24 Development of resistance against cancer drugs

In our last chapter we will discuss the development of resistance to cancer agents. We debated quite an impressive list of cancer drugs in the previous chapters. We deliberately left the development of resistance to the end of this course. In principle, we could have discussed the development of resistance for each drug in the respective chapters, but in our expereince that would have been much more difficult. In our opinion it is much easier to discuss and learn the common underlying principles of resistance, than to memorise the mechanisms of resistance for each drug.

24.1 Introduction to drug resistance

What is drug resistance? “Drug resistance is the insensitivity or reduced sensitivity of cancer cells to drugs that would normally cause cell death.” There are two types of resistance: the first one is termed intrinsic resistance, or also called pre-existent or primary resistance. The second type of resistance is termed acquired resistance, or induced or secondary resistance. One can find all these different terms in the literature. The development of resistance has been observed for both cytotoxic and targeted therapies. With respect to drug resistance there is no obvious advantage of targeted therapies over standard chemotherapy. Why is it important to recognise drug resistance early? Well, if a cancer is resistant to treatment, the medication will not help, but will still lead to toxic side effects for the patient, which are unnecessary if the treatment has no effects, aprt form additional economic considerations. One important point to make is that differences have been observed between mechanisms of resistance in tumour cell lines, compared to real patient tumours. Tumour cell culture models are easier to work with whereas working with patient tumours requires the consent of patients and is genrally more complex and expensive.

One important type of resistance is Multidrug resistance (MDR) or cross-resistance. If the mechanism of resistance in a tumour cell line or tumour is the over expression of the efflux pump P-glycoprotein (PgP), then all cancer drugs that are also substrate for Pgp will be effluxed from the cell and the drugs becomes ineffective. The development of drug resistance is the principal factor for the failure of cancer therapy.

Sometimes, patients do not respond to cytotoxic chemotherapy.The specific mechanism of resistance is in most cases unknown. For example: 20% to 25% of breast cancers are Her2-positive, which means that we can only treat about one quarter of breast cancer patients with Herceptin. However, only 1/3 of these Her2-positive patients respond to Herceptin, which indicates that 65% of Her2-positve patients are intrinsically resistant to Herceptin already at the onset of treatment. In summary, we do not reach 25% of all breast cancer patients but only less than 10%. For Herceptin treatment we would need a novel biomarker, indicating which Her2-positve patients will respond to Herceptin treatment and which ones will not. That would also be significantly more cost effective for health care systems and the money safed could be invested into other treatments.

Another example of primary resistance concerns Philadelphia-Chromosome-negative CML patients. There is a small subset of patients without detectable molecular evidence of Bcr-Abl fusion and these patients will not respond to Gleevec treatment. These patients may be classified as having a myelo-proliferative disorder.

How does secondary resistance develop? Cancer cells are initially sensitive to drug treatment and the tumour may shrink during treatment.However, the tumour develops a way to evade the effects of these drugs and once the tumour does not respond to the drug treatment anymore, it will regrow. The tumour has acquired resistance and will not respond to the original treatment anymore. What has happened? Well, we already learned about this in chapter 10 (Figure 10.4), when we discussed the concept of somatic evolution. There is a variation in the population of cancer cells which is heritable: when a cancer cell divides, both daughter cells inherit the genetic and epigenetic abnormalities of the parent cell, but may also acquire new abnormalities. This variation affects survival and reproduction. In this concept, drug treatment acts as a form of artificial selection, by killing sensitive cancer cells, but leaving a few resistant cells alive. Often, tumours will regrow from those resistant cells. In addition the new tumour will be resistant to the initial treatment. In a tumour with high heterogeneity, the chances of occurrence of cells with resistance are higher than in a tumour with low heterogeneity.

24.2 Tumour cells care capable of developing a variety of mechanisms to escape the effects of cancer drugs

We are now in the position to discuss some common mechanisms of drug resistance (Figure 24.1). To reach its target, a drug has to cross the cell membrane. The main mechanism for permeability is passive diffusion. Cell membranes contain so called transporters, which are able to transport molecules into the cell, in a process called influx, or which can expel molecules from the cell, which is called efflux. This represents two common mechanisms of resistance to drugs. Once in the cell drugs or prodrugs can be inactivated by metabolisation through for example metabolising enzymes. Once the drug reaches and binds to its cellular target, either a protein such as an enzyme or DNA, cancer cells can develop resistance by for example over-expressing or amplifying the target or by the introduction of point mutations in or close to the inhibitor binding pocket which can reduce the affinity of the drug to its target. If the target is DNA,as we have discussed for alkylating agents, platinum containing agents, Doxorubicin or Bleomycin, the cellular damage, in particular DNA damage, can sometimes be repaired through the various DNA repair mechanisms that exist in our bodies.There are five to six major DNA repair pathways.

In summary, a variety of different and sophisticated mechanisms of resistance are at a cancer cell’s disposal to overcome the effects of cancer drugs. As already mentioned, the development of resistance is the major reason for treatment failure.

 Various mechanisms are at the tumour cells disposal to fights cancer drugs.

Figure 24.1: Various mechanisms are at the tumour cells disposal to fights cancer drugs.

Table 20.1 represents an incomplete overview of a few mechanisms of resistance that have been identified for the list of cancer drugs we discussed in the previous chapters. It is not useful to learn all the possible mechanisms for each drug. It makes much more sense to identify common mechanisms and trends. Probably the most common mechanism of resistance, at least in tumour cell lines, not necessarily in tumours, is the over-expression of efflux pumps, as seen in the red coloured column. Another clear trend is increased DNA repair or increased tolerance for drugs that target DNA, including alkylating agents, platinum-containing compounds, Bleomycin and DNA-intercalating agents. Two additional common mechanisms iare the development of mutantions in the protein target and gene amplification leading to the over-expression of the protein target. Finally the inactivation of drugs by glutathione or other anti-oxidants is still another know mechanism. We will now discuss a few mechanisms of resistance as examples for the recently studied drugs Methotrexate, Cisplain, Herceptin and Gleevec in more detail.

** Table 21.1** Common mechanisms of resistance identified for a variety of cancer drugs.

24.3 The role of members of the ABC transporter family as uptake or efflux pumps

We want to give you a brief introduction to the transporter family and in particular Pgp. ATP-Binding Cassette (ABC) transporters constitute a large family of proteins with 48 genes identified in the human genome.These are membrane proteins driven by ATP hydrolysis to actively transport molecules from the cytoplasm to the periplasm or in the reverse direction (Figure 24.2). They can have very broad substrate specificity. Probably the most important transporter is P-glycoprotein(or Pgp, also named MDR1 or ABCB1), present in many tissues and tumour and the most clinically relevant transporter, and Breast Cancer Resistance Protein (BCRP). Pgp’s natural function is to protect tissues and organs from toxic compounds by eliminating them from cells: out of the brain, or out of other organs, into urine or bile, thus mediating the development of resistance to anticancer agents. Unfortunately, this is also true for cancer drugs and Pgp can mediate the development of resistance to many different classes of anticancer drugs. In particular, it can induce MDR. Substrates for Pgp are for example taxanes and vincaalkaloids and several kinase inhibitors, whereas epothilones, MT-targeting agents, have low susceptibility to Pgp.

 Function of membrane proteins of the ABC transporter family. Uptake transporter can either actively transport molecules into cells, whereas efflux transporter can expel xenobiotics from the cell.

Figure 24.2: Function of membrane proteins of the ABC transporter family. Uptake transporter can either actively transport molecules into cells, whereas efflux transporter can expel xenobiotics from the cell.

Transporters in the small intestine can modify the absorption of some drugs (Figure @ref(fig:20.3)). This figure shows an epithelial cell and different types of uptake and efflux transporters identified:

  • Uptake transporters are involved in the uptake of compounds from the lumen through epithelial cells into blood,

  • Efflux transporters are involved in the efflux from the blood into epithelial cells and from the cell membrane back into the gastric lumen.

P-Glycoprotein: Pgp; Multidrug Resistant Protein: MRP; Breast Cancer Resistance Protein: BCRP.

Figure 24.3: P-Glycoprotein: Pgp; Multidrug Resistant Protein: MRP; Breast Cancer Resistance Protein: BCRP.

24.4 Development of resistance against Methotrexate

Folates and antifolates such as Methotrexate use specific transport systems for their cellular uptake. We have already discussed in detail how Methotrexate enters cancer cells and how it reaches and inhibits its target (chapter 14). In brief, Methotrexate enters cells via uptake by two different proteins, either the Reduced-Folate Carrier (1) or the Membrane Folate Binding Protein (2).

One mechanism of resistance against Methotrexate is impaired uptake due to down-regulation and decreased expression of the human transporter protein Reduced Folate Carrier. This mechanism of resistance has been shown in tumour cell lines as well as in tumours.

 Mechanisms of resistance tumour cell develop to evade the drug Methotrexate.

Figure 24.4: Mechanisms of resistance tumour cell develop to evade the drug Methotrexate.

Once Methotrexate entered the cell through active transport it can be expelled from the cell by a variety of ATP-driven efflux transporters such as Multidrug Resistance-associated Protein 1 (MRP1), MRP2 and MRP3. Although this mechanism is not shown in this figure, it represents another powerful mechanism of resistance.

Methotrexate is then polyglutamylated by the enzyme Folypolyglutamate Synthetase (3). Polyglutamylated Methotrexate (glu)n is a potent inhibitor of Dihydrofolate Reductase (DHFR) (4). Polyglutamylation is a form of reversible posttranslational modification of glutamate residues. The γ-carboxy group of glutamate may form a peptide-like bond with the amino group of a free glutamate.

However, Methotrexate polyglutamates can be hydrolyzed to Methotrexate in the lysosome by the enzyme called γ-Glutamyl Hydrolase (GGH) (5). Hydrolysis of the polyglutamate tails by this enzyme makes (anti)-folates exportable again. An additional mechanism of resistance is the increased expression of y-Glutamyl Hydrolase. High expression of this enzyme leads to a decreased ability of the cells to produce methotrexate polyglutamates, which cannot be retained in the cell. Remember: the addition of multiple glutamates is necessary to enable the cell to retain folates and anti-folates.27

Additional mechanisms of resistance against Methotrexate are related to the drug target itself. The first example concerns the enzyme Dihydrofolate Reductase, the target for Methotrexate. Treatment with Methotrexate can lead to gene amplification and subsequent over-expression of DHFR, both in tumour cell lines and in tumours. This changes the ratio between the target protein and the drug resulting in the development of resistance.

Still another mechanism of resistance is the appearance of point mutations in DHFR, such as for example the mutation Leu22Arg. Methotrexate binds to the protein and interacts with key residues in a very defined way. You can easily imagine that by mutating one or several key residues in the inhibitor-binding pocket, one can significantly reduce the affinity of Methotrexate for the enzyme and ultimately conferring resistance. Decreased affinity of DHFR for Methotrexate results from DHFR mutations that markedly decrease the affinity of the enzyme for 4-amino anti-folates but at the same time produce a smaller decrease in the affinity of the enzyme for the natural substrate dihydrofolate. However, this mechanism of resistance has so far only been shown in tumour cell lines, but not in tumours.

 Crystal structure of Dihydrofolate Reductase in complex with Methotrexate. The mutation Leu22Arg is shown as a ball and stick model.

Figure 24.5: Crystal structure of Dihydrofolate Reductase in complex with Methotrexate. The mutation Leu22Arg is shown as a ball and stick model.

In summary, cancer cells are capable of developing a range of distinct mechanisms of resistance against Methotrexate, which may render the cancer drug inefficient.

24.5 Development of resistance against cisplain

As a further example we would like to discuss the development of resistance to cisplain, the platinum-containing drug we discussed in chapter 12. The use of Cisplain is limited by the development of drug resistance. To understand the mechanisms of resistance of cancer cells against cisplain we first have to recapitulate its mechanism of action. DNA binding is the main biological event that triggers anticancer properties of Cisplatin. The formation of cisplain adducts significantly alters the structure of the target DNA. “Cisplain can crosslink DNA in several different ways, interfering with cell division. The damaged DNA elicits DNA repair mechanisms, which in turn activate apoptosis when repair proves impossible.” 1,2-intrastrands d(GpG) and d(ApG) adducts form nearly 90% of the adducts. 1,3-intrastrand d(gpXpG) adducts also occur but are readily excised by the nucleotide excision repair (NER) system, part of the DNA DamageResponse (DDR). A small percentage of interstrand cross-links and monofunctional adducts are also present. As we have already learned for alkylating agents, Cisplain also binds to the N-7 position in guanine bases, because this position is the most reactive one.

We have listed a few mechanisms of resistance including decreased influx, increased efflux, cellular anti-oxidants, increased DNA damage and increased DNA tolerance are summarised in Figure 21.6.

Cisplain concentrations in tumour cells can be reduced by decreased influx, mediated by Copper Uptake Protien 1 (Ctr1), which is responsible for the uptake of platinum containing compounds. A second transporter is Organic Cation Transporter 2 (OCT2).

Increased efflux through efflux pumps can also be an additional mechanism of resistance involving transporters such as Multidrug resistance-associated protein 2 (MRP2) and the copper-transporting ATPases ATP7A andATP7B.

 Summary of distinct mechanism of resistance against cisplatin, including decreased uptake, increased efflux, detoxification by glutathione (GSH), increased DNA repair and increased DNA tolerance.

Figure 24.6: Summary of distinct mechanism of resistance against cisplatin, including decreased uptake, increased efflux, detoxification by glutathione (GSH), increased DNA repair and increased DNA tolerance.

In the cytoplasm Cisplain becomes aquated, which enables them to react with thio-containing molecules, including glutathione (GSH) and metallothioneins. Cisplain is detoxified by GSH through adduct formation.

Cisplain adduct repair occurs primarily through the Nucleotide excision repair (NER) system. Since 1,3 crosslinks are repaired more efficiently than the 1,2 GpG and ApG crosslinks, the hypothesis is supported that the 1,2 intra-strand cross-links are the cytotoxic lesion. Increased expression of several NER genes has been correlated with Cisplain resistance.

Finally, in some cell lines cisplain tolerance can be achieved without the need for DNA repair. Several DNA polymerases have been shown to be able to bypass intra-strand crosslinks.

24.6 Development of resistance against various cancer drugs by DNA repair systems

Various lesions have been discussed that can lead to DNA damage including single-stranded breaks (Bleomycin), double stranded breaks (such as Topoisomerase II inhibitors), covalent binding of bulky adducts (such as platinum-containing agents such as Cisplain) and nucleotide mutations (Figure 24.7). DNA has to be repaired before replication, and if this is not possible, the damaged cell will be targeted for destruction.

Not surprisingly, normal and cancer cells has developed a variety of DNA repair mechanisms, including for example Homologous Recombination, Base Excision Repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair and others.

 Various DNA lesions induced by anticancer agents, and the respective DNA repair systems that cancer cells will employ to repair these lesions.

Figure 24.7: Various DNA lesions induced by anticancer agents, and the respective DNA repair systems that cancer cells will employ to repair these lesions.

Cancer cells have the ability to repair certain DNA damages, by for example over-expressing proteins involved in DNA repair. One important mechanism is NER. NER mainly repairs bulky DNA adducts such as those caused by interaction with Cisplain. Increased levels of several NER genes have been correlated with Cisplain resistance (add ref).

24.7 Development of resistance against Gleevec

In the previous chapter we discussed the targeted therapy Gleevec, which targets the cancer specific Bcr-Abl fusion protein, a fusion protein leading to various leukemias.

Although Gleevec is highly effective initially, relapse is increasingly encountered clinically. While very little is known about the molecular mechanisms responsible for primary resistance, the mechanisms of secondary or acquired resistance are largely understood.

Figure 24.8 shows the development of resistance against Gleevec within two years of starting treatment. We observe that the more the disease has progressed the more likely the development of resistance occurs. Where as primary and acquired resistance have a frequency between 4% and 13 % in the chronic phase, resistance increases to between 24% and 51% during the accelerated phase and peaks between 66% and 88% in the blast phase. This is in agreement with data on other cancer types, which indicated that the more advanced a cancer is, the more difficult it becomes to treat the disease. Again, the take home message is to treat patients early.

 Frequency of Gleevec resistance within two years of starting treatment is highly dependent on the stage of the disease. Whereas the frequency is very low at the early stages of CML, it increases significantly at the later stages.

Figure 24.8: Frequency of Gleevec resistance within two years of starting treatment is highly dependent on the stage of the disease. Whereas the frequency is very low at the early stages of CML, it increases significantly at the later stages.

Figure 24.9 shows the distribution of mechanism of resistance against Gleevec. Mutations in the kinase domain of Bcr-Abl represent the most common mechanism of acquired resistance to Gleevec, occurring in about 75% of cases. More than 40 different mutations have been associated with clinical resistance to Gleevec. Approximately 15% of resistance is associated with the amplification / over-expression of Bcr-Abl. For about 10% of cases resistance occurs through other mechanisms and may include for example the over expression of the efflux transporter P-glycoprotein.

 Various mechanisms are responsible for acquired resistance against Gleevec treatment. The main mechanism however is the development of mutations in the kinase domain of Bcr-Abl.

Figure 24.9: Various mechanisms are responsible for acquired resistance against Gleevec treatment. The main mechanism however is the development of mutations in the kinase domain of Bcr-Abl.

Bibliography

References to be added


  1. Polyglutamylation is a form of reversible post-translational modification of glutamate residues The γ-carboxy group of glutamate may form peptide-like bond with the amino group of a free glutamate.↩︎