1. Acute Myeloid Leukemia
- RUNX , CBFB , KIT
Presentation by,
ATHIRA R G
M.Sc. Biochemistry & Molecular Biology
CENTRAL UNIVERSITY OF KERALA
2. Acute myeloid leukemia
Cancer of the myeloid line of blood
cells.(myeloid blast )
Characterized by rapid growth of abnormal
myeloblasts that build up in bone marrow
and interfere with production of
erythrocytes, platelets and other white blood
cells arising from lymphoid progenitors.
Common acute leukemia affecting the adults
and its incidence increases with age.
Symptoms include fatigue, shortness of
breath, easy internal bleeding & increased
risk of infections.
3. Bonemarrow
normal myeloid blast _ 1- 2 %
Acute leukemia > 20%
Markers
lymphoblast : TdT
Myelo blast : presence of myeloperoxidase
Done by cytoplasmic staining
Auer rods : crystalline form of enzyme can be seen on
microscope.
7. AML with recurrent genetic abnormalities
includes:
With translocation b/w chromosome 8 and 21 t( 8;21) : RUNX 1/RUNX
1T1
With inversion in chromosome 16 or internal translocations in
it:CBFB/MYH11
With translocation in b/w chro 15 and 17 : RARA / PML
With t( 9;11) : MLLT3 / MLL
8. Core Binding Factor [ CBF ]
The Core binding factor (CBF) is a group of heterodimeric
transcription factors.
Core binding factors are composed of:
a non-DNA-binding CBFβ chain (CBFB)
a DNA-binding CBFα chain (RUNX1 , RUNX 2, RUNX 3
RUNX1 and CBFβ are essential for definitive hematopoiesis,
where they regulate expression of genes associated with
proliferation, differentiation, and survival of stem and
progenitor cells
9. The homozygous loss of either RUNX1 or CBFB
alleles, in knockout murine models, resulted in failure
to develop definitive hematopoiesis and in embryonic
death.
In the embryo, RUNX1 and CBFb are required
for the differentiation of definitive
hematopoietic progenitors and hematopoietic
stem cells from a ‘‘hemogenic endothelium.’’
10. RUNX 1/ RUNX 1T1
Runt- related transcription factor
A key regulator of hematopoiesis.
Its functional disruption by point mutations, deletions or translocations
is a major causative factor leading to leukemia.
RUNX1/AML1 directly regulates multiple distinct myeloid and lymphoid
genes that are involved in hematopoietic lineage commitment.
The chromosomal aberration t(8;21)
results in the fusion of RUNX1 on 21q22
with RUNX1T1 (ETO) on 8q22, creating a
chimeric fusion gene, RUNX1/
RUNX1T1 (AML1-ETO).6
11. The translocation fuses the AML1 gene
(also called RUNX1) on chromosome 21
with the ETO gene (also referred to as the
RUNX1T1 gene that encodes the CBFA2T1
protein) on chromosome 8.
The breakpoints affect RUNX1 exon 5 and
RUNX1T1 exon 2.
Rowley J. Identification of a
translocation with quinacrine
fluorescence in a patient with
acute leukemia. Ann Genet.
1973;16(2):109–112
.
12. Altered subnuclear targeting of RUNX1 in patients with
the 8;21 translocation may contribute to the pathology
of AML. Because RUNX1/AML1 mutations that alter
subnuclear routing and fidelity of transcriptional
control result in a differentiation block and increase
proliferation of myeloid progenitors .
13. In addition, a large number of co-factors interact with gene regulatory
domains of RUNX1/AML1, including the C-terminus that is removed in
AML1-ETO .
Through the recruitment of unique co-regulators that interact with the ETO
moiety in lieu of the AML1 C-terminus, the AML1-ETO fusion protein
antagonizes the transcriptional function of native RUNX1/AML.
Thus, there are several plausible mechanisms by which the pathological
formation of the AML1-ETO protein may block differentiation of myeloid
progenitors and promote leukemia.
In addition to regulating hematopoiesis-specific genes, RUNX1/AML1 is also
implicated in the regulation of cell-cycle genes, including p21 WAF1/CIP1,
which encodes a cyclin-dependent kinase inhibitor important for checkpoint
control and terminal differentiation.
14. CBFB/MYH11
Core-binding factor subunit beta is a protein that in humans is
encoded by the CBFB gene.
The beta subunit is a non-DNA binding regulatory subunit; it
allosterically enhances DNA binding by the alpha subunit as the
complex binds to the core site of various promoters and enhancers
Mutations in CBFB are implicated in cases of breast cancer.
The aberrations inv(16) and t(16;16) lead to fusion of CBFB on
16q22 with smooth muscle myosin heavy-chain gene (MYH11) on
16p13, leading to formation of CBFB/MYH11 chimeric gene.
The breakpoint in the MYH11 gene is variable and gives origin to
at least 10 different fusion variants. In contrast,the breakpoints in
the CBFB gene are in intron 5.
15. LeBeau et al4 in 1983
reported the peculiar
association of abnormal
marrow eosinophils in AML
with inv(16).
Le Beau MM, Larson RA, Bitter MA, et al.
Association of an inversion of chromosome
16 with abnormal marrow eosinophils in
acute myelomonocytic leukemia: a unique
cytogenetic clinicopathological association.
N Engl J Med 1983;309(11):630–636.
16. The normal transcriptional activity of RUNX1 is
affected in a negative manner by the chimeric
proteins encoded by RUNX1/RUNX1T1 and
CBFB/MYH11 fusion genes.
Animal studies suggest that the fusion proteins
alone are not able to induce leukemia and that
additional genetic alterations are required for
leukemogenic transformation.
Marcucci and his research group proposed that
AML1/ETO fusion protein recruits histone
deacetylases and DNA methyl- transferase 1 to CBF
target genes.
This leads to increased chromatin deacetylation
and promoter hypermethylation, resulting in gene
transcriptional repression and disruption of normal
pathways of hematopoiesis
17. Fluorescence in situ hybridization (FISH) and reverse transcription
PCR (RT-PCR) are increasingly used for analyzing morphology,
owing to their high sensitivity and rapid turnaround.
Also, FISH and PCR assays are useful to confirm that the
rearrangements actually involve the RUNX1/RUNX1T1 (AML1-
ETO) and CBFB/MYH11 genes in t(8;21) and inv(16),
Patients with t(8;21) frequently present with the French-American-
British (FAB) morphologic subtype M2 or acute myeloid leukemia
with differentiation, while patients with inv(16) more often are
diagnosed with FAB subtype M4Eo or acute myelomonocytic
leukemia with eosinophilia
18.
19. KIT mutations
The KIT gene provides instructions for
making a protein that belongs to a family of
proteins called receptor tyrosine kinases
Cytogenetic Location: 4q12
KIT protein signaling is important for the
development of certain cell types,including
reproductive cells (germ cells), early blood
cells (hematopoietic stem cells),immune cells
called mast cells.
20. The mechanism underlying KIT gene mutations that
adversely affects the prognosis involves phosphorylation of
the KIT receptor after physiologic binding of KIT ligand,
which activates downstream pathways supporting cell
proliferation and survival.
Approximately 50% of patients with CBF-AML remain
incurable, and markers are required to refine the risk
stratification of patients at diagnosis and to optimize their
treatment.
KIT mutations occur in 20% to 25% of t(8;21) cases and in
approximately 30% of cases with inv(16).
In the future, the promising treatment with tyrosine kinase
inhibitors might mandate screening for KIT mutations in
these subgroups of patients with AML.
In patients with CBF AML, KIT exon 8 , 17 mutations are
KIT mutations
