Leukemia is characterized by hyperproliferation of immature white blood cells white blood cell Leukemic patientnormal person red blood cells hyperproliferation of white blood cells
Wikipedia To understand leukemia we need to examine development of the Hematopoietic System myeloidlymphoid self renewal granulocytes
Molecular Cell Biology Lodish et al. Fig Different types of leukemia affect different stem cell types and distinct stages in their development
Chronic myelogenous leukemia (CML) Annual incidence: 1/100,000 people (~15% of all leukemias) Median age: yrs Median survival: 4 yrs with conventional chemotherapy 6 yrs with aIFN therapy; allogeneic bone marrow transplantation may cure the patient
Chronic myelogenous leukemia (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.
Molecular Cell Biology Lodish et al. Fig CML arises in a stem cell that is a granulocyte precursor
“The findings suggest a causal relationship between the chromosome abnormality observed and chronic granulocytic leukemia.” Peter Nowell 1960 Nowell and Hungerford find that one copy of chromosome 22 is extremely short in CML patients
Nature :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 Normal individual Leukemic patient Chr. 9 Chr. 22 9; 22 Translocation The Philadelphia chromosome
Karyotype courtesy of L. J. Beauregard, Eastern Maine Medical Center A characteristic karyotype indicates CML
Acute lymphoblastic leukemia (ALL) Affects precursor of leukocytes (B and T cells) Ph+ chromosomes in 20% of adult ALL 2-5% of childhood ALL In adults prognosis very poor (Only % of adults with ALL survive 2 years) Bone marrow transplant the only long term treatment
Table 4.5 The Biology of Cancer (© Garland Science 2007) Chromosomal rearrangements are a hallmark of leukemia, being present in 70-90% of cases
Table 4.5 The Biology of Cancer (© Garland Science 2007) Why is this the case?
Molecular Biology of the Cell Alberts et al 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 The result: an antibody light chain
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
As we have seen, 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 Chr. 9 Chr. 22 abl bcr Bcr-abl 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
The Cell, G. Cooper, Fig 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
In fact, different breakpoints in bcr Lead to slightly different Bcr-Abl fusion proteins That are found in different cancers
Fluorescence In Situ Hybridization (FISH) a tool for diagnosing CML ablbcr
fusion 9 abl/bcr fusion 22 bcr/abl abl bcr Fluorescence In Situ Hybridization (FISH) a tool for diagnosing CML BCRABL The current methd: PCR
Abelson kinase A fatty-acid modified and actin-binding non-receptor tyrosine kinase SH3 FG SH2 kinase Actin- binding Myristate
Abelson kinase The front end looks a lot like Src!! SH3 FG SH2 kinase Actin- binding Myristate
Oncogenic versions of Abelson FG Gag Abl v-abl SH3 FG SH2 kinase Actin- binding FG Bcr Bcr-Abl
What’s changed?? FG Gag Abl v-abl SH3 FG SH2 kinase Actin- binding FG Bcr Bcr-Abl
Remember this? Src is normally inactive due to intramolecular inhibition
Nagar et al. Cell 112:859 (2003) The structure of Abl reveals a novel mode of intramolecular inhibition
Harrison Cell 112, 737 (2003) 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
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
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??
Abelson has a twin brother SH3 FG SH2kinase Actin- binding FG 89%94%27%34% Abl 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
Focal adhesion proteins are phosphorylated by Abl (mediator of integrin signaling)
Abl phosphorylates regulators of the actin cytoskeleton
Of course it’s even more complicated Than that! Bradley and Koleske jcs.biologists.org/cgi/content/ full/122/19/3441/FIG3
Abl can also directly regulate Cytoskeletal events Using its C-terminal region to bundle actin filaments and Link them to microtubules
But does this all help us understand and treat leukemia? white blood cell Leukemic patientnormal person red blood cells hyperproliferation of white blood cells
BCR-Abl Cytoskeleton/ adhesion defects S G 2 M 1 G G0G0 Apoptosis Stem cell turnover Proliferation & differentiation BCR-Abl affects multiple cell functions Adapted from Jörgensen, 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
To understand this, we must start by learning more about the clinical progression of CML Chronic phase Median 5–6 years stabilization Accelerated phase Median duration 6–9 months Blast crisis Median survival 3–6 months Advanced phases 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+ 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) From Gleevec.com
Data of the Italian Cooperative Study group on Chronic Myeloid Leukemia. Blood 1998: –1548 Until recently interferon-alpha treatment Was the gold-standard in CML Even though its mechanism of action IS STILL NOT UNDERSTOOD IFN=interferon-alpha, CHT= conventional chemotherapy
STI571 Gleevec blocks the ATP binding site of the kinase domain
Abl’s Kinase Domain In complex With the inhibitor Gleevac Kuriyan lab website
0 Months Since Start of Treatment Chronic Phase CML Fraction of patients that responded Major cytogenetic response Complete cytogenetic response Data: Novartis Pharmaceuticals Corporation Gleevec™: in chronic phase CML
0 Months Since Start of Treatment Chronic Phase CML Fraction of patients that responded Major cytogenetic response Complete cytogenetic response Data: Novartis Pharmaceuticals Corporation This is an awesome drug! Most patients survive 10+ years
Drug was discontinued for adverse events in 1% of patients in chronic phase, 2% in accelerated phase, and 5% in blast crisis Of course there are side effects...
$40,000-50,000 per year!! And the cost.....
“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 Unfortunately, natural selection is a powerful process Science 331: March
A possible solution: a new generation of kinase inhibitors that Still inhibit Gleevec-resistant tumors Inject Luciferase-expressing tumor cells Science : aka Dasatinib
Dasatinib FDA approved for patients with relapses Inject Luciferase-expressing tumor cells NCI Cancer Bulletin October 5, 2006 aka Dasatinib
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 Phase II trials suggest Dasatinib effective in ALL patients with Gleevec-resistant tumors NCI Cancer Bulletin May
This is becoming a general approach Science 331: March
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
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
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 Research –3038, October 2002 With Gleevec treatment ~50% of patients respond Tumors shrink in size and disease symptoms are greatly reduced
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
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:
An alternate approach: broader spectrum inhibitors that hit multiple targets Sunitinib: targets Abl/PDGF Receptor, Src, and VEGF Receptor NCI Cancer Bulletin Oct FDA approved after Phase III clinical trial reveal efficacy in GIST patients whose Tumors are resistant to Gleevec George Demetri, MD