Philip RussellN0707900 MSc Biomedical Science Biol45111: Scientificanalysis, review and presentation Elearn 2017/18Assignment 2Developments in the molecular basis ofleukaemia and how this has influenced diagnosis and treatment AbstractWith theadvancement in diagnostics and treatments, the ability to engineer newmolecular techniques to help improve patient experiences. The commonexamination of known mutations and their interactions versus the ability ofnewer technologies to both examine and treat patients have been used to developthese treatments. If understanding on mutations is advanced, then interactionsand outcomes can be predicted to improve treatments. IntroductionLeukaemia is adisease associated with the bone marrow. It is characteristic of an abnormalincrease in white cell entering the peripheral blood stream. These calls canrange from mature to left shifted younger cells and can include blast cells inboth the lymphocytes and myeloid cell line of the disease.
Current testing in ahospital diagnostic setting is the counting of the white blood cells as a firstline screen which will then be accompanied by a blood film to enable theexamination of the cell morphology. The next levelof tests is now looking at the molecular testing of the cells. This can eitherbe cytogenetic, cell markers or molecular tests, Philadelphia chromosome. Theseare general testes and the need for more accurate testing which can be morespecific to the mutation causing the proliferation and the resulting treatmentswhich can be derived from these results. The molecular aspect of this testinghas created future developments which the clinical, scientific and patient canall benefit from.
Molecular interactionsDiagnostic treatmentof leukaemia has developed through the years and with the improvement onmolecular testing new gene groups have been examined to see if the progressionand outcomes of patients can be different from each. Fig 1 illustrates thedifferent mutations and where they occur. By taking looking at examples ofcommon gene mutations, ie: KIT, FLT3 ITD and NPM1, the progression andtreatment of patient’s outcomes can be closely examined. KIT Gene mutationOn aroutine higher level of testing this gene encodes for the receptor tyrosinekinase protein.
This is a transmembrane protein which acts as an intra cellularcommunication for the ATP to phosphorylate and be influence in cellproliferation. On the cell surface this is called CD117 and internally it’scalled cKIT. The activity f the protein is to activate cell proliferation, differentiationand long life mainly seen on stem cell lines and in this case through themyeloid cell line.
This will have an increase in undifferentiated blast cellswhich are then seen to be leukemic in nature which in turn disrupt the normalbone marrow. The presence of higher KIT mutations in core binding factoracute myeloid leukaemia (CBF-AML) or AML carries the risk of a poorerprognosis 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 noeffect on prognosis. (Jay Patel, et al, 2012)A standard treatment which can be usedis a drug called Imatinib. This is an anti-tyrosine kinase drug by stopping theactivity of this protein and causing apoptosis or reduced life span of thecells expressing the cd117. However, this is only effective of cells showing aPhiladelphia positive gene, BCR-ABL1, as this mutation is part of the raisedactivity of CD117 (www.
drugbank.ca). If the mutation is active but is fromanother form of mutation this may make this drug in active. Differentapproaches can be used. An example is the development of oligonucleotides canbe introduced which is specific to the mutation causing a truncated protein orskipping it to prevent its use in the cancerous cells. (Nianxi Zhao, et al,2015) (Nianxi Zhao, et al, 2013)Patientscan gain a long-term remission of from this type of AML due to the treatmentsavailable however the presence of clones in the remaining cells have been seento produce a relapse with higher rates of mutations. This may progress into amore aggressive form of proliferation and development of further treatments maybe required. In first time disease it can stand at 64% in patients, relapse canbe higher at 95% and 10% in blast cells.
(Anjali S Advani, et al, 2006) FLT3 (CD135) (Proto oncogene)Is a second tyrosinekinase receptor located on approx. 70-100% of leukemic cells. As a secondaryGCPD messenger system and is linked to proliferation and anti-apoptosis ofcells. In AML there is two mutations which can affect the cells, internaltandem duplication (ITD) which effects 17-34% of cells and mutations of theactivation loop which effects 7% of cells. These mutations also provide lowerprognoses for the patient as occurrences percentages can be seen in fig 2.These, most common ITD, will cause the proliferation of myeloid cells and cangive a worsening prognosis for patient’s carrying this and the cKIT, NPM1 andCEBPA mutation. (Naval Daver, et al, 2012) Quizartinib isa small molecule receptor tyrosine kinase inhibitor for the treatment of acutemyeloid leukaemia.
Its molecular target is FLT3, also known as CD135 which is aproto-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-stimulatingfactor 1 receptor (CSF1R/FMS), stem cell factor receptor (SCFR/KIT) andplatelet derived growth factor receptors (PDGFRs). (Robert Hills, et al, 2015)(R Motes, et al, 2014)NPM1This is a genewhich encodes for a phosphor-protein which moves between the nucleus and thecytoplasm. This has a function in ribosome biogenesis,centrosome duplication, protein chaperoning, histone assembly, cellproliferation and regulation of tumour suppressor’s p53/TP53. It is theactivity that is important in AML leukaemia’s. The activity on tumoursuppressor’s p53 that causes the increase in proliferation and anti-apoptosison these cells.
The mutation of this gene will cause a change in the c-terminusof the protein expressed and a loose of the nucleolus localization and more ofnucleus export signals leading it to be more localised on the cytoplasm. Thislocalisation will prevent it from activating the p53 tumour suppressor by beingunable to bind to the relevant region in the nucleus. The p53 is a commonprotein in healthy cells, it is a protein which is used to trigger apoptosis bysensing if an error has occurred in DNA or cell damage which is past repair itcan trigger cell death. This mutation isfound in approx. 30% of all AML.
(Sophia Yohe, et al, 2015) (Yanfeng Liu, etal, 2014)CEBPAThis is apromoter/enhancer region which is widely seen as a promoter for transcriptionalfactors for the differentiation of granulocytes cell lines. They are common inthe AML with one third of case showing a mono-allergic presentation a d twothirds the bi-allogenic form. This is important as in normal AML types themono-allergenic form can be seen to give a less favourable outcome to thepatients.
The mutation on this gene lead sot the form mutation of a truncated Cor N terminus on the protein which can prevent its full use or DNA binding inthe cell. (Sophia Yohe, et al, 2015) Inborn genetic mutationsWhen alsoexamine the instances of leukaemia and the treatment plans there is the chancethat inborn genetic mutations can cause a variance in the disease and how thevarious treatments work for them. With Downsyndrome (DS), or trisomy 21, the rate of leukaemia reported to be of a higherrisk then other child. This is more relevant with AML or AMkL in thesepatients, 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 patientshave a predisposed case where there is a transient myeloid-proliferation withthese patients. This is due the mutation in the GATA1 gene.
GATA1 gene is an Xlinked transcription factor which is used in erythroid and megakaryocytedifferentiate. The result is a truncated or shorted GATA1 protein which thenbecomes an uncontrolled proliferation. DS may contribute to this by thelocalised genes contained in chromosome 21 which leads to an abnormal foliatemetabolism uracil accumulation and a rise in oxidative stress causing DNAdamage. These are all linked to cystanimie B synthase and zinc coppersuperoxide dismuatase located on chromosome 21. This however is highlytreatable with metatraxate but as the mutation carriers a higher sensitivity tothis treatment there is a chance of toxicity in the patient and the dose mustbe monitored closely.
(C Xavier, et al, 2010)Due to thedifferences caused in part by the chromosome 21 there is a noticeabledifference in treatment responses for DS patients in AMKL genotypes. The expression of expression of theanit0apoptic genes and proteins such as BCL2 and HSP70 is lower on DS AMkLblast cells compared to non-DS patients. This gives rise to the theory that DSpatients can be more susceptible to chemo induced apoptosis and this is linkedto 18 gene dosage affects to DS AML groups (C Xavier, et al, 2010). However, itis mostly AML and not ALL patients.With DS ALLthere are common proliferation pathways which can be observed. Examine cytokinegrowth with CRFL2 and Jak2 mutations there has been a link developed betweenthe two that indicates that in DS patients this is present but does not carrythe typical lesions associated in non-DS patients as can be seen in fig 3.
Thisis common trait seen in ALL but not AML however it has to be noted the GATA1mutation is present in AML but not ALL patients. As rearrangement s in theCFRL2 gene has an impact on the JAK2 gene for proliferation this has shown innon-DS patients to cause the leucocytosis effect. There are however many mutations associatedwith DS patients, but they are largely excluded from clinical trials and thereis limited range in the information of treatments and effects they can carry. (PLee, et al, 2016) New TechnologyWith thedevelopment and use of CRISP/Cas9 technology a new generation of treatments andscreening tools are being designed for use. These can be very sensitive tospecific gene mutation and can be used as a guided treatment to deliver a dropout gene treatment in leukaemia’s.
In studies screening tool for theidentification of 492, such as DOT1L, BCL2, and MEN, different genes weredesigned. These were both normal genes found in healthy cells and clinicallyactionable AML cells. This was initially done with the traditional technologybut was found to be less sensitive and was thought to be statically not asaccurate. CRISPrtechnology uses a guide RNA (gRNA) built into the CAS9 scaffold to help in theidentification and the removal or dropout of the specific gene. The originalexperiments used mouse stem cells but it was found they are not as sensitive toall genes in the library which had been developed and the dropouts were not assuccessful as hoped. To help in the screening of these genes it washypothesised the library of the gRNA was too large for the scaffolding beingused. A redesign of the scaffolding and refinement of the library helped in thedevelopment of the screening tool. Statistically the on target gene dropout wasseen to be greater in the version two libraries and there was higherspecificity with less off target dropouts compared to the first library.
This was a proof that the technology wascapable to be used in screening as the protocol used specific gene positivecell lines for the identification of dropouts. (Tzelepis Konstantinos, et al,2016)Crispr dropoutsA treatment inexperimental design use has been examined using this technology, KAT2. This isan AML proliferation gene and is associated with the increase of early myeloidcells in humans. Samples of selected cell were introduced to a study of immunecompromised mice. The cells were tagged with florescent dye and were monitoreduntil death to see if the KAT2 dropout had any effect in vivo. It proved thatthe knockout of KAT2A was significant in the halting of the proliferation andpromoting of cell death in AML cells in this study. Following this a human cellline, primary human AML cells, was tested.
The results mirrored the mouse modelbut also showed the treatment had no effect on the surrounding human stem cellsin the experiment. This indicated the treatment was specific to produce a KAT2Adropout and stop the proliferation (Tzelepis Konstantinos, et al, 2016). Thishas the potential to become a new form of targeted treatment as it has beenalready being approved by the FDA for the use in patients own cells as atreatment for ALL with CAR-Ttherapies.
(www.cancer.gov) DiscussionBy examiningthe various mutations that can occur in leukaemia there is a variance in theircombinations and how they will affect the patients. However, the treatments aredifferent to each mutation and combination and this needs to be considered andexamined if the possibility of designing a personalised treatment for eachpatient. Variable differencein the mutations combinations have seen to have different prognosis inpatients.
By taking the examples, it can be seen a combination of FLT3 ITD,NPM1, or CEBPA mutations refines the prognosis of patient with normal tointermediate risk of recovery. FLT3 ITD on its own has a risk value of poorwhile NPM1 and bi-allelic CEBPA mutations will raise the patients risk to amore favourable level. With the presence of KIT mutations in one of the corebinding factors will worsen the prognosis from good to intermediate and maypresent with a more difficult treatment plan (Sophis Yohe, et al, 2015). Fig 3illustrates the difference in mutations and non-mutations between male andfemale patients.These are thecurrent developments in which the genetics of leukaemia’s a are examined.
Theyare a further insight into the expression of the mutations and how they canaffect both the proliferation of the cell; their clones post treatment and theability of the chemotherapy agents to effect a change to the favour of thepatient. Traditional treatments will act in many ways, S phase disruption incell division, while others act a s a pro-drug which can initiate an internaleffect on the cell. This can be by producing a cytotoxic by over expression ofcertain enzymes or by initiating the cell own apoptosis effect.When we examinethe developments of diagnostics and treatments newer technologies are alsoplaying their part. The addition of CRISPr technology to both screen and treatpatients has created a relevantly straight forward route to this. However, ithas shown the gDNA libraries it can act need to refined more and the structurefor the CAS9.
This can be designed to also be used in the same manner as othertraditional drugs. As the technology evolves the differences in the treatmentscan be brought forward and gene editing can show potential in stem cellinfusions or in the localised treatment cells in vivo (www.cancer.gov).Outside ofthis review the potential for further exploration can be seen in research whichcan look at the epigenetic of leukaemia’s. This will give a more overall viewof the genes which encode for proliferation and the potential treatments whichcan be derived from this.
This information can be also being examined inparallel with the gene sequencing and bio-informatics. The larger picture ofmutations for various institutes can help build a library which will developmore accurate diagnostics and staging of the disease and the potential for newtreatments which can be more tailor made for each patient for better outcomes. ConclusionAs leukaemiais a complicated disease there are many variants on mutations and combinationsof these which will affect the patient’s outcome. By examine these genes anddeveloping advanced diagnostics tools to help identifies them there is atreatment which can be built on from these results. These can take the form oftraditional treatment to more up to date technologies which can providedifferent treatment to enable the patient to have a more personalised treatmentand improve their quality of life and prevent future relapses. References1. Anjali S.
Advani, Targeting the c-kit receptor in the treatment of acute myelogenousleukemia, CurrentHematologic Malignancy Reports, June 2006, Volume 1, Issue 2, p 101–107 2. Ana C. Xavier, Jeffrey W. Taub, Acuteleukaemia in children with Down syndrome, Haematologica. 2010 Jul, Issue95, Vol 7, p 1043–1045. 3. D Gonzalez deCastro, P Clarke, B Al-Lazikani, P Workman, Personalized cancer medicine,Molecular diagnostics, predictive biomarkers and drug resistance, TyrosineKinase Inhibitors, Vol 93, 2013, p252 – 259 4.
Gozdecka, Malgorzata ; Ohnishi,Shuhei ; Cooper, Jonathan ; Patel, Miten ; Mckerrell, Thomas ; Chen, Bin ;Domingues, Ana filipa ; Gallipoli, Paolo ; Teichmann, Sarah ; Ponstingl,Hannes ; Mcdermott, Ultan ; Saez-Rodriguez, Julio ; Huntly, Brian j.P. ;Iorio, Francesco ; Pina, Cristina ; Vassiliou, George s. ; Yusa, Kosuke, A CRISPR Dropout Screen Identifies GeneticVulnerabilities and Therapeutic Targets in Acute Myeloid Leukaemia, Cell Reports, 2016, Vol.17,issue 4, p.1193-1205 5. IlariaLacobucci, Cristina Papayanndis, Annalisa Lonetti, Anna Ferrari, MichelsBaccarani, Giovanni Martinelli, Current Haematological Malignancies, Issue 7,2012, p 133-144 6. IrisH.
I.M. Hollink, Marry M.
van den Heuvel-Eibrink, Susan T.C.J.M. Arentsen-Peters,Martin Zimmermann, Justine K. Peeters, Peter J.
M. Valk, Brian V. Balgobind,Edwin Sonneveld, Gertjan J.L.
Kaspers, Eveline S.J.M.
de Bont, Jan Trka, AndreBaruchel, Ursula Creutzig, Rob Pieters, Dirk Reinhardt, C. Michel Zwaan, Characterization of CEBPA mutations and promoter hypermethylation inpediatric acute myeloid leukemia, Haematologica, 2011, Issue 96,p384-392 7. Montes, R ; Ayllón, V ; Prieto, C ; Bursen, A ;Prelle, C ; Romero-Moya, D ; Real, P J ; Navarro-Montero, O ; Chillón, C ;Marschalek, R ; Bueno, C ; Menendez, P, Ligand-independent FLT3 activation does not cooperate with MLLAF4 to immortalize/transform cord blood CD34+ cells Leukaemia, March 2014, Vol.28,Issue 3, p.666-74 8.
NavalDaver, Jorge Cortes, Molecular targeted therapy inacute myeloid leukaemia, Journal Haematology Volume 17, 2012 Pages s59-s62. 9. NianxiZhao, Sung-Nan Pei, Jianjun Qi, Zihua Zeng, Swaminathan P.Iyer, PeiLin, Ching-HsuanTung. YouliZu, Oligonucleotide aptamer-drug conjugates for targeted therapyof acute myeloid leukaemia, Biomaterials, Volume67, October2015, Pages 42-51 10. Nianxi Zhao, Sung-nan Pei, Jianjun Qi, Pei Lin and Youli Zu, TargetedTherapy of Acute Myeloid Leukemia By A CD117-Specific Aptamer-Drug-Conjugate, Blood, 2013, Vol 122, p1446.
11. Patel, Jay P, Levine, Ross L, How do novel molecular genetic markers influence treatment decisions in acute myeloid leukaemia?, American Society of Hematology, 2012,Vol.2012, pp.28-34 12. P Lee, R Bhansali, S Izraeli, N Hijiya J D CrispinoThe biology, pathogenesis and clinicalaspects of acute lymphoblastic leukemia in children with Down syndrome, , Leukemia, 2016, Issue 30, p – 1816–182313. Robert K Hills, Guy Gammon, Denise Trone and Alan K.
Burnett, QuizartinibSignificantly Improves Overall Survival in FLT3-ITD Positive AML PatientsRelapsed after Stem Cell Transplantation or after Failure of SalvageChemotherapy: A Comparison with Historical AML Database (UK NCRI data), , Blood, 2015, Issue 126, p2557. 14. Shen, Yang ; Zhu, Yong-Mei ; Fan, Xing ; Shi, Jing-Yi ; Wang, Qin-Rong ; Yan, Xiao-Jing ; Gu, Zhao-Hui ;Wang, Yan-Yan ; Chen, Bing ; Jiang, Chun-Lei ; Yan, Han ; Chen, Fei-Fei ; Chen, Hai-Min ; Chen, Zhu ; Jin, Jie ; Chen, Sai-Juan, Gene mutation patterns and their prognostic impact in acohort of 1185 patients with acute myeloid leukaemia, Blood, 17 November 2011, Vol.118(20), pp.5593-603 15. Sophia YoheCelalettin Ustun, Lucy A.
Godley, Molecular Genetic Markers in Acute MyeloidLeukemia, Journal Clinical Medicine. 2015, Issue 4, Vol 3, p 460–478 16. TzelepisKonstantinos, Koike Yuas Hiroko, De Breekeleer Etienne, Li Yilong, MetzatopianEmmanouil, Dovey Oliver, Mupo Annalisa, Grinevich Vera, Li Meng, Mazan Milena, ACRISPR Dropout Screen Identifies Genetic Vulnerabilities and TherapeuticTargets in Acute Myeloid Leukaemia, Cell Reports, Vol 17, Issue 4, 2016, p1193 –1205 17. Yanfeng Liu,Pengcheng He, Feng Liu, Lili Shi, Huachao Zhu, Jing Zhao, Yuan Wang, XiaoyanCheng, Mei Zhang, Prognostic significance of NPM1 mutations in acute myeloid leukemia: Ameta-analysis, Molecular Clinical Oncology, 2014, Vol 2, Issue 2, p 275–281. 18.
https://www.drugbank.ca/drugs/DB00619, Imatinib 19. https://www.cancer.
gov/news-events/cancer-currents-blog/2017/tisagenlecleucel-fda-childhood-leukemia.CAR T-Cell Therapy Approved for Some Children and Young Adults with Leukaemia, September2017.