ANTICANCER DRUGS II

Question 1

Chronic Myeloid Leukemia (CML):
1) How does CML arise
2) What is a fusion protein and what is its consequence
3) for this particular fusion protein for CML, what is its activity
4) What is the consequence of its activity

Answer 1

1) Arise from a translocation known as the Philadelphia chromosome
o This translocation leads to a BCR-ABL fusion protein
* ABL gene from chromosome 9
* BCR gene from chromosome 22
2) A fusion protein is:
* Gene + proto-oncogene –> uncontrolled proliferation

3) The BCR-ABL fusion protein has unusual tyrosine-kinase activity
* Tyrosine kinases action is usually tightly controlled
* BRC-ABL instead uncontrollably phosphorylates

4) Activates down-stream activators
* These down-stream activators are genes that inhibit apoptosis and initiate proliferation

Question 2

CML:
1) What is the first agent discovered to target and inhibit BCR-ABL tyrosine kinase activity
2) What does this agent do
3) How does this help CML and what is a downside
4) Will targeted therapy work for everyone?

Answer 2

1) Imatinib
2) Inhibits BCR-ABL by competitive binding at ATP-binding site
3) Increases survival rate in patient with CML
- Resistance can develop
4) Targeted therapy might not work for everyone as it can be dependent on certain oncogenes over others

Question 3

Epidermal Growth Factor Receptor (EGFR)
1) structurally similar to what and whats their importance?
2) upon binding of substrate (EGF) what is the sequence of events
3) What depends on this pathway
4) how do we inhibit this pathway (2 ways)

Answer 3

1) Structurally similar to Human Growth Factor Receptors (HERs)
* EGFRs play a critical role in regulating cell cycle progression and metastasis

2) Receptor dimerizes upon binding of substrate (EGF)
o Stimulation by ligands leads to receptor dimerization
* This activates the kinase domain
* Leads to phosphorylation of itself and downstream effectors gene expression
* This leads to downstream signalling

3) A lot of cancers are dependent on this type of pathway
4) Small molecules and monoclonal antibodies

Question 4

Epidermal Growth Factor Receptor (EGFR)
1) Name an example of a small molecule
2) Explain the pathway of small molecule (what it targets and the consequence)
3) What are pros and cons of small molecules

4) Name an example of a monoclonal antibody
5) Explain the pathway of the Ab (what it targets and the consequence)

Answer 4

1) Gefitinib
2) Inhibit kinase domain/ATP binding site
* Prevents gene expression through phosphorylation of downstream effectors

3) Easy to find kinase inhibitor
* Hard to find specific kinase inhibitor that doesn’t target other kinases

4) Cetuximab
5) Prevents ligand from binding
* Prevents dimerization of receptor –> inhibits downstream
* Ab doesn’t make it to the cytosol of the cell

Question 5

Tyrosine Kinase Inhibitors:
1) what are they used for and what do they target

Answer 5

1)
* Used for all sorts of cancer
* Targets all sorts of tyrosine kinases
o Not just one type
o So, a number of inhibitors made and produced for various cancer types

Question 6

Mechanism of Action of Agents Targeting HER2
1) What is HER2 and what is the pathway between HER2 and gene expression

Answer 6

1) Member of tyrosine kinase family
o Upon ligand binding, the receptor dimerizes
* This activates the kinase domain
o Leads to phosphorylation of itself and downstream effectors gene expression

Question 7

3 Ways to Target HER2:
1) What are the 3 ways

Answer 7

1)
- Single Epitope Monoclonal Antibodies
- Antibody-drug conjugates targeting HER2
- Bispecific antibodies

Question 8

Targeting HER2:
1) How do Single Epitope Monoclonal Antibodies work and their consequence
2) give 2 examples and their function
3) can they be used in combination

Answer 8

1) Inhibition of downstream signaling pathways
* Promotion of receptor internalization and/or degradation

2) EXAMPLES:
*Pertuzumab
* Inhibits receptor dimerization
* Trastuzumab
* Activates antibody dependent cellular cytotoxicity

3) Each can be used in combination with the other due to their different binding cites

Question 9

Targeting HER2:
1) How do Antibody-drug conjugates targeting HER2 work and their consequence
2) what are they used in combination with
2) Give 2 examples

Answer 9

1) Target delivery of highly cytotoxic agents
* A means to deliver the cytotoxic agents to HER2 positive tumor cells and to those around

2) Used in combination with small molecules inhibiting the tyrosine kinase domain for phosphorylation of downstream effectors

3) Trastuzumab + emtansine

Question 10

Targeting HER2:
1) How do bispecific antibodies work and what other antibodies do they mimic in binding
2) what is the structure of the bispecific Ab
3) Give 1 example

Answer 10

1) dual targeting of HER the same way trastuzumab and pertuzumab bind to HER
2) Each arm of Fab region of antibody binds a different site
3) ZW25

Question 11

Possible Mechanisms for Ab to Kill Cancer Cells:
1) what are the 3 mechanisms

Answer 11

1)
- CDC (Complement-Dependent Cytotoxicity)
- ADCC (Antibody-Dependent Cell Cytotoxicity)
- ADCP (Antibody-Dependent Cellular Phagocytosis)

Question 12

CDC (Complement-Dependent Cytotoxicity):
1) describe the mechanism

ADCC (Antibody-Dependent Cell Cytotoxicity)
1) describe the mechanism

ADCP (Antibody-Dependent Cellular Phagocytosis)
1) describe the mechanism

Answer 12

1) CDC (Complement-Dependent Cytotoxicity)
a. Fc portion binds to complement system
i. Leads to complement activation
ii. Lysis of cancer cell

2) ADCC (Antibody-Dependent Cell Cytotoxicity)
a. Activation of NK cells by antibody
b. Once the Ab and the Fc receptor on NK cells bind
i. Release of granzyme and perforin (cytotoxic agents) from NK cell
ii. Causes cytotoxicity of cancer cell

3) ADCP (Antibody-Dependent Cellular Phagocytosis)
a. Activation of macrophage by antibody
b. Once the Ab and the FC receptor on macrophage bind
i. Macrophage phagocytoses the cancer cell

Question 13

Antibody Drug Conjugates:
1) describe the antigen, antibody and drug necessary for a successful antibody drug conjugate
2) describe the optimized drug to Ab ratio

Answer 13

1)
Antigen
* High homogenous expression on tumor
* Low or no expression on healthy tissue

Antibody
* High affinity and avidity for tumor antigen
* Chimeric or humanizes to decrease immunogenicity and rejection
* Long half-life

Drug
* Must be potent
* Not all the antibodies will research the cancer cells therefore, drug must be potent so that the antibodies that do reach are effective in their attack

2)
* Can have more than one drug on antibody
* Too much of a drug causes too much of a change to the Fc portion of the antibody changing its properties
* The Ab will not be as stable
* The Ab will not circulate for as long in the body

Question 14

Timeline of Action of ADC:
1) state the general steps (6)

Answer 14

1)
1) ADC circulate as 3 components
2) As the ADC diffuse slowly towards target cells
3) Antibody engagement with tumor cells leads to payload/cytotoxic agent-independent antitumor activity via several mechanisms
4) Most ADCs are internalized and processes via antigen-dependent pathways
5) The payload is released from the endosome/lysosome
6) Membrane permeable payloads enter neighboring cells

Question 15

Timeline of Action of ADC:
ELABORATE ON STEP 1: ADC circulate as 3 components

ELABORATE ON STEP 2: As the ADC diffuse slowly towards target cells

ELABORATE ON STEP 3: Antibody engagement with tumor cells leads to payload/cytotoxic agent-independent antitumor activity via several mechanisms

Answer 15

1) Due to linker falling apart or poor manufacturing, there is likely
i. ADC
ii. Antibody
iii. Drug

2) ADC can release some of the payload into the tumor microenvironment or elsewhere in the body
b. This is why linker should be stable in circulation

3) Fc-mediated stimulation of immune cells effector function
i. NK release of cytotoxic agents
b. Disruption of receptor dimerization and/or function
c. Disruption of downstream functioning

Question 16

Timeline of Action of ADC:
ELABORATE ON STEP 4: Most ADCs are internalized and processes via antigen-dependent pathways

ELABORATE ON STEP 5: The payload is released from the endosome/lysosome

ELABORATE ON STEP 6: Membrane permeable payloads enter neighboring cells

Answer 16

1) Clathrin mediated endocytosis
i. Antibody is in clarthin coated vesicle
b. This turns into an endosome lysosome
i. Acidic or proteolytic cleavage of the ADC occurs within endosomes and/or lysosomes

2) Payload (cytotoxic drug) then takes its effect on cells cell death

3) Enter neighboring cells regardless of target expression (if they express HER2 or the receptor necessary)
i. Bystander effect
b. Can also kill these cells
i. Targets microtubule or induces DNA damage
ii. Therefore, can also enter nucleus once in the cell

Question 17

Hormonal Treatment:
1) Tumors arising from endocrine organs often express what
2) these tumors maintain what
3) therapies include what

Answer 17

1) Tumors arising from organs such as mammary or prostate often express steroid hormone receptors

2) These tumors maintain the same dependence on these hormones for growth + survival as the tissue of origin

3) So, therapies include
* Androgen deprivation therapy (prostate)
* Endocrine therapy (breast)

Question 18

Breast Cancer:
1) whats the normal context for estrogen signalling
2) What are the 3 anti-hormonal drug treatments
3) explain each and provide an example

Answer 18

1) Estradiol binds ER translocates into nucleus and dimerizes with another ER receptor-ligand complex
* This dimer binds HRE and activates gene expression
* This is key for proliferation
* Inhibiting this inhibits cancer formation

2)
1) Blockade of estrogen receptors with SERMs
*EX: Tamoxifen
* Binds ER
* Induces translocation and dimerization
* Gene expression is different due to differential binding
* Changes expression as in it doesn’t express the genes necessary for proliferation of breast tissue but strengthens bones, etc.

2) Inhibition of estrogen production 
	* EX: Aromatase inhibitors 

3) Selective estrogen receptor down regulators (SERDs)
*EX: Fulvestrant
	* Binds ER 
	* Prevents dimerization and induces degradation 
	* No binding to HRE

Question 19

Aromatase Inhibitors:
1) post menopausal produce high or low estrogen and why
2) Where does the estrogen arise from (precursors)
3) how are the precursors converted into estrogen and where
4) what happens if you block aromatase

Answer 19

1) Postmenopausal women continue to produce low levels of estrogen due to non-functional uterus

2) This estrogen is derived from:
* Adrenal precursors
* Testosterone
* Androstenedione

3) These precursors are converted into estrogen by aromatase activity
* Converted in peripheral tissue and cancer cells

4) By blocking aromatase depletion of hormonal dependence of cancer cells

Question 20

SERMs:
1) give an example
2) what are the metabolites of this drug, how are they made and what are they responsible for
3) mechanism of action of the metabolites

Answer 20

1) Tamoxifen
2) 4-hydroxytamoxifen endoxifen
* CYP450s
* These metabolites are mostly responsible for the activity

3) Hydroxyl group on metabolites mimics estradiol
* Tamoxifen when bound to ER prevents the closing back of the signaling loop
* Induces different types of signaling inside the cell
* The signaling changes depending on tissues
* In breast = complete inhibition of cellular proliferation

Question 21

SERDs:
1) give an example
2) Mechanism of action
3) Route of administration

Answer 21

1) Fulvestrant

2) Pure ER antagonist
- When bound to ER, receptor dimerization is sterically hindered
*ER degradation is induced

3) Low oral bioavailability
* Administered as monthly IM injection

Question 22

Subtypes of Breast Cancer:
1) what are the 4 subtypes of breast cancer
2) describe what to target for each subtype of breast cancer

Answer 22

1)
* HR+/HER-
* HR-/HER2-
* HR+/HER2+
* HR-/HER+

2)
- HR+/HER-
* Dependent on hormone receptor (HR) because negative for HER2

• HR-/HER2-
  
  • Cannot target hormone receptor or HER2
• HR+/HER2+
  
  • Positive for both receptors gives lots of therapeutic options

Question 23

Breast Cancer: Endocrine Combination Therapy
1) what 2 subtypes of breast cancer can combination therapy be used for
2) What kind each therapy involves
3)

Answer 23

1) HR+/HER- breast cancer and HER2+ breast cancer

2) For HR+/HER- breast cancer
* Combination therapy is given and involves
* AI or SERD
* CDK4/6 inhibitors
* Pre-menopausal women –> ovarian suppression/ablation + combination therapy

For HER2+ breast cancer
* Chemotherapy
* Trastuzumab with or without pertuzumab (anti-HER2 antibodies)

Question 24

CDK4/6:
1) what does CDK4/6 promote
2) How does it do so
3) Those resistant to endocrine replacement therapy remain dependent on CDK4/6 do to what
4) what else is expressed with CDK4/6 to drive breast cancer proliferation
5) Whats an approved CDK4/6 inhibitor

Answer 24

1) CDK4/6 promotes cell-cycle entry

2) CDK4/6 does so by inactivating the tumor suppressor Rb by phosphorylation
* Leads to initiate transition from the G1 phase to the S phase
* Cell growth and proliferation

3) Those resistant to endocrine therapy remain dependent on cyclin D1-CDK4/6 to promote proliferation

4) Cyclin-D1 is expressed with CDK4/6 to drive breast cancer proliferation

5) Approved CDK4/6 inhibitors is Palbociclib

Question 25

Triple Negative Breast Cancer:
1) lack expression in what
2) behave in what way compared to other types of breast cancer
3) Because of the way it acts, whats the therapy

Answer 25

1) breast cancers that lack expression of ER, PR and HER2

2) TNBC tends to behave more aggressively than other types of breast cancer

3) Because of this, there is no approved targeted treatments available
* Common regimen of chemotherapy
* Anticancer antibiotic
* Alkylating agent
* Tubulin stabilizer (of polymerized tubulin)

Question 26

Triple Negative Breast Cancer:
1) what is the treatment if the TNBC expresses PD-L1
2) what is the treatment if the TNBC expresses a mutated BCRA

Answer 26

1) Immunotherapy (in combination with chemotherapy) is available
2) PARP inhibitors

Question 27

BCRA Gene:
1) What is BCRA gene involved in

Answer 27

1)
* Involved in DNA repair
* Mutation in BCRA makes you more susceptible to breast/ovarian cancer

Question 28

Synthetic Lethality: PARP + BCRA1
1) What are PARPs
2) What do BCRA mutations do
3) what is used in the treatment of cancer patients with BCRA mutations and what kind of treatment is is
4) Describe what happens when a patient has no mutation in BCRA but takes PARP inhibitors
5) Describe what happens when a patient has mutation in BCRA but takes PARP inhibitors

Answer 28

1) PARPs are enzymes that play part in single-strand DNA break repair

2) BCRA1 or BCRA2 mutation
* Sensitizes the cell to inhibition of PARP activity
* Chromosomal instability
* Cell cycle arrest
* Subsequent apoptosis

3) PARP inhibitors are used in the treatment of cancer in patients with BCRA1 or BCRA2 mutations

4) Without BCRA1/BCRA2 mutation + PARP inhibitors
* Single strand break unrepaired by PARP
* DNA repair by homologous recombination

5) With BCRA1/BCRA2 mutation + PARP inhibitors
* Overwhelm the cell with DNA damage to the point where it reaches cell death
* Leads to cancer in BCRA cancer cells only

Question 29

Immune Checkpoints:
1) What are the mechanisms inhibited in order to re-sensitize cancer cell to immune system

Answer 29

1) CTLA-4 and PD-L1

Question 30

Advanced Melanoma (STAGE III-IV)
1) Whats the difference between stage iii and iv

Answer 30

1)
- Stage III –> cancer reached lymph nodes
- Stage IV –> metastasized and reached peripheral tissue

Question 31

Immune Checkpoint Inhibitors:
1) what happens immune cells are attacking cells
2) What proteins involved in the down regulation of the immune system

Answer 31

1) When immune cells are attacking cells, there are pathways to ensure control of the immune cells/downregulate immune response
2) CTLA-4 + PD-L1

Question 32

ANTI CTLA-4 Ab
1) Where is CTLA-4 found
2) what normally happens with CTLA-4
3) What do ANTI CTLA-4 Ab do

Answer 32

1) on T-cells of lymph node
2) Once T-cell receptor (TCR) recognizes antigen on MHC complex of APC
* Normally, B7 on cell (dendritic) binds CD28 on T-cell
* CTLA-4 binds to B7 on dendritic cell

3) Anti-CTLA-4 antibodies bind to CTLA-4 on the T-cell and inactivates CTLA-4 and the inactivation of the immune system

Question 33

ANTI-PD-L1 Ab
1) where is PD-L1 found
2) what normally happens with PD-L1
3) What do ANTI-PD-L1 Ab do

Answer 33

1) on cancer cells
2) When the T-cell recognizes antigen (PD-L1) on cancer cell
* Eventually leads to interferon production
* Cells senses attack from T-cell/immune system
* Interferon production upregulation of PD-L1 to relax the attack

3) Inhibiting PD-L1 overactive immune cell
* T-cell binds to cancer cell through TCR and MHC

Question 34

1) What can cause the activation of the immune system
2) why can the immune system attack cancer cells
3) do checkpoint inhibitors work in breast cancer?
4) what are checkpoint inhibitors used with in breast cancer
5) what disease requires immune checkpoint inhibitors

Answer 34

1) Sun exposure or smoke
2) cancer cells are immunogenic –> even if self cells are mutated, the cells are recognized as non-self
3) In breast cancer, checkpoint inhibitors don’t work well

4) They are used in combination with:
* Chemotherapy (mutation causing)
* Antibodies

5) Without checkpoint inhibitors, survival rate is lower in TNBC
* Finding the right combination (especially with cancers that are not immunogenic) with checkpoint inhibitors makes the cancer more treatable

Question 35

The Gut Microbiota and Cancer Immunotherapy:
1) how have cancers evolved to avoid the immune system
2) how is this dealt with
3) Why do some patients respond to their and others dont
4) describe the experiment
5) describe the results of the expeiment
6) what is the general conclusion

Answer 35

1) interactions with the PD1 ligand or CTLA-4 that inhibit immune checkpoints

2) Cancer immunotherapy: If PD-1 ligand or CTLA-4 are inhibited then tumors can be resensitized to the immune system

3) Impact of the microbiota on success and failure of immunotherapy
♣ Gut microbiota differ in these 2 patients

4) EXPERIMENT: take microbiome of 2 patients (one that responded to cancer therapy and one that did not)
* Give to mice
* Give mice cancer

5) RESULTS:
* In mice with stool from non-responder –> cancer
* In mice with stool from responder –> no cancer/tumor growth

6) Presumably, the microbiota is interacting with immune system in a favorable way
o By adding this bacteria to mice that developed cancer, because they received stool from non-responders, AND immune checkpoint inhibitors can re-sensitize mice to immunotherapy

Question 36

How can we shape the microbiome of a patient to potentially cure a disease:
1) Give 2 options
2) what are prebiotics
3) what prebiotic was found to work well in cancer immuno therapy in mice transplanted with non-responder fecal matter
4) explain the experimental results
5) what was it more beneficial in combination with

Answer 36

1)
a) Fecal microbiota transplant (Take microbiome of donor (patient that responded well to treatment) and transplant it into patient before the treatment)

b) Prebiotic approaches

2) Molecules that promote growth of some bacteria in gut over others
i. Ensuring that you give the bacteria food that they strive off to create a positive phenotypic response in patients undergoing treatment afterwards.

3) Camu-camu berry

4)
Found to shift gut microbiota composition
- When this was done to mice treated with antibiotics, nothing happened – no response to camu camu

5) More beneficial in combination with immune checkpoint inhibitors