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HLA upregulation via inhibition of the MEK1/2 pathway
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HLA upregulation via inhibition of the MEK1/2 pathway
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Content
HLA UPREGULATION VIA INHIBITION OF THE MEK1/2 PATHWAY
By MICHAEL ANDREW DIAZ
A Thesis Submitted to the Keck School of Medicine of the University of Southern
California In Part of the Requirements for the Degree
MASTER OF SCIENCE
(EXPERIMENTAL AND MOLECULAR PATHOLOGY)
University of Southern California © Copyright by Michael Andrew Diaz,
December 2017
i
ABSTRACT
Selumetinib is a specific non-ATP dependent, small molecule inhibitor against the
mitogen-activated protein kinase kinase 1 (MEK1) isoform. Treatment with Selumetinib has
shown the ability to abate tumor growth to a significant degree in KRAS mutation bearing
tumors in phase II clinical trials. Tumors treated with Selumetinib not only display a marked
reduction in proliferation, but have also been shown to induce an upregulation in surface
expression levels of human leukocyte antigen (HLA) Class I. Loss of HLA class I and II are
commonly observed in carcinomas and represents a significant hurdle in the treatment of solid
tumors. The mechanism by which Selumetinib inhibits cellular growth is well documented, but
how exactly MEK1/2 inhibition leads to HLA upregulation is not known. Understanding the
mechanism by which Selumetinib upregulates HLA would allow for a better understanding of
tumor development as well as superior remedies. Here it is shown that HLA upregulation in
tumors with overactive MAPK signaling is accomplished through ERK1/2 inhibition. By contrast,
inhibition of downstream oncoproteins had little effect on surface HLA levels thereby dispelling
the notion that oncoproteins are involved with HLA downregulation. Furthermore, inhibition of
MEK1/2 and ERK1/2 led to nearly identical induction levels of HLA Class I and Class II. Multiple
cell lines displayed an innate resistance to MEK1/2 inhibition-induced HLA upregulation, despite
carrying the HLA genes. All cell lines analyzed responded to IFNγ with upregulation of HLA class
I and class II, and a synergistic upregulation was noted in dual treatment of Selumetinib and
IFNγ. Interestingly, two populations were noted in the HT29 cell line, one resistant and one
susceptible to MEK inhibition-induced HLA upregulation. Future studies merit elucidation of
specific downstream proteins that mechanistically regulate HLA expression.
ii
ACKNOWLEDGMENTS
The study and research presented in this manuscript would not be possible without the
contributions of numerous researchers, mentors, and friends. Dr. Epstein provided a laboratory
filled with experienced staff and provided invaluable insight himself that allowed my thesis to
come to fruition. I am eternally indebted to Dr. Epstein for his help and guidance over the past
two years. Dr. Epstein also provided a lab full of exceptional people that each had their own
impact on my academic career and research. Dr. Caryn Gonsalves, thank you for guiding my
ability to think critically about experimentation and the implications of my personal data.
Stacey, Ryan, and Brandon thank you for providing a fun and amiable environment to grow as a
person and a researcher. Thank you all for making lab a place I looked forward to attending.
My personal life, which is equally important as my academic career, owes a debt of
gratitude to a great multitude of outstanding humans as well. First and foremost, I would like to
acknowledge my parents for their constant support and encouragement. You have both always
been there for me when I needed it, even when it was not convenient for you. Without your
loving encouragement I may not have had the courage to pursue my dreams. I would also like
to thank my roommates, Russell, Jamal, Alex, and Brian for keeping me sane and always being
around to engage in fun shenanigans. I truly could not afford to live in this place without your
assistance and constant patience with late rent payments (thanks Russ).
iii
TABLE OF CONTENTS
PREFACE
Abstract………………………………………………………………………………………………………………………. i
Acknowledgments………………………………………………………………………………………………….……. ii
List of Tables………………………………………………………………………………………………………………… vi
List of Figures………………………………………………………………………………………………………………. vii
List of Abbreviations……………………………………………………………………………………………………. viii
Declaration of Academic Achievement………………………………………………………………………… ix
CHAPTER 1 - INTRODUCTION
1.1 – HLA AND ITS ROLE IN CANCER……………………………………………………………………………… 1
1.1.1- HLA STRUCTURE AND FUNCTION …………………………………………………………… 1
1.1.2 TWO FORMS OF HLA DOWNREGULATION………………………………………………. 3
1.1.3- HLA DOWNREGULATION AS A MEANS OF IMMUNE ESCAPE…………………. 4
1.2 – MEK1/2 AS AN HLA REGULATOR……………………………………………………………………….. 5
1.2.1 MEK1/2 FUNCTION IN HLA…………………………………………………………………….. 5
1.2.2 MECHANISMS OF RESISTANCE TO MEK INHIBITION BASED
HLA UPREGULATION …………………………………………………………………………… 8
CHAPTER 2 - HYPOTHESIS AND SPECIFIC AIMS
2.1 – HYPOTHESIS…………………………………………………………………………………………………………… 10
2.2 – SPECIFIC AIMS………………………………………………………………………………………………………… 10
iv
CHAPTER 3 – MATERIALS AND METHODS
3.1 REAGENTS AND INHIBITORS…………………………………………………………………………………… 11
3.2 CELL LINES AND CULTURE CONDITIONS……………………………………………………………...… 11
3.3 INHIBITION ASSAYS……………………………………………………………………………………………... 13
3.3.1 FLOW CYTOMETRY………………………………………………………………………………… 13
3.3.2 DATA ANALYSIS……………………………………………………………………………………… 15
CHAPTER 4 – RESULTS
4.1 –SENSITIVITY TO MEK1/2 AND IFNγ DERIVED HLA INDUCTION……………………………… 16
4.2 – ERK1/2 SIGNALING MEDIATES MEK DERIVED HLA EXPRESSION……………….………… 18
4.2.1 – ERK1/2 SIGNALING MEDIATES MEK DERIVED HLA EXPRESSION…………… 18
4.2.2 – GROWTH INHIBITION AND INHIBITOR TOXICITY……………………………………. 21
4.2.3 – EVIDENCE FOR RESISTANT POPULATIONS……………………………………………… 22
4.3 – MEK PATHWAY ONCOPROTEINS AFFECT ON HLA INDUCTION…………………………….. 24
CHAPTER 5 – DISCUSSION
5.1 – HLA UPREGULAITON VIA ERK1/2 INHIBITION……………………………………………….……….. 27
5.2 – HLA UPREGULAITON IS NOT THE RESULT OF DECREASED
ONCOPROTEIN ACTIVITY……………………………………………………………………………………. 28
5.3 – EVIDENCE FOR RESISTANT POPULATIONS……………………………………………………………… 29
5.4 –REGULATION OF BOTH CLASSES OF HLA………………………………………………………………… 30
v
CHAPTER 6 – FUTURE IMPLICATION
6.1 – PROTEINS DOWNSTREAM OF ERK SIGNALING……………………………………………………… 31
6.2 – ADDITIONAL STUDIES………………………………………………………………………………………….. 33
6.2.1 – INCRESED TRANSCRIPTION VERSUS
POST-TRANSLATIONAL MODIFICATIONS………………………………………………. 33
6.2.2 – RNAi TO VALIDATE INHIBITOR FUNCTION………………………………….……….… 34
6.2.3 – IN VIVO EXPERIMENTS………………………………………………………………………… 34
vi
List of Tables
Table 1: P 12 Mutations in the utilized cell lines. Each of the lines studied have
a mutation in the MAPK pathway upstream of MEK1/2.
vii
LIST OF FIGURES
Figure 1 P 2 Protein structure of MHC class I and II
Figure 2 p 6 Graphical depiction of the MAPK pathway.
Figure 3 p. 17 Induction of HLA expression by Selumetinib and IFNγ
Figure 4 p 19 HLA ABC induction upon treatment with MEK pathway inhibitors
and IFNγ
Figure 5 p 20 HLA DR induction upon treatment with MEK pathway inhibitors
and IFNγ
Figure 6 p 21 Growth inhibition by MEK1/2 pathway specific enzyme
inhibition and IFNγ stimulation
Figure 7 p 22 Cell death correlates to increased HLA expression in HT29 cells
Figure 8 P 23 Histograms displaying the resulting MFI associated with
resistant populations in HT29 cells
Figure 9 p 25 HLA ABC induction from oncoprotein inhibition
Figure 10 p 26 HLA DR induction from oncoprotein inhibition
viii
List of Abbreviations
Abbreviation Terminology
α alpha; anti
γ gamma
β Beta
μ micro
IFN Interferon
HLA Human Leukocyte Antigen
MHC Major Histocompatability Complex
TAP Transporter associated with
Antigen Processing
APC Antigen Presenting Cell
MAPK Mitogen-activated protein kinase
EBV Epstein-Barr Virus
LMP Latency Associated Membrane
Protein
B-LCL Human B- Lymphoblastoid
LPS Lipopolysaccharide
cAMP Cyclic Adenosine Monophosphate
PKA Protein Kinase A
MEK Mitogen-activated protein kinase
kinase
ERK Extracellular signal-regulated
kinase
MFI Median Fluorescent Index
CIITA MHC Class II Trans-activator
RFX Regulatory Factor X
NF-Y Nuclear Factor Y
AP1 Activator Protein-1
CREB cAMP Response Element Binding
Protein
FACS Fluorescence Activated Cell
Sorting
r-h Recombinate-Human
PDL1 Programmed Death Ligand 1
ix
DECLARATION OF ACADEMIC ACHIEVEMENT
I conducted the entirety of the following research in the Epstein lab, which has a proven
track record in the field of cancer immunotherapies. The lab had generated numerous papers
dealing with the development of innovative strategies to combat cancer via modulating the
adaptive immune system and creating a more immune permissive environment in tumors
themselves. Dr. Alan Epstein, my thesis advisor, proposed the project of elucidating the
mechanism by which the MEK1/2 inhibitor, Selumetinib, caused the upregulation of surface
HLA Class I molecules. MEK1/2 as a means of HLA induction was a novel idea discovered and
published on within the Epstein lab. The project was proposed in order to better understand
Selumetinib, a drug that is currently in phase III clinical trials for the treatment of Thyroid
cancer [10]. Chapter 1 describes the present knowledge of HLA induction methods as it results
to the MEK1/2 pathway. This knowledge was used to inform my experiments, and was
validated through many of the results. Chapter 3 describes the methods utilized to test my
hypothesis. The experiments were designed by me, along with technical input from Dr. Epstein
and Dr. Gonsalves, a scientist in the Epstein Lab. Each experiment was also run entirely by me.
The results of these experiments are detailed in Chapter 4, with an analysis and discussion in
Chapter 5. I analyzed the experimental data, which was approved by Dr. Epstein. Future
projects and protein targets are discussed in Chapter 6.
1
CHAPTER 1 – INTRODUCTION
1.1 – HLA AND ITS ROLE IN CANCER
1.1.1 – HLA STRUCTURE AND FUNCTION
The Human Leukocyte Antigen (HLA), also known as the major histocompatibility
complex (MHC), is a complex of cell surface proteins necessary for an appropriate acquired
immune response. HLA presentation of antigens begins with cytosolic peptides undergoing
proteolytic degradation, which are then transported into the endoplasmic reticulum via
transporter associated with antigen processing (TAP) where they associate with MHC molecules
that are subsequently transported to the cell surface. Presentation of MHC-associated peptides
and formation of an immune synapse (association of MHC with conjugate receptors on effector
cells) allow immune cells to monitor the processes going on within the cell body [7]. The
synapse interaction allows the cells of the acquired immune system, such as CD8+ T cells, to
identify self versus non-self cells and to identify if the presenting cell has been infected by a
foreign agent [1]. Should the presenting cell display an abnormal peptide to the cytotoxic
effector cell, such as a viral protein not normally expressed in the cell, then the T cell will
release cytokines and other factors that lead to the death of the presenting cell.
MHC peptides are separated into two classes, MHC Class I nd MHC Class II. MHC Class I
is present on all nucleated cells in the body and is responsible for presenting cytosolic peptides
to acquired immune cells. Class I consists of two peptides, the alpha and beta units. The alpha
chain has three separate domains while the beta has only one, β
2
Macroglobulin (Fig. 1). In
humans, the MHC Class I alpha peptide units are coded by three separate major genes, HLA-A,
HLA-B, and HLA-C. The HLA gene complex locus comprises 240 genes spanning 3.6 megabase
2
pairs located on chromosome 6. The β
2
Macroglobulin gene is located on chromosome 15. MHC
Class II peptides are present only on antigen presenting cells (APCs) such as phagocytes, B cells,
or dendritic cells. The major genes are coded for by the HLA-DR, DP, and DQ genes which are
also located within the HLA gene complex on chromosome 6. Class II MHC is composed of two
peptides as well, although the alpha and beta units both have two unique domains unlike in the
Class I receptor (Fig. 1). Class II cell surface proteins are responsible for presenting antigens that
are phagocytosed from the environment, generally presenting peptides derived from
pathogens that are larger than Class I-presented peptides [9] [31]. The specific allele of HLA
presented is unique to each individual. The HLA type carried by an individual can be
heterozygous, which is believed to lead to better disease outcomes through the presentation of
more varied pathogen antigens [30].
Figure 1: Protein structure of MHC class I and II. MHC Class I has β2 microglobulin, while class II
does not. Class II peptides have alpha and beta subunits.
MHC Class I peptides also present abnormal peptides in cancerous tumors that allow the
acquired immune system to identify the tumor, leading to tumor lysis. As a result, many tumors
reduce the surface expression of MHC as a common strategy to avoid immune surveillance [1].
3
Tumor cells that decrease MHC expression are no longer subjected to immune attack and are
likely to proliferate to encompass much of the tumor. A decrease in HLA has been noted in a
high percentage of cancers, including in 70% of head and neck squamous cell carcinomas, 96%
of breast carcinomas, 87% of colon carcinomas, 39% of pancreatic carcinomas, and 63% of
melanomas [10][6]. MHC downregulation is one of many strategies that tumor can employ to
evade cytotoxic immune effects, although complete loss of MHC can lead to cell death via
natural killer cell mediated killing [8] [6].
1.1.2 – TWO FORMS OF HLA DOWNREGULATION
Downregulation of HLA can be either permanent or reversible, depending on the
causative mutation. Tumors bearing irreversible loss of HLA are also known as a “hard” lesion,
while a reversible downregulation is called a “soft” lesion [2]. Irreversible loss of HLA is the
result of DNA deletions that lead to loss of heterozygosity that is impossible for the cell to
reverse [2]. Notable deletions are in the MHC gene family, β2 microglobulin, and TAP protein
defects [3] [8]. Hard lesions have been reported in 30-40% of human cancers [2]. As expected,
Tumors that have irreversible downregulation of their HLA have a poorer prognosis than those
with native expression levels [2][6].
Reversible downregulation is the result of normal intracellular factors that have gone
awry, and these soft lesions have shown the ability to be overcome by cytokine stimulation
[6][2]. Reversible downregulation could be the result of hyper-methylation of HLA genes or
promoters, deacetylation of associated histones, inactivation of transcription factors, or
alterations in glycosylation and transport. These tumors have normal genes intact; however,
4
the protein product is either not transcribed and translated at the normal rate, or the HLA
associated proteins are rendered nonfunctional via post translational modifications or
intracellular sequestering [8]. These factors can be overcome by cytokine stimuli such as IFNγ
binding the IFNγR, thus activating the JAK/STAT pathway leading to the activation of STAT1 [4].
Increases in TNFα signaling and uptake of inhibitors against cellular factors have been shown to
be effective in the restoration of HLA. A synergistic effect of HLA upregulation via inhibition of
the MEK pathway combined with IFNγ treatment has been reported in a variety of different
studies [10][11].
1.1.3 HLA DOWNREGULATION AS A MEANS OF IMMUNE ESCAPE
HLA downregulation is a common form of immune escape employed by cancers, in
addition to expression of immune inhibitory molecules, and invasion of immune suppressive
cells. As previously noted, loss of HLA- ABC has been reported in a high occurrence of a wide
variety of carcinomas and melanomas [10][6]. Loss of HLA prevents the cytotoxic T cells from
effectively recognizing the tumor, allowing the tumor to flourish in the new immunotolerant
environment [29]. In order for adaptive immune therapies to function, the tumor
microenvironment must shift away from an immunotolerant environment to an inflammatory
reaction against the tumor. New strategies, such as antibodies targeting programmed death
ligand 1 (PDL1) (a cell surface protein that suppresses T cell activity), or targeting myeloid
derived suppressor cells (leukocytes that decrease T cell activity) have emerged to counter the
permissive microenvironment [12][35]. These strategies, however, only serves to mitigate T cell
suppression, and may require additional therapies that upregulate HLA in order to restore full T
5
cell functionality. Furthermore, tumor lesions are a heterogeneous mixture of populations with
varied surface expression levels with a selective pressure from T cells to decrease HLA
expression. Cells that initially express native levels of HLA will be selectively eliminated by the
immune cells that remain active within the growing lesions, and those expressing low amounts
of HLA will continue to expand to replace those eliminated [2]. In this manner, even tumors
that express high levels of HLA can progress to low or null expression. Therefore, developing
strategies that restore HLA expression is essential not only in soft lesions, but all carcinomas [6].
1.2 – MEK AS AN HLA REGULATOR
1.2.1 – MEK 1/2 FUNCTION IN HLA
Inhibition of the mitogen associated protein kinase (MAPK) pathway results in HLA
upregulation. This pathway (Fig. 2) is activated by growth factor binding to cell surface tyrosine
kinase receptors leading to cellular proliferation, resistance to apoptosis, and increased
metastasis [23]. Activating mutations in the various stages of the MAPK pathway have been
noted in greater than 50% of carcinomas [15]. Mitogen binding to the surface receptor results
in activating phosphorylation of MAPK that in turn activates several proteins leading to
activation of MEK1/2, rampant cellular growth, and metastatic potential. It has been reported
that inhibition of MEK1/2 with the MEK1 specific inhibitor Selumetinib leads to upregulation of
HLA class I and increased T cell cytotoxicity in cancers [10]. As previously noted, the greatest
induction of HLA occurred when carcinoma cells were treated with both IFNγ stimulation and
Selumetinib inhibition [10]. Exactly which downstream protein deregulated by the inhibition of
MEK, and loss of signaling, was not determined.
6
Figure 2: Graphical depiction of the MAPK pathway. Arrows originate from the effector protein
and point to the protein affected. Horizontal ovals dictate transcription factors, triangles are
kinases, and concentric circles are complexes consisting of multiple proteins (MAP2K1/2 and
ERK1/2 include isoforms of each kinases). Circles, vertical ovals, trapezoids, and diamonds are
other protein types. MAP2K1/2 is another name for MEK1/2, and RPS6KA1 is another name for
RSK. Not all substrates for ERK1/2 are shown. Generated with the Ingenuity Pathway Analysis®
(IPA®, QIAGEN Redwood City, http://www.qiagen.com/ingenuity)
MEK1/2 has only one reported substrate: extracellular signal-regulated kinases 1/2
(ERK1/2). ERK1/2 has over 150 reported substrates that include transcription factors, kinases,
and other regulatory proteins that result in cell growth as well as HLA downregulation [15] [5]
[10] (Fig. 2). Oncoproteins, proteins that cause cells to transform into cancer, have been shown
to be the drivers of several cancerous changes, so their role in the cancerous HLA regulation
7
was investigated. Oncoproteins, such as STAT3 and c-Myc, are active in a majority of human
cancers and are known to be downstream of ERK1/2. These proteins have been shown to
directly influence HLA expression [19][20]. STAT3 and c-Myc are prominent oncoproteins that
are active in a majority of human cancers [19][20]. It stands to reason that the drivers of the
cancerous growth and metastasis, such as STAT3 and c-Myc, may also drive the downregulation
of HLA observed in these tumors.
The c-Myc oncoprotein downstream of MEK1/2 is of particular note as it has been
implicated in both rampant growth as well as downregulation of both classes of HLA. c-Myc is a
transcription factor that controls approximately 15% of all genes, and is also implicated in the
recruitment of histone acetylases to increase transcription. Mutations leading to
overexpression of c-Myc have been observed in 70% of cancers, possibly due to its role in both
growth activation and suppression of p53 [19]. c-Myc activity has been shown to counteract
Epstein-Barr virus latent membrane protein-1 (EBV LMP) induced HLA upregulation in epithelial
cells [16]. This activity was observed in somatic cells, but it stands to reason that the activity
could be replicated in cancers. Work with lymphoblastoid cell line, B-LCL has shown that c-Myc
can also lead to a downregulation of HLA Class II. The B-LCL cells show that high expression
levels of c-Myc led to poor stimulation of CD4+ T cells via downregulation of HLA-DM, an HLA
Class II type. In these cells the native HLA-DM was replaced with HLA-DO, an inhibitory HLA
class II type [17] [18]. c-Myc was not directly responsible for the loss of HLA in this case, but
through its interactions it has been implicated in the loss of both HLA classes.
Much like c-Myc, STAT3 is a transcription factor downstream of MEK1/2 that is
constitutively activated in a wide variety of cancers. The STAT family of protein are cytoplasmic
8
transcription factors that translocate to the nucleus upon phosphorylation. Overactive MEK1/2
signaling leads to increased cytoplasmic STAT3, which is associated with growth, survival,
metastasis, and angiogenesis [20][37]. STAT3 activation in the tumor has also been noted in the
tumor microenvironment as a potent inhibitor of immune function, and, in fibroblasts,
suppresses the LPS induced pro-inflammatory response [20]. A direct link between
constitutively active STAT3 and cancerous changes, poor prognosis has also been described
[37]. While it has not yet been directly shown to reduce HLA specifically, STAT3 is a
transcription factor that has high oncogenic potential and has been shown to modulate
immune activity [20].
1.2.2 MECHANISMS OF RESISTANCE TO MEK INHIBITION BASED HLA UPREGULATION
Targeting the MEK pathway is not a cure-all, as some cells have shown an intrinsic
resistance to MEK inhibition and carcinomas can develop this resistance as well [22] [23]. One
reported cause of intrinsic resistance is through regulation of the cAMP-dependent protein
kinase A (PKA) pathway. Experimental evidence suggests that the PKA pathway is activated by
EGFR, but upstream of MEK, which allows its function to proceed unimpeded by MEK inhibition
[23]. In addition to alternate pathways giving resistance to MEK inhibition, mutations to MEK1
itself have been noted in selumetinib-treated patients. The MEK1/2 specificity of selumetinib
binding is negated by structural mutations to the enzyme itself. Mutations in MEK1/2 do not
grant resistance to ATP-competitive inhibitors, however, as the ATP binding pocket is vital to
enzyme function [22]. Unfortunately, ATP-competitive inhibitors are not as selective as non
ATP-competitive “selective” inhibitors and may have increased off-target effects. A constant
9
supply of inhibitors to the tumor is needed to sustain inhibition effects, as the RAS activity
quickly rebounds after abatement of treatment. This is likely due to ERK1/2-mediated feedback
inhibition of C-Raf, which is lost with MEK inhibition [22].
10
CHAPTER 2 – HYPOTHESIS AND SPECIFIC AIMS
2.1 – HYPOTHESIS
Here in this thesis, I investigate whether or not MEK1/2 based HLA induction is unique
to MEK1/2 inhibition in several human cancer cell lines, or if inhibition of downstream products
also leads to HLA induction. I hypothesize that 1) HLA induction will be similar across each of
the permissive cell lines tested, 2) that MEK1/2 inhibition-induced HLA upregulation is achieved
through reduced ERK1/2 activity, and 3) HLA upregulation will be regulated by the activity of
oncoproteins within the MEK1/2 pathway.
2.2 – SPECIFIC AIMS
To test this hypothesis, flow cytometry was utilized to quantify surface expression of the
HLA-ABC and HLA-DR molecules following treatment of inhibitors against sequential steps in
the MEK1/2 signal transduction pathway.
Specific Aim 1: Assess the susceptibility of cell lines in response to Selumetinib inhibition
and IFNγ stimulation
Specific Aim 2: Determine if ERK1/2 inhibition is equivalent to MEK1/2 inhibition
regarding HLA upregulation.
Specific Aim 3: Determine if downstream oncoproteins in the MEK1/2 pathway are
responsible for HLA reduction by using small molecule inhibitors.
11
CHAPTER 3 – MATERIALS AND METHODS
3.1 REAGENTS AND INHIBITORS
Roswell Park Memorial Institute (RPMI) 1640 cell culture media (Corning-Cellgro;
Tewksbury, MA) was supplemented with L-glutamine-penicillin streptomycin solution (Gemini
Bioproducts; Sacramento, CA) and 10% Fetal bovine serum (FBS) (HyClone Laboratories; Logan,
UT) which will be referred to as complete media hereafter. All cell lines (see 3.2) were grown
and maintained in complete media. Trypsin-EDTA 1X (Corning Cellgro) was utilized to detach
adherent cells from cell culture flasks, for propagation of cultures. Detachin, a gentler cell
detachment reagent, purchased from Genlantis (San Diego, CA) was used to release cells from 6
well dishes prior to flow cytometry.
Inhibitors Selumetinib (MEK1/2), SCH772984 (ERK1/2), Cryptotanshinone (STAT3),
10058-F4 (c-Myc), and Fludarabine (STAT1) were purchased from Selleckchem (Houston, Tx)
and r-hIFNγ was purchased from Peprotech (Rocky Hill, NJ) to be used in flow cytometric
analysis.
3.2 CELL LINES AND CULTURE CONDITIONS
Human cell lines HT29, A431, NCI-H1703, and K1 were purchased from American Type
Culture Collections (ATCC; Manassas, VA). Human fetal lung fibroblasts (FLF) were developed in
our lab from early aborted embryos obtained with IRB approval. HT29 is a colorectal cancer
bearing a BRAF and PI3K activating mutation; A431 is an epidermoid cancer that has high EGFR
expression and null p53; NCI-H1703 is a non-small cell lung cancer that has SOCS3
hypermutation with high FGFR expression; and K1 is a thyroid cancer that is BRAF positive with
12
a missense PIK3CA mutation; FLF is a primary non-cancerous human fetal lung fibroblast cell
strain (Table 1). Cells were maintained in Corning (Corning, NY) 75mm cell culture flasks and
incubated in a light-protected humidified incubator at 37
o
C with 5% CO
2
in complete media.
HT29 A431 NCI-H1703 K1 FLF
BRAF+ EGFR+++ FGFR+++ BRAF+ No known
mutations
PIK3CA+ Null P53 SOCS3
hypermethylation
PIK3CA
missense
Table 1: Mutations in the utilized cell lines. Each of the lines studied have a mutation in the
MAPK pathway upstream of MEK1/2. BRAF is a kinase upstream of MEK1/2, EGFR and FGFR are
surface receptors that trigger the MEK1/2 pathway when bound by mitogens.
Cells were split at approximately 80% confluency according the following protocol:
depleted-culture complete media was discarded into a waste container with 10% bleach. Five
mL of trypsin-EDTA 1X was added to the adherent cell layer and the flasks were placed back
into the light-protected humidified incubator at 37
o
C with 5% CO
2
for 10-15 minutes until the
cell layer detached from the bottom of the flask. Trypsin-cell solution was poured into a 50mL
conical vial (Genesee Scientific; San Diego, CA). Twenty-five to thirty mL of complete media was
added to each conical vial containing trypsin-cell solutions to inhibit trypsinization of the cells.
The trypsin-cell-media solution was centrifuged at 1250 rpm for 7minutes at 4
o
C in a Beckman
Coulter Allegra X-12R centrifuge (Brea, CA). Supernatant was discarded and each cell pellet was
resuspended into a single cell suspension and seeded into Falcon™ 9.6cm 6 well plates
(Corning; Corning, NY) at 7.5 × 10
5
cells for all cell lines except FLF which were seeded at 1 × 10
6
cells. Two million cells from each line were seeded into new 75mm flasks with 45mL of
complete media and all additional cells were discarded into the 10% bleach solution.
13
3.3 INHIBITION ASSAYS
3.3.1 FLOW CYTOMETRY
Cells seeded into 6 well plates were treated with 2mL of complete media and allowed to
adhere overnight. The following day media was replaced with 2mL of complete media
containing various combinations of inhibitors and r-hIFNγ at the following concentrations: IFNγ
100U, Selumetinib 10μM, SCH772984 10μM, Cryptotanshinone 20μM, 10058-F4 10μM,
Fludarabine 10μM, and vehicle control DMSO 2μL. Inhibitor concentrations were chosen based
off of previously published research utilizing Selumetinib [10], SCH772984 [32], r-hIFNγ [10],
Cryptotanshinone [33], 10058-f4 [34], and Fludarabine [25]. When treating with IFNγ and
Fludarabine, the cells were exposed to Fludarabine alone for 48hrs to allow for the inhibitor to
lower the intracellular STAT1 levels prior to stimulating the STAT1 via IFNγ. This treatment was
performed differently because the mechansism of action of Fludarabine inhibits the production
of STAT1 mRNA and protein, while the other inhibitors only inhibit protein activity [25]. Media
and inhibitors were replaced approximately every 48hrs, and after approximately 120hrs in the
complete media-inhibitor solution the cells were detached by discarding the supplemented
media into 10% bleach and adding 1mL of Detachin to the cells. Immediately upon cell
detachment, the detachin-cell mixture was added to a 15mL conical vial (Corning; Corning NY)
containing 3mL of media and centrifuged at 1250 rpm for 7 minutes at 4
o
C in the Beckman
Coulter Allegra X-12R centrifuge. Supernatant was discarded and the cells were resuspended in
1mL FACS buffer (phosphate buffered saline (PBS)(Corning-Cellgro) supplemented with 10%
FBS). Next, cells were counted in an Invitrogen™ (Carlsbad, CA) Countess™ and recorded. One
million cells were transferred into individual 12 x 75mm polystyrene tubes, centrifuged, and
14
resuspended in 100μL of FACS buffer with 20μL of a PE-Cy5 Mouse anti-human HLA-ABC
antibody (Clone G46-2.6; BD Pharmingen; San Diego, CA) and 20μL of a APC Mouse anti human
HLA-DR antibody (custom order, Clone L243; BD Biosciences; San Jose, CA). Cell samples were
incubated with the antibodies for 1hr at 4
o
C. Samples were then washed twice by
centrifugation with FACS buffer and resuspended in PBS for flow cytometry analysis.
Immediately prior to data acquisition cells were treated with 10μL of propidium iodidie (PI)
(Thermo Fisher; Waltham, Massachusetts).
Flow cytometry data were collected and analyzed on an Attune® Acoustic flow
cytometer using Life Technologies’ Attune® software (Life Technologies; Carlsbad, CA). The
dead cell exclusion dye PI was analyzed in the BL2-A channel (Blue laser 2, area) and the highest
intensity peaks were excluded. Density plots of forward scatter height (FSC-H) vs forward
scatter area (FSC-A) were generated to eliminate non-singlet events after exclusion of ‘dead
cells’ via PI. Singlet events were then differentiated into normal light scattering cell events
versus alternate light scattering cell events in a separate density plot of FSC-A vs side scatter
area (SSC-A) and the alternate scattering events were excluded. Normal light scattering events
were analyzed in the channels BL3-A (blue laser filter 3, area) to quantify αHLA-ABC antibody
binding and RL1-A (red laser filter 1, area) to quantify αHLA-DR antibody binding. MFI (mean
fluorescent index) was used as the metric for quantifying HLA expression for each of the culture
conditions. This process was repeated a minimum of three times for each sample with a
representative sample shown.
15
3.3.2 DATA ANALYSIS
Data generated by flow cytometry were analyzed and formatted using GraphPad
Software’s Prism 6, and Attune ® Flow cytometry software. Statistical tests were performed
using GraphPad Software’s Prism 6 and FlowJo software (FlowJo). Results are shown as mean +
SD or mean + SEM as indicated. Unpaired T tests using the Holm-Sidak correction method were
used to compare means and determine statistical significance. Images were generated utilizing
GraphPad Prism 6 and FlowJo software.
16
CHAPTER 4 – RESULTS
4.1 SENSITIVITY TO MEK1/2 AND IFNγ DERIVED HLA INDUCTION.
Prior to elucidating which MEK1/2 pathway proteins are involved in previously observed
HLA induction experiments, the effect on several previously untested cell lines was determined.
A variety of cell lines were tested to understand if the pathway is conserved across multiple
carcinomas or if each responds differently. Cell lines were screened for their response to
Selumetinib and IFNγ-induced HLA-ABC and HLA DR upregulation. Surface expression of HLA
ABC and HLA DR increased a significant amount for each cell line when cells were treated with
IFNγ (Fig. 3). Although some of the lines screened were resistant to Selumetinib treatment
alone, all cell lines displayed a significantly higher HLA upregulation in the combination
treatment of Selumetinib and IFNγ compared to IFNγ alone. Whether the effect was
cooperative or synergistic varied from line to line. The primary cell line, FLF, did not respond to
MEK1/2 inhibition with HLA upregulation but growth was severely inhibited (Fig.3, Fig. 6).
17
Figure 3: Induction of HLA expression by Selumetinib and IFNγ. A) HLA-ABC expression
determined by BL3 MFI. All cell lines used respond with significant HLA upregulation when
treated with Selumetinib and IFNγ. Associated histograms normalized to mode are shown
below. Data shown are mean +SD with significant difference from vehicle control indicated by
*P<0.05, ** P<.01, ***P<.001. B) HLA-DR expression determined by RL1 MFI. Associated
histograms normalized to mode are shown below. All cell lines used respond with significant
HLA upregulation when treated with Selumetinib and IFNγ. Data shown are mean +SD with
significant difference from vehicle control indicated by *P<0.05, ** P<.01, ***P<.001.
18
4.2 – EFFECTS OF ERK 1/2 INHIBITION
4.2.1 – ERK1/2 SIGNALING MEDIATES MEK DERIVED HLA EXPRESSION
As previously noted, ERK1/2 is the only reported substrate for the MEK1/2 enzyme and
therefore SCH772984 was utilized to inhibit ERK1/2 phosphorylation and activation. These data
were compared to MEK1/2 inhibition in the absence and presence of r-hIFNγ to determine if
ERK1/2 inhibition yielded the same results. If Selumetinib and SCH772984 similarly increase HLA
expression then plausibly MEK1/2 kinase diminishes HLA expression by phosphorylating and
activating ERK1/2. Flow cytometry was utilized to assess the effect that Selumetinib,
SCH772984, and dual inhibition of Selumetinib and SCH772984 had on HLA expression.
SCH772984 ERK inhibition alone yielded similar results as Selumetinib MEK1/2 inhibition alone,
but did not surpass Selumetinib-induced upregulation in any of the cell lines. Combination
treatment with SCH772984 and r-hIFNγ yielded a higher induction of surface HLA in the NCI-
H1703 cell lines (Fig. 4, Fig. 5). In the A431 cell line combination treatment with SCH772984 and
r-hIFNγ did not match the induction observed with Selumetinib and r- hIFNγ, but had significant
induction above r-hIFNγ alone for both HLA ABC (Fig. 4) and HLA DR (Fig. 5). In the HT29 cell line
SCH772984 and r-hIFNγ combination treatment yielded highly variable results that did not
surpass r-hIFNγ alone for HLA ABC (Fig. 4). HT29 HLA-DR expression matched untreated stained
(Fig. 5).
19
Figure 4: HLA ABC induction upon treatment with MEK pathway inhibitors and IFNγ. SCH772984
ERK1/2 inhibition yielded comparable results to Selumetinib MEK1/2 inhibition in all lines,
except HT29. HLA ABC expression determined by BL3 MFI. Data shown are mean + SD,
significant difference from vehicle control indicated by *P<0.05, ** P<.01, ***P<.001. A) HT29
cells with associated histogram normalized to mode. SCH772984 treatment decreased MFI and
nearly abolished IFNγ-induced induction. B) A431 cells with associated histogram normalized to
mode. A431 cells respond primarily to IFNγ, and combination treatments yields a moderate
complementary effect. C) NCI-H1703 cells with associated histogram normalized to mode. NCI-
H1703 cells respond to IFNγ and SCH772984 combination treatment with the highest HLA
induction. D) K1 cells and associated histogram normalized to mode. All combinations induce
increased HLA expression. The greatest increase is seen in the IFNγ and Selumetinib combination
treatment. E) FLF cells and associated histogram normalized to mode. FLF cells respond
primarily to IFNγ stimulation. The effects of IFNγ are abated by the combination treatments,
likely due to increased cell death.
20
Figure 5: HLA DR induction upon treatment with MEK pathway inhibitors and IFNγ. SCH772984
ERK1/2 inhibition yielded comparable results to Selumetinib MEK1/2 inhibition in all lines,
except HT29. HLA DR expression determined by RL1 MFI. Data shown are mean + SD, significant
difference from vehicle control indicated by *P<0.05, ** P<.01, ***P<.001. A) HT29 cells with
associated histogram normalized to mode. SCH772984 treatment decreased MFI and nearly
abolished IFNγ-induced induction. B) A431 cells with associated histogram normalized to mode.
A431 cells respond primarily to IFNγ, and combination treatments yields a moderate
complementary effect. C) NCI-H1703 cells with associated histogram normalized to mode. NCI-
H1703 cells respond to IFNγ and SCH772984 combination treatment with the highest HLA
induction. D) K1 cells and associated histogram normalized to mode. All combinations induce
increased HLA expression. The greatest increase is seen in the IFNγ and Selumetinib combination
treatment. E) FLF cells and associated histogram normalized to mode. FLF cells respond
primarily to IFNγ stimulation. The effects of IFNγ are abated by the combination treatments,
likely due to increased cell death.
21
4.2.2 – GROWTH INHIBITION AND INHIBITOR TOXICITY
The decreased MFI observed in flow cytometry can be explained by the result of cell
death, or a heightened reuptake of surface proteins. The utilized inhibitors have been noted for
their ability to induce cell death, which may account for the decrease in surface expression seen
in some treatments [10]. Cell counts using trypan blue at the end of inhibitor treatment were
measured to assess toxicity and death (Fig 6). Cell death in HT29 cells correlated highly with
increased HLA expression (Fig. 7).
Figure 6: Growth inhibition by MEK1/2 pathway specific enzyme inhibition and IFNγ stimulation.
Cells counted on Invitrogen™ Countess™ after the addition of trypan blue and compared to the
vehicle control. Data shown are mean + SD. Percent growth was determined by the formula
[(sample cell count/Vehicle control cell count)-1]*100. HT29 had the greatest sensitivity to
inhibition, particularly SCH772984. Only K1 treatment with IFNγ alone increased cellular
proliferation above the vehicle control.
22
Figure 7: Cell death correlates to increased HLA expression in HT29 cells. Density scatter plot
data acquired by flow cytometry. Gate quadrants set based on the untreated stained sample.
HLA-ABC expression increases as PI incorporation increases for each condition. Very few cells
incorporated PI and had low HLA expression. A) Cells treated with 10μM Selumetnib and PI.
Twenty-one percent of cells with high HLA expression were dead, compared to 3.8% live. B) Cells
treated with 100U IFNγ and 10μM SCH772984, and PI. Nearly all cells expressing high HLA
correlate to death. C) Cells treated with 100U IFNγ and 10μM Selumetinib, and PI. Only 34.3%
were live with low HLA expression, by far the lowest percentage of the treatment conditions.
This is likely due to IFNγ inducing HLA upregulation and Selumetinib having a lower toxicity than
SCH772984.
4.2.3 – EVIDENCE FOR RESISTANT POPULATIONS
As previously noted, there is an intrinsic resistance in colorectal carcinomas to MEK
inhibitors and it appears as though HT29 has a resistant as well as a susceptible population [22]
[23]. HT29 is the only cell line that two apparent populations were noted in, and the susceptible
population increased their HLA expression before inhibition led to cell death. The non-
susceptible population did not increase their HLA in response to inhibition (Fig. 7). Cell death
correlated highly to increased HLA expression, while living cells had lower HLA expression. This
suggests that some cells were susceptible to each inhibitor, both the HLA inducing properties
23
and toxicity, while another population resisted the inhibitors effects. Two distinct and sharp
peaks are noted when treated with Selumetinib (Fig. 8). The higher expressing peaks are dead
cells (Fig. 8A 36% of cells, Fig. 8B 39% of cells), differentiated experimentally by PI, while the
lower peak is all live cells. The decrease in surface HLA is not noted in the combination
treatment of IFNγ and Selumetinib, although there is still a population that appears to resist
Selumetinib inhibition and respond only to the IFNγ stimulation (Fig. 8).
Figure 8. Histograms displaying the resulting MFI associated with resistant populations in HT29
cells. HLA-ABC is shown, although the result was seen with HLA-DR MFI as well. A) Selumetinib
treatment histogram overlaying the vehicle control histogram. Bars and percentages relate only
to Selumetinib 10uM treated cells, not untreated stained. The left peak is 64% of cells, these
cells did not incorporate PI. The right peak 36% of cells did incorporate PI and have significantly
higher MFI. B) IFN Selumetinib combination treatment overlaying IFN treatment alone. Bars and
percentages relate only to IFN 100U + Selumetinib 10uM treatment, not IFN 100U. The left peak
is 61% of cells, these cells did not incorporate PI. The right peak is 39% of cells, which did
incorporate PI. Distinct peaks are visible from the single sample of treated cells giving the
appearance of two populations being present in the cell line. Susceptible cells respond to
treatment with upregulation of HLA while the resistant population retains the same MFI as the
untreated sample.
24
4.3 – MEK PATHWAY ONCOPROTEINS AFFECT ON HLA INDUCTION
The previous results established that at least a majority of the HLA downregulation was
the result of ERK1/2 activation, which is achieved by MEK1/2 phosphorylation (Fig. 4). ERK1/2
has been noted to have greater than 150 substrates [14] making testing each one nearly
impossible. Two downstream oncoproteins, STAT3 and c-Myc, were assessed as they bear a
large effect on tumor progression [19][20][37]. Both STAT3 and c-Myc were inhibited
individually and simultaneously. IFNγ stimulation leading to HLA upregulation is constant across
all cell lines, and this signal is known to be carried out through STAT1 activation [4]. STAT1 was
inhibited to assess if other proteins contributed to the induced upregulation as well as to
validate the method of utilizing inhibitors, which may have off target effects. Neither STAT3,
STAT1, nor c-Myc inhibition led to HLA class I or II induction, except for STAT1 inhibition in the
K1 cell line (Fig. 9, Fig. 10). Each of the conditions, apart from Fludarabine treatment in K1 cells,
led to a significant HLA ABC and DR suppression in HT29, and only DR suppression in A431
instead of induction. Moderate or insignificant change was seen in other conditions for all other
cell lines. r-hIFNγ stimulation based HLA induction was only partially blocked by STAT1
inhibition in the HT29, and A431 cell lines (Fig. 8). STAT1 inhibition led to notable and significant
downregulation of r- hIFNγ stimulation. FLF cells were not tested for oncoprotein inhibition
because they did not respond to ERK1/2 inhibition with HLA upregulation and are not
cancerous, meaning STAT3 and c-Myc have not altered their normal cellular function.
25
Figure 9: HLA ABC induction from oncoprotein inhibition. HLA ABC was decreased upon
treatment with each of the oncoprotein inhibitors in nearly every cell line. HLA ABC expression
was determined by BL3 MFI. Data shown are mean + SEM. Significance verses vehicle control
indicated by *P<0.05, ** P<.01, ***P<.001. A) HT29 cells with associated histograms normalized
to mode. STAT3 and c-Myc inihibition failed to induce HLA ABC expression. Stat1 inhibition failed
to compeltely inhibit the IFNγ response, and elicited almost no effect, although it did decrease
HLA expression in the absence of IFNγ. B) A431 cells with associated histograms normalized to
mode. STAT3 and c-Myc inihibition failed to induce HLA ABC expression. Stat1 inhibition failed to
significantly inhibit the IFNγ response. C) NCI-H1703 cells with associated histograms normalized
to mode. Stat1 inhibition managed to significantly decrease IFNγ-induced upregulation, and . D)
K1 cell line. Fludarabine failed to compeltely inhibit the IFNγ response.
26
Figure 10: HLA DR induction from oncoprotein inhibition. HLA DR was decreased upon treatment
with each of the oncoprotein inhibitors in nearly every cell line. HLA DR expression was
determined by RL1 MFI. Data shown are mean + SEM. Significance versus vehicle control
indicated by *P<0.05, ** P<.01, ***P<.001. A) HT29 cells with associated histograms normalized
to mode. STAT3 and c-Myc inihibition failed to induce HLA DR expression. Stat1 inhibition failed
to compeltely inhibit the IFNγ response, and elicited almost no effect, although it did decrease
HLA expression in the absence of IFNγ. B) A431 cells with associated histograms normalized to
mode. STAT3 and c-Myc inihibition failed to induce HLA ABC expression. Stat1 inhibition failed to
significantly inhibit the IFNγ response. C) NCI-H1703 cells with associated histograms normalized
to mode. Stat1 inhibition managed to significantly decrease IFNγ-induced upregulation, and . D)
K1 cell line. Fludarabine failed to compeltely inhibit the IFNγ response.
27
CHAPTER 5 – DISCUSSION
5.1 – HLA UPREGULATION VIA ERK1/2 INHIBITION
Selumetinib has been previously shown to upregulate HLA in human thyroid carcinomas
and has slowed tumor growth in a wide variety of tumor types [10][22][23]. It was previously
unknown which proteins in the signal transduction cascade were responsible for HLA
downregulation and which proteins would restore HLA expression upon inhibition. In this
manuscript it is shown that cell lines that upregulate HLA following MEK1/2 inhibition also do
so following ERK1/2 inhibition. This is observed for both HLA-ABC and HLA-DR (Fig. 4, Fig. 5).
MEK1/2 inhibition yielded a higher total surface expression than ERK1/2 inhibition, or
MEK1/2 and ERK1/2 combination inhibition (Fig. 4, Fig. 5). This discrepancy can be explained by
two theories. Primarily it has been shown that these inhibitors have an associated toxicity
unique to each and SCH772984 is likely more toxic (Fig. 6). The combination of both may have a
synergistic toxicity as well, which would account for the combination treatment having the
greatest inhibitor effect on growth [14] [15] [11]. It is shown here that each cell line’s growth,
aside from K1, was inhibited to a higher degree when treated with SCH772984 as opposed to
Selumetinib, which corresponded to a decrease in live singlet cells for all cell lines (data not
shown) and an increase in dead cells (only HT29 cells shown) (fig. 6 and 7).
The second explanation is that MEK1/2 is the optimal enzyme to inhibit, which has two
implications itself. Either MEK1/2 has an alternative substrate through which a separate signal
transduction cascade is carried out, and the inhibition of which also increases surface HLA
expression. MEK1/2 expression may optimally regulate ERK1/2 signaling which results in
superior HLA upregulation rather than complete inhibition. ERK1/2 is the only reported
28
substrate for the MEK1/2 enzyme across several studies [11] [24], which makes it more likely
that MEK1/2 inhibition optimally regulates ERK1/2 or that cell death caused the decreased MFI.
The precise reason as to why Selumetinib treatment is superior to ERK1/2 inhibition should be
investigated, but regardless of the reason, it is clear that the main component of Selumetinib-
derived HLA upregulation is through the inhibition of the ERK1/2 enzyme.
5.2 – HLA UPREGULAITON IS NOT THE RESULT OF DECREASED ONCOPROTEIN ACTIVITY
Inhibition of MEK1/2 pathway oncoproteins does not lead to any notable increase in
surface HLA expression, although moderate and significant upregulation is seen in NCI-H1703
(Fig. 9, Fig. 10). In fact, inhibition of oncoproteins led to a decrease in HLA expression for a
majority of conditions tested, possibly due to increased cell death. It is possible that STAT3 and
c-Myc are also necessary for HLA transcription, which merits future considerations. Only STAT1
inhibition in the NCI-H1703 cell line led to a significant increase in surface HLA expression (Fig.
9, Fig. 10). STAT1 has been shown to be vital to the induction of HLA class I and II, making this
result puzzling [4]. This minor increase is likely due to off target effects of the Fludarabine small
molecule inhibitor. STAT1 inhibition also failed to fully inhibit the effects of IFNγ for all cells
except K1, although moderate yet significant decreases are seen in the other lines tested (Fig. 9,
Fig. 10).
STAT1 inhibition occured for two days prior to IFNγ stimulation to ensure that the
inhibitor has had time to downregulate and inhibit STAT1 protein and mRNA [25]. The action of
Fludarabine and inhibition timeline should have ensured that STAT1 would be downregulated
under normal conditions. This makes it notable that HT29 and A431 IFNγ-induced HLA
29
upregulation is not abated by STAT1 inhibition. Both cell lines may carry a resistance to
Fludarabine, as the two other lines (K1 and NCI-H1703) respond with notable and significant
abatement of HLA induction. It is unlikely, although possible, that other proteins play a role in
IFNγ based HLA induction as STAT1 has been heavily reported on in its role in HLA regulation,
although it is certainly possible and should be investigated [25][4][26].
Due to the lack of HLA upregulation after oncoprotein inhibition kinase inhibition should
be given more attention. Each kinase has several substrates that in turn have additional
substrates giving them a wider variety of targets to affect. Analyzing downstream kinases can
also help to narrow down the proteins of interest. ELK is a kinase directly downstream of
ERK1/2 that merits analysis (Fig. 1).
5.3 EVIDENCE FOR RESISTANT POPULATIONS
Each of the kinase inhibitors elicited a notable effect on growth inhibition (Fig. 6) and
HLA upregulation (Fig. 4, Fig. 5) although the effects were not uniform across all lines studied.
In the HT29 cell line two distinct populations of HLA induction are seen (Fig. 8), which was not
observed in a high degree in any other cell line. Most HT29 cells are not resistant to ERK1/2
inhibition, as seen from the sharp decline in proliferation, but resistance to HLA upregulation is
quite apparent. The population of resistant cells may have been relatively small initially and
grew to encompass a larger percentage of the total as the susceptible cells growth was
inhibited. At any rate, two distinct populations are notable in the HT29 cells with one
population being dead and expressing high HLA, while the other is live and expresses little HLA
30
(Fig. 7, Fig. 8). The living cells are likely those that natively expressed less surface HLA and grew
to account for a higher percentage of the total population, as they were able to resist the effect
of inhibition. By this manner the living cells after treatment had lower surface HLA expression
than the untreated cells (Fig. 8). Inhibition seemingly created a selective pressure that drove
the tumor to decrease HLA, which should be considered as HLA inducers move towards human
trials.
HT29 cells are not a homogeneous population, which may have occurred as the result of
a mutation during treatment, although this is highly unlikely as cells were assayed several times
with the same result. A more reasonable explanation is that the HT29 is already a
heterogeneous population upon thawing, some of which have an acquired resistance to HLA
induction via inhibition of the MEK pathway. PIK3CA mutations have been implicated in
resistance to Selumetinib, and HT29 bears an activating mutation in PIK3CA [22]. Resistant cells
may have increased PIK3CA activity, or may have developed a hard lesion while other cells bear
the soft lesion. In order to see if this was an isolated mutation a second vial of HT29 cells were
thawed and assayed, yielding the same results (results not shown). No resistance in any cell line
was noted for IFNγ, and the combination treatment of IFNγ and Selumetinib led to a high and
significant induction in most cell lines including the resistant A431 cells (Fig. 3, Fig.4, Fig. 5).
5.4 –REGULATION OF BOTH CLASSES OF HLA
IFNγ has been described as a regulator of both HLA class types [13]. It is not surprising
then that treatment with IFNγ led to the induction of both types of HLA. It is more substantial
that inhibiting the MEK pathway led to an increase in both HLA classes in each of the studied
31
tumor cell lines and that all cell lines had significant HLA upregulation in the IFNγ and
Selumetinib combination treatment (Fig. 4, Fig. 5). The downstream proteins in the MEK
pathway responsible for HLA induction are likely master regulators in the same vein as STAT1
[4]. HLA class II induction is notable in this case, especially considering that the origin of the cell
lines are not antigen presenting cells. HLA induction via inhibition is likely to be a restoration of
the native HLA profile, as only cell lines with a downregulation of HLA saw an increase in the
presence of inhibitors. This makes the induction of HLA class II more interesting, as the native
expression profile would not bear HLA class II. As HLA class I and class II increase at similar
rates, it is likely that thier expression is linked in the tumor cell lines. Induction of HLA class II
has been noted in tumors not arising from class II presenting cells upon IFNγ treatment [27],
which sets a precedent for cytokine induction of the non-native expression profile. The non-
malignant FLF cells also displayed class II induction when stimulated with IFNγ, but not
inhibitors alone, (Fig. 4, Fig. 5) highlighting that HLA induction from IFNγ is not a restoration of
native HLA expression, but rather an induction of an entirely new profile.
CHAPTER 6 – FUTURE IMPLICATION
6.1 PROTEINS DOWNSTREAM OF ERK SIGNALING
Several downstream proteins have yet to be investigated, and so the causative protein
of HLA indication may have not been identified yet. Additional targets should be examined,
most notably including the RSK enzyme. RSK is another kinase downstream of ERK1/2 that can
translocate to the nucleus upon activation [24]. RSK has not yet been tied to HLA regulation,
but its location in the MEK signal transduction cascade makes it a logical target (Fig. 2). Kinase
32
inhibition is more likely to yield HLA upregulation than investigating individual transcription
factors, as kinases have more varied functions than any individual transcription factor.
Moreover, cancer cells are highly dysregulated systems which makes identifying individual
transcription factors difficult.
That being said, transcription factors are the proteins responsible for transcription of
new genes, which may be the method by which HLA surface expression is induced. One of the
primary transcription factors downstream that has strong ties to HLA expression is class II
transactivating protein (CIITA). CIITA functions as a transcription factor but also has some
kinase activity [21]. Loss or reduction of CIITA protein leads to a downregulation of MHC class II.
Higher MAPK activity leads to an increased level of phosphorylated CIITA, which has been
shown to cause a marked decrease in its function [14]. Activation of CIITA requires STAT1
signaling via IFNγ signaling [28]. CIITA does not bind DNA, but interacts with chromatin
remodeling elements and transcription factors such as RFX, NF-Y, AP1, and CREB to achieve HLA
upregulation. CIITA protein can also be downregulated through histone deactylase enzymes
modifying the histone to reduce transcription, as is the case in diffuse large B cell lymphomas
[13]. CIITA is vital to the expression of MHC Class II, and its contribution to MEK inhibition
induced HLA expression should be investigated [14].
It may be worthwhile to investigate and identify additional molecules with stimulatory
HLA induction. IFNγ stimulation led to a significant increase in HLA, more so than the inhibitors
alone for most of the cell lines tested (Fig. 4, Fig. 5). An increase in HLA via stimulation with
IFNγ also induces a new HLA profile that is significantly higher than the native expression
whereas the inhibitors seem to only restore the native profile. Higher HLA will leads to
33
increased anti-tumor activity by T cells [10][2][6], so inducing a higher expression profile will
likely increase the effect of immunotherapies. IFNγ stimulation also showed no resistance in
any of the lines tested (Fig. 8).
6.2 ADDITIONAL STUDIES
6.2.1 INCRESED TRANSCRIPTION VERSUS POST-TRANSLATIONAL MODIFICATIONS
It should be differentiated whether MEK pathway inhibition-based HLA upregulation is
the result of increased transcription or a change in post translational modifications. Running a
Western blot will reveal if cytoplasmic and surface protein levels change throughout the course
of inhibitor treatment. Running a western blot will also show the effect of each inhibitor on the
level of phosphor-ERK1/2 which will help identify if the superior HLA induction seen with
Selumetinib is the result of ERK modulation. RT-qPCR can determine total mRNA levels for
STAT’s, TAP, and HLA genes throughout the inhibitor treatment, which will show if transcription
levels are altered. Additionally, an in vitro ubiquitination assay will show if the HLA is being
transcribed but degraded through the ubiquitin pathway. This will inform further
experimentation as to the avenue studies should pursue.
If the previously mentioned studies reveal that HLA induction is the result of increased
transcription and translation, then promoter elements will be investigated. Promoter sequence
of the HLA-A, B, and C genes were examined for transcription factor binding utilizing Qiagen®
Biobase Transfac software [36], which revealed Kruppel-like factor (KLF) as a prominent
transcription factor.
34
6.2.2 RNAi TO VALIDATE INHIBITOR FUNCTION
The failure of Fludarabine to cause significant inhibition of the IFNγ-induced HLA
upregulation in HT29 and A431 merits further investigation. Inhibitors commonly have off-
target effects, and cancers can develop resistance to individual inhibitors. Inhibitors also have
toxic effects that confound results (fig. 6, Fig. 7). This is commonly observed across many
inhibitors and therefore each inhibitor should be validated with a corresponding RNAi,
particularly STAT1. To show if STAT1 is not responsible for the IFNγ-induced HLA upregulation,
shRNA can be utilized to knockdown STAT1 mRNA and the subsequent protein definitively,
which is pertinent for the A431 and HT29 cell lines (Fig. 9, Fig. 10). This strategy can be used to
verify the results presented in this manuscript.
6.2.3 IN VIVO EXPERIMENTS
The results presented in this manuscript were all performed in vitro. Ultimately the aim
of these analyses is to determine what happens in each human patient. To determine if in vitro
results are recapitulated in vivo, it is proposed that mouse models will be utilized. Mice will be
inoculated with human tumors shown to have lost HLA expression, treated with a regiment of
inhibitors and r-hIFNγ, sacrificed, and tumors assessed for HLA expression utilizing
immunohistochemistry. This will show if a live system can recapitulate in vitro data in which
soft loss of HLA can be reversed by treatment, thereby optimizing immunotherapy protocols
applied to patients.
35
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Abstract (if available)
Abstract
Selumetinib is a specific non-ATP dependent, small molecule inhibitor against the mitogen-activated protein kinase kinase 1 (MEK1) isoform. Treatment with Selumetinib has shown the ability to abate tumor growth to a significant degree in KRAS mutation bearing tumors in phase II clinical trials. Tumors treated with Selumetinib not only display a marked reduction in proliferation, but have also been shown to induce an upregulation in surface expression levels of human leukocyte antigen (HLA) Class I. Loss of HLA class I and II are commonly observed in carcinomas and represents a significant hurdle in the treatment of solid tumors. The mechanism by which Selumetinib inhibits cellular growth is well documented, but how exactly MEK1/2 inhibition leads to HLA upregulation is not known. Understanding the mechanism by which Selumetinib upregulates HLA would allow for a better understanding of tumor development as well as superior remedies. Here it is shown that HLA upregulation in tumors with overactive MAPK signaling is accomplished through ERK1/2 inhibition. By contrast, inhibition of downstream oncoproteins had little effect on surface HLA levels thereby dispelling the notion that oncoproteins are involved with HLA downregulation. Furthermore, inhibition of MEK1/2 and ERK1/2 led to nearly identical induction levels of HLA Class I and Class II. Multiple cell lines displayed an innate resistance to MEK1/2 inhibition-induced HLA upregulation, despite carrying the HLA genes. All cell lines analyzed responded to IFNγ with upregulation of HLA class I and class II, and a synergistic upregulation was noted in dual treatment of Selumetinib and IFNγ. Interestingly, two populations were noted in the HT29 cell line, one resistant and one susceptible to MEK inhibition-induced HLA upregulation. Future studies merit elucidation of specific downstream proteins that mechanistically regulate HLA expression.
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Diaz, Michael Andrew
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Core Title
HLA upregulation via inhibition of the MEK1/2 pathway
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Experimental and Molecular Pathology
Publication Date
09/25/2017
Defense Date
09/24/2017
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ERK1/2,HLA,major histocompatability complex class I,major histocompatability complex class II,MEK1/2,OAI-PMH Harvest
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ERK1/2
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major histocompatability complex class II
MEK1/2