Cancer therapy strategies - From conventional to personalized medicine

Question 1

Hematopoietic stem cell transplantation

Answer 1

• Performed for patients with certain cancers of the blood or bone marrow, such as multiple myeloma or leukemia
• Autologous: transplantation in which stem cells are removed from a person, stored, and later given back to that same person
• Allogeneic: Allogeneic HSCT involves two people: the (healthy) donor and the (patient) recipient
• Recipient’s immune system is usually destroyed with radiation or chemotherapy before the transplantation

1) Stem cells removed from donor
2) patient receives treatment to destroy blood forming cells
3) patient receives stem cells

Question 2

Stem cell therapy

Answer 2

• replace the patient’s blood and immune system with a healthy one from a donor
• Graft versus tumor effect: newly generated immune cells attack residual cancer cells
• Recovery can take up to 1 year
• Autologous: no graft versus tumor effect; higher risk of relapse
• Allogenic: higher risk for graft versus host disease
• Main side effects:
• Infections
• Graft versus host disease (GVHD)

Question 3

Graft versus host disease

Answer 3

• Inflammatory mechanisms mediated by donor lymphocytes infused into the recipient
• Tissues produce molecules such as proinflammatory cytokines and chemokines
• Increase in expression of key receptors on antigen-presenting cells
• Enhancing cross-presentation of polypeptide proteins to the donor immune cells

1) Host APC activation: TNF alpha, IL1, LPS
2) Donor T-cell activation: Host APC, Donor T cell, Th1, Treg
3) Cellular and inflammatory effectors: CD4 CTL, CD8 CTL, TNF alpha, IL1 -> targeted cell apoptosis

Question 4

Acute GVHD symptoms

Answer 4

• Skin: Maculopapular skin rash
• Upper gastrointestinal tract: Nausea, anorexia, or both, and positive histological findings
• Lower gastrointestinal tract: Watery diarrhoea, severe abdominal pain, bloody diarrhoea ileus (after exclusion of infectious causes)
• Liver: cholestatic hyperbilirubinaemia

Question 5

Chronic GVHD symptoms

Answer 5

• Skin: Dyspigmentation, new-onset alopecia, poikiloderma, Iichen plans-like eruptions, or sclerotic features
• Nails: Nail dystrophy or loss
• Mouth: Xerostomia, ulcers, lichen-type features, restrictions of mouth opening from sclerosis
• Eyes: Dry eyes, sick syndrome, cicatricial conjunctivitis
• Muscle, fascia, joints: Faciliti, myositis, or joint stiffness from contractures
• Female genitilia: vaginal sclerosis, ulcerations
• gastrointestinal tract: Anorexia, weight loss, esophageal web or strictures
• liver: Jaundice, transaminitis
• lungs: restrictive or obstructive defects on pulmonary function tests, bronchiolitis obliterates, pleural effusions
• kidneys: nephrotic syndrome (rare)
• heart: pericarditis
• marrow: thrombocytopenia, anaemia, neutropenia

Question 6

Human leukocyte antigen (HLA) profiling

Answer 6

• HLA molecules control the immune response through recognition of ‘self’ and ‘non-self‘
• HLA molecules present antigens to the T Lymphocytes and initiate specific immune responses encoded by the major histocompatibility complex (MHC)
• Degree of HLA matching is critical in determining the probability of GVHD
• To minimize these alloresponses, the HLA class I (A, B, C) and class II (DRB1) types of the donor and recipient are matched as closely as possible
• Methods: Serology, molecular assays (sequencing)

Class I HLA: almost all nucleated cells of the body at various densities

Class II HLA: mainly expressed on haemopoitiec cells (B cells, dendritic cells and monocytes); but also inducible

Question 7

Hormone therapy

Answer 7

Involves the manipulation of the endocrine system through
- drugs which inhibit the production or activity of steroid hormones
- exogenous or external administration of specific steroid hormones
Hormonal therapy is used for several types of cancers derived from hormonally responsive tissues, including the breast, prostate, endometrium, and adrenal cortex

• In postmenopausal women, approx. 75% of breast tumors are hormone sensitive
• Express the estrogen receptor and/or progesterone receptor
• Stimulated to grow in the presence of estrogen

Question 8

Hormone therapy (example breast cancer)

Answer 8

• Estrogen binds to the estrogen receptor
• Receptors dimerize and translocate to the nucleus
• The complex binds to specific DNA sequences (estrogen response elements) in target genes
• Activating function domains (AF1 and AF2) on the estrogen receptor recruit protein cofactors
• Transcription and expression of the target genes
• Increased cell division and tumor progression

Question 9

Hormone receptor antagonists

Answer 9

Hormone receptor antagonists (e.g. Tamoxifen):
* Selective estrogen receptor modulators (SERM) and degraders (SERD)
* Bind to the normal receptor for a given hormone and prevent its activation
* Premenopausal and Postmenopausal

Question 10

Tamoxifen

Answer 10

• Tamoxifen: first-line treatment for nearly all pre- menopausal women with ER+ breast cancer
• breast tissue, tamoxifen acts as an ER-antagonist
  -> transcription of ERE-targeted genes is inhibited
• Partial agonists: increase estrogen receptor signaling in some tissues (e.g. endometrium)
  -> agonistic and antagonistic effects
  -> full agonist and partial agonist are present: partial agonist will act as an antagonist, competing with the full agonist for the same receptor and thereby reducing the ability of the full agonist to produce its maximum effect
  -> only partial agonist is present: partial efficacy at the receptor (reduced in comparison to a full agonist)

Question 11

Selective estrogen receptor modulators (SERMs)

Answer 11

• Selective estrogen-response modulator and degraders for the treatment of breast cancer
  a) Estradiol
  Receptor dimerization -> Nuclear localization of fully active ER to ERE -> AF1 and AF2 recruit coactivators -> fully activated transcription
  b) Tamoxifen
  Receptor dimerization -> nuclear localization of partially active ER to ERE -> AF1 recruit coactivators -> partially activated transcription
  c) Fulvestrant
  attenuated dimerization or accelerated receptor degradation -> AF1 and AF2 INACTIVE, reduced nuclear localization of inactive ER to ERE or degradation -> no coactivator recruitment -> no transcription

Question 12

Inhibitors of hormone synthesis

Answer 12

Hormone receptor antagonists (e.g. Tamoxifen):
* Selective estrogen receptor modulators (SERM) and degraders (SERD)
* Bind to the normal receptor for a given hormone and prevent its activation
* Premenopausal and Postmenopausal

Inhibitors of hormone synthesis (e.g. Exemestane):
* aromatase inhibitors à inhibits the aromatization step in the synthesis of estrogen; mainly treatment postmenopausal
* Influencing secretion of gonadotropins (GnRH analogs); pre- and postmenopausal

• Estradiol, like other steroid hormones, is derived from cholesterol with androstenedione as the key intermediate
• Aromatase (CYP19A1) is responsible for a key step in the biosynthesis of estrogens (aromatizationàconverts enone ring of androgen precursors to a phenol)
• Aromatase inhibitors: form permanent and deactivating bond with the aromatase or inhibit the synthesis of estrogen via reversible competition

Question 13

Aromatase inhibitors (AI)

Answer 13

• Lowering estrogen production at the site of cancer (i.e. the adipose tissue of the breast) with AI is effective for ER+ breast cancers in postmenopausal women
• Prior to menopause: decrease in estrogen activates the hypothalamus and pituitary axis to increase gonadotropin secretion -> heightened gonadotropin levels stimulate androgen production in the ovary and up regulate aromatase promoter -> increase in total estrogen

Question 14

GnRH analogs

Answer 14

• Analogs of gonadotropin-releasing hormone (GnRH) completely suppress
  -> production of estrogen and progesterone in females
  -> production of testosterone in males

GnRH antagonists:
- Competitive inhibitors of pituitary GnRH receptors
- Blockade directly suppresses secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) and thereby reduces testosterone

GnRH agonists:
* Stimulate GnRH receptors in cells of the anterior pituitary gland
* Constant exposure leads to downregulation of pituitary receptors, inhibition of LH and FSH release, and a concurrent reduction in testosterone

Question 15

Personalized medicine

Answer 15

• Current medicine: one treatment fits all
• Future medicine: more personalized Diagnostics
• Traditionally, treatment strategies have been decided by where cancers arise in the body and how tumors look under the microscope
• Cancers developing in the same part of the body can vary greatly in their genetic make-up
• But also: cancers developing in different parts of the body can share a similar genetic make-up (e.g. vemurafenib (Zelboraf) is effective in melanomas with specific mutation in BRAF gene; hairy-cell leukemia and some forms of thyroid cancer)

The challenges facing personalized medicine:
1. Identifying and understanding the “drivers” of cancer growth
2. identifying drugs that can target these drivers
3. Finding new ways to overcome drug resistance
4. Designing better clinical trials to test new treatments
5. Testing the use of personalized medicine in the clinic

Question 16

Design of personalized clinical trials

Answer 16

• Modern clinical trial strategies (Umbrella or basket strategy) include gene expression profiling into the decision of treatment type

Umbrella clinical trials:
* Multiple drugs tested against multiple genetic mutations within a single cancer type

Basket clinical trials:
* A handful (typically one or two) of drugs tested against a handful of genetic mutations across multiple cancer types

Question 17

How to choose a drug in personalized medicine

Answer 17

Lab tests to assist in treatment decision-making:
1. A patient is enrolled in a clinical trial
2. a) Circulating tumour cells are taken from the patient and grown in the lab
b) Tumour pieces are grown in a mouse (avatar)
c) Tumour pieces are grown in 3D in a dish
3. Different drugs are tested on the cancer cells
4. Drugs that kill the cancer cells are prioritized for testing on the patient

• Circulating tumor cells: Limited cell number
• Mouse avatar: Time consuming, animal testing
• 3D cell culture: growth conditions have to be known

Question 18

Tumor immune surveillance

Answer 18

1. Release of cancer cell antigens (cancer cell death)
2. Cancer antigen presentation (dendritic cells/APCs)
3. Priming and activation (APCs and T cells) -> IMMUNE CHECKPOINT
4. Trafficking of T cells to tumors (CTLs)
5. Infiltration of T cells into tumors (CTLs, endothelial cells)
6. Recognition of cancer cells by T cells (CTLs, cancer cells) -> IMMUNE CHECKPOINT
7. Killing of cancer cells (Immune and cancer cells)

Question 19

Cancer immunotherapy

Answer 19

• Cancer immunotherapy is the artificial stimulation of the immune system to treat cancer, improving on the immune system’s natural ability to fight the disease
• Immunotherapy can be categorized:
• Antibody-Dependent (ADCC)
• BiTE/Bispecofoc Antibody
• Checkpoint Inhibitor
• Genetically Engineered T cell (e.g. CAR-T)
• Macrophage
• NK-Cell
• Activated T cell
• Oncolytic Virus

Question 20

Cancer immunotherapy

Answer 20

• immune system maintains homeostasis in order to get rid of pathogens, yet not elicit autoimmune diseases
• 2018, James P. Allison and Tasuku Honjo received the Nobel Prize in Physiology or Medicine for their discovery of cancer therapy by inhibition of negative immune regulation
• Dendritic cell + T cell + CTLA-4 + Inhibitor = The CTLA-4 checkpoint protein prevents dendritic cells from priming T cells to recognize tumours. Inhibitor drugs block the checkpoint.
• Cancer cell + PD-1 = The PD-1 checkpoint protein prevents T cells from attacking cancer cells. The inhibitor allows T cells to act.

Question 21

Immune checkpoint inhibitors

Answer 21

• Immune checkpoint inhibitors are negative regulatory molecules often found on the surface of T cells
• Regulate responses from self-proteins and protect against autoimmune activity
• Inhibitory immune checkpoint pathways that have drugs approved for use in cancer are cytotoxic T-lymphocyte antigen-4 (CTLA-4) -> Ipilimumab, tremelimumab and programmed death receptor-1 (PD-1) -> Nivolumab, pembrolizumab, as well as the ligand of this receptor (PD-L1) -> Atezolizumab, durvalumab, avelumab
• Adverse effects: autoimmune-related toxicities

Question 22

CAR-T cell therapy

Answer 22

• T cells are isolated, genetically engineered to express chimeric antigen receptors (CAR) directed toward antigens on patient’s tumor cells and then infused into the patient
• CAR-modified T cells can be engineered to target virtually any tumor associated antigen
• Antigens must be highly expressed on the majority of cancer cells, but largely absent on normal tissues

Approved drugs: Tisagenlecleucel and Axicabtagene ciloleucel (2017)
March 2019 : ca. 350 clinical trials
Predominantly blood cancers
Frequent target: CD19
Clinical remission rate: 90 %
Long term survival much lower
-> Antigen escape

Common side effects:
- Cytokine release syndrome
- Neurological toxicity

1) Make CAR T cells in the lab
2) Grow millions of CAR T cells
3) Infuse CAR T cells into patient
4) CAR T cells bind to cancer cells and kill them
5) Remove blood from patient to get T cells

Question 23

Cytokine release syndrome

Answer 23

• Activation of T cells or lysis of immune cells induces release of interferon IFN-γ or TNF-α
• Activation of macrophages, dendritic cells, other immune cells and endothelial cells
• Production of large amounts of IL-6, which in a positive feedback loop manner activates T cells and other immune cells leading to a cytokine storm

Grade 1 = Fever, constitutional symptoms
Grade 2 = Hypotension responding to fluids/low dose vasopressors, Grade 2 organ toxicities
Grade 3 = Shock requiring high dose/multiple vasopressors, Hypoxia requiring >= 40 % FIO2, Grade 3 organ toxicities, grade 4 transaminases
Grade 4 = mechanical ventilation, Grade 4 organ toxicities (excl. transaminases)

Question 24

Sipuleucel-T (APC8015)

Answer 24

• Genetically modified antigen presenting cells (APCs) for treatment of prostate cancer
• incubated with recombinant fusion protein antigen containing PAP (prostatic acid phosphatase) and GM CSF (granulocyte macrophage colony stimulating factor)

Sipuleucel-T treatment includes three courses at two week intervals
Costs: ca. 100.000 / patient
Increase in overall survival rate: 4.1 month
2013: accepted in EU
2015: withdrawal (request by company)

Question 25

Cancer vaccine

Answer 25

• Fundamental principle of mRNA vaccines: ’hijacking’ the cell’s translational machinery to produce pharmacological active proteins Crucial intermediate step in contrast to peptide vaccines: translation
• Minimal structural requirements: 5’ cap, poly(a) tail, open reading frame coding for the protein of interest, start codon, 5’ and 3’ untranslated regions (UTR)
  1) 5’ cap: The efficiency of capping and the cap structure impact innate sensing and protein production.
  2) UTR’s: translational efficiency is regulated by their length, structures and regulatory elements.
  3) CDS (Coding sequence): Modification of sequence, such as codon optimization, have contributed to improved expression.
  4) 3’ Poly-A-tail: properties such as length, are important for translation and protection of the mRNA molecule
  5) Purity: removal of impurities reduces innate sensing promoting expression.
• Barriers that need to be overcome in order for this specialized molecule to perform as intended: Extracellular barriers; endocytic barriers; Intracellular barriers
• mRNA translation in cytosol ➜ protein will be degraded into peptides by the proteasome ➜ loading onto MHC I ➜ shuttled to cell surface for presentation to CD8+ T cells
• MHC class II presentation: fusion of trafficking signals of endosomal or lysosomal proteins residing in MHC class II processing compartments to the encoded antigen
• expression of costimulatory signals is needed for success of vaccine: mRNA functions immunostimulatory ➜ cytokine release

Administration -> mRNA uptake, Stimulation of innate immune system, Target expression and presentation -> Activation of adaptive immune system, Expansion of antigen-specific T- and B-cells -> Balanced cellular and humoral immune response

Question 26

Targeted therapy

Answer 26

• Sustaining proliferative signaling: Dysregulation of the MAPK pathway or its components can lead to tumorigenesis, and cancer cells will be able to sustain proliferative signals and evade regulation of apoptosis and angiogenesis
• Evading growth suppressors: Tumor cell needs to dysregulate this process to grow and survive
• Enabling replicative immortality: telomere ends of the chromosomes shorten with each cell division in normal cells
• While normal cell has limited number of divisions, cancer cell can pass this limit through re-expression or overexpression of more telomerase
• Tumor-promoting inflammation: inflammatory cells can provide the malignant cells with growth factors and other hallmark-facilitating mechanisms
• Activating invasion & metastasis: dysregulation of HGF/c-Met signaling has emerged as a key player in the invasion and metastasis in human malignancies, where HGF can induce epithelial to mesenchymal transition
• MET (c-Met) receptors need the ligand HGF for activation
• Inducing angiogenesis: Angiogenesis is the process of new blood vessel formation and one of the supposed mechanisms by which malignant cells can grow and metastasize
• Survival benefits of targeting this hallmark are disappointing in clinical practice
• Genome instability & mutation: multiple DNA damage and chromosomal abnormalities, such as rearrangement and gain or loss as in aneuploidy
• Poly (ADP-ribose) polymerase (PARP) plays a vital role in repairing single-strand DNA damage in the stabilization of the genome
• Resting cell death: Cancer cells that increase the expression of anti-apoptotic BCL2 or downregulation of pro-apoptotic proteins can dysregulate BCL2-family and evade apoptosis
• BH3-only proteins work through interfering with anti-apoptotic Bcl-2 proteins or stimulating the pro-apoptotic proteins directly
• Deregulation of cellular energetics: Warburg effect
• Cancer cells up regulate glycolysis enzymes
• EGFR inhibitors
• Cyclin-dependent kinase inhibitors
• Immune activating anti-CTLA4 mAb
• Telomerase inhibitors
• Selective anti-inflammatory drugs
• Inhibitors of HGF/c-Met
• Inhibitors of VEGF signaling
• PARP inhibitors
• Proapoptotic BH3 mimetics
• Aerobic glycolysis inhibitors

Question 27

EGFR inhibitors

Answer 27

• Gefitinib is the first selective inhibitor of epidermal growth factor receptor‘s (EGFR) tyrosine kinase domain
• EGFR is overexpressed in the cells of certain types of human carcinomas - for example in lung and breast cancers
• EGFR mutations lead to inappropriate activation of the anti-apoptotic Ras signaling cascade
• Approval: Gefitinib is approved for EGFR positive cancer types in several countries

Activation of signal-transduction cascades (for example, MAPK): Cell proliferation (high), Apoptosis (low), Invasion and metastasis (high), Angiogenesis (high)

Question 28

Imatinib

Answer 28

• Specific inhibitor of tyrosine kinase enzymes
• Binding in the TK domain in abl (the Abelson proto-oncogene), c kit and PDGF-R (platelet-derived growth factor receptor)
• Chronic myolid leukemia: in 95% pathogenesis a consequence of a defined translocation event that creates the philadelphia chromosome with formation of the breakpoint cluster region–abelson tyrosine kinase (BCR-ABL) oncogene
• Imatinib works by binding close to the ATP binding site of bcr-abl, locking it in a closed or self-inhibited conformation

Question 29

CDK4/6 inhibitors

Answer 29

• Palbociclib: first approved selective inhibitor of the cyclin-dependent kinases CDK4 and CDK6 for cancer therapy (Approval 2015)
• CDK4 and CDK6 complex with cyclin D drive the phosphorylation of retinoblastoma protein (Rb) which allows the cell to commit to division
• Palbociclib ensures that the cyclin D-CDK4/6 complex cannot aid in phosphorylating Rb
• Side effects: immunosuppressive -> majority of patients experience neutropenia

Question 30

Imetelstat

Answer 30

• Potent and specific telomerase inhibitor and so far the only drug of its class in clinical trials
• Imetelstat is a 13-mer oligonucleotide complimentary to the template region of the telomerase RNA component
• Binds with high affinity to the template region of the RNA component of telomerase, resulting in direct, competitive inhibition of telomerase enzymatic activity
• Until now imetelstat has failed to show meaningful clinical activity (some clinical trials are ongoing)

Question 31

Celecoxib

Answer 31

• selective reversible inhibitor of the COX-2 isoform of cyclooxygenase
• COX-1 (not inhibited by Celecoxib) plays a role in the protection of the gastrointestinal mucosa, kidney hemodynamics, and platelet thrombogenesis
• COX-2 is extensively expressed in cells involved in inflammation and is upregulated by bacterial lipopolysaccharides, cytokines, growth factors, and tumor promoters

Question 32

Olaparib

Answer 32

• Oral PARP inhibitor used in recurrent ovarian cancer, with or without BRCA mutation
• BRCA1, BRCA2 are important for the repair of DDBs by the error-free homologous recombination repairàmutations can lead to errors in DNA repair
• PARP1 inhibitors cause multiple DDBs and in tumors with BRCA1, BRCA2 or PALB2 mutations these DDBs are not efficiently repaired
• Overall progression free survival: increase of 3 – 19 month depending on tumor type
• Zellen mit BRCA-Mutation: Reparatur
• Zellen mit Medikamenten-induzierter PARP1-Inhibition: Reparatur
• Zellen mit BRCA-Mutation und PARP1-Inhibition: keine Reparatur -> ZELLTOD

Question 33

Venetoclax

Answer 33

• BH3 mimetic inhibitor
• Venetoclax blocks the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein, leading to programmed cell death of CLL cells
• Used in chronic lymphocytic leukemia (CLL) and follicular lymphoma, but it was ineffective in solid tumor

Question 34

Targeting aerobic glycolysis

Answer 34

• Glycolytic pathway comprises a series of ten reactions, which potentially represents targets for anticancer treatment and inhibitors have been developed that target molecular components
• not approved yet