L20- Overcoming resistance and developing novel therapies

how was drug resistance discovered

• Chemotherapy was first introduced in the 1940s

1943 - nitrogen mustard, 1949 – methotrexate

• Patients often had an excellent initial response but on relapse did not respond to chemotherapy -

Acquired Drug Resistance

• Some tumours did not respond to chemotherapy from the outset - Intrinsic Drug Resistance

• Thus, the clinical evidence for drug resistance appeared at the dawn of chemotherapy

What are pharmacokinetics (PK) and pharmacodynamics (PD)?

• PK:

  • Absorption

  • Distribution

  • Metabolism

  • Elimination

• Drug influx and drug efflux

• PD:

  • Drug activation

  • Cellular damage → cell death

  • Drug inactivation

  • Alterations in drug target

  • Adaptive responses

  • Dysfunctional apoptosis

Why is the balance between PK and PD important?

• Balance determines the therapeutic window

  • Minimum effective dose

  • Maximum tolerated dose

• May differ across patients depending on PK and resistance profile

• Example: PK/PD relationship for Gleevec/Imatinib and Iressa (EGFRi)

What is the role of MDR transporters (ABC family) in drug resistance?

• ABC transporters use ATP → ADP to pump drug out of cell

• Leads to reduced intracellular drug levels

• MDR substrates include drugs across:

  • MDR1, MRP1, MXR (overlapping substrates)

• Effects:

  • MDR in tumour = lower drug level

  • Increasing systemic level = toxicity

  • Overall = loss of therapeutic index window

what hallmarks of cancer allows for heterogeneity drug resistance

• genome instability and mutation

Cancer cells employ mechanisms that evolved to enable all living things to thrive and evolve.

• Anticancer drugs are largely aimed at these survival mechanisms

what are the different natures of resistance to cancer therapies

What are the mechanisms of resistance to molecularly targeted agents?

• Generally refers to receptor tyrosine kinases (RTKs) and signal transduction pathways

• Resistance is more specific to that agent and predictable

• Network of signalling cascades means bypass is possible

• Mutation or upregulation of target

what is the relevance of RTK signalling and resistance

there are multiple ways to inginit it so therefore multiple ways to acquire resistance

Resistance to Molecularly targeted agents – EGFR inhibition and V600E B-raf

• resistance mechanism → T790M mutation → third generation EGFR-TKI

• EGFR TKI treatment → bypass pathway activation → c-Met, Her2, B-raf, ALK inhibitors

• cell state change → chemotherapy

• collateral sensitivities

Target Alteration – mutation (Prostate Cancer resistance): role of AR

• Androgen receptor (AR) is a lineage-specific oncogene – prostate cancer is driven by its function

• PSA marker is a good measure of AR driving signalling, hyperproliferation and growth of tumour

• Anti-androgens are an effective but all too often transient therapy in the fight against prostate cancer

Mechanisms of prostate cancer resistance (AR pathway)

• Prostate cancer resistance is largely dependent on the continued adaptive function of the AR

• AR amplifications

• AR mutations

• AR variants

• Alternative signalling:

  • Transcription: AR, c-Myb, EZH2, GR

  • Oncogenic signalling: PI3K/Akt, Autophagy, Stem cell phenotype, NE differentiation

• Novel anti-androgen agents:

  • Abiraterone Acetate

  • AR inhibitors: Enzalutamide, ARN-509

how can AR signalling be targeted

Mutation of AR ligand binding domain-  

Block formation of 2 helices forming and keep it blocked off- effective therapy  

• Mutations can arise in cells and under selective pressure of therapeutic. 

• Therapeutic then may act as an agonist to the receptor- the opposite of original mechanism

what does mutation of AR LBD cause

converts antagonist to agonist

Active drug cant fold over and allow the helices to fold in on it 

• In mutated drug, folding can occur, folds in on itself, may lead to promoted signalling from AR 

Targeting AR signalling to overcome resistance – what does this show?

• Prostate cancer driven by AR → therapies block DHT (abiraterone) or AR (enzalutamide/apalutamide)

• Resistance develops via AR LBD mutations (e.g. F876L)

• Mutations can make antagonists act as agonists

• New drugs (e.g. TRC-253) target mutated AR to overcome resistance

How does apoptosis occur and how do tumour cells evade it?

• DNA damage (not repaired) → triggers apoptosis

• p53 signals damage to BAK, BAX → activates caspases → cleavage of DNA/proteins → cell dies

Tumour cells evade apoptosis by:

• Loss of p53 (function)

• Downregulate BAX, BAK

• Upregulate antiapoptotic proteins (e.g. BCL2)

• Upregulate IAPs (e.g. Survivin)

How does the MDM2–p53 pathway regulate cell death and how do tumour cells evade it?

• p53 guards the genome

• MDM2 and p53 form an auto-regulatory loop to prevent overactivation of p53 (too much cell death)

• Tumour cells with elevated MDM2 enhance p53 degradation → resist cell death and continue to grow

• Active p53 arrests cell cycle (via p21)

How can targeting MDM2–p53 be used therapeutically?

• ASTX295 is a p53/MDM2 antagonist

• Maintains p53 in active state → induces tumour cell death

• ASTX295 developed by Astex Pharmaceuticals and CRUK Newcastle Drug Discovery Unit

• Clinical status: Phase 1/2 study in advanced solid tumours with wild-type p53

• Aim: determine safety, pharmacokinetics and preliminary activity

• No toxicity associated with maintaining active p53

what are the different adaptive resonses of DNA repair

what are the different DNA repair pathways

• Restore fidelity and integrity of DNA to prevent mutation/breakage/rearrangement – parental DNA is largely retained

• DNA repair can go wrong and genetic errors occur

• DNA repair machinery may be dysfunctional OR we can inactivate it

how can we target DNA repair to sensitise to cytotoxic therapy

• Tumours could be sensitised to DNA damage by targeting non-homologous end joining (NHEJ) a double strand break (DSB) repair pathway

• Loss of DNA-PK activity (NHEJ pathway) could result in failure to repair otherwise lethal DSBs

• Focus DNA damage with IR

how was DNA-PK discovered (in newcastle)

• First DNA-PK inhibitors developed at Newcastle

• Original series led to key probes NU7441 and recent NU5455

• Series from which AZD7648 was

developed was part of the Newcastle/AZ collaboration

• First patient dosed Oct 2019

what is base excision repair

• Damage recognition: DNA glycosylases (e.g. OGG1, NEIL) recognise and remove damaged base → creates abasic (AP) site

• AP site processing: APE1 cuts the DNA backbone at the AP site

• End processing: Enzymes (e.g. PNKP) prepare DNA ends for repair

• Gap filling: DNA polymerase (Pol β or Pol δ/ε) inserts correct nucleotide(s)

• Ligation: DNA ligase (LIG3 or LIG1) seals the strand

Key role of PARP:

• Detects single-strand breaks and recruits repair proteins (e.g. XRCC1)

• Inhibition → unrepaired SSBs → collapse into DSBs during replication

how was PARP discovered in newcastle

CRUK Newcastle DDU has driven Drug Discovery projects that have led to the

discovery of clinically relevant medicines and candidate drugs......

• Newcastle pioneered PARP1 inhibition

• First-in class PARP inhibitor

• Defined the BRCA mutation hypothesis

• First administration of a PARP inhibitor to a patient

• Approvals for use in ovarian cancer: FDA Dec 2016 and April 2018; EMA May 2018 and Jan 2019

• FDA Breakthrough Therapy Designation given for use in prostate cancer, Oct 2018

describe resistance to PARP inhibitor therapy

Multiple mechanisms exist for altering expression of the target of the drug or the signalling and repair mechanisms involved in its mechanism of action

Most striking, number one in the list is the reversion of BRCA mutation back to functional protein

What types of resistance occur in cancer immunotherapy?

• Small percentage respond, most have intrinsic resistance (A)

• Some initial responders develop acquired resistance (B–D)

• B: response turned off (e.g. checkpoint)

• C: progression (selection for resistance)

• D: progression – acquired resistance during immunotherapy

What are the main mechanisms of resistance to cancer immunotherapy?

• Lack of sufficient neoantigens or impaired neoantigen processing/presentation

• Insufficient generation of antitumor T-cells

• Impaired T-cell memory

How does DNA-PK inhibition enhance immunotherapy (IO)?

• DNA-PK inhibition (NU7441) → ↑ neoantigens (via DNA damage) and ↑ MHC-I presentation

• ↑ TCR repertoire → stronger T cell responses

• Enhances tumour sensitisation to immune checkpoint blockade (ICB)

• Combination therapy: NU7441 + STING ligand + CD40 agonist (NU-SL40) → improved tumour control

• Overall: increases T cell responses in immune resistant tumours

What is ATT001 and how does it work as a therapeutic?

• ATT001 = radioactive iodine / PARP inhibitor

• Local delivery → isolated damage to tumour

• Kills tumour while sparing patient

• Clinical example: brain tumour shrinks by half in therapy trial

What is virtual screening in drug discovery?

• Designing drugs using the desktop computer

• Uses powerful algorithms to enable pharmacologists to design drugs

Example of virtual screening in drug discovery

• Start from weak affinity (~30 µM)

• ~200,000 known compounds compared computationally

• HTS466284 identified as ATP-binding site inhibitor of TGF-β type I receptor kinase (~27 nM)

• Same inhibitor also found by conventional high-throughput screening

How is AI/virtual screening used in Newcastle DDU?

• Use structure-based drug discovery (SBDD)

• Bind fragments/compounds → evolve into more potent binders

• Computational power could further improve drug discovery capabilities

what is the role role of computational chemistry in AI in drug discovery

1020 molecules are available to screen but would take years to work through without advanced computation

AI to develop novel compounds further expands this chemical universe

How is AI used to predict response to therapy?

• Traditional approach: PSA, biopsy, grade, stage, standard of care, trial and error

• Determine molecular profile of patient’s tumour → choose drugs

• AI-enabled image analysis and disease stratification

• Predict most appropriate (AI-designed) drug for patient

How can AI be used to produce never-before-seen proteins?

• AI (e.g. AlphaFold/AlphaDesign) predicts protein structures and designs new sequences (de novo design)

• Models generate and optimise amino acid sequences for desired properties (fitness, stability, binding)

• Can create novel proteins (monomers, oligomers, binders) with specific functions

• Enabled by breakthroughs in structure prediction (Nobel Prize 2024 – DeepMind + David Baker)

• Applications: structural biology tools and potential therapeutics