CML and Tyrosine Kinase Inhibitors

CML and Tyrosine Kinase Inhibitors

  • CML (Chronic Myeloid/Myelogenous Leukemia)
    • Annual incidence: 1-2 cases per 100,000 people.
    • About 15% of all leukemias.
    • Malignant clonal disorder of hematopoietic stem cells.
    • Initial chronic phase: Increase in committed myeloid progenitors, leading to high numbers of mature granulocytes.
    • Progresses to accelerated phase: Fatal blast crisis with rapid proliferation of immature cells (blasts) and failure of blast differentiation.
    • Bone marrow: Massive increase in myeloid cells at the expense of other cell types.

Historical Treatment

  • Hydroxyurea: Inhibits synthesis of deoxyribonucleotides and G1 to S phase progression.
    • Significant side effects.

Chromosomal Translocation

  • Causative agent in many CML cases.
  • Reciprocal translocation between chromosome 9 and chromosome 22.
    • Modified chromosome 22 is termed the Philadelphia chromosome.
  • ABL (Abelson murine leukemia viral oncogene homolog 1)
    • ABL is located at the distal end of chromosome 9.
    • Part of chromosome 9 encoding ABL translocates to chromosome 22, replacing a part of chromosome 22.
    • Forms a fusion gene called BCR-ABL.
    • The remaining parts of chromosome 9 and 22 combine, resulting in a reciprocal translocation.

BCR-ABL Protein

  • ABL is a non-receptor tyrosine kinase.
  • BCR-ABL, the novel protein from translocation, forms homodimers.
  • Dimerization leads to autophosphorylation of tyrosine residues, causing constitutive activation of the tyrosine kinase.
  • Phosphorylates cytoskeletal proteins, regulating cell adhesion and migration.
  • Recruits adaptor proteins (e.g., GRB2) to activate signaling cascades.
  • Cancer cells are dependent on BCR-ABL activity for survival (oncogene addiction).
    • Without BCR-ABL activity, activated cellular pathways fail, leading to cancer cell death.

GLEEVAC (Imatinib)

  • Poster child of kinase-directed targeted therapy.
  • Also known as imatinib or STI-571.
  • Identified through high-throughput screening for molecules that inhibit Abl kinase activity.
  • ATP-competitive inhibitor: Competes with ATP to inhibit phosphorylation.
  • Binds to the ATP binding pocket of Abl kinase domain, preventing ATP from binding.
  • Rapid clinical trials and FDA approval in May 2001.
  • Clinical impact significantly increased overall survival.
    • Chemotherapy era: ~30% survival after 5-6 years.
    • GLEEVAC era: >90% survival after 5-6 years.

International Randomized Study of Interferon and STI-571 (GLEEVAC) - IRIS Study

  • Overall survival for newly diagnosed chronic phase CML patients treated with GLEEVAC at 5 years = 90%.
  • Mid-1970s five-year survival rate = 22%.

Current & Future Therapies

  • Small molecule inhibitors to block tyrosine kinases.
  • Next-generation sequencing for tumor profiling to develop targeted therapies.
  • Immunotherapy: Monoclonal antibodies to block immune system checkpoints.
  • CAR T cells: Genetically engineered T cells to target tumors.
  • Cell therapies of the innate immune system, including dendritic cells and NK cells.

Timeline of Tyrosine Kinase Inhibitors

  • Imatinib (GLEEVAC): Approved by FDA in 02/2001.
  • Early 2000s: Development of other inhibitors.
  • 2011: Trametinib, a covalent receptor tyrosine kinase inhibitor, approved.
  • 2013: Trametinib often combined with dabrafenib (BRAF inhibitor).
  • 2015: FDA approval of combo receptor tyrosine kinase inhibitors in metastatic melanoma.
  • 2018-2020: Receptor tyrosine kinase inhibitors being combined with immune checkpoints.

Receptor Tyrosine Kinases

  • Receptors on the cell surface that typically bind growth factors.
  • Contain kinase domains in the cytoplasmic region.
  • Ligand binding often causes homo- or heterodimerization.
  • Activate cell signaling pathways:
    • PI3K/Akt/mTOR: Cell growth, metabolism, survival.
    • Ras/MAPK: Metabolism, cell cycle, proliferation, differentiation, migration.
    • JAK/STAT: Signaling downstream of lymphokines, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor. Crucial for viral infection responses.
    • Phospholipase C, calcium calmodulin-dependent protein kinase, protein kinase C (CAMK, PKC) pathways.
  • Downstream effectors of multicellular processes during cancer progression.

Non-Receptor Tyrosine Kinases

  • Downstream of receptor tyrosine kinases.
  • JAK kinases: Involved in cytokine signaling.
  • ABL kinases: Important in CML.
  • Act downstream of many receptor tyrosine kinases, regulating cellular activation and proliferation.

Tyrosine Kinase Mutations in Cancer

  • Six general mechanisms:
    • Activating mutations in cytoplasmic regions of receptor tyrosine kinases or non-receptor tyrosine kinases.
    • Genetic amplifications (e.g., HER2).
    • Activating translocations (e.g., Philadelphia chromosome with BCR-ABL).
    • Antigen activation: Excessive signaling from antigen (e.g., B cell receptor).
    • Autocrine/paracrine activation: Excessive growth factor production.
    • Phosphatase inactivation: Disrupting normal inactive state.

Chemotherapy Overview

  • Used since the mid-1860s.
  • Arsenic trioxide: First chemotherapy used for CML (1865).
  • Nitrogen mustard: First chemotherapy approved drug (1950s).

Chemotherapy - Factors for theraputic approach

  • Choice of therapy depends on tumor location, grade/stage, and patient health.
  • Surgery and radiotherapy: Curative in ~30% of patients (most effective if tumor is benign and non-metastatic).
  • Chemotherapy: Used in 50% of patients, with ~15-20% being cured.
  • Adjuvant chemotherapy: Given after surgery/radiation to destroy micrometastases.
  • Neoadjuvant chemotherapy: Given before surgery/radiotherapy to reduce tumor volume.

Chemotherapy - Limitations

  • Non-selective for cancer cells; targets rapidly dividing normal cells.
  • Side effects due to targeting fast-dividing cells (e.g., lymphocytes, gut cells).
    • Digestive distress, reduced immune system.

Chemotherapy - Cellular Mechanisms

  • Activates the immune system (if not too harsh).
  • Acts directly on hematopoietic system, killing immune cells.
  • Targets different stages/types of cells: nucleic acids, microtubules, mitochondria.

Chemotherapy - Drug Types

  • Cytotoxic drugs: Target dividing cells (not normally cell cycle-phase specific).
    • Alkylating agents: Cell cycle non-specific.
    • Taxanes and vinca alkaloids: M phase.
    • PARP inhibitors and antimetabolites: S phase.

Chemotherapy - Catagories of drugs

  • Anti-metabolites: Gemcitabine and decitabine.
    • Resemble nucleobases, block enzymes for DNA synthesis or induce DNA damage.
  • Bifunctional alkylating agents: Cyclophosphamide.
    • Cause DNA cross-linking.
  • Anti-microtubule agents: Taxanes and vinca alkaloids.
    • Block cell division by impairing microtubule function.
  • Topoisomerase inhibitors: Irinotecan.
    • Block DNA unwinding.
  • Cytotoxic antibiotics: Doxorubicin.
    • Intercalate in DNA and generate free radicals.

Chemotherapy - Additional Drugs

  • Purine and pyrimidine antagonists.
    • Methotrexate inhibits purine ring and DTMP biosynthesis.
    • 5-FU inhibits thymidylate synthase (TDMP) synthesis.
    • Cytarabine inhibits DNA chain elongation.
  • Alkylating agents.
    • Alter DNA structure and function by cross-linking or fragmenting DNA.
    • Dactinomycin intercalates with DNA to disrupt DNA function.

Alkylating Agents

  • Transfer alkyl groups to nucleophilic sites on DNA bases.
  • Cause cross-linkage, abnormal base pairing, and DNA strand breakage.
  • Affect guanine.
  • Nitrogen mustard gas (WWII): Example of alkylating agent that depletes bone marrow cells.

Microtubules and Chemotherapy

  • Microtubules: Polymers of tubulin that provide structural support to cells.
  • Mitotic spindle composed of microtubules.
  • Paclitaxel blocks mitosis by stabilizing tubulin molecules, preventing depolymerization and halting the cell division cycle at metaphase.

Resistance to Chemotherapy

  • Commonly used in stage 3-4 cancers.
  • Initial response is often good, but relapse occurs.
  • Mechanisms of resistance:
    • Limiting chemotherapy drug accumulation by modifying membrane composition and reducing drug transporters.
    • Increasing expression of efflux pumps.
    • Drug inactivation.
    • Drug target modification/loss.
    • Reduced DNA damage/apoptosis due to increased DNA repair genes and pro-survival genes.

Resistance to GLEVAC

  • Resistance occurs due to mutation of the ABL kinase domain (50% of cases).
  • BCR-ABL gene amplification occurs.
  • Increased expression of other tyrosine kinases, for example LYN.
  • Altered expression of drug transporter proteins.
  • Mutation of the gatekeeper residue T315I.
    • Threonine to isoleucine substitution at position 315.
    • Isoleucine cannot hydrogen bond to the drug, reducing GLEEVEC binding.
    • Isoleucine is a bulkier residue and blocks drug access.

Ponatinib

  • Inhibits the mutant form of BCR-ABL (with isoleucine at 315).
  • Binds to and inhibits this mutant form.
  • Approved by the FDA and TGA for CML treatment.
  • Effective in patients who have failed prior tyrosine kinase inhibitor therapy.
  • Not influenced by the isoleucine mutation and works with high efficacy.
  • Further tyrosine kinase inhibitors are in development for other GLEEVAC-resistant mechanisms.

Side Effects of Receptor Tyrosine Kinase Inhibitors

  • Although effective, these drugs can cause significant side effects.
  • Side effects range from cerebral vascular effects to musculoskeletal headaches, etc.
  • Kinase inhibitors can have off-target effects on non-tumorous cells.
  • Imatinib also targets KIT and PDGFR (platelet-derived growth factor receptor).
  • BRAF inhibitors used in melanoma also have side effects on other pathway members.