A new word in cancer therapy
Inhibitors of genetic reprogramming
I.B. Roninson, "First-hand Science"
Igor Borisovich Roninson with his colleague Mengjian Chen in the laboratory of the University of South Carolina (USA).
Cancer has a number of frightening features that give rise to the main problems in the fight against this disease. Firstly, cancer cells are still the same cells of the body, although with altered behavior. Secondly, they are able to change very quickly, adapting to new "niches" during metastasis and acquiring resistance to drugs during therapy. Thirdly, the mechanism of cancer occurrence in different cases is also different. As a result, the disease, like the phoenix bird, can be revived even after the harshest treatment, ricocheting into healthy cells and tissues. The author of this article and his team have discovered a universal mechanism that allows a malignant tumor to survive under stressful conditions due to the activation of "dormant" genes. Moreover, chemical inhibitors of this process have been created and are already undergoing clinical trials, which gives hope to significantly increase the effectiveness of existing methods of combating one of the most formidable diseases of our time.
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When at the end of the last century I began to deal closely with the issues of cancer treatment, all anticancer drugs were, as they are now called, conventional. These classic drugs act on certain molecular objects necessary for both normal and tumor cells. In other words, within the framework of this approach, it is impossible to create a drug that would affect only pathological cells.
The mechanism of action of conventional drugs can be directed at DNA damage either directly (these are the oldest anticancer agents), or by acting on topoisomerase enzymes and nucleotide synthesis, as well as on the perturbation of microtubules that make up the cytoskeleton of a cell, that is, on universal targets necessary to maintain the normal cell cycle of any cell. The reason why such drugs work well in tumor cells is that their control over the cell cycle has been changed, so they are more sensitive to violations of its critical stages.
Of course, such drugs will inevitably be dangerous for the patient – everyone knows about the consequences of chemotherapy. The main thing here is to create a drug that will be effective at a certain moment of application in the most non–toxic dosage, which will reduce the danger to healthy cells. It is important that these drugs bring improvement, since they kill pathological cells so quickly that the tumor does not always have time to start the resistance mechanism. An example of the successful use of conventional means can be testicular cancer therapy, thanks to which up to 90% of patients recover today.
Intracellular targets can be effectively inhibited by small molecules that are easy to synthesize. An example is vemurafenib, which is used for targeted therapy of melanoma with the BRAF V600E mutation. Melanoma, one of the most insidious tumors, is caused by a genetic mutation of BRAF in about half of cases. Creative Common.
But recently, the vast majority of new drugs are aimed at specific targets, which are necessary primarily for tumor cells to survive or maintain the cell cycle. Most of these targets have been identified by genomics methods, and this process continues. Such targeted, targeted drugs are either monoclonal antibodies or "small" molecules of chemical compounds. These include inhibitors of various enzymes, growth factors, receptors, hormone antagonists, etc.
It is important that targeted drugs act only on certain tumors that are targets for these drugs. Therefore, the use of such drugs always requires personalized medicine, that is, careful selection of patients. An example is a very common disease – breast cancer, which today is almost always treated only using a personalized approach.
The development of most subspecies of this cancer is associated with the female hormone estrogen, which binds to the pool of estrogen receptors (ER) inside cancer cells, accelerating their reproduction. On the other hand, the principal "driver" of about 20-25% of breast cancers is tyrosine kinase HER 2. Therefore, before starting treatment for breast cancer, it is necessary to first do a molecular analysis of markers to determine whether tumor cells produce estrogen (or progesterone-dependent) receptors. And if so, such patients are treated with targeted hormone therapy.
If an amplified (repeatedly copied) HER 2 gene is identified in tumors, then these patients are treated with drugs aimed specifically at this protein: antibodies (for example, trastuzumab) or small blocker molecules (for example, lapatinib). If the cancer does not belong to these two groups, then these tumors are classified as triple negative breast cancer. Unfortunately, there is no general successful method of treating such tumors yet.
Genomic analysis is the key to success
Nowadays, the strategy of treating all types of cancer is becoming more and more standard. First, a tumor sample is taken from the patient for analysis for different markers. Genomics methods are used to determine the presence of certain mutations; transcriptomics methods assess expression (activity) of genes; cellular proteins are studied by proteomics methods. Depending on these results, treatment is prescribed. Sometimes it's immunotherapy. In the case of chemotherapy, it is necessary to understand exactly what characteristics of a particular patient's tumor help it grow and survive.
Sometimes we use existing drugs that effectively affect the appropriate targets or, at least, are approved for use for these purposes. Or we develop new ones. In the future, we will only talk about the development of new drugs, which in itself requires genetic analysis. Below I will try to answer the question: "Why?".
Here is a good example of why it is impossible to create new cancer drugs outside the framework of personalized medicine. In the USA in 2004, one of the most popular women of the 1990s, Martha Stewart, the owner of a huge grocery empire and her own television show, was imprisoned. The reason was the appearance of a new cancer drug – cetuximab, a monoclonal antibody that acts on the transmembrane receptor tyrosine kinase EGFR, which plays an important role in stimulating tumor growth, metastasis, etc. ImClone conducted the first clinical trials on different groups of patients without individual selection. The results were mixed, and the Food and Drug Administration (FDA) did not approve the drug. The company's CEO and some other shareholders, including Stewart, sold their shares even before the decision was announced, which is a serious criminal offense.
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A few years later, an article was published where cetuximab was tested against rectal cancer. It turned out that if the tumor cells had an oncogenic K-Ras mutation, which happens in about half of all cases, then the drug did not work at all. But in other cases, it was very effective, and the results of the initial trials would have been completely different if they had been conducted only on patients without the K-Ras mutation. This was the first demonstration that genomic analysis could be the key to the success or failure of an anti-cancer drug.
Another example relates to small molecules: today we can synthesize such molecules that can act very selectively on a specific protein. In our case, this is a protein that is encoded by the BRAF gene – in skin melanoma, the frequency of mutations in this gene is 30-70%. And the creators of the drug vemurafenib intended it for patients with just such a mutation.
At first, the results of treatment with vemurafenib were amazing. For example, during 15 weeks of therapy, a patient with melanoma throughout the body managed to achieve almost complete remission. But after a while, the tumor returned. Why? Because the tumor cells have acquired resistance to the drug. And such resistance, unfortunately, develops in almost all patients who are treated with any targeted drugs, and in most patients who are treated with conventional drugs.
Drug resistance – what is the reason?
How do cancer cells develop drug resistance? This phenomenon is associated with a change in the expression (activity) of genes in cells damaged by chemo-, radio- or immunotherapy. In other words, we are talking about transcription reprogramming – the transfer of genetic information from DNA to an RNA molecule.
This panel of kinase enzymes regulating the stability and activity of many proteins shows their blocking by chemical transcription inhibitors caused by DNA damage. The best inhibitor (SNX14), obtained as a result of high-performance screening, blocks many kinases (a). The inhibitor obtained as a result of chemical modification (SNX2-1-108) blocks only two kinases – CDK8/19 (b). These enzymes are involved in transcriptional reprogramming of damaged tumor cells. Inhibited kinases are marked in red (Porter et al., 2012). Published with the permission of PNAS.
Using transcriptomics methods, we found that many genes encoding proteins begin to activate in damaged tumor cells, which exit the cell and stimulate the growth and survival of other tumor cells, contributing to the appearance of metastases, as well as the formation of drug resistance. We were able to detect some small molecules that suppress this process by developing and applying a high-performance physical screening system that allowed us to evaluate more than a hundred thousand different chemical compounds. At that time it was a huge job – now we can virtually analyze tens of millions of connections using new computer algorithms, which saves time and reduces the cost of research.
According to chemists, our molecules looked structurally like kinase inhibitors – enzymes that regulate the activity and stability of a huge number of proteins that perform various functions, including those involved in the transcription process. Therefore, we tested our molecules on a large (more than 450 in number) set of these enzymes that are found in the human body. And it turned out that our best inhibitor molecule really "disarms" a huge number of kinases.
Then we began to chemically modify this "champion" molecule. At some point, it turned out that its activity in suppressing transcription induced by DNA damage increased dramatically, and some other properties disappeared. And when we tested this modified molecule on the same kinase panel, it turned out that only two "flowers" corresponding to two kinases were inhibited on this powerful "tree" (Porter et al., 2012).
And here the hero of our novel, CDK8 protein (cyclin–dependent kinase 8) and CDK19 closely related to it, which were identified using chemical genomics, comes on the scene "with a drum beat". It turned out that these proteins are the target that needs to be inhibited in order to prevent the "inclusion" in damaged cells of genes associated with the formation of drug resistance and metastasis.
CDK8 and CDK19 proteins are practically "twins", they are 80% identical to each other. These proteins regulate only transcription and have no relation to the regulation of the cell cycle, like some other members of the family of cyclin-dependent kinases. But they interact with a number of different transcription factors, including those associated with cancer (for example, the same estrogen receptors).
Cyclin-dependent kinase 8 (CDK8) and its "twin" CDK19 are included, along with several other proteins, in the CDK module, which interacts with the mediator complex and regulates its activity. The mediator complex, in turn, interacts with the enzyme RNA polymerase II, which is necessary for transcription, the process of RNA synthesis on a DNA matrix (Porter et al., 2012). Published with the permission of PNAS.
But if you ask me what happens in any cell if you suppress CDK8/19 in it, you will not get an answer. The fact is that the effect of this inhibition will depend on the context, that is, the specifics of the cell and its genes. After all, these proteins, as mentioned above, work only in cooperation with other factors. We can only say that inhibition will have little effect on the activity of already working genes. But in this way we can suppress the activity of genes that have "woken up" in the cell using CDK8/19.
But why does our body need enzymes regulating transcriptional reprogramming at all? To answer this question, let's remember when we have changes in the transcription program – during the development of the organism. Moreover, transcriptional reprogramming plays a critical role in this process.
And indeed, if you give pregnant mice a CDK8/19 inhibitor, then the development of embryos will be blocked. When we gave this inhibitor to adult mice for 200 days, nothing happened to them externally, the weight did not change, etc. But when the same drug was given to animals that had breast or prostate cancer (in combination with hormone therapy) or metastases of rectal cancer grew in the liver, the growth of tumors slowed down sharply.
To clinical trials
We tested a large number of already known anticancer drugs in combination with our CDK8/19 inhibitor and were convinced that in this way it is almost always possible to prevent the occurrence of drug resistance or at least slow down this process. The matter remains small: such inhibitors must be approved for practical use. And for this, they must work as single agents, suppress some types of cancer.
To find such targets, we turned back to genomics. Now there are special computer programs with which you can find out how the activation of a particular gene affects the survival of a patient with a certain type of cancer. With the help of one of these programs, we made a selection of different types of cancer for which the CDK8/19 level correlates with the worst prognosis. It includes, for example, prostate cancer, cervical cancer and esophageal adenocarcinomas. Such correlations suggest that our inhibitors can be used in these cases.
A series of CDK8/19 inhibitors discovered by high–performance screening with subsequent chemical modification: SNX2 is the first structure in the series that inhibits many kinases; SNX14 is the most effective structure that inhibits many kinases; SNX2-1-108 – modified structure, selective CDK8/19 inhibitor; Senexin A – modified structure, selective inhibitor CDK8/19 with biological activity; Senexin B is a modified structure, a selective inhibitor of CDK8/19 and the first clinical candidate for a drug.
An indicative history is associated with breast and ovarian cancers. We get different results if we divide these patients into two groups: white Americans and African Americans. Among whites, CDK8/19 has a great effect in breast cancer and very weak in ovarian cancer. For African Americans, everything is exactly the opposite. This is another clear example of how important it is for clinical trials to conduct genomic studies beforehand.
We are now focusing on two types of cancer. The first is androgen-independent prostate cancer (androgens are male hormones). As you know, a common method of treating prostate cancer is antihormonal therapy. As the cancer develops and progresses, CDK19 levels rise. By the time the tumor becomes independent of androgens, it becomes incurable, and at the same time we see a sharp increase in the expression of both CDK8 and CDK19.
In experiments on laboratory mice, our CDK8/19 inhibitor, in combination with hormone therapy, completely suppressed the growth of a tumor in which a gene encoding a variant of the androgen receptor was active, due to which this cancer becomes resistant to all types of modern treatment. The animals did not lose weight and lived much longer.
The second type of cancer is leukemia, a disease of the hematopoietic system, primarily acute myeloid leukemia. This is the only cancer known today, the growth of which in many cases is suppressed by CDK8/19 inhibitors not only in animals, but also in human tissue culture. To find out how well patients would respond to our medicine, we injected mice with cancer cells labeled with the enzyme luciferase, which catalyzes a reaction accompanied by bioluminescence. It turned out that in control animals, leukemia spreads throughout the body after six weeks, and in animals that received a CDK8/19 inhibitor, it almost does not spread.
We tested a large number of tumor cell samples taken from leukemia patients and found that about 40% of them are highly sensitive to the CDK8/19 inhibitor. Thus, if we want to start treating patients with leukemia, we first need to determine for whom such therapy will be effective. Using the methods of genomics and transcriptomics, we found several genes, some of which are most active in tumor cells resistant to the CDK8/19 inhibitor, and the other part in sensitive ones. Thus, we have obtained criteria by which we can judge whether specific patients will respond to our medications.
The effect of SNX631– a CDK8/19 inhibitor, has been tested on laboratory mice with leukemia (a). With continuous administration with food, the inhibitor almost completely suppressed the development of the disease (b), which is clearly noticeable due to the use of bioluminescent imaging.
It is important that acute myeloid leukemia belongs to orphan, that is, rare, diseases, the research of which and the development of appropriate drugs in the United States is supported by the state. This means that we can get approval for our medicine much faster than if we were talking about the treatment of widespread diseases.
Cancer cells, of course, can also acquire resistance to our transcriptional reprogramming inhibitors. Leukemia turned out to be the most sensitive type of cancer to them. Therefore, we conducted a selection, obtaining drug-resistant leukemia cells, and found some things (not yet published) that tell us what to do in such cases. That is, in principle, we already have a workaround strategy.
In any case, resistance to these inhibitors occurs much later than to other drugs, because they themselves suppress its development. In addition, cancer therapy never uses one drug, usually a mixture of them. And if we prove that in the case of a specific cancer (the same leukemia) this medicine significantly improves the survival of patients, then it will be quickly approved for practical use. This means that it can be tested in combination with other anticancer agents. And in this case, we expect an increase in the effectiveness of therapy.
One more important thing should be noted: in some cases (for example, in rectal cancer), our medicine acts selectively on metastatic tumors, which is completely unique. Why? Because metastases are formed where these cells should not grow and they need to adapt to this "wrong" situation, to stressful conditions. And they adapt just through transcriptional reprogramming, the "awakening" of new genes.
Our American company Senex is engaged in the creation of CDK8/19 inhibitors in close cooperation with the Russian company BIOCAD (BIOCAD, St. Petersburg), and this collaboration meant a lot for the progress that we managed to achieve.
It took Senex about six years to find the first molecules that suppressed transcriptional reprogramming. Biocad has conducted clinical trials with the first candidate drug, which we have already developed in cooperation with Russian scientists. Then we finalized it and in a year or two we plan to conduct clinical trials of this improved version. And I hope that by the time we manage to get our drug approved and it can be used in combination with other anti-cancer drugs, targeted therapy will find a new breath.
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The editorial board of the journal appealed to Alexander Albertovich Shtil, head of the laboratory of mechanisms of tumor cell death of the N. N. Blokhin NMIC of Oncology, to tell him about the problems and the place that transcriptional programming studies occupy in a number of works on the study of the molecular nature of malignant neoplasms and the creation of antitumor drugs. A. A. Shtil collaborates with I. B. Roninson with In the 1990s, and in 2018, they organized the laboratory of Molecular Oncobiology at the Institute of Gene Biology of the Russian Academy of Sciences to perform the megagrant "CDK8-mediated Gene Transcription Programming: Biological mechanisms for Medicine"
New hopes in oncology
It is hardly possible to name a more complex, controversial and multifaceted field of medicine than oncology. Today and always cancer has been a problem for doctors and biologists, chemists and physicists, psychologists and sociologists. Among the diseases, and the most severe, this is something special; nothing sounds so serious and alarming. In this area, deep intellectual efforts are combined with selfless actions, universal patience and hope ... Indeed, in the twentieth century. thanks to the discovery of antimicrobial drugs, epidemics have receded – can we expect that a similar revolution will occur in the fight against cancer?
Chemistry and biotechnology offer countless active compounds that are harmful to tumor cells and, one way or another, sparing non-tumor cells. In cell culture, the problem is generally solved, but already in the mouse body it is more difficult: it is required to cure an experimental tumor without killing its "carrier". You reduce the dose of the drug – the tolerance improves, but the tumor also survives – it is "arranged" close to normal. It's easier with microbes: they don't look like their victims at all, and coming up with an antimicrobial drug that doesn't damage mammalian tissues is a feasible task. But this is not the case in oncology… Or is it still possible to find something necessary for them in tumor cells, but not so important for normal cells, and use this feature to create not a general toxic, but a targeted antitumor drug?
This approach – targeted (from target – target) – has been developed in the last 25 years due to the unusually rapid accumulation of knowledge about living matter. To decipher the molecular structure of life, tumor cells are the best object. It turns out that the mechanism that exists in a non-tumor cell along with others, with malignant cell transformation, becomes so necessary for the tumor that its suppression leads to its death with minimal damage to normal cells.
Glivec– an inhibitor of Abl protein kinase, which ensures the survival of tumor myelocytes, is an example of the success of a target-directed strategy. Abl inhibitors have completely changed the situation with chronic myeloid leukemia, a fatal disease of the blood system. The life expectancy of patients has increased, the quality of life has become incomparable: these medications are well tolerated, pills can be taken for months and years. So molecular biology has found a feature that determines the biological essence of the disease – so far for certain types of tumors.
However, this is only the beginning of the path. Medications work, patients live longer, and tumor cells are fighting for their survival. The more precisely our instrument of influence on the molecular target, the more workarounds remain for the tumor – it skillfully uses the biological complexity of the structure of the living and the functional interchangeability of mechanisms… But in this case, we must suppress not one, but several such mechanisms, and moreover those that a normal cell can do without: it is impossible to increase the risk of general toxicity.
Continuing the search leads to the most common problems of the cell structure – gene transcription. Indeed, everything starts with it – but isn't it the same in tumor and non-tumor cells? Once again, molecular science opens up new possibilities: CDK8 and CDK19 protein kinases that are similar in structure and function are vital in special situations and not at all important in others. Cells in which transcription is activated in response to external stimuli cannot do without these kinases. But not all cells and not with any stimuli: again, the general biological mechanisms work differently in each case. Senexins, CDK8/19 inhibitors discovered in Igor Roninson's laboratory, prevent the growth of rectal cancer screenings in the liver, but they do not affect the growth of primary tumors – similar processes are regulated in different ways. But acute myeloid leukemia and certain forms of prostate cancer turn out to be "CDK8 diseases". This selectivity allows us to raise the question of personalized treatment: senexins are indicated in specific clinical cases.
The resistance of tumors to drugs is a problem that accompanies the scientific path of I. Roninson from the first steps, from the discovery of the structure of the gene for multidrug resistance. The logic of his research led him to a completely different mechanism – a kind of regulation of gene transcription in the formation of resistance in response to antitumor drugs. In experiments, it is possible to prevent the development of resistance to modern drugs and enhance their effect on tumor cells. Senexins get a new facility, and oncology gets new hopes.
But we can not flatter ourselves: it is hardly possible to cancel harsh chemotherapy, but targeted "reprogramming" of the disease will expand the possibilities of helping patients.
I am happy that I. Roninson's research has come to Russia. Major work has begun with the company "Biocad" and the Institute of Gene Biology of the Russian Academy of Sciences. The project is Roninsonian in scale, polyphonic. As in any classical symphony, three independent parts form a unity: developmental biology, genetics and cell physiology. The leitmotif is the role of a special gene transcription mechanism and the possibility of its suppression for the treatment of tumors. The conductor has an international consolidated orchestra, a large composition. The hall froze…
Doctor of Medical Sciences A. A. Shtil (Moscow).
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