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Maternal embryonic leucine zipper kinase (MELK) as a novel therapeutic target in the treatment of acute lymphoblastic leukemia

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Maternal embryonic leucine zipper kinase (MELK) as a novel therapeutic target in the treatment of acute lymphoblastic leukemia

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Content 1

MATERNAL EMBRYONIC LEUCINE ZIPPER KINASE (MELK) AS A
NOVEL THERAPEUTIC TARGET IN THE TREATMENT OF ACUTE
LYMPHOBLASTIC LEUKEMIA
by

Rucha Deo

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
MASTER OF SCIENCE
(Molecular Pharmacology and Toxicology)
Graduation: Summer 2017
Degree Conferral: August 2017

ii

Acknowledgments

I am grateful to my advisor, Dr. Houda Alachkar, for her guidance in research and thesis. She provided
extensive training on how to be a scientist and motivated me to think critically and broaden my horizon.
I thank the members of my master’s degree committee, Dr. Curtis Okamoto and Dr. Paul Beringer, for their
guidance.
I am grateful to my lab members: Vijaya Pooja Vaikari, Tian Zhang, Sharon Wu, Lucas Gutirrez and John
Beckford for their support and assistance at all points of time.
Finally, I would like to express gratitude to my family and friends for their love and support, without which I
wouldn’t have been able to study, work and persevere in the challenges faced while pursuing a Master’s
degree.

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Table of Contents
Title Page Number
Acknowledgments ii
List of Figures iv
List of Tables v
List of Abbreviations v
Abstract vi
Introduction 1
Hematopoiesis 1
Acute Lymphoblastic Leukemia 2
Maternal Embryonic Leucine Zipper Kinase 11
Forkhead Box M1 12
β-catenin 13
Materials and Methods 15
Results 17
MELK is overexpressed in Acute Lymphoblastic Leukemia patients 17
MELK is overexpressed in Acute Lymphoblastic Leukemia cell lines 21
Effect of MELK inhibitor on Acute Lymphoblastic Leukemia cell lines 23
Effect of MELK inhibitor on protein expression in Acute Lymphoblastic Leukemia
cell lines
24
Effect of MELK overexpression on β-catenin 27
Interaction between MELK and β-catenin 29
Discussion 30
References 33

iv

List of Figures
A. Hematopoiesis process
B. Age wise incidence of Acute Lymphoblastic Leukemia
C. Genetic abnormalities in childhood and adult leukemia patients

Figure 1. MELK is overexpressed in Acute Lymphoblastic Leukemia patients
Figure 2. Protein expression in Acute Lymphoblastic Leukemia cell lines
Figure 3. Effect of MELK inhibitor on Acute Lymphoblastic Leukemia cell lines
Figure 4. Effect of MELK inhibitor on protein expression in Acute Lymphoblastic Leukemia lines
Figure 5. Effect of MELK overexpression on the expression of downstream target proteins
Figure 6. Interaction between MELK and β-catenin
v

List of Tables

Table 1. Signs and Symptoms of Acute Lymphoblastic Leukemia
Table 2. Cytogenetic abnormalities in Acute Lymphoblastic Leukemia
Table 3. Drugs used in the treatment of Acute Lymphoblastic Leukemia

List of Abbreviations

ALL Acute Lymphoblastic Leukemia
AML Acute Myeloid Leukemia
B-ALL B-cell Acute Lymphoblastic Leukemia
FOXM1 Forkhead Box M1
GSK3B Glycogen Synthase Kinase 3-B
IP Immunoprecipitation
MELK Maternal Embryonic Leucine Zipper Kinase
PGSK3B Phosphorylated Glycogen Synthase Kinase 3-B
Ser/ Thr Serine/ Threonine
T-ALL T-cell Acute Lymphoblastic Leukemia
TCF/ LEF T-cell factor/ Lymphoid enhancing factor

vi

Abstract
Acute Lymphoblastic Leukemia (ALL) is a hematological malignancy that affects children and young
adults. Treatment is possible through chemotherapy and stem cell transplant, however there is still the
risk of relapse and resistance. Although most children are cured, the cure rate in adults is very low.
Hence, there is a need for novel therapeutic targets to improve outcome of ALL in adults. Maternal
Embryonic Leucine Zipper Kinase (MELK) is a protein kinase that is found to be overexpressed in a
variety of solid tumors. A recent study reported MELK to be a therapeutic target in Acute Myeloid
Leukemia (AML). However, the role of this kinase in ALL is not well investigated. Previous studies
showed FOXM1 to be a substrate for MELK. The downstream targets for FOXM1 include the Wnt/ β-
catenin pathway. The Wnt pathway is known to be implicated in T-ALL. Hence, we hypothesized that
MELK may act as a therapeutic target in ALL by inhibiting FOXM1 and subsequently, the Wnt/ β-catenin
pathway. We found that MELK is overexpressed in ALL compared to mononuclear cells obtained from
healthy donors. Inhibiting MELK kinase activity resulted in decreased cell viability. MELK inhibition
decreased p-GSK3B and β-catenin. While these results suggest a possible role of MELK in the β-catenin
signaling pathway, further studies are needed to establish the mechanism by which MELK affects β-
catenin activity. MELK inhibitor is currently in trials for solid tumors as well as hematological
malignancies. Hence, more research is needed to find the exact mechanism by which MELK inhibition is
effective therapeutic approach in ALL.

1

INTRODUCTION
Hematopoiesis
Hematopoiesis is the process of blood formation. All blood cells are derived from hematopoietic stem
cells (HSCs). HSCs are pluripotent stem cells that are in the medulla of the bone marrow. The
hematopoietic stem cells differentiate into myeloid and lymphoid progenitors, which in turn differentiate
and give rise to mature blood cells in the bone marrow or tissues. The figure (xx) shows the process of cell
differentiation from HSCs and the types of blood cells that arise.

The process of cell division initiating from HSCs is called asymmetric division, as some of the daughter cells
do not get converted to lymphoid and myeloid progenitors and remain as HSCs. The lymphoid progenitors
may later differentiate to natural killer (NK) T cells or to B and T lymphocytes. B cell development includes
development initiated at the level of lymphoid-primed multipotent progenitors, common lymphoid
progenitors, pro–B cells, pre–B cells, and mature B cells. This maturation process is tightly controlled by
the hierarchical activation of transcription factors and selection through functional signal transduction
(Zhou et al, 2012)
Apart from their sites of maturation, B and T cells are characterized by the presence of specific receptors
and antigens on the cell surface.
The maturation of bone-marrow progenitor cells to mature B lymphocytes proceeds through stages that
can be identified by the pattern of cellular immunoglobulin protein and cell-surface antigen expression
(Lieben, 2000). The generation of a lymphoid progenitor that expresses CD34, CD10 and low levels of CD19
is the primary step preceding the development of the first identifiable cell committed to B-lineage
Fig. A: Hematopoiesis in humans. The process of formation of different type of blood cells
from hematopoietic stem cells.
Reference: Mikael Häggström, July 2009
2

differentiation. The early pre-B or pro-B cells express CD10, CD19, CD24, CD34, CD45, human leukocyte
antigen (HLA)-DR, cytoplasmic CD22, CD79a, CD79b, and nuclear terminal deoxynucleotidyl transferase
(TDT). Young developing pre-B lymphocytes are the first B-lineage cells to express cell-surface CD22,
followed by the expression of CD20. Lymphocytes in the late stages of pre-B-cell maturation (transitional
pre-B cells) weakly express surface -heavy chains accompanied by the non-covalently linked pseudo-light
chains CD179a and CD179b. The expression of TDT, CD34 and CD10 are lost as the pre-B stage ends.
The maturation of bone-marrow-derived T-cell precursors (pro-T-cell) takes place in the thymus through
stages that can be recognized by the sequential expression of T-cell receptor gene transcripts and cell-
surface antigens. T cells follow a very specific path of maturation in the thymic region. Pro-T cells that
express CD34, CD7, CD33, HLA-DR and possibly CD2 with T-cell receptor genes in germline configuration
arrive in the outer cortical layer of thymic lobules. Following that, they move through the cortex to the
cortico-medullary junction and take up residence in the medulla. During this journey, T-cell precursors
encounter various chemokines and thymic stromal and epithelial cells, and they subsequently undergo
differentiation and maturation (Gill et al, 2003). The earliest cells committed to T-cell development, pre-
T cells are found in outer cortical areas of the thymic lobules. They express CD34, CD7, CD2, cytoplasmic
CD3 and TDT, but not CD4 or CD8 (double-negative cells). As these cells mature, they lose CD34 while
gaining CD4 and then CD8, becoming double-positive early cortical thymocytes that express CD1a, CD10
and low levels of CD3. Most of these cells fail to express a functional T-cell receptor and die by apoptosis.
Thymocytes with functional T-cell receptors gain CD3 expression (late cortical thymocytes) and proceed
through a final stage of differentiation in the medullary of the thymus where they downregulate the
expression of either CD4 or CD8 to become mature single-positive T cells (Pui et al, 2007).
Acute Lymphoblastic Leukemia
Acute lymphoblastic leukemia, also known as Acute Lymphocytic Leukemia (ALL) is a malignant disorder
which affects the cells of the hematopoietic system. ALL is characterized by overproduction of
lymphoblasts in the bone marrow. The antigen-expression profiles of leukemic lymphoblasts parallels the
normal stages of B- and T-cell differentiation and maturation. The immature blasts multiply and
accumulate in the bone marrow causing damage by infiltrating other organs (Crazzolara et al, 2001).

Acute lymphocytic leukemia is most common in children, adolescents, and young adults, or those 15 to
39 years of age. ALL is most common in Hispanics and Whites. The number of new cases of acute
lymphocytic leukemia was 1.7 per 100,000 men and women per year based on 2010-2014 cases. Figure B
represents the number of new cases diagnosed between 2010-2014 (www.seer.cancer.gov) .

3

Every year about about 6000 new cases of ALL are diagnosed in the United States (Pui et al,2006). The
median age at diagnosis is 15 years, and most of the cases are diagnosed in children and young adults, in
whom it represents the most common malignancy. ALL represents 0.4% of all new cancer cases in the US
in 2017. Using statistical models for analysis, rates for new acute lymphocytic leukemia cases have been
rising on average 0.6% each year over the last 10 years. Death rates have been falling on average 1.1%
each year over 2005-2014. (www.seer.cancer.gov)

Signs and Symptoms
Pediatric leukemia often presents with non-specific symptoms mimicking common, self-limiting illnesses.
This complicates the challenges faced by front line clinicians at the time of diagnosis (Dang-tan et al, 2007).
The early diagnosis of leukemia is an important step for the timely and correct treatment.
Clarke et al conducted a meta-analysis in 2016 where they analyzed published data from 33 studies
including 3084 patients for the reported signs and symptoms among pediatric ALL. They grouped the
symptoms into seven distinct clinical categories per the main underlying pathological process or body
system affected: infiltrative, hemorrhagic, infective, systemic, musculoskeletal, gastrointestinal and
cutaneous. The most common infiltrative symptoms were hepatomegaly and splenomegaly. Bruising was
the most common hemorrhagic symptom. Fever was the most common infective symptom, and the most
prominent musculoskeletal features were limb pain and bone pain. Systemic features like pallor and
fatigue were also common. Finally, the most common gastrointestinal feature, anorexia/weight loss, was
present in almost a third of children. Given below is a table of the frequencies of the signs and symptoms
that the authors reported (Clarke et al, 2016).
Table 1: Signs and Symptoms for Acute Lymphoblastic Leukemia
Hepatomegaly 64%
Splenomegaly 61%
Fig. B: Age wise distribution of all
patients (all races, both sexes)
affected by ALL from 2010-2014.
Reference: www.seer.cancer.gov
4

Bruising 52%
Fever 53%
Limb Pain 43%
Bone Pain 26%
Pallor 54%
Fatigue 46%
Anorexia/ Weight Loss 29%
Asymptomatic at diagnosis 6%

Additionally, between a third and half of the children presented with the following additional symptoms
(frequencies in parentheses): Recurrent infections (49%), hepatosplenomegaly (42%), petechiae (42%),
lymphadenopathy (41%), bleeding tendency (38%) and rash (35%).

Molecular subtypes of ALL
B cell precursor leukemia is the most common pediatric leukemia. It is the leading cause of death among
children and young adolescents with almost 4000 new cases being diagnosed every year. Almost 80% of
the patients are cured, however disease relapse is a serious shortcoming in the treatment regimen (Pui et
al, 2008). Usually only 40% adults survive for more than 5 years. A common cause of relapse, among both
children and adults is thought to be poor targeting of leukemia stem cells. (Pui et al, 2007) Leukemic stem
cells are believed to be resistant to normal chemotherapy. (Cobaleda et al, 2009)
T cell acute lymphoblastic leukemia accounts for 15% of all the cases of childhood leukemia, and about
25% in adult patients. (Pui et al, 2004) Patients usually have organomegaly, especially the thymus, which
may cause breathing problems. T-ALL is also known to spread in the cerebrospinal fluid. The European
Group for Immunological Classification of Leukemia (EGIL) recognizes the differentiation stages of the
neoplastic clone: pro-T, pre-T, cortical and mature T-ALL. (Bene et al, 1995) The cortical stage has been
associated with a better outcome. Efforts have been made to identify additional antigens of prognostic
significance that may drive therapeutic decisions.
ALL has many subtypes and can be classified using morphologic, immunologic, cytogenetic, and molecular
genetic methods (Pui et al, 2004). These approaches of classification can identify biologic subtypes that
5

require different treatment approaches. These differences include the specific drug combination, drug
dosages, and duration of treatment required to achieve optimal results. (Pui et al, 2004)
Immunophenotyping is an essential component of the diagnostic work-up of ALL and it can be used to
subclassify cases according to the recognized stages of normal B- and T-cell maturation. Clinically, the only
distinctions with therapeutic importance are those between T-cell and B-cell precursors (including the
early pre-B, pre-B and transitional pre-B immunophenotypes) and between these cell types and mature B
cells. The antigen-expression profiles of leukemic lymphoblasts parallels the normal stages of B- and T-
cell differentiation and maturation.
The clinical heterogeneity of the disease course and outcome, especially when comparing pediatric and
adult populations, reflects different biological subtypes (Zuckerman et al, 2014). Though the frequency of
genetically defined leukemia subtypes differs between children and adults, the underlying mechanisms of
leukemia transformation are the same. They include the aberrant expression of proto-oncogenes,
chromosomal translocations that create fusion genes encoding active kinases and modified transcription
factors, and aneuploidy (Pui et al, 2004; Armstrong et al, 2005). Such abnormalities cause ALL by imposing
an unlimited capacity for self-renewal, a loss of normal proliferative control, a block in cell differentiation
and resistance apoptosis in lymphoid progenitor cells (Pui et al, 2004). Figure C illustrates the genetic
abnormalities in childhood and adult ALL. (Pui 2007)
6

ALL is characterized by gross numerical and structural chromosomal abnormalities, including
hyperdiploidy (>50 chromosomes), hypodiploidy (<44 chromosomes), translocations t{[12;21], [1;19],
[9;22], [4;11]} and rearrangements (MYC, MLL). (Armstrong et al, 2005)
Recurrent genetic abnormalities have prognostic and therapeutic implications, and provide insights into
the mechanisms of leukemogenesis. Studies of global gene expression in ALL have enabled the
classification of the acute leukemias into prognostically and therapeutically important subtypes and have
also identified candidate molecules for targeted therapeutics. (Pui, 2004; Armstrong 2005). The recent
demonstration of distinct mRNA- and microRNA-expression profiles for commonly recognized genetic
Fig C: Genetic abnormalities in
childhood and adult ALL
Ref: Nature Reviews, Pui et al,
2007

7

subtypes of ALL has substantiated the specificity of the genetic abnormalities (Ferrando et al, 2002; Yeoh
et al, 2002, Haferlach et al, 2005).
Primary oncogenic events, such as those triggered by chromosomal rearrangements, are generally
insufficient by themselves to cause leukemia and require secondary cooperative mutations to generate a
fully transformed cell. For example, rearrangements such as t(12;21), ETV6-RUNX1, comprising 22% of
pediatric ALL, are present years before the development of leukemia (Moorman et al, 2010; Ma et al,
2013). Many of the genes involved encode proteins with key roles in lymphoid development. It is
suggested that the initial event confers self-renewal coupled with mutation, leading to developmental
arrest and a secondary cooperative event in cell cycle regulation, tumor suppression and chromatin
modification, eventually leading to establishment of the leukemic clone (Lo Nigro L, 2013; Greaves et al,
2002).
Secondary genetic alterations identified most often in leukemic cells include the overexpression
of FLT3 receptor tyrosine kinase in mixed-lineage leukemia gene (MLL)-rearranged or hyperdiploid B-
lineage ALL (Yeoh 2002, Armstrong et al 2002), NOTCH1-activating mutations in T-cell ALL (Weng et al,
2005) and mutations in components of interrelated pathways controlled by the tumor suppressor
proteins like p53 ( Sherr et al, 2002; Krug et al, 2002, Vousden et al, 2002).
Although genetic abnormalities of leukemic cells do not entirely account for treatment outcome, they still
provide essential prognostic information. In general, the presence of the Philadelphia chromosome with
BCR–ABL fusion, the t(4;11) with MLL–AF4 fusion, and hypodiploidy (less than 45 chromosomes per
leukemic cell) confer a poor outcome, whereas hyperdiploidy (more than 50 chromosomes) and TEL–
AML1 fusion are associated with a favorable prognosis. The high frequency of unfavorable genetic
features and low incidence of favorable genetic abnormalities in adult cases of ALL partly explain their
inferior overall outcome as compared with childhood cases. (Pui et al, 2007)
Table 2 details major known abnormalities in B- and T-ALL and their prognostic significance. (Zuckerman
et al, 2014)
Recent discoveries in the genomic landscape of ALL and its influence on prognosis include: the BCR-ABL1-
like ALL, cytokine receptor-like factor 2 (CRLF2) over expression and Janus kinase (JAK) mutations,
hypodiploid ALL and T-ALL (Asselin et al, 2013; Mullighan CG, 2013).

Table 2: Major Cytogenetic abnormalities in Acute Lymphoblastic Leukemia (Ref: Zuckerman et al, 2014)

Cytogenetic
abnormality
Target gene Frequency in
pediatrics %
Frequency in
adults %
Prognosis
t(1;19)(q23;p13) E2A-PBX1 4-6 2-3 standard
t(9;22)(q34;q11) BCR-ABL1 3-5 25-30 unfavorable
8

Small-molecule inhibitors have already been developed to target some of these primary and secondary
molecular genetic abnormalities. studies are underway to ascertain the precise events that take place in
the genesis of ALL, to enhance the clinical application of known risk factors and antileukemic agents, and
to identify treatment regimens that might boost the generally low cure rates in adults and subgroups of
children with high-risk leukemia. (Pui et al, 2008)
The cure rate for children with ALL is almost 80%, leading most patients to be healthy long term cancer
survivors. (Pui et al, 2006). However, leukemia cannot be added on to the list of ‘completely cured’ cancers
owing to the poor prognosis in adults and the complexity, expense and toxic effects of contemporary
multiagent treatment regimens. (Pui et al, 2008)
Unfortunately, the cure rates in adult ALL seldom exceed 40 percent, despite the use of hematopoietic
stem-cell transplantation in many cases. The poor outcome in adult ALL has been variously attributed to
an increased frequency of high-risk leukemia with greater drug resistance, poorer tolerance of and
compliance with treatment, reluctance to accept certain temporary toxic effects, and less effective
treatment regimens, as compared with childhood ALL (Pui et al, 2006).

Treatment:
The earlier ALL is detected, the more effective the treatment. The aim is to induce a lasting remission,
defined as the absence of detectable cancer cells in the body (usually less than 5% blast cells in the bone
marrow). Treatment for acute leukemia can include chemotherapy, steroids, radiation therapy, intensive
combined treatments (including bone marrow or stem cell transplants), and growth factors. The 5-year
survival rate for children who have ALL has improved from zero six decades ago, to 85% currently, largely
because of clinical trials on new chemotherapeutic agents and improvements in stem cell transplantation
(SCT) technology. (Park KD, 2014)
t(4;11)(q21;q23) MLL-AF4 2-3 3-7 unfavorable
High hyperdiploid

20-30 7 favorable
Hypodiploid

5-6 3 unfavorable
t(12;21) ETV6-RUNX1 25 – favorable
T-ALL TCR

T(7;14)(14q;7q34 or
7p14)
Non-TCR(NOTCH1,HOX11,
JAK1)
60%

favorable
9

Table 3: Commonly used drugs to treat Acute Lymphoblastic Leukemia
Drug Class Mechanism of Action Mechanism of Resistance
Asparaginase Inhibition of protein synthesis by
catalyzing the hydrolysis and
hence depletion of asparagine,
and perhaps glutamine
Induction of asparagine
synthetase; increased
uptake of
glutamine/aspartic acid or
decreased efflux of
asparagine by
transmembrane
transporters
Cyclophosphamide Alters structure and function of
DNA by cross-linking or
fragmenting of DNA strands
Increased degradation by
aldehyde dehydrogenase;
increased conjugation with
glutathione or glutathione
transferase; increased
nucleotide-excision repair
Cytarabine Incorporating into DNA and
inhibiting DNA polymerase
Decreased activity of
deoxycytidine kinase;
decreased drug uptake;
increased deamination;
increased pool size of
competitive deoxycytidine
triphosphate
Anthracyclines
(Doxorubicin, Daunarubicin)
Forms a complex with DNA and
topoisomerase II , leading to
double-stranded DNA breaks;
forms free-radicals that cause
DNA damage; activation of
caspases and other apoptotic
signalling molecules
Increased drug efflux by
transmembrane
transporters; increased
topoisomerase II enzymes;
increased detoxification of
reactive oxygen species;
increased DNA repair;
blockade of apoptotic
signaling
Methotrexate Blocks DNA synthesis by
inhibiting dihydrofolate
reductase (DHFR) and
thymidylate synthase, enzymes
involved in the folate
metabolism; and by inhibiting de
novo purine synthesis
Decreased membrane
transport due to
polymorphisms in the
reduced folate carrier (RFC);
increased drug efflux by
transmembrane
transporters; gene
amplification of DHFR;
polymorphisms in
thymidylate synthase;
decreased formation of
methotrexate
polyglutamates (active
metabolites)
10

(Ref: Pui et al, 2007)

Most of the conventional drugs used to treat ALL either target DNA directly or inhibit nucleic acid
synthesis. Some block protein synthesis by hydrolyzing an amino acid essential for leukemic cell growth
or by interfering with the mitotic spindle apparatus. Such drugs have a narrow therapeutic index and often
produce adverse cytotoxic effects in various normal tissues due to their non-specificity. Efforts to improve
the effectiveness of commonly used antileukemic drugs continue at a substantial pace. Encapsulating
drugs into liposomes is one such strategy used with increasing frequency. Following are the commonly
used drugs in the treatment of ALL along with their modes of action and resistance. (Pui et al, 2007)
To overcome the side effects and resistance, a new class of compounds, via targeted molecular therapy
was recently introduced to treat cancer. Many kinases have been found to be involved in the processes
leading to tumor cell proliferation and survival. The deregulation of kinase function has emerged as a
major mechanism by which cancer cells evade normal physiological constraints on growth and survival.
Currently, several therapeutic kinase inhibitors, including antibodies or small molecules that block the
interactions between kinases and substrates, have already been developed and approved for cancer
treatment (Wu et al, 2015; Gross et al, 2015). For example, Imatinib (Tyrosine kinase inhibitor), Dasatinib
(Tyrosine kinase inhibitor) , Ponatinib (Multi-targeted tyrosine kinase inhibitor) are currently used as
targeted therapy for ALL.
It is therefore a promising strategy to comprehensively identify and functional characterized oncogenic
kinases associated with ALL to enable the development of small molecule kinase inhibitors for the
molecularly-targeted treatment of ALL

Glucocorticoids
(Prednisone, Dexamethasone)
Transactivation or
transrepression of genes by
binding glucocorticoid–
glucocorticoid receptor complex
(GCR) to glucocorticoid
responsive elements in
promoters of the genes
Decreased binding to GCR;
impaired translocation of
GCR to nucleus; reduced
transrepression or
transactivation capacity of
GCR; increased drug efflux
due to overexpression of
transmembrane
transporters
Vincristine Inhibition of mitotic spindle
formation by binding to tubulin
Increased drug efflux by
transmembrane
transporters; mutant
tubulin with decreased
avidity of vincristine binding
11

Maternal Embryonic Leucine Zipper Kinase (MELK)
Maternal Embryonic Leucine zipper Kinase (MELK), also known as MPK38, belongs to the AMP activated
serine threonine protein kinase family. (Heyer et al,1997 ; Boduel et al, 2010). MELK is a member of the
Snf1/AMPK family of kinases, but is unique in that it is not regulated by LKB1 kinase. The kinase activity of
other AMPK/Snf1 family members is dependent on phosphorylation in their activation loop by upstream
kinases (such as LKB1 or CaMKK2). Interestingly, MELK is activated by auto-phoshorylation in
vitro [Lizcano et al, 2004]; a unique mechanism among the AMPK/Snf1 family members. In addition to a
Ser/Thr kinase domain, MELK contains an unstructured C-terminal ubiquitin associated (UBA) domain that
prevents ubiquitin-mediated degradation (Boudrez et al, 2002), a leucine zipper motif and a C-terminal
kinase-associated (KA1) domain.
MELK is found to be upregulated in many types of cancer, however it’s expression is undetectable in
normal tissues except testis (Lin et al, 2007). It is shown to be commonly expressed in poorly differentiated
aggressive cancers (Hemmati et al, 2003; Rhodes et al, 2004). During development, MELK is expressed in
tissues that contain normal progenitor cells (Heyer et al, 1999) and in adult brain progenitor cells (Terskikh
et al, 2001; Geschwind et al, 2001). The MELK gene was first cloned from a human myeloid leukemic cell
line (Nagase et al, 1996).
The in vivo function of MELK is currently unknown, however, CDC25B phosphatase, a critical G2/M
checkpoint protein, and apoptosis signal-regulating kinase 1 (ASK1) were suggested as potential MELK
targets (Mirey et al, 2005; Jung et al, 2008). MELK was found to control cell cycle progression and
proliferation likely through phosphorylation of CDC25B. (Gray et al,2005). In glioblastoma, MELK was
found to regulate the activity of its substrate FOXM1 a transcriptional factor and a master regulator of
mitosis in cancer stem cells (Joshi et al, 2013). MELK RNA is dramatically upregulated in glioblastomas and
is correlated with malignancy grade in human astrocytomas (Liu et al, 2006; Marie et al, 2008). It has been
demonstrated in vitro that MELK siRNA inhibits the growth of primary glioblastoma cell lines (Nakano et
al, 2008). Elevated expression of MELK was found to be associated with the poor prognosis of glioblastoma
patients (Nakano et al, 2008) and breast cancer patients (Pickard et al, 2009). MELK is included in three
different multi-gene expression profiles that predict breast cancer aggressiveness, prognosis, and therapy
response in the clinical setting (Koleck et al, 2016). Additionally, in multipotent neural progenitors (MNPs),
MELK is considered to be a marker of self-renewal [Nakano et al, 2005]. MELK depletion was also found
to sensitize colorectal cancer cells to radiation or 5-FU treatment [Choi et al, 2011]. MELK was found to
physically interact with and phosphorylate pro-apoptotic Bcl-G. The over expression of wild-type (WT)
MELK, but not a kinase-dead mutant, was reported to suppress Bcl-G–induced apoptosis promoting
mammary carcinogenesis (Lin et al, 2007). Furthermore, targeting MELK decreased FOXM1
phosphorylation and subsequently downregulation of FOXM1 downstream targets such as CyclinB1 and
CDC25B in acute myeloid leukemia (Alachkar et al, 2014). MELK function was demonstrated to be required
for mammary tumor growth in vivo (Hebbard et al, 2010). Based on the growth inhibition of several cancer
cell lines in vitro, MELK was proposed to be a promising target for multiple cancer types.
We hypothesized that MELK can be a novel target in the treatment of Acute Lymphoblastic Leukemia,
based on previous studies showing it to be a viable target for the treatment of solid tumors.
A competitive type I kinase inhibitor, OTSSP167 (OTS167) has been designed to inhibit MELK activity, and
its efficacy has been explored in several cancers (Chung et al, 2012). OTS167, is shown to be effective in
treating various cancers like kidney cancer (Kato et al, 2016) and Acute Myeloid Leukemia (AML) in vitro
12

(Alachkar et al, 2014). Several Phase I clinical trials with OTS167 are in process for solid cancers, different
types of leukemias, and triple negative breast cancer (clinicaltrials.gov). FOXM1 was identified as
transcription factors whose targets were differentially expressed upon treatment with OTS167. Previous
research has demonstrated that FOXM1 phosphorylation is influenced by MELK (Joshi et al, 2013). FOXM1
activity was found to be reduced after OTS167 treatment. (Simon et al, 2017)

Forkhead Box M1 (FOXM1)
Forkhead Box M1 (FOXM1) is a member of the Forkhead family of transcription factors, which are
identified by an evolutionarily conserved Forkhead/winged-helix DNA-binding domain (Korver et al, 1997).
The FOXM1 protein exists in three different isoforms. While FOXM1a is transcriptionally inactive, the
FOXM1b and FOXM1c are transcriptionally active (Yao et al, 1997).
A comparative microarray analysis identified FOXM1 as one of the most commonly overexpressed genes
in solid tumors of the prostate, lung, bladder, ovary, colon, liver, breast, kidney, stomach and pancreas
(Pilarsky et al, 2004). Aberrant expression of FOXM1 has been found in a variety of other cancers including
cervical cancer, malignant mesothelioma, glioblastoma (GBM), malignant peripheral nerve sheath tumors
(MPNST), medulloblastoma, meningioma, head and neck squamous cell carcinoma (HNSCC), oral cavity
squamous cell carcinoma (OCSCC), esophageal squamous cell carcinoma (ESCC), oropharyngeal squamous
cell carcinoma (OPSCC), papillary thyroid carcinoma (PTC), laryngeal squamous cell carcinoma (LSCC),
nasopharyngeal carcinoma (NPC) and acute myeloid leukemia (AML) (Halasi et al, 2013).
Overexpression of FOXM1 in various tumors indicates a strong dependence of the tumor cells on FOXM1,
and that is explained partly by its role in cell proliferation. FOXM1 is actively involved in regulating the cell
cycle through many processes. (Wang et al, 2005)
FOXM1 stimulates expression of SKP2 and CKS1, which are involved in the proteolysis of p27Kip1 and G1–
S progression. FOXM1 also stimulates expression of several genes that are critical for the G2–M
progression like Plk1, Aurora B, Cyclin B1, CDC25B, CENP-A, and Survivin (Wang et al, 2005). FOXM1
expression is restricted to proliferating cells. Interestingly, FOXM1 itself is regulated during the cell cycle.
The transcriptional activation function of FOXM1 depends upon phosphorylation by cyclin and cyclin-
dependent kinases (CDK) and by the Plk1 kinase. FOXM1 is phosphorylated in the C-terminal activation
domain by cyclin/CDKs, which serves as priming phosphorylation for further phosphorylations by Plk1
(Chen et al, 2009; Fu et al, 2008). Mutations of the cyclin/CDK or the PLK1 phosphorylation sites render
FOXM1 transcriptionally inactive (Chen et al, 2009; Fu et al, 2008). The transcriptionally active
phosphorylated FOXM1 accumulates as the cells progress through the cycle (Chen et al, 2009; Park et al,
2008). At the end of M-phase, FOXM1 becomes dephosphorylated (Fu et al, 2008), and in early G1-phase
of the next cycle, it is polyubiquitinated by APC/C-Cdh1 for degradation by the proteasome. The
degradation of FOXM1 in the early G1-phase is important for regulated entry into S-phase (Park et al,
2008). Thus, in proliferating cells, FOXM1 is synthesized and degraded in every cycle of cell division.
MELK regulates the cell cycle progression and mitosis-related genes through FOXM1 pathway (Xia et al,
2016).
FOXM1 expression is also induced by oncogenes. RAS increases expression of FOXM1 by inducing the
cellular levels of the reactive oxygen species (ROS), which were shown to activate FOXM1 even on their
13

own. Following induction by ROS, FOXM1 functions in a negative feedback loop to reduce the levels of
ROS by stimulating expression of the antioxidant genes Superoxide Dismutase (SOD), Catalase, and
Peroxiredoxin 3 (PRDX3). This ROS-regulatory function of FOXM1 protects proliferating normal or tumor
cells from oxidative stress and promotes survival. Consistent with that notion, tumor cells expressing ROS-
inducing oncogenes (such as RAS or AKT) are addicted to FOXM1 for their survival (Park et al, 2009).
Interestingly, the tumor cells overexpressing FOXM1 are resistant to apoptosis or premature senescence
induced by oxidative stress, which has strong implications in resistance to chemotherapy.
The downstream target pathways of FOXM1 include the vascular endothelial growth factor (VEGF), matrix
metalloproteinase-2 (MMP-2) and Wnt/ β-catenin pathways. These are all known to be involved in tumor
formation and progression (Joshi et al, 2013). FOXM1 enhances cancer stem cell self-renewal through
direct binding to β-catenin inducing nuclear localization and transcriptional activity (Zhang et al, 2011)

β-catenin
Wnt Signaling is the fundamental mechanism directing cell proliferation, cell polarity and cell fate
determination during embryonic development and tissue homeostasis. Hence, mutations in the Wnt
pathway are often linked to birth defects, cancer and other diseases in humans (MacDonald et al, 2009).
The canonical Wnt pathway is found to be impacted in many solid tumors like breast, glioma, ovarian and
lung cancer. (Morin, 1999) The phosphorylation and degradation of cytosolic Β-catenin , and its regulation
by Wnt are the essence of Wnt signaling.
β-catenin accumulates in the cytoplasm through the Wnt signaling pathway and gets translocated to the
nucleus and acts as a transcriptional co-activator for TCF/LEF transcription factors. In the absence of Wnt,
β-catenin does not accumulate in the cytoplasm as it gets degraded by the destruction complex consisting
of Axin, GSK3B, CK1A, APC. The APC/β catenin pathway is highly regulated and includes players such as
GSK3-b, CBP, Axin, Conductin, TCF. c-MYC and cyclin D1 were identified as key transcriptional targets of
this pathway (Morin, 1999). Axin interacts with with GSK3, CK1α, and β-catenin and coordinates the
sequential phosphorylation of β-catenin at serine 45 by CK1α and then at threonine 41, serine 37 and
serine 33 by GSK3 (Kimelman and Xu, 2006).
β-catenin phosphorylation at serine 33 and 37 creates a binding site for the E3 ubiquitin ligase β-Trcp,
leading to β-catenin ubiquitination and degradation (MacDonald et al, 2009). Studies in colon cancer
suggest that GSK3beta is required for β-catenin down-regulation. (Rubinfield et al, 1996).
Mutations of β-catenin at and surrounding these serine and threonine residues are frequently found in
cancers, generating mutant β-catenin that escapes phosphorylation and degradation (MacDonald et al,
2009).
Deregulation of β-catenin signaling is an important event in the genesis of several malignancies, such as
colon cancer, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer and prostate
cancer (Morin, 1999). Βcatenin mutations appear to be a crucial step in the progression of a subset of
these cancers, suggesting an important role in the control of cellular proliferation or cell death. Increased
levels of beta catenin are found to be correlated with a poor prognosis in breast cancer. The Wnt/β-
catenin pathway is implicated in mammary tumorigenesis (Liu et al, 2004).
14

Wnt and NOTCH pathways are often known to be dysregulated in T-ALL. A previous study demonstrated
the overexpression of active beta catenin caused leukemia development even in the absence of NOTCH
mutations, in a mouse model (Guo et al, 2007). A human study also reported patients showing high levels
of Wnt in T-ALL without activating NOTCH mutations (Ng et al, 2014). This suggests that Wnt, like NOTCH,
could be a leukemia initiating event (Staal et al, 2016).

Considering the need of suitable targeted therapy for ALL, the aim of this study was to understand the
role of MELK in ALL and establish it as a viable target for therapy. We were also interested in elucidating
a pathway for the mechanism of action for MELK inhibitor OTS167 in ALL.

15

MATERIALS AND METHODS
Cell lines
All the ALL cell lines were bought from American Type Culture Collection (ATCC), Manassas, VA. The cell
lines were passaged and maintained in our laboratory. The cells were supplemented with Roswell Park
Memorial Institute (RPMI) 1640 medium (Life Technologies, Grand Island, NY) enriched with 10% fetal
bovine serum (Life Technologies, Grand Island, NY) and 1% anti-anti (Life technologies, Grand Island,
NY). The cells were maintained in a humidified incubator at 37 degrees and 5% CO2 level.
Reagents
MELK inhibitor drug OTS167 was purchased from EMD Millipore (Temecula, CA, USA). Stock solution was
prepared by dissolving the drug in Dimethyl Sulfoxide (DMSO) (Acros Organics, New Jersey). The stock
was diluted to desired concentrations using RPMI medium.
Western Blot Analysis
Western blot analysis was performed by normalization to GAPDH expression level. Cells were lysed with
IP lysis buffer (Thermo Scientific, Waltham, MA) containing protease inhibitor (Thermo Scientific,
Waltham, MA). The proteins were separated by electrophoresis using 4-20% or 7.5% SDS-PAGE gel, and
transferred onto PVDF membrane (Biorad). The membranes were incubated with the primary antibody,
respectively: MELK antibody, FOXM1 antibody, Β-catenin anti body, GSK3B antibody, Phospho GSK3B
antibody (Cell Signaling Technology) and AKT antibody (Santa Cruz Biotechnology, CA) using 5% non-fat
dry milk or 5% Bovine Serum Albumin (BSA) (VWR, Solon, OH) per manufacturer specified conditions
and concentration. Finally, the membrane was incubated with horseradish peroxidase-conjugated
secondary antibody and protein bands were visualized by enhanced chemiluminescence detection
reagents WestDura Extended Duration Substrate (Thermo Scientific, Rockford, IL). The membranes were
visualized with the help of ChemiDoc system.
Cell Viability Assay
50,000 cells were plated in triplicates, in a flat bottomed 96 well plate (VWR, Radnor, PA) and treated
with MELK inhibitor at varying concentration. 10 ul alamarBlue (Invitrogen, Eugene, OR) was added to
each well 48 hours after treatment. The cells along with alamarBlue were incubated for 4 hours.
alamarBlue measures the amount of metabolically active cells in the solution. Metabolically active cells
have reducing properties. The alamarBlue reagent has Resozurin, a non-fluorescent and blue dye, which
upon reduction turns to Resorufin (highly fluorescent and red in color). Thus, by measuring the
fluorescence at specified wavelengths (Excitation 540 nm, Emission 580 nm), the amount of
metabolically active cells can be measured. Fluorescence was measured using BioTek Synergy H1
Multiplate reader.
The second experiment was carried out by plating 10,000 cells, in triplicates, in each well of a 96 well
late. The cells were treated with 100 nM MELK inhibitor. alamarBlue was added immediately after
treatment and the cells were incubated for 6 hours after which the fluorescence was measured.
(Excitation 540 nm, Emission 580 nm)
Transfection Experiments
293T cells were transfected with MELK and Β-catenin plasmids. pCAGGS MELK WT, MELK D150A
(mutated) and Β-catenin vectors were used for transfection. Empty pCAGGS vactor was used as control.
Calphos Mammalian Transfection kit (Takara Clontech, Mountainview, CA) was used for carrying out
transfection in 293T cells. The cells were plated in flat bottomed 6 well plates and grown in RPMI
medium with 2% FBS. Cells were then transfected by using Calcium solution, HBS and plasmid. The
16

culture medium was changed to 10% FBS the next morning. The cells were collected 48 hours post
transfection and Western blot was performed to check transfection efficiency.
Immunoprecipitation
MELK (WT and mutated) were overexpressed in 293T cells. The cells were collected 48 hours post
transfection. The pellet was lysed with IP lysis buffer. The proteins in the lysate were pulled down with
primary antibodies for MELK, Β-catenin (Cell Signaling Technology) and IgG (Santa Cruz Biotechnology,
CA). 20 ul of Agarose A/G bead solution (Santa Cruz Biotechnology, CA) were then added to the lysate
and the solution was incubated overnight with shaking at 4 degrees. As the lysate was pre-treated with
antibodies, the protein complex gets pulled down and attached to the beads. The bead pellet was
collected and washed with PBS solution. The proteins were separated by Western Blot. The anti-MELK
pull down was stained for Β-catenin antibody and Anti-Β-catenin was stained with MELK antibody. IgG
was used as a control.
Online resources
Publicly available database Oncomine was used to access datasets. Andersson Leukemia (GSE7186),
Zhang Leukemia (GSE33315) and Haferlach Leukemia (GSE13159) were analyzed for MELK and FOXM1
expression.
Statistical Analysis
Students T test was used to determine statistical significance. Microsoft Excel and Graphpad Prism 7
were used to analyze the data. Statistical significance was set at p < 0.05

17

RESULTS
MELK expression in ALL patients
We checked MELK expression in patients with ALL using publicly available gene expression database
Oncomine. We analyzed the Andersson Leukemia dataset (GEOID: GSE7186) for the expression of MELK.
This dataset contains the data from 121 pediatric ALL patients and 6 samples of PBMCs from healthy
donors. Both B-ALL and T-ALL patients showed an increased expression of MELK compred to monocytes
from healthy donors. T-ALL patients showed a significant increase in MELK expression compared to the
healthy PBMCs (p < 0.0001) (Fig 1A).
MELK was found to phosphorylate FOXM1 in glioblastoma (Joshi et al, 2013). FOXM1 is a key
transcription factor in cell proliferation and differentiation, and was found to be aberrantly expressed in
solid tumors. (Zhang et al, 2011). Additionally, Alachkar et al have reported that MELK mRNA expression
was correlated with FOXM1 mRNA expression in AML patients. Therefore, we examined the expression
of FOXM1 in patients with ALL. The Andersson leukemia dataset showed higher FOXM1 expression in
both B-ALL and T-ALL patients compared with cells from healthy donors (p = 0.0014) (Fig 1B).
We also analyzed data MELK expression from two other datasets, Zhang Leukemia (GEOID: GSE33315)
and Haferlach Leukemia (GEOID: GSE13159). The Zhang Leukemia dataset had the gene expression data
for 575 patients including 567 pediatric ALL patients and 8 healthy donors. Both MELK and FOXM1 were
expressed more in the healthy donors than ALL patients. The mean ± SEM values were higher for both,
albeit only slightly (Fig 1C and Fig 1D). The mean ± SEM value for MELK expression in healthy donors was
1.96 ± 0.19, the MELK expression values in B-All and T-ALL were 1.22 ± 0.03 and 1.182 ± 0.11
respectively. There was no statistical significance in the values of MELK expression. The p value of B-ALL
was 0.008 compared to healthy donors and was 0.76 in T-ALL. A similar trend was found in the
expression of FOXM1 where the mean for FOXM1 expression was 1.84 ± 0.14 in healthy donors and 0.87
± 0.44 in B-ALL and 1.428 ± 0.11 in T-ALL. There was no statistical significance in FOXM1 expression.
The Haferlach leukemia dataset was much larger with 2096 samples that included various subtypes of B-
ALL, T-ALL and healthy donors. MELK was shown to be expressed lesser compared to healthy donors in
all the subtypes of B-ALL. The expression values were 1.97 ± 0.06 in healthy donors and 1.88 ± 0.05 in B-
ALL, 1.46 ± 0.03 in childhood B-ALL and 1.58 ± 0.09 in pro-B-ALL. MELK was found to be expressed higher
in T-ALL, however this increased expression was not statistically significant. The values of expression
were 1.97 ± 0.06 in healthy donors and 2.17 ± 0.07 in T-ALL patients. The p value was 0.077 (Fig 1E). We
also analyzed the expression of FOXM1 in the Haferlach dataset and found it to be expressed more in
healthy donors than ALL patients (Fig 1F). Statistical significance was not observed. The mean expression
values were 1.85 ± 0.05 in healthy donors, 1.42 ± 0.05 in B-ALL, 1.14 in Childhood B-ALL, 1.21 ± 0.08 in
Pro B-ALL and 1.59 ± 0.06 in T-ALL.
However, even though the expression values for MELK were lower in ALL patients compared to healthy
donors, MELK gene was among the overexpressed genes in these studies (Zhang et al, 2012; Haferlach et
al, 2010).

18

A

B

19

C

D

20

E

F

Fig 1: MELK and FOXM1 expression in ALL patients
A) MELK is overexpressed in ALL patients compared to healthy donors (Andersson Leukemia)
B) FOXM1 is overexpressed in ALL patients compared to healthy donors (Andersson Leukemia)
C) MELK expression in ALL patients and healthy donors (Zhang Leukemia)
D) FOXM1 expression in ALL patients and healthy donors (Zhang Leukemia)
E) MELK expression in ALL patients and healthy donors (Haferlach Leukemia)
F) FOXM1 expression in ALL patients and healthy donors (Haferlach Leukemia)

21

MELK is overexpressed in ALL cell lines
MELK is known to be overexpressed in a variety of solid tumors. To validate that MELK is a viable
molecular target in ALL, we measured MELK expression in ALL cell lines. We compared MELK expression
in 6 ALL cell lines (3 B-ALL and 3 T-ALL) with 3 samples of peripheral blood mononuclear cells (PBMCs).
MELK expression was absent in PBMCs from healthy volunteer, whereas, the ALL cell lines exhibited
variable levels of MELK expression (Fig 2A). I also assessed the expression of FOXM1 in ALL cell lines. 10
ALL cell lines (4 B-ALL and 6 T-ALL) were chosen for this purpose. Fig 2B shows the immunoblot for
FOXM1 and MELK expression in ALL cell lines. Certain T-ALL cell lines like SUPT1, SUPT3, SUPT11, CEM
and MOLT4 express higher FOXM1 compared to other cell lines. B-ALL cell line REH also showed higher
FOXM1 expression. I also analyzed the expression of proteins associated with the canonical Wnt
signaling pathway like GSK3B and Phosphorylated GSK3B. Fig 2C shows the expression of these proteins.
GSK3B is known to be highly conserved across species and was expressed in all the cell lines.
Phosphorylated GSK3B, however, showed endogenous expression in the majority of the B-ALL and in
few T-ALL cell lines. The T-ALL cell lines that expressed PGSK3B were SUPT3, SUPT11, Jurkat and
MOLT16.

A

22

B

C

Figure 2: Protein expression in ALL cell lines
Western blots showing the expression of various proteins in ALL cell lines and PBMCs
A) MELK is overexpressed in B-ALL and T-ALL cell liens compared to PBMCs
B) MELK and FOXM1 are expressed in ALL cell lines
C) MELK, GSK3B are overexpressed in ALL cell lines compared to PBMCs. GSK3B is expressed in all
ALL cell lines. PGSK3B is not expressed in all ALL cell lines endogenously.

23

Effect of MELK inhibitor on ALL cell lines
As described previously, OTS167 is a potent MELK inhibitor that has been proven effective in solid
tumors and AML. We tested the effect of OTS167 on ALL cell lines. 4 ALL cell lines (2 B-ALL (KOPN-26,
REH) and 2 T-ALL cell lines (Jurkat, CEM)) and PBMCs from healthy donor were treated with increasing
concentration of MELK inhibitor at concentrations of 0 nM, 50 nM, 100 nM and 500 nM. Viability assay
was performed after 48 hours using alamarBlue reagent (Invitrogen, Eugene, OR). Fig 3A shows the
number of viable cells 48 hours after treatment. The effect of the inhibitor on PBMCs was was only
observed at high concentration (500 nM). (Fig 3A)
Since substantial cell death was seen at 100 nM, we plated fewer cells per well and for the above cell
lines and treated them with 100 nM MELK inhibitor. A reduction in the number of metabolically active
cells across all cell lines was observed 24 hours post treatment (Fig 3B). A significant decrease was seen
in Jurkat, CEM (T-All) and KOPN-26 (B-ALL) where the p values were found to be p= 5.044E-07, 0.005,
0.0002 respectively. Although there was a reduction in the number of metabolically active cells in REH, it
was not statistically significant (p= 0.113)

A

B

0
0.2
0.4
0.6
0.8
1
1.2
1.4
Jurkat CEM KOPN-26 REH
Cell viability 100 nM
Control Treated
Figure 3: Antileukemic effect of
OTS167
A) Viability assay in ALL cell lines and
PBMCs 48 hours after treating with
varying concentrations of OTS167. X
axis shows cell viability relative to
control (0 nM).
B) Viability assay 6 hours after
treatment with 100 nM OTS167. X
axis shows cell viability relative to
control (untreated cells).
24

Effect of MELK inhibitor on protein expression in ALL cell lines

We treated T-ALL cell line Jurkat with 100 nM MELK inhibitor and collected cells at 3 and 6 hours. The
cells were then analyzed for MELK expression by Western Blot. Cells from the untreated group were
found to express more MELK than treated ones (Fig 4A). Jurkat cells were also collected at 3, 6 and 18
hours and analyzed for Β-catenin expression. Β-catenin expression was found to be reduced in treated
cells, 3 hours after collecting (Fig 4B). To better understand the mechanism of action of MELK inhibitor,
and to check whether it affects the Wnt signaling pathway, we analyzed the expression of GSK3B and
Phosphorylated GSK3B in the cells. We selected 2 T-ALL cell liens, Jurkat and CEM, for this purpose. The
cells were collected at 3 and 6 hours after treatment with 100 nM MELK inhibitor. GSK3B was found to
be expressed consistently across both cell lines before and after treatment. Phosphorylated GSK3B,
however, was found to be reduced after treatment (Fig 4C).
The next step was to confirm whether MELK inhibitor plays a similar role in B-ALL cell lines. We treated
REH cells with 100 nM MELK inhibitor and collected the cells at 3 and 6 hours. MELK and FOXM1
expression were found to be reduced after treatment (Fig 4D). To verify the role of MELK in Wnt
signaling in ALL, we treated 2 cell lines, REH and KOPN-26 with 100 nM inhibitor. Cells were collected 3
and 6 hours post treatment and analyzed for the expression of GSK3B and Phosphorylated GSK3B.
Similar to T-ALL, GSK3B showed similar expression before and after treatment. However,
phosphorylated GSK3B showed reduced expression after treatment (Fig 4E).
These results show that MELK might have a role to play in the Wnt signaling pathway, and the proteins
involved in Wnt are downstream of MELK.

A

25

B

C

D

26

E

Figure 4: OTS167 inhibits the expression of downstream targets of MELK
Immunoblots showing the effect of OTS167 on the expression of MELK and its downstream targets
A) OTS167 inhibited MELK expression in Jurkat cells
B) OTS167 inhibited Β-catenin expression in Jurkat cells 3 hours after treatment. Β-catenin expression
was unchanged between control and treatment groups 6 and 18 hours after expression.
C) T-ALL cell lines Jurkat and CEM showed reduction in expression of PGSK3B after treatment with
OTS167. GSK3B expression was unchanged after treatment
D) OTS167 inhibited MELK and FOXM1 expression in B-ALL cell line REH
E) B-ALL cell lines showed a reduction in expression of PGSK3B after treatment with OTS167. Similar to T-
ALL, GSK3B expression remained unchanged.
27

Effect of MELK overexpression on Β-catenin
As treatment with MELK inhibitor showed a reduction in the expression of Β-catenin and
Phosphorylated GSK3B, it is possible that Β-catenin is a downstream target of MELK. To confirm this
hypothesis, we checked the effect of MELK overexpression on the expression of Β-catenin . 293T cells
were transfected with MELK plasmids (Wild type and kinase dead mutant). The kinase dead mutant is
known to have a significant loss in functionality (Lin et al, 2007). The transfected cells were collected 48
hours later and protein expression was checked by Western Blot.
Overexpression of MELK resulted in an increased expression of Β-catenin in both WT and mutant
transfection (Fig 5A). We also analyzed the expression of non-phosphorylated active Β-catenin and
phosphorylated Β-catenin (Ser 552). Though both the transfections showed overexpression compared
to the cells transfected with empty vector, the WT transfection showed slightly higher levels of both
proteins (Fig 5B).
To establish a link between MELK and the Wnt signaling pathway, we also checked the expression of
GSK3B and Phosphorylated GSK3B after MELK overexpression. MELK overexpression had no effect on
the expression of GSK3B, which was found to be expressed equally across the cells transfected with
empty vector, MELK WT and mutant MELK. PGSK3B, however, showed no expression in cells transfected
with empty vector, indicating it to be absent endogenously. The cells transfected with MELK WT showed
a slightly higher level of expression compared to mutant MELK (Fig 5C).
These results indicate that the kinase activity of MELK plays a role in the regulation of Β-catenin
expression through the Wnt signaling pathway.

A B

28

C

Figure 5: MELK overexpression increased expression of Β-catenin and proteins in the Wnt Signaling pathway
Immunoblots showing increased expression of downstream targets of MELK after MELK overexpression
A) Shows expression of MELK and Β-catenin in 293T cells transfected with empty vector, MELK WT and kinase dead
MELK D150A
B) Shows the expression of Non-phosphorylated active Β-catenin and Phosphorylated Β-catenin (Ser 552). The
expression of the proteins is lesser in mutated MELK transfected cells compared to cells transfected with MELK WT.
C) Shows the expression of GSK3B and Phosphorylated GSK3B. GSK3B is unchanged after MELK overexpression.
PGSK3B is expressed slightly more in cells transfected with MELK WT than mutant MELK.

29

Interaction between MELK and Β-catenin
After previous results confirmed Β-catenin to be a downstream target of MELK via the Wnt signaling
pathway, we checked whether MELK and Β-catenin could interact with each other.
Immunoprecipitation was performed after overexpressing MELK (WT and mutant) and Β-catenin in
293T cells. Cells were collected 48 hours post transfection and subjected to lysis. The lysate was pulled
down with anti-MELK and anti-Β-catenin antibodies. IgG pull-down was used as control. The protein
complexes were pulled down on Agarose beads and separated by Western Blot. 293T cells which had
MELK (both variants) and Β-catenin , that were not pulled down with antibodies, were used as input.
The anti-MELK lysate was stained with primary Β-catenin antibody. The IP protein complexes showed
binding to the Β-catenin antibody, indicating possible interaction between the two proteins. Β-catenin
binding with the empty vector indicates the presence of endogenous Β-catenin (Fig 6A).
The lysate treated with anti-Β-catenin antibody was separated by Western Blot and stained with
primary MELK antibody. The immunoblot showed bands only at the IP lane with MELK WT. There were
no IP bands with the empty vector and the cells transfected with mutant MELK (Fig 6B).
This can further the hypothesis that the kinase ability of MELK plays a role in regulating Β-catenin .

A B

Figure 6: MELK and Β-catenin interact with each other
Immunoprecipitation was performed on cells expressing MELK (WT and mutant) and Β-
catenin
A) Shows Β-catenin binds to protein complex pulled down with anti-MELK antibody in cells
transfected with empty vector as well as both variants of MELK
B) Shows MELK binds to protein complex pulled down with anti-Β-catenin antibody in cells
transfected with MELK WT
30

DISCUSSION
Acute lymphoblastic leukemia is the most common cause of death among children and young
adolescents in the US. Almost 6000 new cases are diagnosed each year (Pui et al, 2006). Though, it is
curable to an extent in children, there is a lack of suitable treatment in adults. It is reported that while
80% of children with ALL are cured, the cure rate for adults is less than 40%. The poor outcome in adult
ALL has been attributed to higher drug resistance, poorer tolerance of and compliance with treatment,
reluctance to accept certain temporary toxic effects, and less effective treatment regimens, as
compared with childhood ALL (Pui et al, 2004). Relapse is known to occur even after treatment in
several cases. The lack of suitable treatment for adult leukemia and the cases of relapse in childhood
leukemia warrant for newer therapy regimens. Several tyrosine kinase inhibitors are currently in trials
and a few like Dasatinib are in use for the treatment of ALL. Several new kinases have recently come into
light as viable therapeutic targets for treating cancer. A promising new target appears to be maternal
embryonic leucine zipper kinase (MELK).
MELK elevation has been reported in various solid tumors. It is known to be correlated to worse
prognosis in many solid tumors (Gray et al, 2005). Analysis of publicly available databases revealed that
MELK is overexpressed in subsets of acute myeloid leukemia and acute lymphoblastic leukemia. The
MELK gene was found to be upregulated and hypomethylated in pediatric B-ALL patients (Almamun et
al, 2015). Researchers believe that aberrant expression of MELK due to methylation may provide a
survival advantage to malignant cells and play a role in the progression of pediatric ALL (Almamun et al,
2015). As MELK is found to be overexpressed in ALL, we wanted to investigate its role and establish this
kinase as a novel target for treating ALL. A potent MELK inhibitor, OTS167, was developed to target
MELK in solid tumors preclinical studies. The aim of the study was to find the role of MELK in ALL and
establish a possible mechanism of action for OTS167 in ALL. Previous studies have successfully
demonstrated the efficacy of MELK inhibitor in treating AML and kidney cancer. However, our study for
the first time shows that MELK can be targeted in ALL. We used potent MELK inhibitor, OTS167 to treat
ALL cell lines. OTS167 is already in clinical trials for solid tumors.
Our study was the first to report the involvement of MELK in ALL. We hypothesized that MELK could be a
target in the treatment of ALL. MELK has been reported to be overexpressed in ALL patients on publicly
available gene expression databases. MELK was found to be overexpressed in ALL cell lines compared to
PBMCs, showing its inhibition will not have an effect on healthy cells. MELK inhibitor OTS167 has been
proved effective in various types of cancers (Chung et al, 2013). We treated ALL cells with OTS167 and
analyzed the protein expression. We observed that the expression for Β-catenin and PGSK3B was
reduced. This observation linked MELK with the regulation of Β-catenin regulation. To corroborate the
observation, we co-expressed Β-catenin and MELK (WT and kinase dead mutant) and carried out
immunoprecipitation. This showed an interaction between the two proteins, validating our earlier
finding. To conclusively state whether MELK is indeed involved in Β-catenin regulation, we checked the
effect MELK overexpression had on Β-catenin expression. Overexpression of two variants of MELK (WT
and kinase dead mutant) resulted in increased expression of Β-catenin and PGSK3B. However, the
kinase dead mutant showed lesser expression compared to WT, indicating a possible role for the kinase
activity of MELK in the regulation of Β-catenin pathway.
The interaction between MELK and Β-catenin is particularly interesting, as Β-catenin is known to be
dysregulated in a variety of cancers. Β-catenin accumulates in the cytoplasm and gets translocated to
31

the nucleus via the Wnt signaling pathway and acts as a transcriptional co-activator for TCF/LEF
transcription factors. In the absence of Wnt, Β-catenin does not accumulate in the cytoplasm as it gets
degraded by the destruction complex consisting of Axin, GSK3, CK1A, APC. GSK3 phosphorylates Β-
catenin and makes it more prone for ubiquitination and successive proteolysis. GSK3B is an integral in
making Β-catenin more prone to proteolysis, its under-expression or inactivation will lead to an increase
in the level of Β-catenin. Phosphorylation of GSK3B at the Serine-9 site renders it inactive (Fang et al,
2000).
Wnt and NOTCH pathways are often known to be dysregulated in T-ALL. Wnt could be a leukemia
initiating event in T-ALL based on previous studies (Staal et al, 2016). In the absence of canonical Wnt
signaling pathway, Β-catenin will not get degraded in the cytoplasm and get translocated to the
nucleus. As Β-catenin is translocated to the nucleus, it activates the TCF/ LEF transcription factors that
are known to be associated with oncogenes like c-Myc and cyclinD1. The activation of TCF/ LEF offers a
survival advantage to stem cells. Hence, it is important to limit Β-catenin getting translocated to the
nucleus.
Our studies indicated MELK that MELK plays a role in the regulation of Β-catenin via PGSK3B. Wnt
pathway appears to be downstream of MELK, hence inhibiting MELK decreased expression of Β-catenin
and PGSK3. Whether MELK effect on β catenin occurs directly or through the inhibition of GSK3B
phosphorylation is unclear, and further mechanistic studies are still needed. Previous studies have
shown that FOXM1 interact with Β catenin (Zhang et al, 2011) and that FOXM1 is a downstream target
of MELK (Joshi et al, 2013). Thus it is plausible that the MELK effect on Β-catenin is mediated by FOXM1
inhibition.
Our studies indicate that MELK having kinase activity will cause an increase in the production of PGSK3B.
Kinase dead mutant MELK or MELK inhibition will cause a reduction in PGSK3B expression. A reduction
in the expression of PGSK3B, will cause reduced Β-catenin translocation, as GSK3B will make Β-catenin
more prone for proteolysis. Hence, Β-catenin will not translocate to the nucleus, and thereby not
activate the target genes associated with Wnt pathway.
Whether the effect of MELK on cancer progression occurs via regulation of Β-catenin and/or by
inhibiting PGSK3B requires further studies. However, this effect is a plausible mechanism by which MELK
contribute to cancer growth, and therefore inhibiting MELK kinase activity may provide a therapeutic
approach to target cancers associated with Wnt/β-catenin activation.
One of the shortcomings of this study is that the mRNA expression of MELK and other downstream
targets was not analyzed. Getting the mRNA data for the target genes of Β-catenin like c-myc, cyclin D1
after treating cells with MELK inhibitor would help ascertain the exact genes that MELK affects. Another
way to add value to this study is to study the activation of Β-catenin target genes by using luciferase
assay. The study provides a basic idea about the possible interaction between MELK and Β-catenin .
However, it fails to address the effect of this association has on target gene activation.

MELK inhibitor has been investigated in ongoing and finished clinical trials. The trials demonstrating
safety and efficacy of the drug in solid tumors finished Phase I in 2016. Additionally, another trial to
study the oral bioavailability of MELK inhibitor in healthy volunteers was also carried out in 2016.
Currently trials are ongoing for triple negative breast cancer and various types of refractory or relapsed
32

leukemias. (www.clinicaltrial.gov) Since MELK inhibitor is already in clinical trial for leukemia, it would
be interesting to complement the clinical findings with a functional and mechanistic studies.
Additionally, the preclinical studies are important to identify biomarkers that can be utilized to assess
the effectiveness of the treatment.
In closing, we conclude that targeting MELK will result in anti-leukemia activity. MELK plays a role in
acute lymphoblastic leukemia via the regulation of Β-catenin . Further research is needed to validate
these findings and identify the exact mechanism by which MELK is effective in acute lymphoblastic
leukemia.

33

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Abstract (if available)

Abstract Acute Lymphoblastic Leukemia (ALL) is a hematological malignancy that affects children and young adults. Treatment is possible through chemotherapy and stem cell transplant, however there is still the risk of relapse and resistance. Although most children are cured, the cure rate in adults is very low. Hence, there is a need for novel therapeutic targets to improve outcome of ALL in adults. Maternal Embryonic Leucine Zipper Kinase (MELK) is a protein kinase that is found to be overexpressed in a variety of solid tumors. A recent study reported MELK to be a therapeutic target in Acute Myeloid Leukemia (AML). However, the role of this kinase in ALL is not well investigated. Previous studies showed FOXM1 to be a substrate for MELK. The downstream targets for FOXM1 include the Wnt/ β-catenin pathway. The Wnt pathway is known to be implicated in T-ALL. Hence, we hypothesized that MELK may act as a therapeutic target in ALL by inhibiting FOXM1 and subsequently, the Wnt/ β-catenin pathway. We found that MELK is overexpressed in ALL compared to mononuclear cells obtained from healthy donors. Inhibiting MELK kinase activity resulted in decreased cell viability. MELK inhibition decreased p-GSK3B and β-catenin. While these results suggest a possible role of MELK in the β-catenin signaling pathway, further studies are needed to establish the mechanism by which MELK affects β-catenin activity. MELK inhibitor is currently in trials for solid tumors as well as hematological malignancies. Hence, more research is needed to find the exact mechanism by which MELK inhibition is effective therapeutic approach in ALL.

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Creator Deo, Rucha (author)

Core Title Maternal embryonic leucine zipper kinase (MELK) as a novel therapeutic target in the treatment of acute lymphoblastic leukemia

School School of Pharmacy

Degree Master of Science

Degree Program Molecular Pharmacology and Toxicology

Publication Date 08/02/2018

Defense Date 08/02/2017

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

Tag acute lymphoblastic leukemia,FOXM1,MELK,novel,OAI-PMH Harvest,β-catenin

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Okamoto, Curtis (committee chair), Alachkar, Houda (committee member), Beringer, Paul (committee member)

Creator Email rdeo@usc.edu,ruchadeo04@gmail.com

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