Lecture Summaries

The summaries below provide an overview of what was covered in each session of this course. Detailed lecture notes are not available for this course.

1 Introduction We will get to know each other, discuss the syllabus and hear about how to read scientific articles. The instructors will give an introduction to hematology, the branch of medicine that studies blood and blood diseases. Introduction to some of the techniques to be discussed throughout the course.
2 From Stem Cells to Blood

Hematopoiesis is the process of the continuous formation of the blood system. It is estimated that 2 x 1011 red blood cells and 7 x 1010 neutrophilic leukocytes (the most abundant type of white blood cells) are produced every day in the human body, which sums up to several thousand kilograms during a human lifespan. This enormous output of mature blood cells is derived from a very rare cell type named the hematopoieteic stem cell (HSC). By definition a single HSC is capable of producing all cell types of the hematopoietic system, including new HSCs and all classes of mature cells in the circulating blood. This clonal development of the blood system has enabled researchers and physicians to transplant HSCs, and thus entire hematopoietic systems, from one individual to another.

A strict clonal hierarchy of differentiation stages from the HSC to mature blood cells is important to maintain control over hematopoiesis. When a HSC divides it can give rise to zero, one or two HSCs, or to zero, one or two more mature multi-potent progenitor cells that in turn can divide into even more mature progenitor cells. After a few more cell divisions, depending on signals from the environment, morphologically recognizable blood precursor cells are formed that can give rise to mature blood cells that are able to leave the bone marrow. During this long chain of events every cell division is accompanied by a decision of the fate of the progeny. It is believed that no daughter cell can ever become more primitive after mitosis, but the cells can choose to go into apoptosis (programmed cell death), to differentiate, or in some cases to self-renew. Today we probably know more about blood development than about development of any other organ in our body.

3 Gene delivery vehicles engineered from viruses Viruses belonging to the retrovirus group are probably the most studied viruses in molecular biology, in part because of a famous family member, human immunodeficiency virus (HIV-1). Retroviruses encode an RNA-dependent DNA polymerase called reverse transcriptase. This protein is essential, since the retroviral genome consists of RNA, while viral replication depends on expression from a reverse transcribed DNA copy that is integrated into the host genome as a cellular gene. Since these viruses naturally integrate their genomes into infected cells, viral vectors based on these viruses are ideal mediators of permanent gene transfer. A viral vector is simply a replication-incompetent virus particle in which most of the viral genes have been substituted by the gene or genes desired to integrate in the target cell. In a simple vector, genes coding for viral proteins are replaced by the desired transgene. Numerous modifications of the early vectors have been made to improve titers, transgene expression, safety and tropism. The retrovirus-based vectors can be divided into two groups: oncoretroviral and lentiviral vectors. Oncoretroviral vectors are based on oncoretroviruses such as the Moloney murine leukemia virus, while lentiviral vectors are based on lentiviruses such as human or simian immunodeficiency viruses. Lentivirus (lenti- means "slow" in Latin) is a retrovirus subgroup characterized by a long incubation period before onset of disease. Lentiviral vectors have an important advantage over oncoretroviral vectors, since they can transduce non-dividing cells. Since few HSC are in mitosis at any given time, lentiviral vectors would be suitable for gene transfer into these cells.
4 Treating genetic disorders by fixing the bad gene Instead of transplanting HSCs from a healthy donor, the patient s own HSCs are transplanted after being transduced ex vivo with a vector mediating expression of the deficient gene product. Permanent introduction of new genes into HSCs and long-term engraftment of these cells are fundamental requirements for gene therapy of monogenic blood disorders. One such disorder is X-linked severe combined immunodeficiency (X-SCID), which we discussed during the second week. This week we will discuss two papers that describe the outcome of a clinical trial for gene therapy of X-SCID. The outcome of this clinical trial will take us into a discussion about ethics in medical research. The Declaration of Helsinki states that biomedical trials (such as those needed to develop clinical gene therapy protocols) must have an objective that is in proportion to the risks to the subjects. To develop gene therapy, which has potentially lethal side effects, a researcher must show that the amount of benefit outweighs the amount of risk considering the probability and utility of different outcomes. A study can be considered ethical only if there is a clearly advantageous risk-benefit ratio. Is it ethical to continue gene therapy trials for X-SCID?
5 RNAi: Using an ancient defense against viral infection to turn off disease genes

In 2006 the Nobel Prize in Physiology or Medicine was awarded to Andrew Fire and Craig Mello for their discovery of RNA interference - gene silencing by double-stranded RNA. The biological pathways underlying double-stranded RNA (dsRNA)-induced gene silencing depend on the enzyme Dicer and the nuclease complex RISC (RNA-induced silencing complex). Dicer cleaves dsRNA into ≈22 nucleotide fragments called small interfering RNAs (siRNAs). The siRNAs are then incorporated into RISC, which unwinds the siRNA and incorporates one of the strands into the protein complex. RISC then uses single stranded RNA as a template to find single stranded RNAs (i.e. mRNAs) with which it can hybridize. If there is a complete match the targeted mRNA will be degraded.

One efficient method to deliver siRNA to cells is through expression from viral vectors. Viral vectors enable inexpensive, efficient induction of RNAi in cells that are very difficult to transfect with dsRNA.

6 Future of personalized medicine using induced pluripotent stem (iPS) cells IPS cells are believed to be identical to natural pluripotent stem cells, such as embryonic stem cells, in many but probably not all respects. IPS cells were first produced in 2006 from mouse cells and in 2007 from human cells. This important advance in stem cell research allows researchers to obtain pluripotent stem cells, which are important for many research purposes and potentially have therapeutic uses as well, without the controversial use of embryos.
7 Field trip to the Whitehead Institute and to the Whitehhead Flow Cytometry Facility

Instead of a usual class this week we will take a field trip to the Whitehead Institute and to the Whitehhead Flow Cytometry Facility. We will show you how to use a light microscope and let you examine slides of stained blood, fetal liver and bone marrow cells.

Next we will stain hematopoietic cells from mouse with fluorescent antibodies against cell surface antigens CD71 and ter119 to analyze the level of red cell maturation using Fluorescence-Activated Cell Sorting (FACS). FACS is one of the corner stones of hematological research and leukemia diagnosis and is used in several of the papers discussed in the course. The analysis will be performed at the Whitehead Flow Cytometry Facility. You will learn how this technique allows multi-parametric analysis and separation of thousands of single cells per second using single wavelength lasers and detectors.

About Whitehead

Week 8 - Week 13: The journey of a 'wonder' drug, Gleevec

Case 1: On December 1995, 51 years old Judy Orem was diagnosed with chronic myeloid leukemia (CML). Bone marrow transplantation is the only known cure, but the procedure is highly dangerous and has less than a 30% success rate. Alternative treatments such as interferon and chemotherapy can extend her life by up to two years, but comes with serious side effects. [Read more about Judy Orem.]

Case 2: Marco Nese, 33 years old, was also diagnosed with CML. Since he has no siblings, bone marrow transplantation was not feasible. Although interferon could potentially extend his life from 5 to 7 years, he would live those years with serious side effects, often feeling tired and depressed. He began to work only part time. As his health continued to deteriorate, he started to work from home. Then he developed pneumonia and stopped working altogether. [Read more about Marco Nese.]

There are many patients like Judy and Marco who are in desperate need of a miracle drug. A drug that can save their lives or at least improve their quality of life. We will spend the remaining six sessions learning about the 'wonder' drug Gleevec.

8 Identification of bcr-abl translocation and its oncogenic properties

In late 1950s, Peter Nowell of the University of Pennsylvania School of Medicine, and David Hungerford, of the Insiutute for Cancer Research identified an abnormal human chromsome, later named the Philadelphia chromosome, that was abnormally short. This chromosome was seen consistently in blood samples from patients suffering from chronic myeloid leukemia (CML). In 1973, Janet Rowley, a biologist at the University of Chicago, discovered that a missing piece of the abnormal chromosome 22 was 'translocated' to chromosome 9 and that a piece of missing DNA in chromosome 9 was found in chromosome 22. She proposed that a reciprocal chromosomal translocation is common in CML.

It took another 10 years before scientists from National Cancer Institute and Esramus University identified c-abl, originally located in chromosome 9, as translocated to chromosome 22. Meanwhile, they found that the breakpoint cluster region (bcr) from chromosome 22 is the site that fused to c-abl. These two genes formed a novel bcr-abl fusion. It was not known if the bcr-abl fusion was the cause or by-product of CML. David Baltimore, at the Whitehead Institute for Biomedical Research and MIT showed that infected bone marrow cells with a retrovirus expressing fusion gene rapidly induce CML in a mouse model.

9 Drug development for inhibiting kinase activity in the bcr-abl fusion

In 1986 and 1987, David Baltimore and Owen Witte identified the bcr-abl protein as a tyrosine kinase, a type of enzyme that plays a critical role in regulating cell growth and division. After 30 years of basic research, we now have a drug target for CML.

In the 1990s, the development of drugs that target kinases was considered a risky project because many scientists were worried that kinase inhibitors may not be specific enough and would inhibit a wide range of kinases, creating devastating side effects. Scientists Alex Matter and his team members Nick Lydon, Jurg Zimmermann and Elizabeth Buchdunger took this challenge. When the CEO of Novartis, Daniel Vasella told Alex that fellow scientists had thought him crazy to pursue kinases, Alex replied, 'Am I responsible for the narrow-mindedness of my colleagues?'

The teams began by drawing chemical structures that might fit the 'pocket,' the catalytic domain of the bcr-abl enzyme. Each week, they came up with ten compounds that they then screened for activity. Many chemicals indeed inhibited bcr-abl kinase, but also other kinases. After many trials and failures, they eventually found a specific compound, STI 571, later named Gleevec.

10 Testing the efficacy of Gleevec in cell lines, mouse and human

Brian Druker, who is now at Oregon Health & Science University, collaborated with Matter's group to test the efficacy of STI571. Druker was extremely enthusiastic about this project, since CML was one of the only cancers in which the genetic cause was known. In their principle experiment, STI571 effectively reduced the colony formation of bcr-abl transformed bone marrow cells by more than 90%, while leaving normal colonies intact, suggesting that the compounds might be specific and safe.

It's possible for drugs to work effectively and safely in small animals and even primates, but still cause extreme toxicity in humans. Therefore, the performance of a drug in human trials is crucial. Many issues needed to be considered, including drug dosage, level and schedules and choice of patients, etc., The Phase I clinical trial began on June 22, 1998. Usually, phase I studies simply aim to test toxicity. In this case, all 31 tested patients experienced complete remission i.e. their blood count returned to normal. Subsequent clinical trials produced equally astonishing results. The Food and Drug Administration (FDA) approved the use of STI571 (Gleevec) for CML on May 10, 2000, less than three years after the start of the first phase clinical trials, an extraordinary short time.

11 Evolving resistance to Gleevec While the mass media was ecstatic about Gleevec, two articles in Science reported two mechanisms of resistance to Gleevec in blast crisis, the terminal acute phase of CML, in 2001. CML often progresses from chronic phase (3-6 years) to blast crisis (less than 1 year), which eventually caused the deaths of patients. Unfortunately, in CML blast crisis, most patients develop resistance to Gleevec, usually through at least a mechanism such as bcr-abl over-expression, gene amplification, and, most notably, mutations in the bcr-abl kinase domain that prevent binding of Gleevec.
12 Overcoming Gleevec resistance Since the problem of Gleevec resistance became apparent, pharmaceutical companies have been testing previously synthesized kinase inhibitors to see how well they can inhibit Gleevec resistant CML cells. One compound, BMS-354825, later named as Dasatinib, was found and shown to inhibit the tyrosine kinase activity and cell growth of cell lines expressing 14 of 15 different Gleevec-resistant bcr-abl mutant. Moreover, the drugs worked equally well in a mouse model of Gleevec-resistant CML. In HIV and other infectious disease, multiple drugs are often used to slow down the development of resistance that occurs in single-drug therapy. Therefore, it would be interesting to see if multiple drugs that target different conformations and sites of bcr-abl would be a very effective treatment.
13 Use of Gleevec in other diseases The huge success of Gleevec in treating CML raised the possibility that the drug can be used to treat other diseases with activated kinases. The c-kit proto-oncogene encodes a type III receptor tyrosine kinases which binds to its ligand, stem cell factor. Their binding are essential for the development of melanocytes, erythrocytes, mast cells and stromal cells. Activating mutations of c-kit gene have been found in mast tumor cells. Kitamura's group identified a gain of function of c-kit gene in a rare subset of soft tissue sacromas, called gastrointestinal stromal tumors (GISTs). Since Gleevec also shows relatively specific and potent activity towards c-kit receptor, clinical trials were launched to test Gleevec in treating patients with advanced case of GIST. The results showed that Gleevec induced a sustained objective response in more than half of the patients.