Cancer Biology - Molecular Targets and Therapies

A Diverse, Heterogenous Tumor Cell Landscape Requires a Diverse Approach

Stratification of Breast Tumor Genetics

  • Analysis of 70 bioinformatically determined prognostic genes in 295 primary breast cancer tumors from women 53 years and younger.
  • A Kaplan-Meier plot was generated from 151 breast cancer patients tracked for 10 years post-diagnosis.

Stratification of Diffuse Large B-Cell Lymphomas (DLBCLs)

DLBCLs are categorized into three subtypes:

  1. Primary Mediastinal B-Cell Lymphomas (PMBCLs)
  2. Germinal-Center B-Cell (GCB) DLBCLs
  3. Activated B-Cell-Like (ABC) DLBCLs

Molecularly Targeted Therapies

  • Recent therapies target molecular pathways:
    • Inhibition of oncogenic pathways.
    • Activation or rescue of tumor suppressor function.
    • Proliferation pathway targeting.
    • Differentiation pathway targeting.
    • Angiogenesis targeting.

Dysregulated Cell Division Regulators as Potential Targets

  • Tumor Suppressors: p53, PRB
  • Oncogenes: Ras, Myc

Considerations for Targeting Molecular Mechanisms in a Subset

  • Drugs are typically low molecular weight biochemical inhibitors.
  • Restoring function of large proteins like tumor suppressors is difficult logistically.
  • Targets must be "druggable", possessing catalytic clefts for strong and specific interactions with low molecular weight molecules.
  • Treatment should yield substantial therapeutic indices, affecting more tumor cells than normal cells.

Drawbacks to Molecular Targeting

  • Synthesis of stable therapeutic molecules is size-restricted.
  • Smaller size increases access to small spaces and more cancer cells.
  • Synthesized small molecules are more effective at inhibiting rather than enhancing biochemical functions.
  • Molecular targeting is therefore relegated to oncoproteins.

Potential Targets in the EGF Proliferative Inducing Molecular Pathway

  • PI3-Kinase

Effects of Shutting Down Expression of a Tumor Initiating Transgene

  • Permanent regression after shutdown of transgene is observed in various oncogenes:
    • H-ras: melanoma regressed
    • K-ras: lung adenocarcinoma regressed
    • bcr-abl: B-cell leukemia regressed
    • myc: T-cell lymphoma, acute myelogenous leukemia, and epidermal, mammary, and islet cell carcinomas regressed
    • fgf-7: lung epithelial hyperplasia regressed
    • SV40 large T: salivary gland hyperplasia regressed if transgene expressed < 4 months
    • Wnt1: mammary adenocarcinoma regressed

Molecular Target Selection Requirements

  • Structural vulnerability to interaction with other molecules.
  • A functional domain capable of strong and specific interaction with a small drug molecule.
  • Preference for proteins with measurable enzymatic function underlying oncogenic effect.

Tarceva as an Inhibitor of the EGFR Kinase Domain

  • Targets ATP-binding cavity within the N-terminal and C-terminal lobes.

Druggability of Transcription Factors

  • Potential structural targets:
    • Nuclear hormone receptors (e.g., estrogen) with ligand-binding domains.
    • Non-receptors with transactivation domains.
    • DNA binding domains.
    • Domains facilitating interactions with other regulatory proteins.

GTPase Inhibiting Dilemma

  • Inhibiting the catalytic activity of a GTPase will sustain an active state.

Targeting the Monomeric GTPase Ras

  • Non-small-cell lung carcinoma features smoker-induced Glycine to Cytosine mutation at the 12th amino acid position (G12C) residing in the catalytic cleft.
  • Molecular screening identified a targetable pocket interacting with Cytosine when this mutant Ras is bound to GDP.
  • A small molecule locks this mutant Ras in a GDP-bound state.

Ras G12C Mutant Inhibitors in Clinical Trials

  • Clinical trials involving solid tumors.
  • Compound 12 at 10μM10 \mu M induces apoptosis.

Kinases as Drug Targets

  • Kinases are still the most sought-after target.
  • Due to evolutionary conservation, acquiring a specific targeting drug is difficult.
  • Current approaches utilize high-throughput screening of small molecules.

Drug Candidate Testing

  • Drug candidates undergo various tests before human trials:
    • Effective pharmacokinetics.
    • Effect on cancer models in vitro.
    • Effect on animal models.

Dose-Response Curves

  • Show protein-specific inhibitory effects.

Drug Candidate Affect on Cancer Model in Cell Culture

  • Demonstrates substantial effect.

Phase I Trials

  • Primary goal: test toxicity.
  • Subsequent battery of tests:
    • Pharmacokinetics: Is the drug reaching the intended target?
    • Pharmacodynamics: Is the drug affecting the intended cancer cell?

Phase I Trial with Iresa

  • A small molecule inhibitor of EGFR tyrosine kinase.
  • IHC of a skin biopsy of a hair follicle shows drug entering skin cells and inhibiting kinase.

Phase I Trial with Iresa Dosage Study

  • Determination of therapeutic window: efficacy, minimum effective dose, maximum tolerated dose, and toxicity.

Phase II and III Trials

  • Larger groups are used in both phases.
  • Low toxicity and acceptable effects lead to larger group tests in Phase II.
  • Clear signs of efficacy in Phase II lead to Phase III.
  • Phase III trials enroll a much larger population of patients.
  • Clear improvement in patient outcome results in FDA licensing.
  • Phase IV trials may be instigated to test drugs over an extended period of time.

Phase III Trial of Gemcitabine for Pancreatic Cancer

  • A small increase in overall patient health will move drug into licensing by FDA.

A Short History of a Famous Molecular Target (BCR-Abl)

  • 1960: Cytologist in Philadelphia noted an abnormally short chromosome 22 associated with cases of Chronic Myelogenous Leukemia (CML).
  • Researchers in Chicago uncovered the chromosomal identity as a product of a reciprocal translocation between chromosome 9 and 22.
  • Genes involved in the translocation were identified in 1982: abl (Ableson mouse leukemia virus) and later BCR.
  • The BCR-Abl fusion protein was characterized as a constitutively active tyrosine kinase.

Distinct Oncogenic Molecular Profile: Chronic Myeloid Leukemia

  • 15-20% of all Leukemias.
  • bcr-abl Fusion Molecular abnormality.
  • Constitutively active tyrosine kinase ABL.
  • Fusion dependent.

Features of the BCR-Abl Fusion Protein

  • Domains activate Ras pathway, PI3 kinase–Akt/PKB pathway, Jak–STAT pathway, transcription factors such as Jun, Myc, and NF-κB.
  • The Tyrosine Kinase Domain played a key role in leukemogenesis.

BCR-Abl as a Druggable Target

  • 1990s research program commenced.
  • Developed a low molecular weight molecule capable of binding to and antagonizing the catalytic cleft of the BCR-Abl fusion gene.
  • Named imatinib mesylate and later commonly known as Gleevec.

Imatinib (Gleevec) Action

  • Imatinib binds to and nullifies the catalytic activity of tyrosine kinase domain.

Mechanism of Imatinib

  • Imatinib competitively binds to the bcr-abl site and inhibits phosphorylation of substrates.
  • Tumor cell cannot proliferate.
  • Remission observed in 96% of early-stage patients.

Blood Cell Analysis Before and After CML Gleevec Treatment

  • Many leukemic cells with large dark nuclei before treatment.
  • Normal granulocytes observed after treatment.

Successful Treatment Inspired an Onset of Targeting Therapeutics

  • Bcl-2 targeting
  • EGF receptor antagonists
  • Proteasome inhibitors

Cancer Vaccines

  • Moderna teamed up with Merck in 2017 to develop an RNA-based vaccine creating an immune response to 34 AI-selected proteins.

Cancer Vaccines Results

  • 68 percent of patients with stage 3 and 4 melanomas responded positively to the vaccine.

Treatment Considerations

  • Diagnosis
  • Tumor size, metastases, etc.
  • Phenotypical Variant
  • Molecular Variant
  • Cancer Properties
  • Age
  • Patient Profile

Cancer Stage Assignment

  • Plays critical role in determining the treatment.
  • Evaluation: Collection of clinical and pathological sampling, use of standardized metrics.
  • Stage Outcome: Bring all evidence to assign a stage, proceed with therapeutic approach.

TNM System of Initial Staging

  • T: Primary Tumor Information - Size and Spread
  • N: Lymph Node condition - Number of carcinogenic lymph nodes
  • M: Metastasis - Extent of distant organ tissue colonization
  • Once T, N, & M are assigned, an overall stage is prescribed for the cancer (Stage I – IV).

Breast Cancer Staging

  • Uses initial TNM in addition to a multi-level staging process.
  • Characteristic Consideration:
    • ER Status: Estrogen receptor expression
    • PR Status: Progesterone receptor expression
    • Her2: Her2 overexpression
    • Grade of Cancer: Level of cellular differentiation

Immediate Herceptin Treatment

  • Targets HER2 receptor, preventing dimerization and auto-phosphorylation, inhibiting downstream pathways such as PLC Gamma, P13k, RAS/MAPK, ultimately reducing proliferation and promoting apoptosis.

Breast Tissue Composition

  • Made up of ducts and glands, including lobes, ducts, and fatty tissue.

Carcinoma in Situ

  • Tis - Carcinoma in situ designation is pivotal in defining overall stage, particularly Invasive Ductal Carcinoma.

IB Stage Outcome Example

  • TNM:
    • Cancer larger than 5 cm (T3).
    • Spread to 4 to 9 lymph nodes under the arm or to any internal mammary lymph nodes (N2).
    • No spread to distant organs (M0).
  • Additional Criteria:
    • Grade 2
    • Her2 positive
    • ER positive
    • PR positive

Stages of Breast Cancer

  • 0: Abnormal cells are present but have not spread to nearby tissue.
  • I: Early Stage: Cancer has spread to other tissue in a small area.
  • II: Localized: Tumor is between 20-50mm and some lymph nodes are involved or a tumor larger than 50mm with no lymph nodes involved.
  • III: Regional Spread: Tumor is larger than 50mm, with more lymph nodes involved across a wider region. In some cases, there is no tumor present at all. Cancer may have spread to skin or chest wall.
  • IV: Distant Spread: Cancer has spread beyond the breast to other parts of the body.

Breast Cancer Staging and Treatment

  • Treatment options depend on the stage:
    • Stage I and IIA: Conservative or radical treatment.
    • Stage IIB and III: Conservative or radical treatment with/without radiotherapy.
    • Stage IV: Palliative treatment.