Lecture 20 - Drug Resistance, Discovery, and Precision Medicine

What is the biggest challenge in cancer treatment?

Cancers developing resistance to chemotherapies

Why does genetic instability make cancers ‘moving targets’

Because the treatment will only work for a time until the cancer mutates, then another treatment has to be utilized

How many resistance mechanisms occur in a single tumor and at what stages can resistance occur?

Tumors have multiple resistance mechanisms that can occur at any stage in cancer cell

What is the evidence for the genetic basis of drug resistance?

1. daughter cells will inherent mutations if they are beneficial

2. resistance cells arise spontaneously at rate consistent with natural mutation

3. genetic instability/gene altering compounds increase resistance clones

4. drug’s target proteins often mutated

5. resistance is reversed if normal functioning gene is added back to cancer cell

List the 6 cellular mechanisms of drug resistance

1. impaired drug influx

2. enhances drug efflux (by ABC carriers)

3. Altered Drug Activation/Inactivation

4. Modification of Drug Target

5. Altered regulation of DNA repair

6. Altered control of cell death

what is the most common resistance mechanism of tumors

enhanced drug efflux by ABC carriers

What is impaired drug influx

Drug intake by diffusion, facilitated diffusion or receptor transport is decreased

What mechanisms do the cancer cells take to impair drug influx

gene mutations and polymorphisms

Give an example of a transport protein that cells mutate to prevent drug influx

Solute ligand carriers

• to prevent mutation, drugs must resemble natural ligands

What role do ATP binding cassettes (ABC) play in enhancing drug efflux

they actively transport chemo molecules across membrane out of cell

What three members are part of the ABC family?

1. P-glycoprotein

2. ABCG2

3. Multidrug Resistance Proteins (MRPs)

What drugs to P-glycoproteins remove from cell and how is their drug resistance activated?

Removes: small molecules - tyrosine kinase inhibitors and heterocyclic hydrophobic drugs in natural substances

Resistance Mechanism: gene upregulation and point mutations

What drugs to ABCG2 remove from cell and how is their drug resistance activated?

Removes: Similar drugs to P-glycoproteins (tyrosine kinase inhibitors)

Resistance mechanism: gene upregulation

What drugs to multidrug resistance proteins (MRPs) remove from cell and how is their drug resistance activated?

Removes: organic anions, Phase II metabolites (sulfate conjugates)

Resistance mechanism: gene upregulation, polymorphisms

How do cancer cells alter drug activation and inactivation

decrease expression of drug-activating enzymes and increase expression of drug-detoxifying enzymes

• turns off prodrug activating enzymes or turns on excretion enyzymes

What drugs are affected by altered enzyme activity

Anti-metabolites and platinum compounds

How do cancer cells modify the drug target?

Either increase expression of the target so the drug can’t bind to every single target (like anti-folate methotrexate), or mutate the drug binding site of the target gene (This is how BCR-ABL and Topoisomerase are inhibited)

How do cancer cells alter their regulation of DNA repair?

Mutate the cell cycle checkpoint proteins or increase efficiency of DNA repair

• Ex: cancer cells increase MGMT to repair alkylated DNA damage from akylating agents

How do cancer cells alter the control of their cell death?

Change the expression or function of BCL2 caspases or mutate p53

Primary apoptosis

Drug-induced apoptosis: drug is created to turn on apoptosis

Secondary Apoptosis

Damaged induced apoptosis: The cell is so badly damaged by chemo drug, that the cell will turn on the apoptosis pathway

• this is what most drugs do

Drug Resistance in Vivo

How does Cell-Cell contact contribute to drug resistance

cell-cell contact regulates the ability of the drug to access cells and affects cell’s proliferation rate

• the denser the cells are together, the drug can only diffuse a certain distance (like oxygen)

How does hypoxia contribute to drug resistance?

1. hypoxia increases proliferation by promoting survival of mutant p53

2. hypoxia increases ROS

3. Hypoxia decreases extracellular pH, making basic drugs useless

Why are stem cells less sensitive to drugs and often develop resistance easier?

They have enhanced DNA repair, increased oncogenic signaling, and increased expression of ABC transporters

What three questions do chemists/pharmacists need to ask when developing cancer drugs?

1. What is a good target?

2. What is a good endpoint to evaluate the drug?

3. How can a drug be kept working (resistance)?

How is drugability of a cancer target determined?

• Structural/functional comparison to allow for selectivity from normal cells

• frequency of altered structure/function in patients

• characteristics of a molecule that could block the active site

What physical processes are done to select a druggable target

DNA sequencing and gene expression to identify genes with different variants

Gene knockdown screening to see which cells express tumor properties when mutated

What types of libraries can be used to help select drug candiates

1. Chemical libaries: shows compounds and their structure

2. Natural Compound Libraries: shows physical/chemical properites of natural compounds

3. Known Drug libraries: shows drugs used for other purposes

You have selected a few drugs from the library, how do we screen potential drug candiates

add the drug to a cell and look for cell reaction

add the drug to purified proteins and see how they react

What is Structure-Based Computer Modeling / Rational Drug Design?

Using the structure of the target molecule to synthesize a drug to block it’s function

• need crystal structure of target and knowledge of potential binding sites

What criteria must new drug candiates be compatible with?

ADME criteria

• will it get in

• where will it go

• will it remain intact

• will it stay in

How do we test new drugs in preclinical studies?

Start with cell cultures to see efficacy and mechanism

then use xenograft mice to evaluate toxicity and understand biological complexity

transgenic mice are used to see mechanism of action in biological animal

start phase 1 trials

What is the population and purpose of Phase 1 drug trials?

Population: random healthy people who are recruited to try drug

Purpose: determine the max tolerated dose and test safety in humans

What is the population and purpose of Phase 2 drug trials?

Population: a small sample of people with the disease you are testing the drug for

Purpose: does the drug treat the disease I am interested in? (must use placebo)

What is the population and purpose of Phase 3 drug trials?

population: same subset of population in phase 2, just more of them

purpose: compare the efficacy of your drugs to other drugs already on the market

What is the population and purpose of Phase 4 drug trials?

Population: several hundred thousand people who have been prescribed medication

Purpose: drug is highly available to public, now we ask questions about interactions with other disease/therapies/pregnancy

Without regulations and quality control, what happened to herbal supplements?

Half of the vitamins on the mark don’t even contain what they are supposed to, and often they contain contaminants or fillers

What could potentially be true about herbal supplements?

Natural compounds are bioactive and likely to exert some effect on cancer cells.

• Proper controlled studies into these could yield drugs of huge benefit.

What is precision medicine?

An approach to disease treatment that takes into account an individual’s variability in genes, environment and lifestyle

Goal of precision medicine

implement the best available treatment strategy for every patient by measuring samples to determine patient’s health, disease risk, disease prognosis, best course of treatment, outcome, and side effects

What are companion diagnostic tests?

prescreening tests used to predict if a patient will respond to a specific targeted therapy

• Ex: HER2 IHC staining to see if patient can use trastuzumab

What questions are asked for precision medicine development, and their answers (don’t memorize, not worth it)

1. What measurements allow for individualization

   • need to use biomarkers

2. How can biomarkers be used to maximize their predictive value for the patient

   • use modeling

3. How can validated models be applied on a large scale?

   • use a bench-to-bedside transition

4. How can information be safely managed

   • quantum computing? Security measures

What is the role of predictive modeling in precision medicine

to use biomarkers to determine the optimal treatment and dosing strategy ahead of time

The first step in creating a model in precision medicine is to identify good biomarkers. Why is it important?

because any sort of biological parameter can be an indication of some physiological state, we need to choose a marker that can be easily obtained and reliable for the disease we are looking at

this marker should also differ in physiological state (like PSA and prostate cancer), and it should be sensitive and specific

Step 2: how do we determine that the biomarker relates to the disease?

functional genomics: use gene data to study the gene and protein expression in an organism

• correlate genotype with disease phenotype

Step 3: how do we determine biomarkers work together to cause the disease?

Systems biology: study interactions and pathways to see how they contribute to organism’s function

• relies heavily on computational modeling

We can use two types of models in predictive medicine? What are they?

1. Phenomenological (Descriptive) Models —> top down

2. Mechanistic (Explicactive) Models —> bottom up

Phenomenological (descriptive/top-down ) Model

Uses a bird’s eye view of a system without focusing on why it functions as it does

• I know my drug’s half-life is x, but what does it mean for my patient?

Mechanistic (explicative/bottom-up) Model

Focuses on causal relationships in a system

• why is my drug’s half-life x?

There are Six Challenges in using models and biomarkers, describe the first challenge “Cancer is not a monogenic disease”

mutations don’t always lead to functional changes in RNA and protein expression, they may be passenger mutations

Challenge 2: Scientific Understanding Lags Behind Data Acquisition Technologies

we can’t predict why passenger genes become drivers, and we can’t always identify driver mutations

Challenge 3: Across Patient Heterogeneity

Different patients will have different mutations within each type of cancer

Challenge 4: Within-Patient Heterogeneity

There are genetic differences between metastases and the primary tumor.

There are also different subclones within a single tumor!

overtime mutation pattern in cancers may change as well!

• makes finding best treatment a pain in the ass

Challenge 5: Confounding Variables

Is race a significant determinant of cancer risk or disease progression?

• no, socioeconomic factors are more important

Challenge 6: Ownership, Security, and Management of Data Problems

Data management problems: data storage takes a lot of memory, databases are incomplete and poorly integrated

Ownership: the patient doesn’t own their data, by legislation it belongs to AHS

Data Security: how do you balance data completeness with patient anonymity

Is precision medicine effective?

Yes, a meta-analysis of 570 studies showed a universal benefit for cancer patients treated with a personalized regimen