FLT3 and KIT in Acute Myeloid Leukemia

105 Pages·2016·10.94 MB·English

by  Oscar Lindblad

FLT3 and KIT in Acute Myeloid
Leukemia
Translational studies on oncogenic signaling from
receptor tyrosine kinases

Oscar Lindblad

DOCTORAL DISSERTATION
by due permission of the Faculty of Medicine, Lund University, Sweden.
To be defended at Hörsalen, Medicon Village, Lund, 29th of April 2016 13.00.

Faculty opponent
Professor Bjørn Tore Gjertsen
University of Bergen

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Organization Document name
Division of Translational Cancer Research Doctoral dissertation
Department of Laboratory Medicine
Faculty of Medicine, LUND UNIVERSITY
Date of issue
April 29, 2016
Author: Oscar Lindblad Sponsoring organization
Title and subtitle: FLT3 and KIT in Acute Myeloid Leukemia. Translational studies on oncogenic signaling from
receptor tyrosine kinases.
Abstract

Acute myeloid leukemia (AML) is a life-treatening disease with an uncontrolled expansion of immature blasts in
the bone marrow. Current forms of therapy are conventional chemotherapy and in some cases allogeneic stem
cell transplantation. New techniques have recently promoted new insights into the complex genetic
background of the disease. Around 60% of the genetic aberrations found in leukemia patients involve signaling
pathways. The aim of this thesis has been to investigate the altered singnaling properties of two receptor
tyrosine kinases, KIT and FLT3, which are frequently mutated in AML.
In our first study, we demonstrated that the ligand-independent activation of the KIT/V560D mutation is totally
dependent on the binding to PI3-kinase, and like the KIT/D816V mutation, the contribution of PI3-kinase to
ligand-independent activation of KIT/V560D is independent of its lipid kinase activity.
Next, we investigated how BEX1 and the FLT3/ITD mutation interact. By using gene expression data of
primary AML patient samples, we found that loss of BEX1 expression reduced the median survival by around
50% (HR 1.697, p = 0.0452) in FLT3/ITD-positive patients. Furthermore we found that expression of BEX1
significantly reduced FLT3/ITD-dependent cell proliferation, enhanced apoptosis and reduced the number of
colonies in semi-solid medium of both Ba/F3 and 32D cells. Moreover, BEX1 expression significantly reduced
tumor volume and tumor weight in Ba/F3 as well as in 32D cells in xenografted mice.
In the third study we examined the role of HOX proteins in AML. We found that HOXB2 and HOXB3 were up-
regulated in FLT3/ITD positive patients and the expression levels of HOXB2 and HOXB3 correlated to
prognosis in AML. Lower expression levels correlated to enrichment of oncogenic pathways and induced
overexpression of HOXB2 or HOXB3 inhibited FLT3/ITD downstream signaling as well as FLT3/ITD-induced
biological events.
In the last study we showed that secondary resistance to sorafenib in FLT3/ITD-positive AML may be caused
by up-regulation of the PI3K/mTOR signaling pathway even though the FLT3 receptor stays responsive to
inhibition. Furthermore, the highly selective dual PI3K/mTOR inhibitor gedatolisib can override this resistance
and block colony formation, decrease cell proliferation, induce apoptosis and block tumor growth in vivo.
To conclude, our studies have gained new insights into the role of mutated receptor tyrosine kinases in AML
and suggested some new ways to counteract their oncogenic properties.

Key words: acute myeloid leukemia, KIT, FLT3, tyrosine kinase receptors, FLT3/ITD, HOX, BEX
Classification system and/or index terms (if any)
Supplementary bibliographical information Language
English

ISSN and key title 1652-8220 ISBN
978-91-7619-244-3
Recipient’s notes Number of pages 105 Price
Security classification
I, the undersigned, being the copyright owner of the abstract of the above-mentioned dissertation, hereby grant to all
reference sourcespermission to publish and disseminate the abstract of the above-mentioned dissertation.

Signature Date 2016-03-22
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FLT3 and KIT in Acute Myeloid
Leukemia
Translational studies on oncogenic signaling from
receptor tyrosine kinases

Oscar Lindblad

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Cover photo: Sloe berries at Måryd, Skåne.

Copyright Oscar Lindblad

Faculty of Medicine | Department of Laboratory Medicine

ISBN 978-91-7619-244-3
ISSN 1652-8220

Printed in Sweden by Media-Tryck, Lund University
Lund 2016

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“In theory, theory and practice are the same. In practice, they are not.”
-Albert Einstein

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Content
Content 8
List of papers 9
Introduction 10
List of Abbreviations 11
Background 12
The genomic landscape of AML 12
Receptor tyrosine kinases 12
Signaling pathways 13
The KIT receptor 15
KIT inhibition 16
PI3K inhibition 17
Dual PI3K/mTOR inhibition 18
The FLT3 receptor 18
FLT3 in normal and malignant hematopoiesis 19
The clinical significance of FLT3 mutations 22
FLT3 inhibition 22
BEX proteins 25
The physiological role of BEX proteins 25
BEX proteins in malignant disease 27
HOX proteins 28
HOX proteins in AML 29
Present investigation 30
Aims 30
Results and discussion 31
Acknowledgements 39
Populärvetenskaplig sammanfattning 40
References 42

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List of papers

I. PI3 kinase is indispensable for oncogenic transformation by the V560D mutant
of c-Kit in a kinase-independent manner.
Lindblad O, Kazi JU, Rönnstrand L, Sun J. Cell Mol Life Sci. 2015 72(22):4399-
407.
Reprinted by permission from Elsevier.

II. BEX1 acts as a tumor suppressor in acute myeloid leukemia.
Lindblad O, Li T, Su X, Sun J, Kabir , Levander F, Zhao H, Lu G, Rönnstrand L,
Kazi JU. Oncotarget. 2015 6(25):21395-405

III. The role of HOXB2 and HOXB3 in acute myeloid leukemia.
Lindblad O, Chougule RA, Moharram SA, Kabir NN, Sun J, Kazi JU, Rönnstrand
L. Biochem Biophys Res Commun. 2015 467(4):742-7.
Reprinted by permission from Springer.

IV. Aberrant activation of the PI3K/mTOR pathway promotes resistance to
sorafenib in AML
Lindblad O, Cordero E, Puissant A, Macaulay L, Ramos A, Kabir NN, Sun J,
Vallon-Christersson J, Haraldsson K, Hemann MT, Borg Å, Levander F, Stegmaier
K, Pietras K, R önnstrand L and Kazi JU. Oncogene, advance online publication,
March 21, 2016
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Introduction
Acute myeloid leukemia is a quite uncommon disease with around 350 new cases
every year in Sweden. However, it can affect persons from early childhood to high
ages and it is always a life-threatening condition requiring acute treatment. At
present there is a tremendous development of new targeted drugs in hematology but
unfortunately this is not true for AML where the standard treatment has been the
same for over 50 years. The only real improvement of survival among these patients
has been the introduction of allogeneic stem cell transplantations in the 1980’s.
This thesis is based on the concept of translational research where findings in the
clinical practice should be easily transferred and investigated in laboratory models.
In our studies we have worked both ways. In three studies we started with clinical
findings which were transferred and mimicked in the laboratory in order to
investigate the underlying principles. In one study we compared two cell lines in the
laboratory and found differences that we could confirm also existed between
different patient groups.
It is today impossible to have a clear overview of the rapidly accumulating data on
cellular signaling. Therefore an improved cooperation between clinicians and
laboratory researchers is essential in order to know where to focus. The goal is to
find cellular mechanisms that many patients share despite different genetic
backgrounds of their diseases and to efficiently inhibit these mechanisms without
causing too much side-effects.
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List of Abbreviations
AML Acute Myeloid Leukemia
FLT3 Fms-like Tyrosine Kinase 3
ITD Internal Tandem Duplication
RTK Receptor Tyrosine Kinase
TKI Tyrosine Kinase Inhibitor
JM Juxtamembrane
TKD Tyrosine Kinase Domain
PI3K Phosphoinositide 3-Kinase
MAPK Mitogen-activated Protein Kinase
ERK Extracellular Signal-regulated Kinase
SCF Stem Cell Factor
FL FLT3 Ligand
mTOR Mechanistic (or Mammalian Target of Rapamycin)
STAT5 Signal Transducer and Activator of Transcription 5
CBF Core-Binding Factor
GIST Gastrointestinal Stromal Tumor
BEX Brain-Expressed X-linked
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Background
The genomic landscape of AML
As around 50% of all AML cases are cytogenetically normal when using classical
cytogenetic methods, the introduction of high-throughput sequencing has revealed
the role of smaller-scale genomic abnormalities. Compared to solid tumors AML
has a low average number of mutations1. The disease is characterized by clonal
heterogeneity at the time of diagnosis, including both a founding clone and at least
one subclone. During disease progression dynamic clonal evolution occurs and
probably contributes to resistance to therapy2. The Cancer Genome Atlas Research
Network has recently examined the genomes of 200 AML patients using whole-
genome or whole-exome sequencing together with RNA and microRNA sequencing
and DNA-methylation analysis. Genes that were significantly mutated were
classified into eight categories according to their function: Mutations in signaling
genes (i.e. FLT3, KIT) enhance proliferation through aberrant signaling pathways
whereas mutations in myeloid transcription factors (i.e. RUNX1) or transcription
factor fusions by chromosomal rearrangements (i.e. t(8;21)(q22;q22); RUNX1-
RUNX1T1), deregulate transcriptional activity and impair hematopoietic
differentiation. Mutations of the nucleophosmin (NPM1) gene induce an aberrant
cytoplasmic localization of NPM1 and NPM1-interacting proteins whereas
mutations of spliceosome-complex genes (i.e.SF3B1) confers deregulation of RNA
processing. Mutations of cohesin-complex genes (i.e. STAG2) causes impairment
of chromosome segregation and transcriptional regulation. Mutations of genes
responsible for the epigenetic control of cells (i.e. ASXL1) impairs chromatin
modification whereas DNMT3A and TET2 mutations, as well as IDH1/IDH2
mutations, promote deregulation of DNA methylation. Mutations in tumor-
suppressor genes such as TP53 leads to impairment of transcriptional regulation and
degradation of TP533.
Receptor tyrosine kinases
Around 60% of the identified mutations in AML are found in genes responsible for
cell signaling3. Two of the most recurrently mutated genes, KIT and FLT3, are part
of a family called receptor tyrosine kinases (RTKs). A kinase is defined as an
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