT)
M + 4-OHT

Fenretinide 10
M + 4-OHT

Fenretinide 20
0
2000
4000
6000
8000
Fig. 9 shows the effect of Fenretinide on NFkB activity
in K562- NF-κB luc K13-ER at indicated doses for 15h
23

Fenretinide is not selectively toxic towards any cell line that expresses K13 and possesses
increased NF-κB activity.

Table 3

Cell line Disease IC
50 72h (µM)

BC-1 Primary effusion lymphoma 11
BC-3 Primary effusion lymphoma 9.23
BJAB Burkitt’s Lymphoma 9.9
Namalwa Burkitt’s Lymphoma 12.7
MM1S Multiple Myeloma 0.45
U266 Multiple Myeloma 1.1
Fenretinide ( M)
% Viability
0 5 10 15 20 25
0
25
50
75
100
125
BC-1
BC-3
BJAB
Namalwa
MM1S
U266
Fig. 10 shows dose response of fenretinide BC-1, BC-3, BJAB, Namalwa, MM1S and U266
cells at indicated concentrations for 72h
24

DISCUSSION
Interleukin 6 (IL-6) is one of the best characterized myeloma growth factors [27]. IL-6 is
produced by bone marrow stromal cells and binds to IL-6 receptors on myeloma cells, promoting
their survival and proliferation [27]. However, as myeloma progresses, the disease becomes
independent of micro-environmental support and IL-6, which allows the myeloma cells to enter
the peripheral circulation (plasma cell leukemia) and form extramedullary tumors or
plasmacytomas. The best therapeutic approach for advanced myeloma, including plasma cell
leukemia, remains unknown so far and the prognosis is still poor [28]. For example, plasma cell
leukemia patients treated with standard chemotherapy have a median survival of few months
[28]. Novel approaches targeting signaling pathways involved in IL6-independent growth of
plasma cells are needed for the treatment of this disorder.
In this study, we carried out a synthetic lethal screen to identify drugs that can selectively
kill plasma cells with increased NF-κB activity. After screening 4607 chemical compounds, we
identified Fenretinide as a compound with synthetic lethal activity against T1165 cells
expressing K13 and possessing increased NF-κB activity. The synthetic lethality of Fenretinide
against plasmacytomas with increased NF-κB activity was demonstrated against an independent
clone of T1165 cells expressing K13-ER fusion construct.
Although Fenretinide showed synthetic lethality against plasmacytomas with increased
NF-κB activity, it did not block K13-induced NF-κB activity. Furthermore, Fenretinide did not
show selective cytotoxic towards PEL cell lines. These results suggest that rather than blocking
NF-κB activation directly, it blocks a downstream target of NF-κB pathway that is crucial for the
survival of IL6-independent plasmacytoma cells.
25

FUTURE EXPERIMENTS
Future studies should explore the mechanism of synthetic lethality of Fenretinide against
NF-κB-dependent plasma cell disorders. In addition to plasma cell disorders, NF-κB pathway
plays a key role in the pathogenesis of a number of hematologic malignancies, such as Hodgkin's
and non-Hodgkin's lymphomas [29]. As such, future studies should also examine whether
Fenretinide has synthetic lethality against other NF-κB-dependent hematologic malignancies.
Based on the results of the above studies, Fenretinide should be tested in animal models of NF-
κB dependent hematologic disorders. Fenretinide has been already tested in human clinical trials
[30]. As such, in case of positive results in animal studies, it could be immediately tested in
human clinical trials.

26

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Abstract (if available)

Abstract Nuclear factor- kappa B (NF-κB) was first described as a transcription factor in B cells that binds to the enhancer element controlling immunoglobulin kappa light chain expression. Since its discovery in 1986, NF-κB and its role in inflammatory responses, immune reactions, and tumorigenesis has been extensively studied. Although the abnormal activation of the NF-κB pathway plays a key role in the pathogenesis of several cancers, inflammatory and autoimmune disorders, no specific NF-κB inhibitor is currently in clinical use or in clinical trial. Additionally, to date, efforts to target NF-κB signaling have been focused mostly on IKK2-based, in vitro screening. However, NF-κB pathway activates the expression of a large number of genes, and it is possible that distal downstream components of the NF-κB-signaling network could be targets for drug discovery. A strategy that could be used for revealing such NF-κB-linked targets is synthetic lethal screening. To test this hypothesis, we carried out a screen with an isogenic pair of cell lines derived from IL6-dependent T1165 mouse plasmacytoma. A derivative of T1165 cells stably expressing Kaposi's sarcoma associated herpesvirus encoded viral FLICE inhibitory protein (vFLIP) K13 possess increased NF-κB activity and is independent of IL6. We screened T1165-K13 and T1165-vector cells using libraries of small molecule compounds and identified Fenretinide as a compound with synthetic lethality against plasmacytomas with increased NF-κB activity.

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Asset Metadata

Creator Halabi, Waiel (author)

Core Title A synthetic lethal screen for NF-κB-dependent plasma cell disorders

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 11/28/2012

Defense Date 10/21/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag hematology,OAI-PMH Harvest,plasma cell disorders

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair), Chaudhary, Preet M. (committee member), Hong, Young-Kwon (committee member)

Creator Email wael_halabi@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-120349

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