Drug+Therapies+for+treating+childhood+ALL

Introduction
toc In this project, we will be focusing on two chemotherapeutic drugs used to treat Acute Lymphoblastic Leukemia (ALL) in children. Specifically, we will be focusing on one targeted therapy, Methotrexate, and one non-targeted therapy, Gleevec. While Methotrexate is a derivative of one of the first chemotherapeutic drugs used to treat ALL, Gleevec is one of the newer drugs on the market. Additionally, we will discuss the relative advantages and disadvantages of both of these drugs, how the two can be used in combination, and where the future treatments for ALL are headed.

**What Is Acute Lymphoblastic Leukemia (ALL)? **
There are four different types of leukemia, ALL being one of them. Acute Lymphoblastic Leukemia is a cancer of the blood and the bone marrow. The childhood form is the most common type of cancer in children, accounting for 33% of all pediatric cancers (1). Stem cells are produced in the bone marrow where immature stem cells, known as lymphoblasts, mature either into myeloid stem cells or lymphoid stem cells. Lymphoid stem cells mature into lymphoblasts and then into a type of white blood cell (Figure 1). The problem in ALL is that lymphoid cells accumulate in the bone marrow and fail to mature into lymphocytes. Additionally, the lymphoid cells do not function as optimally and cannot fight off infection, making the body extremely susceptible to illness. When these blast cells accumulate, they crowd the space where red blood cells, platelets, and healthy white blood cells normally reside, leading to the symptoms of ALL such as joint pain and anemia.



Symptoms of ALL (Acute Lymphoblastic Leukemia)
 The accumulation of non-functional blast cells can lead to a whole host of symptoms. When these leukemic cells accumulate, there is not enough room for other cells essential to the body, red and white blood cells, and platelets, to exist (1). Red blood cells, the cells responsible for carrying oxygen throughout the body, decrease in number, causing symptoms such as fever, anemia, bruising, fatigue, pallor, loss of appetite, pain below the rib cage, and shortness of breath (2). Healthy white blood cells, which are responsible for fighting off infection, also decline. Due to the weakened immune system, the body is also much more susceptible and sensitive to illness and infection. Along with red and white blood cells, platelet levels in ALL patients also decline. These cells are what help the blood clot, thus when they are in shortage, it is much more difficult for the body to heal wounds. One may also experience bruising, or red spots beneath the skin called petechiae (1). If ALL is left untreated for too long, it can affect the Central Nervous System and if it reaches the brain, can cause symptoms such as headaches and vomiting (1).

**Diagnosis of ALL **
 As previously mentioned, fever, shortness of breath, joint pain, and anemia are all potential signs of ALL. Unfortunately, these symptoms pertain to a broad range of other diseases and conditions, and therefore specific tests need to be done to ensure ALL is the correct prognosis. These tests include a CBC (complete blood count) test, bone marrow aspiration and biospy, lumbar puncture (spinal tap) and lymph node biospy. These procedures look at white blood cell and platelet levels in the blood and the presence of leukemia cells in the bone marrow. Leukemia cells have a distinct morphology. There are also numerous cytogenetic/chromosome tests that can be done via bone marrow or blood to classify the type of leukemia once the cancer is detected (1).

**Specific Diagnosis: Philadelphia Chromosome (Ph+ ALL)**
 The Philadelphia Chromosome is a chromosomal abnormality present in 25-30% of adults and 2-10% of pediatrics with ALL. This abnormality refers to a translocation between chromosomes 9 and 22, which generate the Philadelphia chromosome.  In Philadelphia chromosome positive (Ph+) ALL patients, the ABL gene (on chromosome 9) breaks off and gets transferred next to the BCR gene on chromosome 22, generating the Philadelphia chromosome. As a result of this translocation, the gene gets transcribed and two proteins become fused together. The BCR-ABL fusion protein as a result of this translocation is oncogenic. Under normal conditions, these proteins work in isolation. However, the BCR-ABL fusion activates many cell cycle-controlling proteins and inhibits DNA repair.  More information about the Philadelphia chromosome can be found here.

**Methotrexate **
<span style="font-family: Arial,Helvetica,sans-serif;"> <span style="font-family: Arial,Helvetica,sans-serif;"> Methotrexate is a chemotherapeutic drug, specifically an antineoplastic antimetabolite that competitively binds to the enzyme dihydrofolate reductase (DHFR), preventing the formation of tetrahydrofolic acid (THF), the active form of folate. The drug is a competitive inhibitor that binds allosterically to DHFR with 1,000-fold greater affinity than that of folate. The binding of methotrexate inhibits the activity of DHFR, leading to folate deficiency. DHFR reduces dihydrofolic acid to tetrahydrofolic acid (THF). THF levels and metabolism are necessary for the synthesis and repair of DNA as the central role of folate is as a precursor in DNA synthesis. Derivatives of folate are responsible for single carbon methylations, which generate biomolecules such as thymidine, purine, and guanine.

**<span style="font-family: Arial,Helvetica,sans-serif; font-size: 1.1em;">Mechanism of Action: **
<span style="font-family: Arial,Helvetica,sans-serif;"> Methotrexate acts during the S phase of the cell cycle. It enters the cell via a reduced folate carrier. Once it has entered the cell, the drug is polyglutamated by an enzyme; this extends the activity of the drug. Methotrexate then binds to DHFR and blocks the catalytic reaction of DHFR preventing the formation of THF. The diminished levels of THF reduce thymidine synthesis and therefore compromise DNA synthesis. This has a cytotoxic effect on rapidly dividing cells, especially.

<span style="font-family: Arial,Helvetica,sans-serif;"> **Five Year Event Free Survival**

<span style="font-family: Arial,Helvetica,sans-serif;"> <span style="font-family: Arial,Helvetica,sans-serif;"> Methotrexate is still one of the most commonly used drugs in the treatment of pediatric ALL and is very effective. Overall five year remission rates Methotrexate are nearly 85%. This Kaplan-Merier Curve (1986) shows a five-year complete remission rate of 65% and about 75% hematological remission rate with use of high-dose Methotrexate. Nonetheless, there are downsides to this drug. Being that Methotrexate is a non-targeted chemotherapeutic, it targets all cells, cancerous and non-cancerous, and thus induces many undesired side effects. Methotrexate is cytotoxic during the S phase of the cell cycle as it acts specifically during DNA and RNA synthesis. Because of this, it is more toxic on rapidly dividing cells like gastrointestinal cells, which leads to side effects such as abdominal pain, loss of appetite, and nausea.

<span style="font-family: Arial,Helvetica,sans-serif;"> **Gleevec**
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-family: Arial,Helvetica,sans-serif;">Treats Philadelphia-positive (Ph+) ALL <span style="font-family: Arial,Helvetica,sans-serif;"> **Figure 5** shows the Bcr-Abl protein and the pathway it activates. Imatinib targets the Bcr-Abl fusion protein. Since Imatinib inhibits this protein, the proteins that depend on the activation of Bcr-Abl also get inhibited. Specifically, as this diagram illustrates, when Bcr-Abl is inhibited, the Ras pathway is similarly inhibited because the Ras depends on Bcr-Abl as one of its constituent proteins in order to properly get activated. Since the Ras pathway has been disabled, the leukemic phenotype is similarly inhibited because the Ras pathway (through Raf-->MEK-->ERK-->ERK-->ELK) leads to cell proliferation, cell differentiation, etc. Due to this direct correlation between the Bcr-Abl fusion protein and the one the hallmarks of cancer (cell proliferation), Imatinib is able to be used quite effectively in many cases (see following section for efficacy rates and similar details).

<span style="font-family: Arial,Helvetica,sans-serif;">**Gleevec**
<span style="font-family: Arial,Helvetica,sans-serif;"> In general, the five-year relative survival rates for pediatric ALL have increased significantly since 1964. As can be seen in Figure 6, the survival rate from 1999-2006 is 89.2%. This figure shows that unlike in the past, where pediatric ALL prognosis was incredibly poor, there is now a high chance of remission for children diagnosed with ALL. One fairly new area that is being heavily researched is targeted drug therapies. A new drug that seems to show promise is Gleevac (Imatinib). Research on the use of Gleevac for ALL has shown combination therapy is currently the best treatment plan, providing the greatest survival outcomes. More specifically, it is recommended by physicians and pharmacists that Gleevac should be used in conjunction with normal chemotherapy. This is because initially, Gleevac was used by itself and resulted in a modest increase in survival rate (58 days) (Lee et al. 1585). However, after integrating Gleevac with traditional chemotherapy, which helped to reduce the side effects, the survival rate increased dramatically. In the same review paper mentioned earlier (Lee et al.), after integrating Gleevac and chemotherapy, complete remission rose to an outstanding 95%. Due to the promising success rate of Gleevac and other chemotherapeutics, different combinations of chemotherapy and Gleevac have been studied. The results are shown in Table 1 below. <span style="font-family: Arial,Helvetica,sans-serif;"> <span style="font-family: Arial,Helvetica,sans-serif;"> Although Gleevec has only been studied a short amount of time compared to chemotherapeutics, it has shown much better event-free survival than historical controls. The graph below provides an event-free survival plot over time and shows that the five-year survival rate for Gleevec is 80% while it is only about 30% for historical controls.

<span style="font-family: Arial,Helvetica,sans-serif;">Advantages and Disadvantages of Targeted Therapies and Traditional Therapies
<span style="font-family: Arial,Helvetica,sans-serif;">Imatinib is not a perfect drug, there are cases where the leukemia becomes resistant to Imatinib and reappears. As many researchers have noted, this is due to the accumulation of point mutations that results in inefficient/poor binding of Imatinib at the catalytic site. This causes Imatinib to "fall off" of the BCR-ABL protein and prevent the drug from inducing inhibition of the various pathways mentioned above. Examples of point mutations that lead to Imatinib-resistance is shown below in Figure 8. <span style="font-family: Arial,Helvetica,sans-serif;">

<span style="font-family: Arial,Helvetica,sans-serif;"> There have been various strategies that have been used to circumvent this issue with ALL, some of these are discussed in a review paper in the New England Journal of Medicine. As discussed in the review paper, the 3 main strategies that currently undergoing clinical trials or in the development stage include: using second-generation targeted therapies that are more potent and active that could target the BCR-ABL protein, using drugs that denature the permanently such that it can't be used all together, and combine targeted therapies with cytotoxic or general chemo-therapies or other targeted therapies that help to bring down "the defenses" of the tumor and destroy it. <span style="font-family: Arial,Helvetica,sans-serif;"> Since 2nd generation drug therapies are based on the highly successful survival rates of 1st generation drug therapies (ie Imatinib), this paragraph will discuss the potential of a few of these 2nd generation drug therapies that are in clinical trials or in active development. The drug therapies that are currently being researched upon include Nilotinib, Bosutinib, and Dasatinib. The difference that is readily apparent upon viewing Figure 5, which illustrates the specific molecular pathways that these drugs target, is that they target a variety of pathways. These make the drugs more potent for longer periods of time because it is less likely that the cancer will simultaneously develop mutations that will make it resistant to all of these pathways at once. As a result, the efficacy rates for these new 2nd generation targeted therapy treatments are higher than those when only Imatinib was taken by the patient.<span style="font-family: Arial,Helvetica,sans-serif;">

<span style="font-family: Arial,Helvetica,sans-serif;">**<span style="font-family: Arial,Helvetica,sans-serif;">Conclusion **
<span style="font-family: Arial,Helvetica,sans-serif;">In conclusion and with the eye towards the future, it seems that targeted drug therapies are the way forward. Due to its specificity and lack of prolonging side-effects, these drugs are more desirable when compared to regular chemotherapeutic drugs. In this project, we illustrated the advantages and disadvantages that general chemotherapy and targeted cell therapy have. Since targeted cell therapies are generally less impactful towards the patient, this type of treatment is slowly being more frequently recommended by oncologists across the country. In a similar notion, when ALL becomes resistant to Gleevec, 2nd generation targeted therapy treatment options have been developed and going through clinical trials to neutralize this disadvantage of targeted cell therapy. It is recommended by many doctors that targeted cell therapy drugs should taken alongside general chemotherapeutic drugs to ensure that the cancer is completely removed.