hyperproliferation of white blood cells Leukemia is characterized by hyperproliferation of immature white blood cells normal person Leukemic patient red blood cells white blood cell hyperproliferation of white blood cells

To understand leukemia we need to examine development of the Hematopoietic System self renewal myeloid lymphoid self renewal self renewal granulocytes Wikipedia 2

Different types of leukemia affect different stem cell types and distinct stages in their development Molecular Cell Biology Lodish et al. Fig. 24.1

Chronic myelogenous leukemia (CML) Annual incidence: 1/100,000 people (~15% of all leukemias) Median age: 30-60 yrs Median survival before onset of new therapies: 4 yrs with conventional chemotherapy 6 yrs with aIFN therapy; allogeneic bone marrow transplantation may cure the patient

CML Arises in a particular bone marrow stem cell = The granulocyte precursor Gives rise to neutrophils , basophils & megakaryocytes. Neutrophils-- fight infection by phagocytosis Basophils-- release immune modulators, e.g., histamines, Prostaglandins Platelets- cell fragments of megakaryocytes.

CML arises in a stem cell that is a granulocyte precursor Molecular Cell Biology Lodish et al. Fig. 24.1

1960: Nowell and Hungerford find that one copy of chromosome 22 is extremely short in CML patients “The findings suggest a causal relationship between the chromosome abnormality observed and chronic granulocytic leukemia.” Peter Nowell 7

Upon receiving the Lasker Award Nature 1973 243:290-3 “A new consistent chromosomal abnormality in CML identified by quinacrine fluorescence and Giemsa staining.” Rowley JD. Janet Rowley in 1998 Upon receiving the Lasker Award

A chromosomal translocation triggers CML Leukemic patient Normal individual Chr. 22 Chr. 9 9; 22 Translocation The Philadelphia chromosome

Here’s the characteristic karyotype of CML Karyotype courtesy of L. J. Beauregard, Eastern Maine Medical Center

Acute lymphoblastic leukemia (ALL) affects precursor of leukocytes =B +T cells) Ph+ chromosomes in 20% of adult ALL 2-5% of childhood ALL

Acute lymphoblastic leukemia (ALL) affects precursor of leukocytes =B +T cells) In adults prognosis is less good (80-90%of will have remissions but half will experience a relapse, making overall cure rate ˜40%) Bone marrow transplant the only long term treatment

Chromosomal rearrangements are a hallmark of leukemia, being present in 70-90% of cases Table 4.5 The Biology of Cancer (© Garland Science 2007) 13

Why is this the case? Table 4.5 The Biology of Cancer (© Garland Science 2007) 14

just to make antibodies. What’s the solution? We cannot dedicate all 25,000 genes in the genome just to make antibodies. What’s the solution? Put antibodies together by a mix-and match approach! Molecular Biology of the Cell Alberts et al

requires rearranging the DNA Molecular Biology of the Cell Alberts et al

requires rearranging the DNA Molecular Biology of the Cell Alberts et al

Molecular Biology of the Cell Alberts et al The result: an antibody light chain Molecular Biology of the Cell Alberts et al

Since there are multiple types of each gene segment, there are thousands of possible V-D-J combinations Each B cell gets a unique combination This slide gives current best numbers for human antibody segments. You could do some simple calculations like those in the notes of slide 25, “A unique recombination occurs in each B cell” to determine how many combinations are possible based only on the number of different segments.   K and λ refer to two distinct forms of light chains that exist in most vertebrates. An IgG molecule may have two K chains or two λ chains, but not both.

Since there are multiple types of each gene segment, there are thousands of possible V-D-J combinations Each B cell gets a unique combination Isn't that amazing! This slide gives current best numbers for human antibody segments. You could do some simple calculations like those in the notes of slide 25, “A unique recombination occurs in each B cell” to determine how many combinations are possible based only on the number of different segments.   K and λ refer to two distinct forms of light chains that exist in most vertebrates. An IgG molecule may have two K chains or two λ chains, but not both.

However sometimes this goes wrong, and other genes are juxtaposed to the Ig or TCR genes

Rearrangement mistakes can also juxtapose Other genes with oncogenic consequences

The Philadelphia chromosome translocation fuses the bcr and abl genes normal individual Leukemic patient bcr Bcr-abl Chr. 22 abl Chr. 9 9; 22 Translocation fuses Bcr and Abl De Klein et al. Nature 300, 765 (1982) Groffen et al. Cell 36, 93 (1984)

Abelson was first identified as the oncogene carried by Abelson leukemia virus, which causes pre-B cell Lymphoma in mice Abelson and Rabstein, Cancer Res 30, 2213 (1970)

The v-abl containing retrovirus was recovered from a tumor found in mice infected by Moloney Leukemia virus

In CML the translocation results in production of a fusion protein that joins the amino-terminal end of the BCR protein to most of the Abl protein The Cell, G. Cooper, Fig. 15.25

In fact, different breakpoints in bcr lead to slightly different Bcr-Abl fusion proteins that are found in different cancers 27

Fluorescence In Situ Hybridization (FISH) provides a tool for diagnosing CML abl bcr 28

Fluorescence In Situ Hybridization (FISH) a tool for diagnosing CML fusion 22 bcr/abl fusion 9 abl/bcr abl bcr The current methd: PCR BCR ABL 29

Abelson kinase A fatty-acid modified and actin-binding non-receptor tyrosine kinase Actin-binding Myristate SH2 F G SH3 kinase

Abelson kinase The front end looks a lot like Src!! Actin-binding Myristate SH2 F G SH3 kinase

Oncogenic versions of Abelson Abl Actin-binding SH2 F G SH3 kinase v-abl Gag F G Bcr-Abl Bcr F G

What’s changed?? Abl v-abl Bcr-Abl Actin-binding SH2 SH3 kinase Gag F G SH3 kinase v-abl Gag F G Bcr-Abl Bcr F G

Remember this? Src is normally inactive due to intramolecular inhibition

The structure of Abl reveals a novel mode of intramolecular inhibition Nagar et al. Cell 112:859 (2003)

Distinct yet analogous modes of regulation Src and Abl Distinct yet analogous modes of regulation Harrison Cell 112, 737 (2003)

A multistep mechanism for activating Src Harrison Cell 112, 737 (2003)

A proposed mechanism for activating Abl Harrison Cell 112, 737 (2003)

But what does Abl normally do?

Insights from the mouse model • abl mutant mice are viable but runted and have a shortened lifespan They also have problems with: male fertility B cell maturation osteoblasts and bone formation • Truncation of C-terminus leaving an intact kinase has same phenotype as the null mutant

Why so mild?? Insights from the mouse model • abl mutant mice are viable but runted and have a shortened lifespan They also have problems with: male fertility B cell maturation osteoblasts and bone formation • Truncation of C-terminus leaving an intact kinase has same phenotype as the null mutant

Abelson has a twin brother Abl Actin-binding SH3 SH2 kinase F G 34% 89% 94% 27% F G Arg

Are Abl and Arg redundant? • arg mutant mice have behavioral defects (Arg is expressed in the brain at high levels)

Are Abl and Arg redundant? • arg mutant mice have behavioral defects (Arg is expressed in the brain at high levels) • abl; arg double mutants have defects in neural tube Wild-type abl; arg

Now let’s turn to Abl’s cell biological role 45

Remember Focal adhesions? Alberts et al. Fig. 16-75

Focal adhesion proteins are phosphorylated by Abl (mediator of integrin signaling) 47

Abl also phosphorylates regulators of the actin cytoskeleton 48

Of course it’s even more complicated Than that! Bradley and Koleske jcs.biologists.org/cgi/content/ full/122/19/3441/FIG3 49

bundle actin filaments Abl can also directly regulate cytoskeletal events using its C-terminal region to bundle actin filaments and link them to microtubules 50

hyperproliferation of white blood cells But does this all help us understand and treat leukemia? normal person Leukemic patient red blood cells white blood cell hyperproliferation of white blood cells

BCR-Abl affects multiple cell functions Proliferation & differentiation Stem cell turnover S G 2 M 1 G0 BCR-Abl Cytoskeleton/ adhesion defects Apoptosis Adapted from Jörgensen, 2001. Hem. Onc.

Abl may play roles in the nucleus in response to DNA damage •ATM can phosphorylate Abl in response to DNA damage •Abl may stabilize p53 Van Etten, TICB 9 179-186

To understand leukemia, we must start by learning more about the clinical progression of CML Advanced phases Chronic phase Median 5–6 years stabilization Accelerated phase Median duration 6–9 months Blast crisis Median survival 3–6 months Clinical Course: Phases of CML CML progresses through 3 phases characterized by increasing refractoriness to therapy and worsening clinical features and laboratory findings. These stages include chronic phase, accelerated phase, and blast crisis. Although the majority of patients present in chronic phase and then progress to accelerated phase, 25% to 40% of patients progress directly from chronic phase to the terminal blast crisis phase without evidence of a transitional accelerated phase.1,2 Chronic phase. In the chronic phase, there are less than 10% blasts in peripheral blood and bone marrow, and the white blood cell (WBC) count at presentation is typically elevated to 20 x 109/L. Signs and symptoms may be mild initially and develop as the disease progresses. The chronic phase of CML may last 5 to 6 years before the disease accelerates.1,3 Accelerated phase. There are more than 10% to 15% (but less than 30%) blasts in either peripheral blood or bone marrow. Symptoms may increase and include unexplained fever, bone pain, splenomegaly, and hepatomegaly. Basophilia, decreased platelet counts, and cytogenetic progression may also be observed. The accelerated phase may last 6 to 9 months.1,3 Blast crisis. There are more than 30% blasts in peripheral blood or bone marrow and symptomatology is increased, especially relating to anemia and infection, central nervous system (CNS) disease, lymphadenopathy, and bleeding. Approximately 50% of patients have myeloid blast crisis, 25% have lymphoid blast crisis, and 25% are mixed.3 Patients with CML in blast crisis have a poor prognosis owing to the lack of effective therapy. This phase is rapidly fatal, with a median survival of 3 to 6 months.2,3 References 1. Hill JM, Meehan KR. Chronic myelogenous leukemia. Curable with early diagnosis and treatment. Postgrad Med. 1999;106:149-152, 157-159. 2. Faderl S, Kantarjian HM, Talpaz M. Chronic myelogenous leukemia: update on biology and treatment. Oncology. 1999;13:169-180. 3. Cortes JE, Talpaz M, Kantarjian H. Chronic myelogenous leukemia: a review. Am J Med. 1996;100: 555-570. Provided by: Gleevec.com

Blast crisis is thought to involve additional genetic changes that are only beginning to be characterized Suggested events: •Mutations in p53 •MSI2/HOXA9 fusion protein •AML1/EVI-1 fusion protein •Ras mutations •Deletion of the Ikaros transcription factor

Therapy for CML: how do you evaluate whether a drug is working? Hematologic Response Cytogenetic Response – Complete: – Major: Normal peripheral blood count Complete: 0% Ph+ No immature cells Partial 1-35% Ph+ – Minor: 36%–95% Ph+ Goals of Therapy for CML: Response Criteria Response criteria in CML have been defined in terms of hematologic and cytogenetic responses.1,2 The first goal of therapy in patients with CML is to reduce the WBC count to near-normal levels. Hematologic responses are defined by whether (1) peripheral cell counts have normalized and (2) immature myeloid cells are no longer present. Hematologic improvement must be maintained for 1 month to qualify as response. Disappearance of signs (eg, splenomegaly or hepatomegaly) and symptoms of disease is also required. The second goal is to achieve a cytogenetic remission by reducing or eliminating cells that carry the Ph chromosome. Cytogenetic responses are based on bone marrow analyses of dividing myeloid cells in metaphase to quantify the number of detectable Ph+ cells. Molecular techniques such as fluorescence in situ hybridization (FISH) and quantitative reverse transcription polymerase chain reaction (RT-PCR) are also used for detection of bcr-abl transcripts.2 Achieving a cytogenetic response has been associated with improved survival in interferon-alpha (IFN-) patients.3 A therapy that is well tolerated and has no or few side effects is also an important parameter. References 1. Faderl S, Talpaz M, Estrov Z, et al. Chronic myelogenous leukemia: biology and therapy. Ann Intern Med. 1999;131:207-219. 2. Sawyers CL. Chronic myeloid leukemia. N Engl J Med. 1999;340:1330-1340. 3. Kantarjian HM, Smith TL, O’Brien S, et al. Prolonged survival in chronic myelogenous leukemia after cytogenetic response to interferon- therapy. Ann Intern Med. 1995;122:254-261. Modified from Gleevec.com

Therapeutic Options for CML Allogeneic stem cell transplantation (SCT) Interferon-alpha (IFN-)–based treatments Chemotherapy with hydroxyurea, busulfan Gleevec™ (imatinib mesylate, = STI571) Therapeutic Options for CML The current treatment options for patients with CML include allogeneic stem cell transplantation (SCT), IFN-–based regimens, chemotherapy with hydroxyurea or busulfan, and Gleevec™ (imatinib mesylate, formerly STI571). Allogeneic SCT is currently the only therapy with the potential to “cure” CML. Access to the procedure is limited by availability of a suitable donor and by patient age (many centers do not accept candidates >55 years of age). Transplantation within 1 year of diagnosis is preferred.1 Approximately 15% to 20% of patients with CML meet these criteria for SCT.2 Five-year survival for patients transplanted during the chronic phase of the disease ranges from 54% to 70%; survival rates decrease and morbidity and mortality increase dramatically with advanced stages of disease. IFN- induces hematologic responses in most patients and cytogenetic responses in some patients. The effects are dose-related; higher doses of IFN- correlate with higher rates of hematologic and cytogenetic response as well as more severe adverse events.3,4 Approximately 25% of patients discontinued therapy because of major side effects.5 Chemotherapy is of limited value in inducing cytogenetic responses, but it is effective in lowering WBC counts. These agents are generally considered to be palliative.6 Gleevec is a tyrosine kinase inhibitor that has shown very promising results in phase I and phase II clinical trials. References 1. Faderl S, Talpaz M, Estrov Z, et al. Chronic myelogenous leukemia: biology and therapy. Ann Intern Med. 1999;131:207-219. 2. Sawyers CL. Chronic myeloid leukemia. N Engl J Med. 1999;340:1330-1340. 3. Faderl S, Kantarjian HM, Talpaz M. Chronic myelogenous leukemia: update on biology and treatment. Oncology. 1999;13:169-180. 4. Silver RT, Woolf SH, Hehlmann R, et al. An evidence-based analysis of the effect of busulfan, hydroxyurea, interferon, and allogeneic bone marrow transplantation in treating the chronic phase of chronic myeloid leukemia: developed for the American Society of Hematology. Blood. 1999;94:1517-1536. 5. Guilhot F, Chastang C, Michallet M, et al. Interferon alfa-2b combined with cytarabine versus interferon alone in chronic myelogenous leukemia. N Engl J Med. 1997;337:223-229. 6. Hill JM, Meehan KR. Chronic myelogenous leukemia. Curable with early diagnosis and treatment. Postgrad Med. 1999;106:149-152, 157-159. From Gleevec.com

Until recently interferon-alpha treatment Was the gold-standard in CML Even though its mechanism of action IS STILL NOT UNDERSTOOD Data of the Italian Cooperative Study group on Chronic Myeloid Leukemia. Blood 1998:92 1541–1548 IFN=interferon-alpha, CHT= conventional chemotherapy

Gleevec blocks the ATP binding site of the kinase domain STI571 59

Abl’s Kinase Domain In complex With the inhibitor Gleevac Kuriyan lab website

Months Since Start of Treatment Gleevec™: in chronic phase CML Chronic Phase CML 1.0 Major cytogenetic response 0.9 0.8 Complete cytogenetic response 0.7 Fraction of patients that responded 0.6 0.5 0.4 Gleevec™: Rapid Hematologic and Cytogenetic Response1 More than 50% of patients with MCR achieved their response within 3 months (median = 2.9 months) in chronic phase CML. In accelerated phase and blast crisis phase CML, similar results were obtained. The median time to a hematologic response was 1 month, with a range of 0.9 to 9.3 months in the accelerated phase CML. The median time to a hematologic response was also 1 month for the other phases of CML. Reference 1. Data on file. Novartis Pharmaceuticals Corporation, East Hanover, NJ. 0.3 0.2 0.1 0.0 1 2 3 4 5 6 7 8 9 10 Months Since Start of Treatment Data: Novartis Pharmaceuticals Corporation

Months Since Start of Treatment This is an awesome drug! Most patients survive 10+ years Chronic Phase CML 1.0 Major cytogenetic response 0.9 0.8 Complete cytogenetic response 0.7 Fraction of patients that responded 0.6 0.5 0.4 Gleevec™: Rapid Hematologic and Cytogenetic Response1 More than 50% of patients with MCR achieved their response within 3 months (median = 2.9 months) in chronic phase CML. In accelerated phase and blast crisis phase CML, similar results were obtained. The median time to a hematologic response was 1 month, with a range of 0.9 to 9.3 months in the accelerated phase CML. The median time to a hematologic response was also 1 month for the other phases of CML. Reference 1. Data on file. Novartis Pharmaceuticals Corporation, East Hanover, NJ. 0.3 0.2 0.1 0.0 1 2 3 4 5 6 7 8 9 10 Months Since Start of Treatment Data: Novartis Pharmaceuticals Corporation

This is an awesome drug! Patients who achieve a stable cytogenetic response have overall survival rates of 95.2% after 8 years, similar to the rate in the general population. Only 1% of patients died because of leukemia progression Gleevec™: Rapid Hematologic and Cytogenetic Response1 More than 50% of patients with MCR achieved their response within 3 months (median = 2.9 months) in chronic phase CML. In accelerated phase and blast crisis phase CML, similar results were obtained. The median time to a hematologic response was 1 month, with a range of 0.9 to 9.3 months in the accelerated phase CML. The median time to a hematologic response was also 1 month for the other phases of CML. Reference 1. Data on file. Novartis Pharmaceuticals Corporation, East Hanover, NJ. Journal of the National Cancer Institute 103 (7): 553–561 (2011)

Of course there are side effects... Drug was discontinued for adverse events in 1% of patients in chronic phase, 2% in accelerated phase, and 5% in blast crisis

And the cost..... Still $30,000-50,000 per year!!

Started at $30,000-50,000 per year And rose from there And the cost..... Started at $30,000-50,000 per year And rose from there Now >75,000/year!! BUT it lost patent protection and became generic in 4 January 2015

BUT it loses patent protection and becomes generic in 2014

Unfortunately, natural selection is a powerful process “We now know of over 30 different mutations that can cause BCR-ABL to become resistant to imatinib,” says Dr. Charles Sawyers of UCLA’s Jonsson Cancer Center. In patients with newly diagnosed disease, we are seeing resistance to imatinib in about 4%of patients per year. The further the disease has progressed before initiating imatinib treatment, the greater the chances are that resistance will arise.”

Unfortunately, natural selection is a powerful process About 17% of all patients develop resistance in 5 years Science 331: 1542-44 March 25 2011

A possible solution: a new generation of kinase inhibitors that Still inhibit Gleevec-resistant tumors aka Dasatinib Inject Luciferase-expressing tumor cells Science 2004 305:399-401

Dasatinib FDA approved for patients with relapses NCI Cancer Bulletin October 5, 2006 aka Dasatinib Inject Luciferase-expressing tumor cells

Dasatinib FDA approved for patients with relapses NCI Cancer Bulletin October 5, 2006 Leads to 73% progression free survival for 3 years

Dasatinib FDA approved for patients with relapses NCI Cancer Bulletin October 5, 2006 Phase II trials suggest Dasatinib effective in Blast-Crisis Patients with Gleevec-resistant tumors NCI Cancer Bulletin May 2 2007 Phase II trials suggest Dasatinib effective in ALL patients with Gleevec-resistant tumors NCI Cancer Bulletin May 29 2007

This is becoming a general approach Science 331: 1542-44 March 25 2011

How much do you think that costs?

For CML Add another $70,000/year!

Gleevec also has promise in other tumors e.g., Gastrointestinal Stromal Tumors 90% of malignant GISTs harbor a mutation in c-kit leading to KIT receptor autophosphorylation and ligand-independent activation http://www.answers.com/topic/gist-2-jpg-1

Gleevec also has promise in other tumors e.g., Gastrointestinal Stromal Tumors 90% of malignant GISTs harbor a mutation in c-kit leading to KIT receptor autophosphorylation and ligand-independent activation Does not respond to chemotherapy (<10% response) Only can be effectively treated if the entire tumor Can be removed surgically Without this median survival 1-2 yrs

With Gleevec treatment ~50% of patients respond Tumors shrink in size and disease symptoms are greatly reduced Report from the FDA Approval Summary: Imatinib Mesylate in the Treatment of Metastatic and/or Unresectable Malignant Gastrointestinal Stromal Tumors Dagher et al. Clinical Cancer Research3034 3034–3038, October 2002

Gleevec treatment also reduces risk or recurrence April 17, 2008 Gleevec treatment also reduces risk or recurrence After surgical removal of GISTs 97% of patients treated with Gleevec had no recurrence after 1 year Versus 83% of those receiving placebo

Gleevecxtends median survival GIST Gleevecxtends median survival from 15 months to 5 years

But..... Long term outcome ? Many patients who initially respond develop secondary resistance to Gleevec and relapse Cause: second site mutations in c-kit!

GIST Long term outcome ? Many patients who initially respond develop secondary resistance to Gleevec and relapse Cause: second site mutations in c-kit! Approach: Develop new drugs targeted against c-kit e.g., AMG706, SU11248 Current Oncology Reports (2005) 7: 293-299

An alternate approach: broader spectrum inhibitors that hit multiple targets Sunitinib: targets Abl/PDGF Receptor, Src, and VEGF Receptor FDA approved after Phase III clinical trial reveal efficacy in GIST patients whose Tumors are resistant to Gleevec George Demetri, MD NCI Cancer Bulletin Oct. 31 2006