The+Dual+Role+of+NOTCH1+in+Varying+Cancers

toc Stereotypically college students consume high levels of alcohol and tobacco. Unfortunately, alcohol and tobacco are two major risk factors in head and neck squamous cell carcinoma (HNSCC). = = This table was taken from a study that took 521 experimental and 599 control individuals that averaged around the age of 60-61. In general, what their study found was that tobacco and alcohol have a direct correlation to a person's risk of attaining HNSCC. As you can tell by this table, heavy drinking (>26 drinks per week) and heavy smoking (35-58 packs per year) increase an individuals risk by about 2x and 3.5x respectively. However, what is very interesting to note is the risk when an individual partakes in both. From this study, an individual who is both a heavy drinker and a heavy smoker as about a 17x likely chance of acquiring HNSCC (Peters 2005). Understanding these facts, we thought that studying at HNSCC would be a relevant to us and the student body as a whole. However, when looking at this disease, we found something even more interesting. This was one of the commonly mutated genes in HNSCC: // NOTCH1 //.
 * [A statement of ** **purpose]**
 * Background: **

**I. Introduction**
In our cancer project, we explore the mutated //NOTCH1// gene and how it can act either as a tumor suppressor gene or an oncogene, which we will ultimately use to observe how genes like these with seemingly contradictory functions alter how cancer should be perceived. //NOTCH1//'s involvement in cancer was first observed in T-Cell acute lymphoblastic leukemia (T-ALL) as an oncogene, but recent research has proven that it can also act as a tumor suppressor gene in some types of cancer such as HNSCC as seen in the Figure 1 (Lobry 2011). We used T-ALL and HNSCC to compare and contrast the complexity of //NOTCH1//'s character, and ask the question: how can //NOTCH1// act as both a tumor suppressor gene and an oncogene?



//**II. Overview of the Notch1 Protein Pathway **//
In general, NOTCH1 codes for a transmembrane receptor protein called Notch1 that plays a prominent role in cell-fate determination. This includes cell proliferation, apoptosis, and cell-differentiation. As seen in figure 2 (Radtke and Raj 2003), when a ligand attaches to the Notch1 receptor, the protein is left exposed to two proteolytic cleavages from y-secretase and one proteolytic cleavage from TACE, which release the cytoplasmic end of the protein, Notch1-IC, into the cytoplasm. Notch1-IC then travels to the nucleus to act as a transcription factor that promotes numerous genes that code for proteins that effect the cell cycle and cell differentiation (Radtke and Raj 2003). Notch1 receptors are expressed significantly during tissue development due to its ability to regulate the differentiation fate of cells. Because of its role in cell proliferation, cell death, and cell growth suppression, the mutated gene protein product can act as a oncoprotein or a tumor suppressor. Whether the Notch1 protein acts like an oncoprotein or a tumor suppressor is contingent to “the cellular context and the crosstalk with other signal-transduction pathways” (Radtke and Raj 2003). In other words, the Notch1 receptor's actions are dependent on the cellular environment and the cell type. Because Notch1 directly and indirectly regulates gene expression by acting as a transcription factor when it is activated, it has the ability to control cell differentiation based on cell context. Because of this, it is possible that it can act as an oncogene in some cells and as a tumor suppressing gene in other cells.

//**III. Protein Physiology**//
To understand how mutated Notch1 can act as either an oncogene or a tumor suppressor, we first must look the physiological features of the Notch1 protein. For starters, Notch1 is a transmembrane protein with an extracellular, transmembrane, and cytoplasmic domain.

This figure gives us a general diagram of the different sub-domains in Notch1. For our study on specifically T-All and HNSCC, we will look at the EGF like repeats domain, the HD domain, the TM domain, the RAM domain, the ANK domain, and the PEST domain. As seen in the diagram above, taken from //The Mutational Landscape of Head and Neck Squamous Cell Carcinoma,// the EGF like repeats is widely known for ligand binding, and the HD and TM domains are both significantly important for proteolytic cleaving (Stransky 2011). However, the physiological importance of the RAM, ANK, and PEST domains were not so easily presented.

To understand the RAM and ANK domain better, we found data that presented their physiological affects. Figure 4 is taken from a study titled, //[|Role of the Ram Domain and Ankyrin Repeats on Notch Signaling and Activity in Cells of Osteoblastic Lineage]//. This chart shows the 12xCSL-Luc transactivation by the Notch Intracellular Domain (NICD), Notch Intracellular Domain without Ankyrin repeats (NICD ΔANK), and Notch Intracellular Domain without the RAM domain (NICD ΔRAM). 12xCSL-Luc is a product of the Notch pathway that is required for proper CSL signaling to take place. CSL signaling of Notch is essentially how Notch performs its variety of functions. However, as seen from this chart, deletion of the ankyrin and RAM domains decreased the 12xCSL-Luc levels significantly. With the complete absence of 12xCSL-Luc levels in NICD ΔANK and the significantly lower levels in NICD ΔRAM, it’s concluded that the Ankyrin repeats are absolutely necessary for the activation CSL signaling and that the RAM domain is only partially necessary for this same process ( Deregowski 2006).



Combined with analysis of the Ankyrin repeat crystal structure , figure 5 has proposed [Fig 5 doesn't do any proposing] a very reasonable mechanism of the physiological features of both the ANK and RAM domains. The original BT/RHR-N/RHR-C complex acts as an inhibitor for the HES genes. These genes are what triggers or inhibits cell differentiation. However, when a ligand binds to a Notch receptor, NCID is released where the RAM domain creates an interaction between NCID and this CSL inhibitor while the ANK domain creates a groove between the CSL inhibitor and NCID such that MAML can now bind and transform the inhibitor into an activator (Nam 2006).



Interestingly, taken from the same study, it was found that the Ankyrin’s and RAM’s domain play a crucial role in inhibiting the Wnts pathway ( Deregowski 2006). The Wnts pathway creates products such as Cyclin D that induces cell cycling. Relative to figure 6, WISP is a primary gene that is encoded with the activation of this gene. Therefore this graph measures the transcription levels of this gene over 4 weeks in cultured cells that are presented with NICD, NICD ΔANK, and NICD ΔRAM. As seen in the results for week 3 and 4, deletion of both the Ankyrin or RAM domains decreases the inhibition of the Wnts pathway, which is usually inhibited by complete NICD. 

Lastly, we will look at the PEST region. When Notch1 activates its target genes by making them transcriptionally active, a ubiquitylation mechanism ensures that it is degraded quickly after it completes this task. The PEST domain, which is mutated frequently in T-ALL, is targeted as a signal for degradation, which allows it to maintain the intensity and duration of NOTCH1 activation. After the RNA polymerase II holoenzyme is recruited to the transcriptionally active complex (Notch1-IC, MAML, BT RHR-N, RHR-C), the PEST domain is phosphorylated, which allows it to be targeted by FBX7-SCF ubiquitin ligase complex so that it be degraded (Bhanushali 2010).

[So what can we conclude from this section?]

//**IV. NOTCH1 as a Tumor Suppressor Gene**//


To understand how Notch1 can act as a tumor suppressor, we must look at its signaling pathways that give it this title. Figure 7 does a good job at summing this up (Dotto 2008). Since the figure is somewhat complicated, the first thing to notice is both the upper and lower parts of the figure. The upper part diagrams Notch’s tumor suppressor role through inhibitions of stem cell renewal and cell differentiation signaling pathways and the lower part portrays Notch’s tumor suppressor role by either inhibition or activation of apoptotic pathways. A few of the most important pathways to notice are the Notch1, p63, p53, p21, and Wnts pathway along with the FoxO3a and NFkB/Akt pathway. In the Notch1, p63, p53, p21, and Wnts pathway, Wnts triggers the activation of products such as Cyclin D1, which is a main component that causes a cell to enter the cell cycle and go from G1 phase into S phase. Therefore the Wnts inhibition by Notch1, p63, p53, and p21 has a big role in maintaining a proper level of cell proliferation. On the other hand, FoxO3a is a pro-apoptotic gene and NFkB/Akt are pro-interferon genes and play major roles in cell survival, inflammation, cell cycling, cell adhesion, and migration. Notch inhibits FoxO3a and activates NFkB, which essentially creates this balance between stopping cell death and promoting many other pathways that maintain proper cell proliferation (Dotto 2008). This balance is important for regulating cell’s such that they don't enter a hyperplastic or neoplastic state while still maintaining appropriate levels of cell growth.

In order to figure out if //NOTCH1// acts as a tumor suppressor gene in HNSCC, genomic analysis was required. Genomic analysis has made it possible for researchers to compare the genomes of healthy cells compared to cancerous cells, which allows them to evaluate the differences between the two and ultimately observe the mutations present in cancer cells. More importantly, genomes of tumors of the same type of cancer can be compared, which allows researchers to observe which mutations are common among that particular type of cancer.



In the article, //Exome Sequencing of Head and Neck Squamous Cell Carcinoma Reveals Inactivating Mutations in NOTCH// 1, the authors used whole-exome sequencing to sequence 18,000 protein-encoding genes in 32 patients who had head and neck squamous cell carcinoma. DNA of the tumors and non cancerous cells on each patient were purified and sequenced, which was used to compare the genome of the tumor to the genome of a normal cell. DNA was sequenced by massive parallel sequencing initially, which found 911 total somatic mutations in the tumors. In order to verify the accuracy of the sequencing, the DNA was evaluated again by Sanger sequencing, which confirmed 609 mutations. If a mutated gene was observed in more than 2 different patients, it would be selected for further analysis. In this analysis, the genomes of tumor cells and normal cells in 88 more patients were sequenced. This analysis found that 15% of patients displayed loss of function mutations in NOTCH1. In total, there were 28 NOTCH1 mutations in 21 out of 120 patients (32 patients from the first analysis and 88 from the second analysis). 34% of the mutations reduced the size of notch1 through nonsense and deletion mutations (Table 1). The rest of the patients had missense mutations of NOTCH1 (Table 2). In order to complement the data found through sequencing, the authors utilized Affymetrix [|SNP6.0 microarrays] to observe the copy number of the genes most mutated in head and neck squamous cell carcinoma tumors. The authors analyzed the number of NOTCH1 gene copies of 3 tumors with NOTCH1 mutations, which displayed that 2 of the 3 genomes suffered from a loss of heterozygosity. Because the data suggests that NOTCH1 either suffers from a loss of function mutation or a complete deletion of the gene when it is mutated in head and neck squamous cell carcinoma, the authors concluded that it acts as a tumor suppressor gene in this type of cancer. The results also showed that NOTCH1 mutations are the second most frequent mutation to appear in this type of cancer (Agrawal 2011).

We are satisfied with the methods that the authors used for determining the presence of mutations in head and neck squamous cell carcinoma tumor genomes, however, we are skeptical of the validity of the results of this study because of the sample sizes used throughout their experiment [? explain.]. The authors did a an excellent job of confirming which genes have a significant role in head and neck squamous cell carcinoma by using both massive parallel sequencing and Sanger sequencing techniques, however, they lacked an adequate sample size (32 patients at the time) to make these assumptions [Don't confuse the number of patients analyzed with the statistical rigor needed to associate a gene to a cancer; I think if you go back and look at the statistics you'll find a strong correlation. you need some a statistical argument here]. Also, when the authors sequenced 88 more patients and emphasized observing the genes that were mutated most frequently in the tumors, the results were still obscured by the lack of patients (120 patients in total at the time), but by using both massive parallel sequencing and Sanger sequencing techniques, the results had more credibility. In short, we believe that using both massive parallel sequencing and Sanger sequencing to verify the amount of mutations gives robust results, but the inadequate sample sizes hinders the conclusions that can be drawn from the data. Also, when the authors were testing for the amount of NOTCH1 genes in the genome tumors, they solely used 3 patients, which makes the results of this portion of the experiment lack adequate support to draw conclusions from. We can infer that the small sample sizes for this experiment was contingent to the time and money that are necessary to be invested into whole-exome sequencing and gene number copy analysis. As time progresses, hopefully such techniques will become more inexpensive and available to more research facilities and hospitals. Overall, we cannot fully entrust this study, but its results definitely suggest that NOTCH1 acts like a tumor suppressor gene in head and neck squamous cell carcinoma.

When looking at the actual mutations found in Notch1 in HNSCC, we came across 2 figures that depicted these mutations well. Each of these figures is created from their own individual study. The study for figure 9, taken from //[|The Mutational Landscape of Head and Neck Squamous Cell Carcinoma]// (Stransky 2011), looked at 92 different HNSCC tumor/normal cell pairs, which about 12 of these tumors had NOTCH1 mutations. Likewise, Diagram C in figure 8 of NOTCH1 is taken from study listed above, //[|Exome Sequencing of Head and Neck Squamous Cell Carcinoma]// (Agrawal 2011). As seen in both figures, in HNSCC, the mutations are very sporadic and located in regions where their physiological roles would result in a loss of function. In figure 9, the majority of the mutations in // NOTCH1 // occur in the extracellular domain within the EGF-like repeats. This region is the ligand-binding region. Therefore these mutations cause a loss of function of NOTCH1 mostly by the inability of ligand-binding.

There are some cases too, with nonsense, splice, and deletion mutations where there is either a no presence of NICD or no cleavage site. Similarly in figure 8 under diagram C, the sporadic and high concentration of mutations in the EFG-like region suggests the same idea. However, in this study, more mutations were found, mainly in the Ankyrin repeats domain. The Ankyrin repeats domain is a domain that is essential for activating NICD's signaling pathway and inhibiting the Wnts pathway. Therefore, these mutations also create a loss of function in //NOTCH1// by essentially creating a defective NICD. If ligand binding is still operable, but NICD is not functional, Notch1's tumor suppressing functions are still inhibited.

Judging from these studies and analyses of HNSCC's genomic mutations, it is reasonable to see why Notch1 acts as a tumor suppressor gene in HNSCC. Although there are other mutations in HNSCC tumors, they occur at very low percentages and have not shown any significant data that Notch1 could act as a oncogene in these tumors. We are not certain as to why mutations in HNSCCs occur mostly in the EFG like repeats and ANK domains, but we believe that cellular environment and head and neck squamous cell's functionality are factors that lead to these specific mutations. Understanding that Notch1 contains loss of function mutations in HNSCC, it makes treatment and therapy that targets this protein much more complicated. Unlike oncogenes which usually has a "target" that most therapies can attack, loss of function mutations takes this "target" away. Therefore in the future, it seems most logical for therapies treating HNSCC to look at other pathways that Notch1 induces or inhibits and use those pathways as central focus points HNSCC therapy invention.

//**V. NOTCH1 as a Oncogene**//
In T-Cell acute lymphoblastic leukemia (T-ALL) researchers have observed //NOTCH1// acting as an oncogene, because 60% of all T-ALL cases have a gain of function mutation in NOTCH1 (Ferrarando 2009). Because NOTCH1 is an oncogene in T-ALL, the mutations induce an increase in activity of the Notch1 receptor, which occurs by either allowing ligand-independent activation or decrease in ability of Notch1-IC to be degraded.



In a study titled, //NOTCH1 Mutations in T-Cell Acute Lymphoblastic Leukemia: Prognostic Significance and Implication in Multifactorial Leukemogenesis// 14 //,// 77 patients with T-ALL were investigated for Notch1 mutations. Of the 77 patients, a total of 32 mutations were found in 29 of those patients. The figure above is taken from this study and depicts the types of mutations and the location of where they were located on the Notch1 protein. Diagram A in this figure shows the types of mutations in selected regions of the protein. However, when looking at all the mutations found in Notch1 throughout this study, diagram B allows us to see where these mutations most likely took place in T-ALL. The most common location of mutations is within the HD region. Out of 32 Notch1 mutations found, 23 were located within the HD region. These mutations in the HD region are known to disrupt the cleavage sites enough such that their protecting groups essentially lose their ability to protect these cleavage site and proteolytic cleaving constantly occurs. It is this mutation that essentially gives Notch1 ligand-independent activity in Notch1. The next most common mutated region of Notch1 is the PEST carboxyl-terminal domain. Out of 32 mutations, 5 were recorded in the PEST region. These mutations inhibit the phosphorylation of PEST such that the FBX7-SCF ubiquitin ligase complex can no longer degrade NICD. This in turn can cause an over expression of NICD, which leads to T-ALL. Similarly, mutations affiliated with Notch1 have also been seen in FBW7, an E3 ubiquitin ligase that degrades Notch1-IC and discontinues Notch1 signaling. Notch1 signaling is critical to T-Cell development and essential for lymphoid progenitors to commit to normal T-cell lineage, which has been known among researchers for a significant amount of time. However, it is unknown how exactly Notch1 acts to induce tumorigenisis when it is overactivated. Because //NOTCH1// mutations, which was solely a rare translocation mutation, were solely noticed in less than 1% of all T-ALL cases before 2004, research on its influence on cellular pathways when it is hyperactivated was relatively unknown (Mascarenhas, 2006). In 2004, research suggested that //NOTCH1// has a gain of function mutation in 60% of all T-ALL cases, which ultimately instigated a significant increase in research on how this gene acts when it is overactivated in T-ALL ( Mascarenhas, 2006). Research since 2004 has suggested that promotion of the G1/S cell cycle progression, upregulated MYC transcription, activation of PI3K/AKT/MTOR pathway, and increasing NF-κB activity (Mascarenhas, 2006). The most prominent and robust research on Notch1's influence in these pathways are in its relationship with the NF-KB pathway.

Research has found that constitutively active Notch1 activates the NF-KB pathway transcriptionally and by activating the (IKK) complex (Mascarenhas, 2006).The NF-kB pathway has a major role in regulating cell survival, inflammation, the cell cycle, cell adhesion, and migration. Because of these functions, it is apparent that the deregulation of NF-kB pathway constructs an environment that is conducive for tumorigenesis. When an aberrant NOTCH1 gene was first recognized in T-ALL, a translocation mutation, which moved the 3' region of NOTCH1 into the [|TCRβ] locus mutations resulting in a overexpression of the active form of Notch1 (Notch1-IC) (Mascarenhas, 2006). However, this mutation was solely found in less than 1 percent of all T-ALL cases, which discouraged researchers from further investigating this gene. 13 years after this discovery, a researcher discovered that NOTCH1 was mutated in 56% of TALL cases studied. (Mascarenhas, 2006).



In order to observe if Notch1 promotes the NF-KB pathway, the authors of [|NOTCH1 Mutations in T-Cell Acute Lymphoblastic Leukemia: Prognostic Significance and Implication in Multifactorial Leukemogenesis] infected two hematopoietic progenitor populations-- a population of uncommitted bone marrow progenitor 4 days after they were infected and another population that was cultured for an additional 7 days on the OP9 stroma line consisting primarily of CD25+CD44low pro-T cells-- with a retrovirus containing a gene coding for Notch1-IC. For a control, they used hematopoietic progenitors infected with a retrovirus with a gene that coded for a empty, enhanced green fluorescent protein. In order to quantitatively evaluate the transcriptional activity of of genes related to NF-KB, the authors used [|RT-PCR] on the samples studied. The authors found that many of the genes involved in the NF-KB pathway, especially Relb and NFkb2, were upregulated. Most of these genes remained upregulated in the late population. By using [|chromatin immunoprecipitation] analysis, which allows one to observe which genes proteins attach to, the authors found that the Notch1-IC attaches to RELB and NFKB2 promoters. This is consistent with the results of the RT-PCR, which indicated that transcripts of these genes were more abundant when it was under the influence of Notch1-IC.

Besides inducing the expression of RELB and NFKB2, the authors thought that Notch1 influenced the increase in activity of the IKK complex, a major component of the NF-KB pathway. In order to investigate the ability for Notch1 to influence the IKK complex, the authors inhibited the Notch1 signaling pathway in CEM cells, which are derived from a cell line suffering from T-ALL, by using two different types of γ-secretase,zLLNLA and LY411575, and observed the amount of phosphorylated IkBα by densitometry analysis. Because IKBKA and IKBKB, which are both major components of the IKK complex, phosphorylate IkBα when they are activated, IkBα phosphorylation is indicative of NF-KB activation. The authors observed that as the CEM cells were exposed to higher concentration of γ-secretase, less IkBα were phosphorylated, which reflects that Notch1 does effect this KF-KB pathway by increasing the activity of the IKK complex as well. Despite the difference in magnitude of the decrease of activity in IKBKA and IKBKB in each increasing treatment, as the concentration of γ-secretase increases, the amount of kinase activity for both of the proteins decreases (figure 12). This correlation indicates that as Notch1 activity is decreased in T-ALL cells, the NF-KB pathway decreases, thus reflecting a direct correlation between the two cellular pathways. The truly anomalous data point here is at the 25 concentration in the LY411575 treatment because it is the only data point where the IKBKA activity is lower than the IKBKB activity (figure ). I can infer that because of the lack of adequate sample size (n=1) for each treatment, this data point could not accurately reflect how the NF-KB activity is effected by this treatment. The sample size is inadequate to derive statistical evidence to back up this study, however, the consistency of both of these different treatments (LY411575 & zLLNLA) to decrease the activity of the NF-KB pathway by inhibiting Notch1 illustrates that the Notch1 pathway could positively influence the activity of the NF-KB pathway.

It is apparent that Notch1 can have mutations, such as HD domain and PEST domain mutations, that are conducive to increasing the activity of Notch1, thus allowing the intracellular domain to be unattached from the transmembrane protein more often or the intracellular domain is degraded less easily. When Notch1 in an hyperactive state, researchers are not sure exactly which pathways Notch1 influences to induces tumorigenisis, however, promising evidence, especially evidence supporting that an increase in NF-KB pathway activity can be influenced by an increase in Notch1 signaling pathway, suggesting that the Notch1 signaling pathway can increase the amount of MYC transcribed, overactivate PI3K, promotion of rapid progression from G1 phase to S phase, and. Ultimately, more research should be conducted to observe how exactly Notch1 effects other cellular pathways when it is hyperactivated in T-ALL patients, because if we learn more about which pathways Notch1 influences, then one could potentially fabricate a therapeutic that targets this pathway in conjunction with Notch1 targeted therapy, thus allowing an increase in the chance of eliminating T-ALL cells or reducing progression of the disease.

//**VI. Possibly Therapy Targeting Notch1 **//
Researchers have attempted to fabricate targeted therapy against Notch1 in cancers, such as TALL, where //NOTCH1// acts as an oncogene. In order to inhibit Notch1, researchers targeted γ -secretase, the enzyme that cleaves Notch1 and releases the Notch1 intracellular domain (Notch1-IC) into the cytoplasm. Gamma secretase inhibitors (GSIs), especially GSI I, are popular inhibitors among these researchers. Pre-clinical research indicates that GSI's provide anti-tumor therapy by arresting the cell cycle and inducing apoptosis (Grosyeld, 2009). Unfortunately, because γ-secretase inhibitors induce intestinal toxicity the inhibitors must be administered with dexamethasone, which counteracts the toxic side effects (Grosyeld, 2009). This cocktail treatment has reaped successful results in mice, however, this combination still needs significantly more modification before it can ever reach clinical trials. Dexamethasone treatment has known side effects of hypertension, oseteopenia, and muscle atrophy (Grosyeld, 2009).Besides causing intestinal toxicity, γ-secretase cleaves over 30 other different transmembrane proteins. γ-secretase inhibitor treatment could possibly cause more damage than benefits because of its ability to inhibit 30+ proteins besides Notch1 (Grosyeld, 2009). If these proteins become disabled by γ-secretase inhibitors, there could be major negative physiological ramifications (Grosyeld, 2009). Innstead of targeting γ-secretase, drugs targeting Notch1 should focus on blocking the cleavage site on the protein, thus allowing γ-secretase to function fully while still remaining incapable of cleaving Notch1.

In the article Notch1 as a Potential Therapeutic Target in Cutaneous T-cell Lymphoma, the authors researched the possible therapeutic propertiesof inhibiting the Notch1 pathway. In their study, the observed the ability of of GSI I in inducing cell apoptosis and stalling the cell cycle in cutaneous T-Cell lymphoma, which is a cancer similar to T-ALL that also has Notch1 acting as an oncogene. The authors assessed the influence of GSI I oninducing apoptosis in the cancerous cell lines by examining the activity of caspase 3 and caspase 7, which both have crucial roles in apoptosis. To do this, the authors incubated cells infected with cutaneous T-cell lymphoma, which were MyLa, SeAx, and Hut78 cell lines, with increasing amounts of GSI's (GSI I, IX, XX, and XXI) for 48 hours (figure 13). Out of all the GSIs, the authors found that GSI I was the only inhibitor to truly have any significanteffects on any of the cell lines, which is apparent because for the other GSI IX, XX, and XXI, there is never a high induction of caspase 3 or caspase 7 (figure 13 & 14). The GSI I induced apoptosis the greatest at concentrations of 1 µM and higher (Figure 13). In figure 13, this graph shows how after 1µM of GSI I treatment, the induction of caspase 3 and caspase 7 remain consistently near a 5 fold increase.The big error bars on the 1µM point indicate that 1 µM of GSI I treatment can induce unpredictable results in relation to apoptosis, which could range from a major induction of apoptosis to a minor induction of apoptosis. Because the error bars overlap on all of thedata points after the 1 µM data point, I can infer that these data points are very similar.Also, the time of induction of caspase 3 and caspase 7 were also evaluated, and the results indicated that the induction of these enzymes is most apparent at 24 hours (Figure 13). After 24 hours, the amount of caspase 3 and caspase 7 declines (Figure 13). In order to gain more insight on the effects of GSI I on apoptosis and the cell cycle, the authors used nuclear isolation medium-4,6-diamidino-2-phenylindole dihydrochloride NiM DAPI staining on the DNA of the three cell lines that were incubated with GSI I for 48 hours and used flow cytometry to observe the proportion of cells in each phase of the cell cycle. The authors found that after 24 hours of treatment, there was a significant increase in the number of cells in both the G0 and G1 phase (Figure 14),which was "indicative of apoptosis" (Kamstrup, 2508). This is consistent with their previous data because they found that the highest induction of apoptosis, which is displayed by the highest peak (8 fold increase) of caspase 3 and caspase 7 is at the 24 hour data point of GSI I treatment (figure 2), is also after 24 hours of treatment. To ensure that GSI I inhibited γ-secretase, rather than the inhibitor influencing other signal pathways that would lead to cell-cycle arrest or apoptosis, the authors down regulated the amount of Notch1 proteins by using [|SiRNA] nucleofection in the SeAx cell line.The authors did this by transfecting SeAx cells with Notch1 SiRNA, which causes Notch1 RNA tobe broken down and eventually degraded, thus disabling the translation of Notch1. After they performed this transfection, the authors evaluated the cell line’s induction of apoptosis by observing the activity of caspase 7 and caspase 3. The results were consistent with the conclusions the authors drew from this data on GSI's possible therapeutic effects, which suggests that the inhibition of Notch1 induces apoptosis and cell cycle stalling in cutaneous T-cell lymphoma cells. (Kamstrup, 2010).This data indicates that about 1 µM--5 µM of GSI I treatment is an adequate amount of treatment, and the most therapeutic results will occur occur after 24 hours of treatment, which will induce about an 8 fold increase in apoptosis and about a 25 fold increase of cancerous cells stalled in the G1 and G0 phase of the cell cycle (figures 13, 14, & 15).

This was a well done pre-clinical study that reveals a possible therapeutic method of counteracting the oncogenic effects of //NOTCH1//. I am disappointed with the small sample sizes of the cell line experiments because they were derived and cultured from solely a few patients. Besides the Hut-78 cell lines, which used 4 patients, all of the cell lines were derived from one patient. Because of the heterogeneity of the genomes of cancer cells between patients who have the same cancer type, more cell lines should have been derived and tested for each type of cell line evaluated to have results that are more representative of cutaneous T-cell lymphoma cells in humans who are suffering from this disease. Besides this, the authors did each experiment in triplicate and repeated each experiment 2-3 times, which allowed the authors to back up their evidence with statistical evidence. Also, the authors did many different experiments to confirm each of their conclusions drawn, thus giving their data more credibility. Because this study was used to merely infer how future treatment can be developed against //NOTCH1//, I believe this was a robust pre-clinical study.

Besides inhibiting γ-secretase, T-ALL has strived to be inhibited by inhibiting Notch1 induced pathways, such as the NF-KM pathway. In [|NOTCH1 Mutations in T-Cell Acute Lymphoblastic Leukemia: Prognostic Significance and Implication in Multifactorial Leukemogenesis], the authors proposed a method of treatment against patients with overactivated //NOTCH1// mutations in T-ALL that inhibited the NF-KM pathway and observing the potential therapeutic effects of this. In order to inhibit this pathway they used bortezomib, a broad spectrum pharmaceutical that is a proteasome inhibitor, to inhibit the preastosomal degradation of Nfkbia, which has a major influence in the function of NF-KM pathway (Mascarenhas, 2006). By incubating T-ALL cell lines in 5nM of bortezomib and monitoring the proportion of viable cells compared to the control, which were not exposed to bortezomib, of each cell line. In figure 16, one can observe how the proportion of viable T-All cell lines (all of the arms except Jurkat and SCI) decline significantly after 24 hours, except for the RPMI 8402 cells, and continue to decline. As seen in figure 16, the results indicated that that 5 out of 6 Notch-dependent T-ALL cell lines (All-SIL, BE13, DND41, KOP-TK1, RPMI8402, T-ALL1) were sensitive to bortezomib, which is apparent because of the rapid decline of percent of cell viability in T-ALL cell lines after 24 hours of treatment. Because bortezomib does not inhibit proteases, such as γ-secretase and TACE, it cannot directly inhibit the Notch1 pathway, so I can infer that it this induction of apoptosis is dependent to the drug’s ability to inhibit the NF-KM pathway. This graph also displays how T-ALL cells begin to obtain resistance against this therapy after 48 hours, which is apparent in both the T-ALL1 arm and RPMI 8402 arm--both increase in the amount of viable cells after 48 hours. Ultimately, this data suggests that therapies targeting the NF-KB pathway can induce cancer cell apoptosis, however, we need to fabricate a more aggressive pharmaceutical than bortezomib, because cancerous cells would become resistant to this drug merely after two days, thus obscuring its therapeutic effects.

Ultimately, as research has been reflecting, inhibiting the transmembrane protein through γ-secratase inhibitors has had successful results in counteracting //NOTCH1//'s oncogenic effects. Pre-clinical trials of interventions using cell lines and mice are good mechanisms to extrapolate information from, but to truly develop a robust therapeutic strategy targeting //NOTCH1//, clinical trials must be done. Similarly to the ability of, because of its ability to inhibit tyrosine kinases, to have therapeutic effects on both gastrointestinal stromal tumors and chronic myeloid leukemia, γ-secretase inhibitors can be utilized to target both T-cell acute lymphoblastic leukemia and cutaneous T-Cell lymphoma because they both inhibit the the proteolytic cleavage of Notch1. The ability for these type of similar drugs to have therapeutic effects on different types of cancers truly illustrates how physicians should use therapy that targets the aberrant genes, rather than provide interventions that target the type of tissue that is cancerous. Similarly to what Bradner explained in his lecture, once we become more advanced in targeted cancer therapy and genomic analysis becomes less expensive, cancers should be treated more based on the mutations that cause them, rather than the type of organs, tissues, cells that become cancerous.

**VII. Conclusion**
Now that we acknowledge that //NOTCH1// can act as either an oncogene or a tumor suppressor gene, we should become extremely cautious when targeting the Notch1 protein with therapy because if one provides an intervention that inhibits Notch1 when it acts as a tumor supper protein, then one could unintentionally promote tumorigenisis. Because of the dual properties of //NOTCH1//, researchers should not be stubborn with their classifications of genes that are driver mutations when they are mutated. When //NOTCH1// expresses tumor suppressor gene functions, one cannot truly provide targeted therapy against it because there simply is no protein to target. On the other hand, when //NOTCH1// acts an oncogene, the Notch1 protein product can be targeted by gamma secretase inhibitors to hinder γ-secretase's ability to cleave Notch1 and release Notch1-IC. However, gamma secretase inhibitors induce intestinal toxicity and can only be utilized in humans by using extremely low doses, which do not reap any positive therapeutical effect. Even if researchers can create a therapeutic window with gamma secretase inhibitors that would allow a high efficacy of the drug at non-toxic levels, one still could not target translocation mutations that allows the //NOTCH1// gene to express a truncated Notch1 protein with solely its intracellular domain because the active portion of the protein is already detached from the protein, thus inhibiting proteolytic cleavages would simply be useless because this step does not even need to occur to active the Notch1 pathway. Because of these fallacies, therapy targeting Notch1 influenced pathways, such as the NF-KB pathway, seems to be another possible way we can counteract this aberrant gene, however, due to the lack of research on how Notch1 induces neoplasia when it acts as an oncogene, researchers are far from producing therapy against the potential pathways Notch1 effects. We believe that more research should be done to confirm exactly which pathways Notch1 enhances as an oncogene, so that therapy can target these pathways in conjunction with therapy targeting overactive Notch1.

**Works Cited:** [references have to match citations: either number citations put references in alphabetical order. Link out to the references]
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