Possible Therapy Targeting the NOTCH1 gene

    As our class has been studying the treatments of cancer and how drugs are developed in the clinical and pre-clincal settings, I have been interested in researching therapy targeted against NOTCH1. While I was doing this, I stumbled upon one article, Notch1 as a Potential Therapeutic Target in Cutaneous T-cell Lymphoma, that researched the possible therapeutic properties of inhibiting the Notch1 pathway (see blog 1 paragraph 2 for simple pathway) in cutaneous T-cell lymphoma.
In this article, the authors observed the effects of γ-secretase inhibitors on cell lines of patients who had cutaneous T-cell lymphoma with overexpressed Notch1 proteins. It is unknown whether NOTCH1 plays a role in cutaneous T-cell lymphoma, however, due to the oncogenic effects of NOTCH1 in T-cell leukemias and other solid malignancies, the authors speculated that it could have a role in cutaneous T-cell lymphoma. Sure enough, their research indicated that  NOTCH1 acts as an oncogene in cutaneous T-cell lymphoma, which allows it to have a gain of function mutation. In this study, the authors researched the effects of γ-secretase inhibitors on cell lines in the two most common forms of cutaneous T-cell lymphoma, which are mycosis fungoides and Sezary syndrome. When the authors used γ-secretase inhibitors on the cell lines of mycosis fungoides and Sexary syndrome, they observed that one particular inhibitor, GSI I, inhibited the Notch1 protein significantly, which ultimately induced cell cycle arrest and apoptosis of the cancerous cells.

      In their research, the authors used tissue samples of 40 patients who had cutaneous T-cell lymphoma to see if Notch1 was overexpressed in this type of cancer. Of these 40 patients, 35 patients suffered from mycosis fungoides, while the rest had Sezary syndrome. In this analysis, the results reflected that the Notch1 receptors were expressed highly in cutaneous T-cell lymphoma cells, while the non cancerous T-cells lacked a significant concentration of them. In order “to address whether lymphoma cells in vivo resemble cutaneous T-cell lymphoma cell lines with respect to the expression of Notch” (2506)³, the authors sampled the 40 patients by using immunohistochemical staining of skin specimen. They performed this to see if the Notch1 expression in their cultured cell lines would accurately reflect the expression of Notch1 in humans who have cutaneous T-cell lymphoma. In order to evaluate the presence of Notch1, trained pathologists (L.M.R.G. and E.R.) scored the labeled Notch1 receptors. Out of these 40 patients, 21 of them tested positive for the presence of Notch1, and the more advanced cases of cancer displayed more Notch1 expression.  8 out 9 of the mycosis fungoides patients who were at the tumor stage, which ranges from IIB-IVB stages, and 7 out of 10 mycosis fungoides patients, who were in the transformed cancer position, which ranges from stages II-IVB, had Notch1 expressed. On the other hand, 1 out of 6 cases of patients within the patch stage, which was all I-B stage patients, had Notch1 in tumor cells, and 2 out of 10 plaque stage cancers, which are in stage IA and IB, expressed Notch1 in their tumor cells. For Sezary syndrome, half of the patients, who were in stages IV, IVA, and IVB, had Notch1 expression. After observing that most of the mycosis fungoides patients who where in later stages of cancer had Notch1 expressed, I thought it was odd to see that only half of the Sezary syndrome patients had Notch1 expressed. However, all of this data still displays that Notch1 expression is observed more in later stages of cutaneous T-cell lymphoma. After observing this data, it is apparent that Notch1 is expressed significantly in cutaneous T-cell lymphoma, which allows the authors to have faith that their cell lines of cutaneous T-cell lymphoma will accurately reflect how cancerous cells in vitro will truly respond to γ-secretase inhibitors.

       After the authors concluded that Notch1 is overexpressed in cutaneous T-cell lymphoma, the authors attempted to inhibit Notch1 by exposing the cancer cell lines to gamma secretase inhibitors. Gamma secretase inhibitors (GSIs) inhibit γ-secretase from performing proteolytic cleavages, which allows Notch-IC, the cytoplasmic portion of the transmembrane protein, to be released into the cytoplasm and act as a transcription factor. Without releasing Notch-IC into the cytoplasm, Notch1 is unable to influence cell proliferation, cell death, and cell growth suppression. First, they attempted to see if gamma secretase inhibitors truly hindered γ-secretase’s ability to release Notch-IC into the cytoplasm. The authors exposed SeAx cells to different concentrations of GSI I for 18 hours and observed the concentrations of the cyoplasmic domains of Notch1, Notch2, Notch3, and Notch4 in the cytoplasms of the cancerous cells with each treatment of GSI I. They found that the concentration of GSI I was inversely proportional to the amount of cytoplasmic domains of Notch1, Notch2, Notch3, and Notch4 present in the cytoplasm (Figure 1). As the concentration of GSI I

Figure 1. Effect of different treatments of GSI I on 

             the concentration of Notch-IC in the cytoplasm of SeAx cells. 

Western blot analysis of cells treated with GSI I

 at different concentrations (0-5M). 
Notch-IC of Notch 1,2,3, and 4 analyzed.

increased, the concentrations of Notch-ICs of all Notch receptors decreased (Figure 1). Because there were not a significant amount of Notch 2, Notch3, or Notch 4 in SeAx cells, the results of this experiment were most significant with Notch1 (Figure 1). In order to confirm that GSI I is responsible for these results, the authors observed the expression of STAT3, which would indicate, if its concentration increased, that cytotoxicity and protein degradation are to blame for the observed results. This experiment resulted in confirming that GSI I was responsible for the decrease of the concentration of Notch-IC in the cytoplasm. Ultimately, the results of these experiments indicate that GSI I can inhibit the pathway of Notch1 by inhibiting the proteolytic cleavage of Notch1 from its cytoplasmic domain. 

Figure 2. Induction of apoptosis of MyLa cells

 treated with various concentrations of GSIs (0-40 uM) 

        and different GSI treatments (GSI I, IX, XX, and XXI)

 over 48 hours. Magnitude of apoptosis evaluated by measuring 

fold increase of caspase 3/7 activity of 

treatments compared to control.

Figure 3. Induction of apoptosis of MyLa cells treated with 

a 5 uM concentration of GSI I or a 20 uM concentration 

of either GSI IX, XX, or XXI. Magnitude of apoptosis

 evaluated by measuring fold increase of caspase 3/7

 activity of treatments compared to vehicle control.

Figure 4. Proportion of MyLa, SeAx, and HuT cells in either

 G1 or G0 of cell cycle that were treated with 5uM GSI for

 48 hours. Cell cycle phases were evaluated by NiM-DAPi

 staining of the DNA, and the amount of cells in each 

phase were analyzed by flow cytometry.

    Next, the authors assessed the influence of GSI I on inducing 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 MyLa, SeAx, and Hut78 tissues with increasing amounts of GSI's (GSI I, IX, XX, and XXI) for 48 hours. Out of all the GSIs, the authors found that GSI I was the only inhibitor to truly have any significant effects on any of the cell lines (figure 2 and figure3). The authors found that GSI I induced apoptosis the greatest at concentrations of 1 µM and higher (figure 2). In figure 2, this graph shows how after 1µM of GSI I treatment, the activity 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 the data 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 3). After 24 hours, the amount of caspase 3 and caspase 7 declines (Figure 3). 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) stainings 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 cycles. 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 4), which was "indicative of apoptosis" (2508)3. 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 to be 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 results observed from the GSI I data, which suggests that the inhibition of Notch1 induces apoptosis in cutaneous T-cell lymphoma cells.

         I thought 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, rather than using all 40 of the patients in the study. 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, I believe 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.

       The therapeutic effects of targeting NOTCH1 by using γ-secretase inhibitors have not only been noticed in cutaneous T-cell lymphoma, but their positive effects have also been acknowledge by researchers in other cancers that utilize NOTCH1 as an oncogene.  In another article, γ-secretase inhibitors: Notch so bad, the author describes how the use of γ-secretase inhibitors has had positive therapeutic results when used against mice suffering from T-cell acute lymphoblastic leukemia. However, because y-secretase inhibitors induce intestinal toxicity the inhibitors must be administered with dexamethasone, which counteracts the toxic side effects. 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. 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. If these proteins become disabled by γ-secretase inhibitors, there could be major negative physiological ramifications. I believe that instead 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. It is evident that creating a targeted therapy against NOTCH1 is a very complicated situation that will require a cocktail treatment and more selective drugs. However, it is much more complicated to create a drug targeting NOTCH1 when it acts as a tumor suppressor gene because “in the case of inactivated or lost tumor suppressor proteins, inhibitors are of no use, and reactivation is complex or impossible” (Brakenhoff 1103). This illustrates that a therapeutic strategy targeting NOTCH1, whether it acts as a tumor suppressor gene or an oncogene, is incredibly difficult to develop.

           Ultimately, as research has been reflecting, inhibiting the Notch1 transmembrane protein through γ-secratase inhibitors has had successful results in both T-cell acute lymphoblastic leukemia and cutaneous T-Cell lymphoma where the NOTCH1 gene acts as an oncogene. 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 Gleevec, 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 give 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.

Resources:

Brakenhoff, R. H. "Another NOTCH for Cancer." Science 333.6046 (2011): 1102-103. Web. 13 May 2014.

Grosveld, Gerard C. "γ-secretase Inhibitors: Notch so Bad." Nature Medicine15.1 (2009): 20-21. Web. 13 May 2014.

Kamstrup, M. R., L. M. R. Gjerdrum, E. Biskup, B. Thyssing Lauenborg, E. Ralfkiaer, A. Woetmann, N. Odum, and R. Gniadecki. "Notch1 as a Potential Therapeutic Target in Cutaneous T-cell Lymphoma." Blood116.14 (2010): 2504-512. Web. 13 May 2014.