Analyzingdrug resistance patterns to understand the evolution of therapeuticresistance in medulloblastoma and neuroblastoma cancer cell lines
Definition of Terms
Apoptosis-programmed cell death
“Chemotherapy”: chemical compounds given orally or intravenouslyto treat various
cancers.
Genetic mutation-change in gene structure
in vitro”: experiments on cultured cells.
“in vivo”: experiments on intact organisms.
Tumor heterogeneity-diversity in tumor properties
Metastasis-spread of cancerous cells from one location to another inthe body
Background
Medulloblastomaand Neuroblastoma
Medulloblastoma has been listed as a common brain tumor amongchildren contributing to 20% of the cases reported (Johnson et al.,2014). The condition has been perceived to be second after pilocyticastrocytoma in brain tumors affecting children. Medulloblastomas havebeen determined to occur during the first ten years of life and peakswhen one attains the age of 20 (Zyrianova, Alexander, & Faruqui,2015). The genetic tumor syndromes associated with medulloblastomasand Gorlin (nevoid basal cell carcinoma) and Turcot (familialadenomatous polyposis). Medulloblastomas are categorized as theembryonal tumor of the brain and are analogous to neuroblastoma ofthe adrenals and Wilms tumor of the kidney. The condition has a highassociation with the infants owing to its embryonal nature. Thetumors have an undifferentiated and an immature appearance that isrepresentative of a developing neural tissue. Medulloblastoma has ahistological and biological distinction separating it from the restof the tumors (Rey-Casserly, & Fennell, 2016).
Medulloblastomas arise in the midline, in the posterior vermis andare adjacent to the fourth ventricle. The tumor is commonlyassociated with the cerebellum. Medulloblastomas have been determinedto grow rapidly over a period of weeks and present with symptoms ofan increase in intracranial pressure and dysfunctioning of thecerebellar (Louis et al., 2015). Four primary histological variantsof medulloblastomas are recognized the classic, large cellanaplastic, nodular-desmoplastic and medulloblastoma that hasextensive nodularity. Classic medulloblastomas are highly cellularand composed of diffuse masses of undifferentiated, small oval cells(Moxon-Emre, 2013). The neuroblastoma shows glial, neuronal and otherforms of differentiation. Neuronal differentiation is characterizedby the formation of neuropil and rosette. Glial differentiation ischaracterized by GFAP-positive cells. Further differentiation may bemanifested in oligodendroglia or the ependymal lines. Desmoplasmicmedulloblastoma is nodular and permeated by collagen fibers givingthem a firm consistency. The tumors are less cellular compared to thesurrounding packed small cells (Swanton, 2012). Desmoplasmicreactions are witnessed as the tumor extends to the subarachnoidspace. The tumors are superficially located and are circumscribedmaking them easier to resect. Desmoplasmic tumors are common ininfants and have a high chance of prognosis compared to the classicforms (Moxon-Emre, 2013). Medulloblastomas with extensive nodularityare difficult to distinguish from desmoplastic but also have a goodprognosis. Large-cell anaplastic medulloblastomas are defined bylarge nuclei that have a high rate of apoptosis and mitosis (Swanton,2012). The variant, however, has a poor prognosis compared to theother forms stated. Medulloblastomas are highly malignant. Theyinfiltrate, destroy the brain tissue and can seed in the subarachnoidspace resulting in its spread to the walls of ventricles. An analysisof cerebrospinal fluid indicates a high amount of protein, but lowglucose with many tumor cells. Treatment of the condition entails thecombination of resection to help in the reduction of tumor mass whiledecompressing the fourth ventricle (Lammers, Kiessling, Hennink, &Storm, 2012). Other modes of treatment that have been employed in thetreatment are radiation of tumor bed, shunting lateral ventricles andapplication of intrathecal chemotherapy.
Neuroblastomas represent a common form of extracranial solid tumorsof infants (Rey-Casserly, & Fennell, 2016). It is characterizedby embryonal malignancy of a sympathetic nervous system that arisesfrom the neuroblasts. As the embryo develops, the cells invaginate,migrating to the nuraxis followed by its population to thesympathetic ganglia and the adrenal medulla. The pattern ofdistribution of cell lines dependent on the site of primaryneuroblastoma presentation. Treatment of neuroblastomas is dependenton understanding the age, stage and biological features of the tumor.Differences exist regarding the outcome of patients suffering fromneuroblastomas. Those with a lower risk and intermediate riskneuroblastoma portray an excellent prognosis and outcome. Contrary tothe same, those at high risk depict poor results despite theintensive therapy methods applied for treatment. Statistics indicatethat 70-80% of patients older than eighteen months have a chance ofpresenting with metastatic disease in regions such as the liver,lymph nodes, bone and bone marrow (Louis et al., 2015). Less thanhalf of the patients who present with the condition are cured despitemeasures that include the application of high-dose therapy. Childrenunder the age of five years are the most affected and in 70% of thecases, the tumor starts in the adrenal gland that is located in theabdomen (Ward, DeSantis, Robbins, Kohler, & Jemal, 2014). Thecondition could be present at birth but rarely is it detected.However, it could be detected by performing an ultrasound test on thefetus. Cases of children suffering from the condition are high amongchildren aged below one year, with most diagnoses being made beforeone reaches six months of age. Neuroblastomas have however beendetermined to regress by themselves with researchers not yet able toexplain the rationale behind the phenomena. The most appropriatetreatment option for the condition is the application of a bonemarrow transplant. However, chemotherapy has also been employed whenit comes to the treatment of the condition. The drugs used to serveto kill the cancer cells while at the same time shrinking the tumors(Zyrianova, Alexander, & Faruqui, 2015). The method relies on theapplication of powerful anti-cancer drugs through injection into theveins or muscle. Different stages of neuroblastomas have beendepicted to exist. They include localized resectable, localizedunresectable, regional, disseminated (Stage IV), Stage IVS andrecurrent. The form of treatment employed for each of the typesvaries.
Precision Medicines in Cancer Treatment
The application of precision medicine in the treatment of cancerentails giving the most efficient form of treatment for everyindividual’s disease. Customization of the form of therapy would bedependent on understanding the individual’s genetic setup,non-genetic factors such as lifestyle and the molecularcharacteristics of the given disease. The primary goal of applicationof precision medicine is to ensure patients get the most efficientform of treatment for the illness they are suffering. Precisionmedicine can be applied to treatment and management ofmedulloblastoma and neuroblastoma among those affected. Developmentof therapies that target the specific tumors stands as the bestpathway of managing the conditions resulting in growth of the tumor(Lammers, Kiessling, Hennink, & Storm, 2012). Precision medicineaims at ensuring that treatment guarantees the patient of bestresults, especially for medulloblastomas and neuroblastomas. Further,the choice of treatment employed ought to aim at ensuring that sideeffects emanating from the treatment are avoided since they result inadded costs of management of the disease. Understanding the childaffected by both medulloblastomas and neuroblastomas serves as aprerequisite for the management of the condition. Further, by gaininga broad knowledge about the tumors, it is possible to employprecision treatment to facilitate their management. Particularly, itis crucial to understand the prognosis of the disease to help in thepersonalization of the treatment. The goal is to ensure that patientsbenefit the most out of the treatment that they have been subjected(Wicki, Mandalà, Massi, Taverna, Tang, Hemmings, & Xue, 2016).Further, the genetic profile of both medulloblastoma andneuroblastoma cells have a significant impact when it comes todetermination of the mode of treatment employed.
Examples of Treatment Employed
Different therapeutic agents have been used when it comes totreatment and management of the tumor cells. RAF and MEK inhibitorshave been determined to play a fundamental role when it comes to theinhibition of proliferation of cancer cells (Wicki et al., 2016).Particularly, RAF inhibitors are essential in the prohibition ofBRAFV600E . The ability of the drugs to curtail theproliferation of tumor cells, particularly in medulloblastomas andneuroblastomas, may serve as a ground to for treatment. MEKinhibitors have also been determined to play a fundamental role incurbing the proliferation of the cells. Notably, MEK inhibits ERKphosphorylation in tumor cells (Maves et al., 2013). Overall, the twodrugs BRAF and MEK are essential since they affect the expression ofgenes involved in the proliferation of the tumor cells. Studiesconducted by Joseph et al. (2010) illustrate that MEK and RAFinhibition affects the expression of same genes as those of BRAFV600Ein melanomas.
Further, a prime target for the tumor cells is the introduction ofinhibitors that seek to prohibit the epidermal growth factorreceptors. EGFR have been determined to be overexpressed or have adysregulated function in various solid tumors. The introduction ofagents that target the EGFR-mediated signaling pathways may serve asan appropriate therapeutic target for medulloblastomas andneuroblastomas. The decision to use the drugs emanates from the factthat they are active against malignancies and could be useful indealing with the cancers. Apoptosis in tumor cells is ofteninactivated (Jha et al., 2016). The use of BRD inhibitorsserves as important agents to help in prohibiting the proliferationof the tumor cells in medulloblastomas.
Clonal evolution and intratumoral heterogeneity often result indrug resistance to cancer therapies
Different tumor cells have distinct morphological and phenotypiccharacteristics. Particularly, there are variations in cellularmorphology, metabolism, gene expression, motility, metastaticpotential and proliferation (Swanton, 2016). The phenomenon regardingthe distinction in the morphological and the phenotypic traits isheterogeneity. The variation in phenotypic and morphologicalcharacteristics presents a challenge the process of designing anefficient treatment strategy. Despite the difficulties in drugdesign, extensive research is being conducted to help understand andcharacterize the heterogeneity to comprehend the causes and progressof a disease. It results in the potential to guide the process ofcreating a refined form of treatment strategy through theincorporation of knowledge gained from the same to yield a highefficacy. Tumor heterogeneity has been observed in medulloblastomasand neuroblastomas.
Clonal evolution proposed by Peter Nowell suggests that tumorsemanate from a cell that has been mutated resulting in theaccumulation of additional mutations as they progress (Greaves, &Maley, 2012). The changes lead to the other subpopulations with eachof them having the ability to mutate and divide further. Theheterogeneity could result in the development of subclones thatpossess an evolutionary advantage over others in the tumorenvironment. The subclones could become dominant within the tumorover time. The model provided room for the understanding of thegrowth of tumors, failure in treatment methods and tumor aggressionthat occurs in the natural process of its formation. Initial tumorsthat evolve could do so through two different methods.
Linearexpansion
Mutations that are sequentially ordered accumulate in the drivergenes, DNA repair enzymes and tumor suppressor genes resulting in theclonal expansion of tumorigenic cells. Linear expansion has a lowerchance of reflecting the endpoint of a malignant tumor because theaccumulation is stochastic in the heterogenic tumors.
Branchedexpansion
Branched expansion occurs through the splitting mechanism. Mutationacquisition is random and could result in an increase in genomicinstability with successive generations. The long-term accumulationconfers a selective advantage in the stages of tumor progression.Tumor expansion may be affected by the tumor microenvironment becauseit is endowed with the ability to changing the selective pressureswhere the tumor cells are exposed.
Intratumoral heterogeneity exhibits variations in the sensitivitiestowards cytotoxic drugs. The rationale behind the differentsensitivity is associated with the clonal interactions that couldinhibit or alter therapeutic efficacies in treatment depictingsignificant challenges in the management of cancers. Administrationof drugs in the treatment of heterogenic tumors has not managed tokill them. The heterogenic tumor cells have developed resistant celllines that survive in the presence of the cancer cells. Through thesame, there is a chance of the resistant tumor populations to growand replicate through the branching evolution mechanisms. Theresulting tumor cells are diverse morphologically and phenotypicallyaccompanied by a resistant trait toward the drug that is applied fortreatment.
Cancer therapies have often failed to achieve the goals for whichthey are meant because of the development of resistant cancer celllines. Medulloblastomas and neuroblastomas have continued to cause asignificant amount of suffering especially on those affected becausethe cancer cells are not responsive toward the form of treatment thatis employed (Gupta et al., 2015). Resistant clones have a selectiveadvantage and are endowed with the ability to replicate and growfurther. Replication occurs through branching evolution resulting intumor heterogeneity. Repopulated tumors could be more aggressivebecause of the drug-resistant selective advantage of tumor cells.
DrugResistance
Drug Resistance has been witnessed with drugs such as BRAF inhibitors(Sebolt-Leopold, & English, 2006). Vemurafenib and Dabrafenibwere initially active against the melanomas but experiencedresistance approximately seven months after commencement of treatment(Girotti et al., 2015). The application of the drugs presented anadequate response rate and was crucial in the improvement ofsurvivability of patients having the BRAFV600E metastaticmelanoma. The acquisition of drug resistance and issues of toxicitypresented a significant challenge when it came to the use of drugs. According to studies, the majority of the resistance towards the BRAFinhibitors results in the promotion of reactivation of MAP kinasesignaling pathways in their presence (Mandal, Becker, &Strebhardt, 2015). An example is the case of mutational activation ofMEK1 or MEK2 that could reactivate MAP kinase pathway in the presenceof the BRAF inhibitor. Further, an elevation of CRAF proteins plays acrucial role in conferring resistance towards BRAF inhibitors in thecell culture models of the melanomas.
However, further analysis is needed to ascertain the role of the CRAFprotein in enhancing the resistance towards the drugs. In otherstudies, it was shown that serine/threonine MAP kinase COT kinasethat was encoded by MAP3K8 activated MEK in the presence of BRAFinhibition (Gruosso et al., 2015). An increase of COT copy number andthe mRNA expression is seen in biopsy specimens of the melanomasafter treatment with Vemurafenib (Lito, Rosen, & Solit, 2013).Further, there is the aspect of the overexpression of the mutant formof BRAF protein that is essential in cancer drug resistance. Also,identification of the BRAFV600E splice variants that endowproteins the ability to dimerize in RAS-independent manner increaseskinase activity of proteins resulting in the resistance mechanismthat entails a structural change to BRAF (Sullivan, & Flaherty,2013). Such mechanisms are crucial for the role they play inpromoting the resistance seen by the cancer cells towards the drugs.
Challenge
Drug resistance has detrimental effects on the patients. Criticalamong them being death. Chemotherapy has failed to achieve its targetof curing cancer patients suffering from advanced diseases (Lammers,Kiessling, Hennink, & Storm, 2012). Patients withmedulloblastomas and neuroblastomas are at a risk of losing theirlives because of the evident failure of available drugs in the marketto help in dealing with the tumor cells. Presence or development ofresistance toward anticancer agents is a significant cause of thefailure of the drugs. However, much focus has shifted toward gainingan understanding of the mechanisms that underlie drug resistance tothe cytotoxic drugs. Over the past two decades, researchers haveemployed in vitro drug-selected cancer cell lines to understand theconcept of resistance against the anticancer cells. Because of theinability of drugs to tackle the drugs, the results is patientssuccumbing to the cancerous disease. Medulloblastomas andneuroblastomas that account for approximately 70% of the patientdeaths witnessed among infants have managed to countering the effectof the drugs used in their management (Ward, DeSantis, Robbins,Kohler, & Jemal, 2014). Because of the continued loss of liveswitnessed due to the cancerous cells, it becomes mandatory to developchannels through which the resistance can be comprehended to helpreduce the mortality rates that are observed because of the cancers.However, various attempts have been made that seek to deal with theissues with some physicians recommending the use of a wide array ofdrugs to help in dealing with the menace. The dangers of such aprocedure, however, is the likelihood of development of multidrugresistance.
Processof the evolution of drug resistance
Medulloblastomas and neuroblastomas have the ability to developresistance to the traditional therapies employed in their treatment(Markant et al., 2013). Further research is needed to facilitate theunderstanding of resistance and proper channels of drug developmentto come up with therapies endowed with the ability to tackle thediseases. Drug resistance is a common phenomenon resulting indiseases becoming tolerant to the pharmaceutical regimens employed.The concept of drug resistance came to be considered with bacteriabecoming resistant to antibiotics developed toward their treatment.However, similar characteristics of drugs have been since to occur indiseases including cancers. Medulloblastomas and the neuroblastomasmainly have developed a resistance toward the drugs used in theirtreatment (Markant et al., 2013). Different methods of drugresistance exist with some being disease-specific and others beingdrug efflux as seen in the human drug-resistant cancers. Cancers areinitially susceptible to therapeutic methods, with time, thecancerous cell lines develop resistance through mechanisms such asDNA mutations and metabolic changes that seek to promote drugdegradation and inhibition (Alexandrov et al., 2013).
The activation of a particular drug used for therapy entails a seriesof complex mechanisms through which substances interact with avariety of proteins. The interactions could modify, degrade orcombine with proteins resulting in the drug becoming activated toexecute its functions. Anticancer drugs have to undergo the processof metabolic activation for them to acquire the clinical efficacy toexecute the purpose for which it is meant (McGuigan, 2016). In casesof drug resistance, there is a failure of the drugs to becomeactivated because of the effect of cancerous cells. An example is inthe case of the medulloblastomas and neuroblastomas where cancercells are active against the drugs used to fight them.
Evolutionary models of drug resistance can be understood through theapplication of population genetic models (Al-Lazikani, Banerji, &Workman, 2012). The target of the drug is a protein or enzyme thathas evolved to conduct its biological functions. For example, akinase could bind and phosphorylate the specific proteins. Intricatecontrol applies where the drug target becomes activated throughdifferent interactions as it activates the others. Mutations couldmake the protein active by curtailing the control measures againstit. After the drug has targeted the particular protein, it isincapable of executing the functions that it is meant. Particularly,the protein would not be able to execute its tumorigenic properties.Mutations that result in drug resistance can be divided into twogroups. The first serves to interfere with the ability of the drug tobind to the target protein. The second allows the drug to executeefficiently the functions for which it is meant but enables the tumorto become resistant. Mutations that entail binding of the drug do sothrough the alteration of the active domain of a target protein. Itcould be the catalytic or regulatory domain (Alexandrov et al.,2013). The direct modification involves the interference of residuesat a target binding site. Indirect alteration involves thedestabilization of the inactive site state which binds to the givendrug. The effect of such activities is the ability of the cancercells to cope with the drug making it ineffective. After survivingthe presence of the drug, cancerous cells proceed to multiply throughgrowth and reproduction. The changes witnessed become difficult tomanage efficiently the cancerous cells resulting in them spreadingand continuing to cause more harm.
PreexistingDrug Resistance
Drug resistance can be categorized as being pre-existing. Mutationsresult in the particular cell lines gaining the capability not to besusceptible to the drugs used in their treatment. Specifictumorigenic cells develop resistance to drugs because of thepreexisting conditions. Particularly, there is the aspect of originalcell lines gaining the ability to adapt to a particular drug. Havingacquired the traits that confer to it the resistance patterns, thecharacteristics are passed to the next generation of cell lines. If asimilar drug was to be used in the treatment of the particular cancerdisease, the chances are that it would not be effective. Therationale behind the inability of the drug to adapt to the changes inthe cancerous proteins.
Preexisting drug resistance emanates from the fact that initially,cells that have the ability to resist the drugs originated from thosethat were originally susceptible. It is out of the evolutionarychanges that the cells have gotten the capacity to withstand thecytotoxic effects of drugs that were employed against them. Forexample, initially, BRAF inhibitors were active againstmedulloblastomas and the neuroblastomas (Suijkerbuijk, 2015).However, through the evolutionary changes, the cell have becomeactive against drugs that are currently employed in their treatment.The cancer diseases that develop the resistance pass on the acquiredtraits to the subsequent generations (Wicki et al., 2016). The effectof such a feature is difficulty in management of the disease. Thesituation is compounded by the fact that the drugs that are developedare not endowed with the ability to cope with the transformationswitnessed in the new cell lines. Because of the same, cancer cellsincluding the medulloblastomas and neuroblastomas continue to causesuffering on those affected.
Identificationof Drug Combinations to Disrupt Drug Resistance Patterns
Drug resistance continues to present a significant challenge when itcomes to treatment of cancer diseases (Fedele, Tothill, &McArthur, 2014). The existing therapies used for treatment have notbeen efficient when to curb the effects of drugs efficiently. Theincrease in cases of drug resistance negates the need to come up withbetter treatment strategies that seek to enhance the process oftherapy. Cancer causing diseases cause a significant amount of painto patients (Abrahm, 2014). Because of the same, it could be crucialto try alternatives that would promote treatment. A viable optioncould be the possibility of identification of drug combinations thatseek to disrupt the drug resistance patterns (Sun, Bao, Nelson, Li,Kulik, & Zhou, 2013). Evidently, as cancer causing disease getsto adapt in the presence of the drug, identification of methodsthrough which the patterns interfere with serves as a good ground ofdealing with the resistance. Through the same, it would be anopportunity to curb the detrimental effects of drug resistance whilerestoring the ability of the regimen to deal with the disease.
The understanding of molecular mechanisms that underlie synergistic,antagonistic and potentiating effects of the drug combinations serveas an opportunity to address the cancer disease. Particularly, itcould be crucial to understanding the molecular interactions ofdifferent drugs with a specific protein involved in the proliferationof illness (Bozic, et al., 2013). Through the comprehension of thesame, it would be prudent to conduct an analysis of various drugcombinations that have been identified to ascertain the possibilityof using them as potential regimen in the treatment of cancers suchas medulloblastomas and neuroblastomas. Research should focus ongaining an understanding of molecular interaction profiles tofacilitate treatment of the particular disease.
Conventional chemotherapeutic methods that have been employed in thetreatment of cancers incorporate various strategies to achieve thegoal of therapy. The selection of the most appropriate drug to fightthe cancerous disease may be a challenge (Dietel, 2016). However,through the identification of the most efficient combination, it canbe possible to reduce the burden of disease brought about by cancer.Various models have been employed to identify the best combinationthat can be employed to facilitate the working of the drug againstthe cancerous disease.
The first technique that can be used in the identification of themost effective drug combination is an understanding of the geneticmakeup of the illness causing cancer. Drugs that have previously beenintroduced to tackle cancer have focused on a particular protein thatfacilitates the proliferation of the cancerous cell (Panowski,Bhakta, Raab, Polakis, & Junutula, 2014). The drugs work byinhibiting the particular target thereby resulting in apoptosis ofthe cells. However, with time, the technique has proven not to beeffective because mutations endow the cells to thrive in the presenceof the drug. It is mandatory to consider that knowledge about thebest combination could serve to increase chances of efficientlymanaging cancer since the disease is attacked from different angles.
Drug combinations could include the use of about three to fourdifferent drugs that target various parts of the disease-causingpathogen (Crystal et al., 2014). For example, the drugs couldseparately inhibit different metabolic pathways of cells responsiblefor the proliferation of the disease. DNA synthesis, cellular energyproduction, and signaling pathways are some of the metabolic routesthat each of the drugs used could inhibit resulting in the death ofthe particular cancerous cells. Different drugs employ a variety ofmodes of actions to counteract the disease. An understanding of themechanisms through which the drugs work against the disease couldserve as a basis to determine the most appropriate combination tohelp in dealing with the disease. The reasoning could be employed tooffer an efficient therapeutic outcome for the disease compared towhen the drug was used singly.
Medulloblastomas and neuroblastomas can efficiently be managedthrough the application of combination drug treatment while at thesame time eliminating the chance of drug resistance and reduction ofthe side effects (Wojtalla et al., 2012). The combination of a seriesof regimens is non-trivial due to a high number of possibilities.Studies conducted in other forms of cancer such as angiogenic haveshown greater potential in reducing the rate of proliferation of thedisease. The decision to combine different types of drugs couldparticularly be useful since it presents with the opportunity oftargeting different pathways. The drugs could be combined based onthe ability to efficiently attack a particular target of the disease. Through the application of a combination of the drugs, the chancesare that the there is a high level of toxicity that the cancer cellswould have to face. The effect is a reduction in the detrimentalimplications of the cancer-causing disease. Much evidence points tothe possibility of employing a variety of drugs to help in themanagement of cancer. However, it is essential to assess the level oftoxicity that could arise if both cells were to be subjected to thecombination. Normal cells must be shielded from the toxicityresulting from the combination of the drugs.
II.
The Research Problem
Significant progress has been made in the context of understandingthe molecular mechanisms of drug resistance to cancer therapies, butvery little is known regarding the evolution of resistancemechanisms. It is quite common for patients receiving treatment todevelop resistance to the inhibitors. For instance, BRAFinhibitors prompt fairly rapid antitumor responses when treatmentis administered, however, after approximately seven months (mediantime observed) drug resistance develops and the disease advances(Flaherty, et al., 2010 Hauschild, et al., 2012).Another common therapeutic target in cancer is the epidermal growthfactor receptor (EGFR) gene using tyrosine kinase inhibitors(TKIs), where tumor cells eventually develop resistance after 10 –16 months of drug therapy, resulting in disease progression(Maemondo, et al., 2010). Despite the initial response totreatment, disease relapse due to drug resistance is a primarysubject in cancer research. While several escape mechanisms totreatment have been discovered, there is still insufficient evidencerevealing resistance patterns, their evolution, and how resistancecan be prevented. If we can identify an ideal combination of drugtreatments that can disrupt the patterns of resistance, patientrelapse could be prevented.
Some patterns of resistance can be pre-existing, while others appearto be stochastic and arise due to epigenetic changes. Drug resistancemight also be acquired by tumor cells attempting to evade treatment,forcing new mutations to emerge in the targeted molecule or in thegenes of the downstream pathways (Easwaran, et al., 2014).Generating resistant cell lines is one way to recapitulate the drugresistance observed in cancer patients (Wacker, et al., 2012Jha, et al., 2016). As a result, these cell lines can be usedas models for testing various inhibitors and compounds to understanddrug action mechanisms, and in turn, resistance mechanisms andpatterns.
To better understand the process of drug resistance evolution, weneed to determine whether resistance patterns are pre-existing,stochastic, or plastic (malleable). One way of doing this is togenerate drug resistant cell lines
MainHypothesis
Observingselection patterns directly will reveal that drug resistanceevolution is malleable (preexisting and stochastic)
Aim #1
Hypothesis
The compounds being tested will individually yield different patterns of resistance.
Experimental plan
Use barcoded medullo- and neuroblastoma cell lines
Compounds to test:
BETi
PI3Ki
CDK 4/6-i
BCL2i
CDK7
I-BET (negative control)
Aim #2
Hypothesis
The resistance patterns in combination will guide us to the most efficient therapeutic target(s).
Experimental plan
Analyze resistance patterns resulting from drug combinations to determine an ideal combination(s).
Research Methods
The study analyzes the drug resistance patterns to understand theevolution of therapeutic resistance in medulloblastoma andneuroblastoma cell lines. Medulloblastoma and neuroblastoma celllines will be cultured. The need is to come up with viable cell linesupon which selected drugs would be tested. Susceptibility of the celllines towards the drugs would be assessed alongside the patterns ofresistance. Compounds that would be tested include BETi, P13Ki,CDK4/6-I, BCL2i, and CDK7. I-BET will be used as a negative control.The drug susceptibility would be tested through culturing a fixedamount of the cell lines of medulloblastoma and neuroblastoma celllines in the presence of the inhibitory drugs highlighted.Concentrations of drug needed to inhibit the replication and growthof cell lines by 50% or 90% would be employed as the most commonmeasure of the susceptibility to the drug. Standard amounts of thecell lines would be used in conducting the experiment to ascertainthe viability of each of the drugs.
Following treatment with the inhibitory drugs, medulloblastoma andneuroblastoma cell lines would be assessed for a defined duration oftime to check on how susceptible the cell lines are in the presenceof the inhibitory drugs. Cell lines that survive in the presence ofthe drugs would be assessed for changes in morphological traits togain a broader understanding of drug resistance patterns.
ResearchLimitations
Multiple interactions within the cell lines determine the cancerproperties. A variety of genes is expressed in the medulloblastomaand neuroblastoma cells that undertake the duties of apoptosis,metabolism, repair and detoxification of the drugs. Similarly,different drug combinations used in the experiment produce variedindividual responses among the cell lines. Because of the same, itcould be difficult to monitor the changes in the cell lines andattribute them to a particular drug.
The experimental conditions used in the assessment of the patterns ofdrug resistance may vary significantly from the typical human model.The environment with which the drugs would be working is different.Because of the same, the chances are that the variations inexperimental conditions could affect the results. For example, thedrugs could be active against the medulloblastomas and neuroblastomasin vitro but fail to show the same level of activity in vivo. Itcould be essential to proceed with human trials to determine theefficacy of the drugs. Further, such variations could affect theability efficiently to understand the patterns of drug resistanceamong medulloblastomas and neuroblastoma cell lines.
Further, maintaining pure cultures of medulloblastomas andneuroblastomas may present a significant challenge to those executingthe experiment. Skill and expertise are needed to ensure thatcultures that would be employed to test for patterns of resistanceare pure to help in yielding the best results.
Conclusion
Analysis of drug resistance patterns among medulloblastomas andneuroblastoma cell lines serves is essential in helping come up withan efficient strategy to deal with the issue. Because of thecontinued evolution of cancerous cells, it is fundamental to have aproper framework through which mechanisms through which the processtakes place. Knowledge about the patterns of resistance serves as anopportunity of introducing effective therapeutic strategies to helpdeal with the menace. Owing to the increase in number of children whosuffer from the tumors, an understanding of the drug resistancepatterns could act as a potential method to identify the besttechnique to deal with the challenge. An investigation on theresistance patterns could promote measures that seek to introducetherapeutic techniques to help in killing of cancerous cells.
Annotated Bibliography
Al-Lazikani, B., Banerji, U., & Workman, P. (2012). Combinatorialdrug therapy for cancer in the post-genomic era. Naturebiotechnology, 30(7), 679-692.
Researchers elaborated on aspects of combining different drugs to be used for cancer therapy.
Acknowledges potential of drug combination as basis for cancer treatment.
Abrahm, J. L. (2014). A physician`s guide to pain and symptommanagement in cancer patients. JHU Press.
Paper elaborates management of paper for cancer patients undergoing therapy.
Draws from sentiments of physicians as patients are taught on best practices to cope with the pain.
Alexandrov, L. B., Nik-Zainal, S., Wedge, D. C., Aparicio, S. A.,Behjati, S., Biankin, A. V., … & Boyault, S. (2013). Signaturesof mutational processes in human cancer. Nature, 500(7463),415-421.
Researchers focus on aspects of mutation among the cancer cells and provide an overview of how the process takes place.
Hallmarks of mutation discussed in relation to cancer.
Bozic, I., Reiter, J. G., Allen, B., Antal, T., Chatterjee, K., Shah,P., … & Lipson, E. J. (2013). Evolutionary dynamics of cancerin response to targeted combination therapy. Elife, 2,e00747.
Researchers state increase in cases of resistance toward available cancer therapies.
Combinational therapy identified as best chance of dealing with the mutagenic cancer cells.
Employ dual therapy to prove efficacy of the same.
Crystal, A. S., Shaw, A. T., Sequist, L. V., Friboulet, L., Niederst,M. J., Lockerman, E. L., … & Lee, D. (2014). Patient-derivedmodels of acquired resistance can identify effective drugcombinations for cancer. Science, 346(6216), 1480-1486.
Cases of drug resistance a common occurrence in cancer therapy.
Pharmacological profiling of patients could serve as best way to deal with drug resistance as patients are customized to a given drug that is efficient for them.
Dietel, M. (2016). Personalized Medicine Challenges the Health CareSystem. In Boundaryless Hospital (pp. 143-155). SpringerBerlin Heidelberg.
Explores possibility of using molecular based interventions not only for diagnostics but treatment to help curb rising cost of healthcare service provision.
Easwaran, H., Tsai,H., Baylin, S.B. (2014). Cancer epigenetics: tumor heterogeneity,plasticity
of stem-likestates, and drug resistance. Molecular Cell, 54, 716-727.
Figure 4 demonstrates the idea of pre-existing resistance or resistant mutations arising due to epigenetic changes.
Fedele, C., Tothill, R. W., & McArthur, G. A. (2014). Navigatingthe challenge of tumor heterogeneity in cancer therapy. Cancerdiscovery, 4(2), 146-148.
Researchers acknowledge intertumoral heterogeneity as a primary challenge in efficient cancer management.
Computational model explored as a potential to check on heterogeneity and provide room for intervention.
Flaherty, K.,Puzanov, I., Kim, K., Ribas, A., McArthur, G., Sosman, J., O’Dwyer,P.J., Lee,
R.J., Grippo, J.F.,Nolop, K., Chapman, P.B. (2010). Inhibition of mutated, activated
BRAF in metastaticmelanoma. N Engl J Med 363(9), 809-819.
In patients with V600E BRAF mutations, researchers show that response to PLX4032 treatment ranges from 2 – 8 months, before displaying drug resistance.
Girotti, M. R., Lopes, F., Preece, N., Niculescu-Duvaz, D., Zambon,A., Davies, L., … & Suijkerbuijk, B. M. (2015).Paradox-breaking RAF inhibitors that also target SRC are effective indrug-resistant BRAF mutant melanoma. Cancer cell, 27(1),85-96.
BRAF and MEK inhibitors identified as potential chemotherapeutic agents but patients seen to relapse as resistance toward drugs erupts
Resistance mediated by reactivation of pathway RTKs
Greaves, M., & Maley, C. C. (2012). Clonal evolution in cancer.Nature, 481(7381), 306-313.
Gruosso, T., Garnier, C., Abelanet, S., Kieffer, Y., Lemesre, V.,Bellanger, D., … & Mechta-Grigoriou, F. (2015).MAP3K8/TPL-2/COT is a potential predictive marker for MEK inhibitortreatment in high-grade serous ovarian carcinomas. Naturecommunications, 6.
Genetic diversity identified to play a crucial role in diversification of cancers resulting in development of mutants.
Gupta, M., Dahiya, J., Marwaha, R. K., & Dureja, H. (2015).THERAPIES IN CANCER TREATMENT: AN OVERVIEW. International Journalof Pharmacy and Pharmaceutical Sciences, 7(4).
Identifies possibility of understanding genes and signaling pathways to help deal with cancer diseases efficiently.
States need to conduct further research on cancer stem cells to facilitate development of therapies.
Hauschild, A., Grob, J., Demidov, L.V., Jouary, T., Gutzmer, R.,Millward, M., Rutkowski, P., Blank, C.U., Miller Jr., W.H., Kaempgen,E., Martin-Algarra, S., Karaszewska, B., Mauch, C., Chiarion-Sileni,V., Martin, A., Swann, S., Haney, P., Mirakhur, B., Guckert, M.E.,Goodman, V., Chapman, P.B. (2012). Dabrafenib in BRAF-mutatedmetastatic melanoma: a multicentre, open-label, phase 3 randomisedcontrolled trial. Lancet 380, 358-365.
Research shows that the median progression-free survival for patients receiving dabrafenib was 5.1 months.
Jha, S., Morris,E.J., Hurza, A., Mansueto, M.S., Schroeder, G., Arbanas, J.,McMasters, D.,
Restaino, C.R.,Black, S., Elsen, N.L., Mannarino, A., Cooper, A., Fawell, S., Zawel,L.,
Jayaraman, L.,Samatar, A.A. (2016). Dissecting therapeutic resistance to ERK
inhibition. MolCancer Ther 15, 548-559.
Demonstrates the use of an established cell line as a model for ERK inhibitor resistance.
Johnson, K. J., Cullen, J., Barnholtz-Sloan, J. S., Ostrom, Q. T.,Langer, C. E., Turner, M. C., … & Schwartzbaum, J. A. (2014).Childhood brain tumor epidemiology: a brain tumor epidemiologyconsortium review. Cancer Epidemiology Biomarkers &Prevention, cebp-0207.
Focus is on understanding risk factors associated with childhood cancers to help address the problem.
Lammers, T., Kiessling, F., Hennink, W. E., & Storm, G. (2012).Drug targeting to tumors: principles, pitfalls and (pre-) clinicalprogress. Journal of controlled release, 161(2),175-187.
Explores routes employed in targeting of tumors and identifies them as potential for application of therapy.
Lito, P., Rosen, N., & Solit, D. B. (2013). Tumor adaptation andresistance to RAF inhibitors. Nature medicine, 19(11),1401-1409.
Explores adaptation of tumors to treatment regimen such as RAF inhibitors contributing to the issue of resistance.
Louis, D. N., Perry, A., Reifenberger, G., von Deimling, A.,Figarella-Branger, D., Cavenee, W. K., … & Ellison, D. W.(2015). The 2016 World Health Organization classification of tumorsof the central nervous system: A summary. Acta neuropathologica,1-18.
Tumors are classified through the use of molecular parameters to help in diagnosis of the disease.
Medulloblastomas and neuroblastomas classified under such basis.
Maemondo, M., Inoue, A., Kobayashi, K., Sugawara, S., Oizumi, S.,Isobe, H., Gemma, A., Harada, M., Yoshizawa, H., Kinoshita, I.,Fujita, Y., Okinaga, S., Hirano, H., Yoshimori, K., Harada, T.,Okura, T., Ando, M., Miyazawa, H., Tanaka, T., Saijo, Y., Hagiwara,K., Morita, S., Nukiwa, T., North-East Japan Study Group (2010).Gefetinib or chemotherapy for non-small-cell lung cancer with mutatedEGFR. N Engl J Med 362(25), 2380-2388.
Authors compare progression-free survival rates in patients receiving gefitinib and chemotherapy treatment for metastatic, non-small-cell lung cancer.
Mandal, R., Becker, S., & Strebhardt, K. (2015). Stamping out RAFand MEK1/2 to inhibit the ERK1/2 pathway: an emerging threat toanticancer therapy. Oncogene.
RAS-RAF-MEK1/2-ERK1/2 pathway identified to be crucial in cancer development and could serve as potential targets for therapeutic interventions.
Markant, S. L., Esparza, L. A., Sun, J., Barton, K. L., McCoig, L.M., Grant, G. A., … & Remke, M. (2013). Targeting sonichedgehog-associated medulloblastoma through inhibition of Aurora andPolo-like kinases. Cancer research, 73(20), 6310-6322.
Explores options of treatment and management of medulloblastomas after failure and detrimental side effects of chemotherapy, radiation or surgery.
Mayes, P. A., Degenhardt, Y. Y., Wood, A., Toporovskya, Y., Diskin,S. J., Haglund, E., … & Maris, J. M. (2013). Mitogen‐activatedprotein kinase (MEK/ERK) inhibition sensitizes cancer cells tocentromere‐associatedprotein E inhibition. International Journal of Cancer, 132(3),E149-E157.
(MEK/ERK) activity identified as a potential biomarker and could be applicable in dealing with neuroblastomas.
McGuigan, C. (2016). Drug metabolism: manipulating the microbiome.Bipolar disorder, 11, 00.
Explores aspects of drug metabolism and the need to understand the process of activation to help in dealing with the disease.
Moxon-Emre, I. (2013). Neuropsychological Outcome followingCranio-spinal Radiation in Medulloblastoma Patients: A LongitudinalAnalysis of Predictors (Doctoral dissertation).
Illustrates side effects of radiation therapy on medulloblastomas on children and possibility of exploring other options in management of the disease.
Panowski, S., Bhakta, S., Raab, H., Polakis, P., & Junutula, J.R. (2014, January). Site-specific antibody drug conjugates for cancertherapy. In MAbs (Vol. 6, No. 1, pp. 34-45). Taylor &Francis.
Explains the application of antibodies to target tumors responsible for cancers.
Potential for management of disease owing to increase in cases of resistance.
Rey-Casserly, C., & Fennell, E. B. (2016). 12 Brain tumors inchildren. Textbook of Clinical Neuropsychology, 171.
Identifies different types of brain tumors among children and the treatment approaches employed in their treatment.
Sebolt-Leopold, J. S., & English, J. M. (2006). Mechanisms ofdrug inhibition of signalling molecules. Nature, 441(7092),457-462.
Explores the new trend of cancer therapy where focus is on signaling molecules and potential they have in treatment.
Sullivan, R. J., & Flaherty, K. (2013). MAP kinase signaling andinhibition in melanoma. Oncogene, 32(19), 2373-2379.
Mutations in NRAS and BRAF have significant effects on treatment.
Acknowledges role of MAPK pathway as a potential therapeutic route.
Sun, X., Bao, J., Nelson, K. C., Li, K. C., Kulik, G., & Zhou, X.(2013). Systems modeling of anti-apoptotic pathways in prostatecancer: psychological stress triggers a synergism pattern switch indrug combination therapy. PLoS Comput Biol, 9(12),e1003358.
Elaborates the use of drug combination therapy in treatment of cancers with reference being made on prostate.
Similar approach could be employed for medulloblastomas and neuroblastomas to facilitate their management.
Swanton, C. (2012). Intratumor heterogeneity: evolution through spaceand time. Cancer research, 72(19), 4875-4882.
Cancer genomes analyzed at the single nucleotide level to gain better understanding.
Helps understand tumor heterogeneity and develop ways of treatment.
Wacker, S.A., Houghtaling, B.R., Elemento, O., Kapoor, T.M. (2012).Using transcriptome sequencing to identify mechanisms of drug actionand resistance. Nature Chemical Biology 8, 235-237.
Illustrates the generation and use of drug resistant clones in an established cell line to analyze resistance to multiple drug treatments.
Ward, E., DeSantis, C., Robbins, A., Kohler, B., & Jemal, A.(2014). Childhood and adolescent cancer statistics, 2014. CA: acancer journal for clinicians, 64(2), 83-103.
Provides statistics on cases of cancers among children with emphasis being on issues of mortality and incidences.
Wicki, A., Mandalà, M., Massi, D., Taverna, D., Tang, H., Hemmings,B. A., & Xue, G. (2016). Acquired Resistance to Clinical CancerTherapy: A Twist in Physiological Signaling. PhysiologicalReviews, 96(3), 805-829.
Explores acquisition of resistance by cancers while acknowledging challenges imposed by such a trait.
Wojtalla, A., Salm, F., Christiansen, D. G., Cremona, T., Cwiek, P.,Shalaby, T., … & Arcaro, A. (2012). Novel agents targeting theIGF-1R/PI3K pathway impair cell proliferation and survival in subsetsof medulloblastoma and neuroblastoma. PloS one, 7(10),e47109.
RTK/P13K pathway seen to be essential for cancer cell proliferation and is altered in development resulting in tumors.
Zyrianova, Y., Alexander, L., & Faruqui, R. (2015).Neuropsychiatric presentations and outcomes in children andadolescents with primary brain tumours: Systematic review. Braininjury, 1-9.
Explores literature on neuropsychiatric symptoms and development of tumors among children.