Philip on stem cell lines and in this case

Philip Russell

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MSc Biomedical Science

 

Biol45111: Scientific
analysis, review and presentation Elearn 2017/18

Assignment 2

Developments in the molecular basis of
leukaemia and how this has influenced diagnosis and treatment

 

 

Abstract

With the
advancement in diagnostics and treatments, the ability to engineer new
molecular techniques to help improve patient experiences. The common
examination of known mutations and their interactions versus the ability of
newer technologies to both examine and treat patients have been used to develop
these treatments. If understanding on mutations is advanced, then interactions
and outcomes can be predicted to improve treatments.

Introduction

Leukaemia is a
disease associated with the bone marrow. It is characteristic of an abnormal
increase in white cell entering the peripheral blood stream. These calls can
range from mature to left shifted younger cells and can include blast cells in
both the lymphocytes and myeloid cell line of the disease. Current testing in a
hospital diagnostic setting is the counting of the white blood cells as a first
line screen which will then be accompanied by a blood film to enable the
examination of the cell morphology.

The next level
of tests is now looking at the molecular testing of the cells. This can either
be cytogenetic, cell markers or molecular tests, Philadelphia chromosome. These
are general testes and the need for more accurate testing which can be more
specific to the mutation causing the proliferation and the resulting treatments
which can be derived from these results. The molecular aspect of this testing
has created future developments which the clinical, scientific and patient can
all benefit from.

Molecular interactions

Diagnostic treatment
of leukaemia has developed through the years and with the improvement on
molecular testing new gene groups have been examined to see if the progression
and outcomes of patients can be different from each. Fig 1 illustrates the
different mutations and where they occur. By taking looking at examples of
common gene mutations, ie: KIT, FLT3 ITD and NPM1, the progression and
treatment of patient’s outcomes can be closely examined.

 

KIT Gene mutation

On a
routine higher level of testing this gene encodes for the receptor tyrosine
kinase protein. This is a transmembrane protein which acts as an intra cellular
communication for the ATP to phosphorylate and be influence in cell
proliferation. On the cell surface this is called CD117 and internally it’s
called cKIT. The activity f the protein is to activate cell proliferation, differentiation
and long life mainly seen on stem cell lines and in this case through the
myeloid cell line. This will have an increase in undifferentiated blast cells
which are then seen to be leukemic in nature which in turn disrupt the normal
bone marrow. The presence of higher KIT mutations in core binding factor
acute myeloid leukaemia (CBF-AML) or AML carries the risk of a poorer
prognosis for the patient due to the higher chance of proliferation of blasts.
However, this under debate as some studies show this and others argue it has no
effect on prognosis. (Jay Patel, et al, 2012)

A standard treatment which can be used
is a drug called Imatinib. This is an anti-tyrosine kinase drug by stopping the
activity of this protein and causing apoptosis or reduced life span of the
cells expressing the cd117. However, this is only effective of cells showing a
Philadelphia positive gene, BCR-ABL1, as this mutation is part of the raised
activity of CD117 (www.drugbank.ca). If the mutation is active but is from
another form of mutation this may make this drug in active. Different
approaches can be used. An example is the development of oligonucleotides can
be introduced which is specific to the mutation causing a truncated protein or
skipping it to prevent its use in the cancerous cells. (Nianxi Zhao, et al,
2015) (Nianxi Zhao, et al, 2013)

Patients
can gain a long-term remission of from this type of AML due to the treatments
available however the presence of clones in the remaining cells have been seen
to produce a relapse with higher rates of mutations. This may progress into a
more aggressive form of proliferation and development of further treatments may
be required. In first time disease it can stand at 64% in patients, relapse can
be higher at 95% and 10% in blast cells. (Anjali S Advani, et al, 2006)

 

FLT3 (CD135) (Proto oncogene)

Is a second tyrosine
kinase receptor located on approx. 70-100% of leukemic cells. As a secondary
GCPD messenger system and is linked to proliferation and anti-apoptosis of
cells. In AML there is two mutations which can affect the cells, internal
tandem duplication (ITD) which effects 17-34% of cells and mutations of the
activation loop which effects 7% of cells. These mutations also provide lower
prognoses for the patient as occurrences percentages can be seen in fig 2.
These, most common ITD, will cause the proliferation of myeloid cells and can
give a worsening prognosis for patient’s carrying this and the cKIT, NPM1 and
CEBPA mutation. (Naval Daver, et al, 2012)

 

Quizartinib is
a small molecule receptor tyrosine kinase inhibitor for the treatment of acute
myeloid leukaemia. Its molecular target is FLT3, also known as CD135 which is a
proto-oncogene. The presence of this mutation is a marker of adverse outcome.
Quizartinib is a selective inhibitor of class 3 receptor tyrosine kinases.
These include FMS-related tyrosine kinase 3 (FLT3/STK1), colony-stimulating
factor 1 receptor (CSF1R/FMS), stem cell factor receptor (SCFR/KIT) and
platelet derived growth factor receptors (PDGFRs). (Robert Hills, et al, 2015)
(R Motes, et al, 2014)

NPM1

This is a gene
which encodes for a phosphor-protein which moves between the nucleus and the
cytoplasm. This has a function in ribosome biogenesis,
centrosome duplication, protein chaperoning, histone assembly, cell
proliferation and regulation of tumour suppressor’s p53/TP53. It is the
activity that is important in AML leukaemia’s. The activity on tumour
suppressor’s p53 that causes the increase in proliferation and anti-apoptosis
on these cells. The mutation of this gene will cause a change in the c-terminus
of the protein expressed and a loose of the nucleolus localization and more of
nucleus export signals leading it to be more localised on the cytoplasm. This
localisation will prevent it from activating the p53 tumour suppressor by being
unable to bind to the relevant region in the nucleus. The p53 is a common
protein in healthy cells, it is a protein which is used to trigger apoptosis by
sensing if an error has occurred in DNA or cell damage which is past repair it
can trigger cell death.  This mutation is
found in approx. 30% of all AML. (Sophia Yohe, et al, 2015) (Yanfeng Liu, et
al, 2014)

CEBPA

This is a
promoter/enhancer region which is widely seen as a promoter for transcriptional
factors for the differentiation of granulocytes cell lines. They are common in
the AML with one third of case showing a mono-allergic presentation a d two
thirds the bi-allogenic form. This is important as in normal AML types the
mono-allergenic form can be seen to give a less favourable outcome to the
patients. The mutation on this gene lead sot the form mutation of a truncated C
or N terminus on the protein which can prevent its full use or DNA binding in
the cell. (Sophia Yohe, et al, 2015)

 

Inborn genetic mutations

When also
examine the instances of leukaemia and the treatment plans there is the chance
that inborn genetic mutations can cause a variance in the disease and how the
various treatments work for them.

With Down
syndrome (DS), or trisomy 21, the rate of leukaemia reported to be of a higher
risk then other child. This is more relevant with AML or AMkL in these
patients, but these patients have a survival or event free rate of approx.
80-100%. However, it is a worse prognosis for ALL patients.  (C Xavier, et al, 2010)

DS patients
have a predisposed case where there is a transient myeloid-proliferation with
these patients. This is due the mutation in the GATA1 gene. GATA1 gene is an X
linked transcription factor which is used in erythroid and megakaryocyte
differentiate. The result is a truncated or shorted GATA1 protein which then
becomes an uncontrolled proliferation. DS may contribute to this by the
localised genes contained in chromosome 21 which leads to an abnormal foliate
metabolism uracil accumulation and a rise in oxidative stress causing DNA
damage. These are all linked to cystanimie B synthase and zinc copper
superoxide dismuatase located on chromosome 21. This however is highly
treatable with metatraxate but as the mutation carriers a higher sensitivity to
this treatment there is a chance of toxicity in the patient and the dose must
be monitored closely. (C Xavier, et al, 2010)

Due to the
differences caused in part by the chromosome 21 there is a noticeable
difference in treatment responses for DS patients in AMKL genotypes.  The expression of expression of the
anit0apoptic genes and proteins such as BCL2 and HSP70 is lower on DS AMkL
blast cells compared to non-DS patients. This gives rise to the theory that DS
patients can be more susceptible to chemo induced apoptosis and this is linked
to 18 gene dosage affects to DS AML groups (C Xavier, et al, 2010). However, it
is mostly AML and not ALL patients.

With DS ALL
there are common proliferation pathways which can be observed. Examine cytokine
growth with CRFL2 and Jak2 mutations there has been a link developed between
the two that indicates that in DS patients this is present but does not carry
the typical lesions associated in non-DS patients as can be seen in fig 3. This
is common trait seen in ALL but not AML however it has to be noted the GATA1
mutation is present in AML but not ALL patients. As rearrangement s in the
CFRL2 gene has an impact on the JAK2 gene for proliferation this has shown in
non-DS patients to cause the leucocytosis effect.

 There are however many mutations associated
with DS patients, but they are largely excluded from clinical trials and there
is limited range in the information of treatments and effects they can carry. (P
Lee, et al, 2016)

 

 

 

New Technology

With the
development and use of CRISP/Cas9 technology a new generation of treatments and
screening tools are being designed for use. These can be very sensitive to
specific gene mutation and can be used as a guided treatment to deliver a drop
out gene treatment in leukaemia’s. In studies screening tool for the
identification of 492, such as DOT1L, BCL2, and MEN, different genes were
designed. These were both normal genes found in healthy cells and clinically
actionable AML cells. This was initially done with the traditional technology
but was found to be less sensitive and was thought to be statically not as
accurate.

CRISPr
technology uses a guide RNA (gRNA) built into the CAS9 scaffold to help in the
identification and the removal or dropout of the specific gene. The original
experiments used mouse stem cells but it was found they are not as sensitive to
all genes in the library which had been developed and the dropouts were not as
successful as hoped. To help in the screening of these genes it was
hypothesised the library of the gRNA was too large for the scaffolding being
used. A redesign of the scaffolding and refinement of the library helped in the
development of the screening tool. Statistically the on target gene dropout was
seen to be greater in the version two libraries and there was higher
specificity with less off target dropouts compared to the first library.  This was a proof that the technology was
capable to be used in screening as the protocol used specific gene positive
cell lines for the identification of dropouts. (Tzelepis Konstantinos, et al,
2016)

Crispr dropouts

A treatment in
experimental design use has been examined using this technology, KAT2. This is
an AML proliferation gene and is associated with the increase of early myeloid
cells in humans. Samples of selected cell were introduced to a study of immune
compromised mice. The cells were tagged with florescent dye and were monitored
until death to see if the KAT2 dropout had any effect in vivo. It proved that
the knockout of KAT2A was significant in the halting of the proliferation and
promoting of cell death in AML cells in this study. Following this a human cell
line, primary human AML cells, was tested. The results mirrored the mouse model
but also showed the treatment had no effect on the surrounding human stem cells
in the experiment. This indicated the treatment was specific to produce a KAT2A
dropout and stop the proliferation (Tzelepis Konstantinos, et al, 2016). This
has the potential to become a new form of targeted treatment as it has been
already being approved by the FDA for the use in patients own cells as a
treatment for ALL with CAR-T
therapies.  (www.cancer.gov)

 

 

Discussion

By examining
the various mutations that can occur in leukaemia there is a variance in their
combinations and how they will affect the patients. However, the treatments are
different to each mutation and combination and this needs to be considered and
examined if the possibility of designing a personalised treatment for each
patient.

Variable difference
in the mutations combinations have seen to have different prognosis in
patients. By taking the examples, it can be seen a combination of FLT3 ITD,
NPM1, or CEBPA mutations refines the prognosis of patient with normal to
intermediate risk of recovery. FLT3 ITD on its own has a risk value of poor
while NPM1 and bi-allelic CEBPA mutations will raise the patients risk to a
more favourable level. With the presence of KIT mutations in one of the core
binding factors will worsen the prognosis from good to intermediate and may
present with a more difficult treatment plan (Sophis Yohe, et al, 2015). Fig 3
illustrates the difference in mutations and non-mutations between male and
female patients.

These are the
current developments in which the genetics of leukaemia’s a are examined. They
are a further insight into the expression of the mutations and how they can
affect both the proliferation of the cell; their clones post treatment and the
ability of the chemotherapy agents to effect a change to the favour of the
patient. Traditional treatments will act in many ways, S phase disruption in
cell division, while others act a s a pro-drug which can initiate an internal
effect on the cell. This can be by producing a cytotoxic by over expression of
certain enzymes or by initiating the cell own apoptosis effect.

When we examine
the developments of diagnostics and treatments newer technologies are also
playing their part. The addition of CRISPr technology to both screen and treat
patients has created a relevantly straight forward route to this. However, it
has shown the gDNA libraries it can act need to refined more and the structure
for the CAS9. This can be designed to also be used in the same manner as other
traditional drugs. As the technology evolves the differences in the treatments
can be brought forward and gene editing can show potential in stem cell
infusions or in the localised treatment cells in vivo (www.cancer.gov).

Outside of
this review the potential for further exploration can be seen in research which
can look at the epigenetic of leukaemia’s. This will give a more overall view
of the genes which encode for proliferation and the potential treatments which
can be derived from this. This information can be also being examined in
parallel with the gene sequencing and bio-informatics. The larger picture of
mutations for various institutes can help build a library which will develop
more accurate diagnostics and staging of the disease and the potential for new
treatments which can be more tailor made for each patient for better outcomes.

 

Conclusion

As leukaemia
is a complicated disease there are many variants on mutations and combinations
of these which will affect the patient’s outcome. By examine these genes and
developing advanced diagnostics tools to help identifies them there is a
treatment which can be built on from these results. These can take the form of
traditional treatment to more up to date technologies which can provide
different treatment to enable the patient to have a more personalised treatment
and improve their quality of life and prevent future relapses.

 

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