Resistance to Antineoplastic

Drugs
p53? easy as ABC? or is the answer still
in the VAULT?
John T. Wiernikowski, PharmD
McMaster Children’s Hospital
McMaster University
Hamilton, CANADA
Introduction

Drug Resistance: significant barrier to cure.

Many cancers (NB, Osteo, Ewings, Leukemias &
Lymphomas, Breast, Colon, Lung, Ovarian)
show a good response initially; but eventually
become resistant

Certain adult cancers (Renal, Pancreas,
Melanoma, GBM, Esophagus) have low
response rate to begin with

Clinically; Resistance is both

Intrinsic

Acquired
Introduction

Intrinsic resistance

Present at time of diagnosis.

Poor response to treatment.

Acquired resistance

From ongoing chemotherapy or radiotherapy.

Changes within cancer cells.

Sensitive cells die off leaving a resistant
clone.
Causes of Drug Resistance

Inadequate drug exposure

Dose modifications resulting in a sub-
therapeutic dose.

Poor drug distribution.

Limited blood supply.

Impaired diffusion into the tumor.

High Protein Binding.

Alterations in the cancer cell affecting drug
sensitivity.
Causes of Drug Resistance

Alterations in the cancer cell affecting drug
sensitivity

Increased Drug Efflux (Multidrug resistance)

Decreased cancer cell death

DNA repair

Anti-apoptotic proteins

Enhanced intracellular drug metabolism.

Altered Drug Target.
Increased Drug Efflux

ATP Binding Cassette (ABC) Family of
proteins

P-Glycoprotein

MDR related proteins (MRP1-6)

Breast Cancer Resistance Protein (BCRP)

ABCG2

Irino/Topotecan, flavopiridol, mitoxantrone,
methotrexate
P-glycoprotein

Most clinically relevant (to date) MDR
Factor identified.

Belongs to the MDR multigene family.

Situated on long arm of chromosome 7
(7q21.1)

Codes a 1280 amino acid protein that
forms a transmembrane pore.
P-glycoprotein

2 ATP binding sites intracellularly.

Drug binding sites are in the
transmembrane layer close to the
cytosolic layer.

Actively ‘pumps’ drugs out of the cell.
P-glycoprotein
P-glycoprotein

“Normal” expression on surfaces of
epithelial cells in kidney, liver, GI tract;
endothelial cells of brain and testes,
adrenal glands, bone marrow stem cells
and peripheral blood lymphocytes.

A ‘protective protein’

Also shows a great deal of homology
between many species of plants,
protozoa, bacteria and fungi.
P-glycoprotein

Endogenous Substrates

Cortisol

Aldosterone

IL-2,3,4,6

Interferon (gamma)

Sphingolipids
P-glycoprotein
MDR Related Proteins (MRP’s)

6 related proteins identified so far; gene
locus on chromosome 16.

All members of ABC family.

Don’t interact with drugs directly; drug
requires some extra intracellular
‘processing’ before these proteins can
handle them.

Correlation with outcomes much less
clear.
Clinical Significance

P-Glycoprotein Expression shown to

portend poor outcome either at diagnosis
or relapse in:

Neuroblastoma (linked with MYC-N
expression)

Hodgkin's and NHL.

Brain Tumors.

Conflicting Data in Bone Tumors and AML.

Relapsed ALL.
VAULTS

First described in mid 1980’s

Ribonucleoprotein particles found in the
cytoplasm.

Composed of a Major Vault protein (MVP)
and 2 minor vault proteins.

Why VAULT? On 3-D structural analysis
they have the shape of a “vaulted’ ceiling.
Structure of VAULTs
VAULTS

Like p-glycoprotein, present in diverse
species, and shows a great degree of
structural homology (60-90%).

Present in ALL human tissues, unlike P-
glycoprotein.
How do they work, what do they
do?

Normal ‘role’ unclear.

Drug resistance mechanism, thought to be
related to drug packaging or trapping; and
keeping the drug from it’s intracellular target.

? Transport out of the cell or to an ABC
Transporter.

Drug resistant cells show up regulation of
VAULTS. (up to 15 fold )

However, evidence suggests that they work in
concert with other factors.
Lung Resistance Protein (LRP)

A major VAULT protein.

Up regulation in certain tumors shows
some correlation with poor response

AML (adult)

Lung Cancer

Ovarian

Wilms’
P-53 and Cancer

Functional p-53 is needed for cells to proceed to cell
death.

P-53 mutations are the most common mutation in
human cancer and occur in about 50% of cancers.

Lack of functional p-53 contributes to resistance to
both radiation therapy and certain drugs, especially
platinum compounds.

P-53 mutations are correlated with poor response
and tend to have worse clinical outcomes. It’s not
clear if this genotype predicts for a poor response to
chemotherapy or portends a more aggressive tumor
type.
Decreased Cell Death: Apoptosis
and p53

Apoptosis occurs in
response to DNA
damage and the p-53
protein usually binds to
DNA and then regulates
other pro-apoptotic genes

p-51, p-21, GADD45

NOXA, BAX, PUMA

Death Receptors: CD95,
TRAIL-R1 & R2
Apoptosis: Strategies to
circumvent drug resistance

Restoration of p-53 activity

Gene transfer therapy

Onyx 015 is a gene therapy that transfects wild-
type p-53 into tumor cells. Initial studies are
negative

Small molecules that convert p-53 mutant
strains back to wild-type p-53

CP-31398 (styrylquinazoline)

Increases BAX expression independently of p-53
DNA Repair mechanisms

Platinum, anthracyclines and alkylating
agents produces DNA damage.

Normal response to DNA damage is
apoptosis

Some cancers (Bone, Brain, Ovarian)
become tolerant to DNA damage and
fragmentation by generating a deficiency
in mismatch repair (MMR) proteins that
recognize damage.
DNA Repair—Mismatch Repair
(MMR)

MMR deficiency causes cells to not recognize
DNA damage.

Decitabine, an azacytidine analog, re-sensitizes
malignant cells to cisplatin, carboplatin, and
epirubicin in vitro and xenograft models, while
not being cytotoxic.

Phase II trials in adults are encouraging.

Phase III studies are about to begin.
Altered Drug Targets:

Mutations in Topoisomerase I/II genes
leads to production of functional enzymes
that show lower affinity for inhibitors like
Etoposide, Teniposide and anthracyclines.

Both mutation and up regulation of DHFR
reductase results in resistance to Anti-fols
such as methotrexate.
Can we fight back?

P-glycoprotein and MRP Inhibitors

Colchicine

Cyclosporin A

Verapamil and Dexverapamil (Cardiotoxic)

PSC-833 (Valspodar) decreased RR and had no
survival benefit in ovarian Ca.

Tiamulin (antibiotic)

Indirect inhibitors

Rapamycin, Sirolimus

Potent Inhibitor of mTOR, an effector of Akt in B-cell lmphomas

Modifying our drugs

Epirubicin.

Go after more specific targets (Imitanib, Herceptin, Mibs
& Mabs)
Can we fight back?

Fast growing panel of biologic and
molecularly-targeted agents.

Proteosome Inhibitors

Bortezomib

EGFR Inhibitors

Gefitinib (Iressa)

Cetuximab

VGFR Inhibitors

Bevacizumab

Genotypic profiling and pharmacogenomics
will drive the development of new therapies.
Cyclosporin to overcome MDR
in AML Becton D et al: Blood 107(4):1315-24, 2006
Cyclosporin to overcome MDR
in AML Becton D et al: Blood 107(4):1315-24, 2006