Lecture 14 – Cancer treatments and drug resistance

How do most chemotherapy drugs work?

Work by killing rapidly dividing cells

• This causes toxicity to highly replicative tissues

What are the three main types of chemotherapies?

Antimetabolites

Spindle inhibitors

Genotoxic drugs

How do antimetabolites work?

• Interfere with formation of key biomolecules 

  • Generally affect synthesis of DNA precursors  

• Some are analogues of normal metabolites 

  • E.g. methotrexate  

How do spindle inhibitors work?

• Interfere with cytoskeletal components necessary for cell division  

• Hinder microtubule formation needed for mitotic spindle  

  • Halts division during early mitosis  

How do genotoxic drugs work?

Damage DNA

What are the three ways Genotoxic drugs can damage DNA?

Alkylating agents

• Modify bases of DNA

Intercalating agents

• Wedge into spaces between bases and interfere with replication/transcription

Enzyme inhibitors 

• Inhibit key enzymes involved in DNA replication

  • E.g. topoisomerases 

What is dose limiting toxicity?

With cytotoxic drugs, the therapeutic index and therapeutic window are both very small  

• This limits the dose you are able to give to people

What is the therapeutic index?

Toxic dose/Therapeutic dose

What is the therapeutic window?

Range of useful doses

How can dose limiting toxicity be overcome?

• Repeated drug doses with recovery periods in between

  • Allows toxic effects to be treated 

  • Allows normal cells to recover 

  • May also recapture G0 cancer cells

• Appropriate support to the patient

  • E.g. blood platelet transfusion for treatments which induce bone marrow toxicity

What is complete remission?

Total removal of the disease

• No evidence of it

What is Long term remission?

5 years of no evidence of the disease

• Disease could return after those 5 years 

What is partial remission?

Tumour is still there and visible but it has gotten smaller and treatment is working somewhat

What is stable disease?

Following treatment the tumour hasn’t grown or shrunk. 

• Tumour has stayed stable

What is progressive disease?

Following treatment the tumour has continued to progress

What is relapse of disease?

Following remission, the tumour comes back at a later time

• Often associated with resistance to therapy 

What factors can affect a successful clinical response to treatment?

• Polymorphisms of genes

  • Patient may have more active enzymes which can clear the drug faster 

  • This will affect the delivery of the drug

• Intrinsic and acquired cancer cell factors

  • E.g. acquired resistance to a treatment

What are the two cellular mechanisms of therapy resistance?

• Tumour heterogeneity

• Microenvironment

What are the three molecular mechanisms of therapy resistance?

• Reduces access to drug target

• Alterations to target

• Altered cellular response

What is an example of heterogeneity in ALL?

Dormant Ki67 negative cells can predominate in chemoresistant leukaemia cells

• These are present at diagnosis and evade drug killing

What is an example of the microenvironment providing therapy resistance?

• Chronic lymphocytic leukaemia cells were treated with bruton’s tyrosine kinase inhibitor 

  • Ibrutinib 

• Was then shown that CLL cells were able to evade therapy by sheltering inside fibroblasts within the microenvironment 

What is an example of reduced access to target?

P-glycoprotein 

• An ATP-binding Cassette membrane transporter protein family member

• Use ATP to mediate drug transport out of the cell

• Cancer cells which express high levels of PGP can have multi-drug resistance as a result 

How does PKCε overexpression cause drug resistance?

PKCε overexpression drives drug resistance by upregulating PGP expression

• PGP then exports drugs out of the cell

How is TMP synthesised from dUMP?

Thymidylate synthetase (TS) which transfers a methyl group from methylate tetrahydrofolate to dUMP turning it into TMP 

What does TMP synthesis rely upon?

The folate cycle.

How does the folate cycle produce TMP?

• Thymidylate synthetase (TS) transfers a methyl group from methylate tetrahydrofolate (CH2-FH4) to dUMP turning it into TMP 

• This forms dihydrofolate (FH2) which is turned into tetrahydrofolate (FH4) by dihydrofolate reductase (DHFR) 

• This tetrahydrofolate is then turned back into methylate tetrahydrofolate (CH2-FH4) 

What is necessary for methotrexate inhibition of DHFR?

Methotrexate must be polyglutamated

• Increased half life from 12 minutes to 2 hours 

• This increases inhibition 

What is methotrexates mechanism of action?

• Acts as a substrate for the DHFR enzyme preventing formation of more tetrahydrofolate 

• Causes increased dUMP levels and decreased TMP levels 

  • Uracil is incorperated into DNA abberantly 

• Uracil-N-glycolase removes uracil causing double strand breaks 

  • This triggers apoptosis 

What is the role of Folypolyglutamate synthetase (FPGS)?

Performs polygutamation of molecules

• E.g. Methotrexate 

What is the role of Gamma glutamyl hydrolase (GGH)?

Undoes polyglutamation of molecules

• E.g. Methotrexate 

What is an example of changes of the drug target in cancer resistance?

Increased DHFR levels/Mutated DHFR providing resistance against Methotrexate

• Some mutated DHFR are able to allow substrate binding while preventing methotrexate binding despite how similar the molecules are 

What is the mechanism of action of Melphalan?

Nitrogen mustard alkylating agent

• Two DNA binding sites

• Cross-links DNA strands

What is one way Melphalan can act via alkylation?

• Attaches alkyl groups to guanine bases 

• DNA fragmented by repair enzymes 

• Remaining alkylated bases prevent DNA synthesis/transcription 

What is one way Melphalan can act via cross-linking?

• Formation of cross-links between DNA bases 

• Intra or Inter strand cross-links 

• Cross-linking prevents DNA synthesis or transcription 

  • Major cytotoxic lesion   

How can cancer cells develop Melphalan resistance?

Increase in repair mechanisms 

• Increased repair of DNA following melphalan-mediated cross-linking

Why can p53 mutations result in therapy resistance? 

• Cells are more likely to repair or tolerate DNA damage 

• Causes damaged cells to survive and harbour further mutations which affect responses of the tumour to treatment 

Chemotherapy can perhaps induce p53 mutations due to them being frequently seen at relapse but not diagnosis.

True or False.

True.

Perhaps chemotherapy has induced the p53 mutation 

• Although other studies have shown some subsets of cancer cells in tumour have mutations 

What strategies can be used to avoid resistance?

• Early detection 

• Debulking surgery 

• Maximum drug dose possible 

• Combination therapy 

• Target MDR cells 

  • Can evade multiple drug types  

How does drug resistance develop?

Random chance

• Often from pre-existing sub-clones 

• The larger the tumour, the greater the chance of drug resistant mutations 

Genetic instability

Mutagenic chemotherapies 

What can APR-246 be used for?

APR-246 can reactivate mutated p53

• Stabilizes the protein