WHITEPAPER

Expert Q&A: Tackling Drug Resistance with Combinational Therapy

Introduction

The application of immunotherapy – specifically immune checkpoint inhibitors – for cancer treatment has burgeoned over the past decade and revolutionized the management of certain types of cancer. Yet, it has become well-understood that most patients do not respond to immunotherapy, or they develop resistance to such treatment. This realization paved the way for current research efforts to find companion therapies that will enhance the efficacy of immunotherapy in unresponsive patients. Epigenetics research is leading the way in these efforts.

Dr. Baylin’s research focuses on the epigenetics of cancer, particularly DNA methylation changes in the chromatin events that subserve those for changing the epigenome and gene expression. In this article, Dr. Baylin shares insights on immunotherapy applications and research, the status of epigenetic therapy research, and where he and his research team are focusing their efforts now and looking toward the future.

PerkinElmer: Dr. Baylin, could you summarize the impact that anti-cancer immunotherapies, specifically immune checkpoint inhibitors, have had on cancer treatments?

Dr. Baylin: Immunotherapy for cancer has long been a sought-after goal. It wasn’t until the 1990s and early 2000s that Nobel Prize winners Allison and Honjo identified the T-cell proteins CTLA-4 and PD-1 and their involvement in reduced T-cell activity. The identification of the ligands and cancer cell proteins that activate CTLA-4 and PD-1 set the stage for the first immune checkpoint inhibitor therapies. By breaking the interaction between the T-cell proteins and their ligands, you create a less tolerant immune setting.

Those discoveries stoked the entire field of immunotherapy and have led to many checkpoint inhibitors that target PD-1 and CTLA-4. It revolutionized the treatment of certain cancers including non-small-cell lung cancer, renal cell cancer, Hodgkin lymphoma, and others. Today, for almost all types of cancer, there is some FDA-approved scenario where these immunotherapies can be used.

PerkinElmer: What prerequisites exist to induce a response to immune checkpoint inhibitors?

Dr. Baylin: You need to know what the patient's tumor contains in terms of the immune cells that are in it and the status of those immune cells. Are they tolerant “exhausted” cells, or are they primed to be competent cells that are more likely to respond to an immune checkpoint inhibitor?

You also want to know the levels of receptors and ligands within the tumor, and to understand the tumor cells’ mutational burden. For example, in lung cancer that is cigarette-induced, the cancer cells contain a lot of mutations, and that high mutational burden seems to indicate a good response to immune checkpoint inhibitors. These are some of the prerequisites you want to know in your effort to predict what the patient’s response might be. But it is very dependent on the type of cancer, subgroups within cancer type, and the individual patient.

PerkinElmer: Can you describe the basic principles of epigenetics and the role those processes play in regulating immune cell function and mediating anti-tumor immunity?

Dr. Baylin: Epigenetics means above and beyond genetics. You can think of the DNA in a cell as the hard drive. Theoretically, it has every piece of information needed to manage cell function. But that hard drive doesn’t know what to do with the information without a software package. The cell’s epigenetics can be viewed as the software package. It’s all about how the epigenome packages the DNA to make some areas of it exposed for expression of genes.

This means that “functional” DNA expression is in a plastic state regulated by different epigenetic states. In immune cells, and tumor cells, if the epigenetics of the cell can be manipulated, then gene expression can be altered to adjust the immune response. This is the key to using epigenetics to mediate anti-tumor immunity. The epigenome is very complicated. It's a three-dimensional structure with many layers, so it provides many potential therapeutic targets.

PerkinElmer: What effects do epigenetic mechanisms have on T-cell invasion and exhaustion?

Dr. Baylin: There are at least four important ways in which epigenetic therapies show promise and all involve a critical intersection of effects on both tumor and immune cells. First, is an upregulation of tumor suppressor genes which can influence cell signaling pathways central to tumor immune responses.

This process involves reversing cancer-specific, abnormal DNA methylation at the start site of such genes which otherwise would normally be induced to express as an anti-tumor mechanism. In normal cells, this on-off gene expression in embryonic development, and in adult cell renewal is controlled by chromatin proteins and not by presence of DNA methylation at the gene start sites. But if DNA methylation is added in such genes in cancers, a loss of tumor suppressor gene function then ensues. This can be associated with failure to suppress oncogenic cell pathways, such as KRAS which are immune suppressive.

Second, in our laboratory in 2015, and simultaneously in the labs of Peter Jones and Daniel De Carvalho, it was discovered that the drugs we used to upregulate gene expression produced a series of tumor cellular responses associated with repeat elements in the human genome, many of which were from endogenous retroviruses that had been incorporated into the genome over the years. When those sequences were upregulated, the cell saw this as viral RNA and mounted an immune response.

This viral mimicry gave us the idea to stimulate a tumor to upregulate signal production which, in turn, would reach out from the tumor to create an immune response and draw immune cells into the tumor environment. If you bring these latter cells into the tumor in this manner, then immune checkpoint therapy may activate their anti-tumor activity. In essence, the epigenetic therapy is then using the tumor cell to activate its own inflammatory signaling to signal the immune system.

Third, in concert with these drug-induced changes in tumor cells, all immune cell subsets have key properties under epigenetic control of gene expression, and which can be affected by epigenetic therapy. DNA methylation for these events is different than in the cancer and involves acquisition and removal of this DNA modification which occurs normally. These changes help allow conversions of immune cells from an inactivated to an activated state, and then back again so continued immune activity does not cause autoimmunity.

However, when immune cell interaction with cancer cells is chronic, the above type of DNA methylation can be “locked in” and the epigenetic state is altered such that this helps the cells develop resistance to activation and stay in a state of exhaustion. Epigenetic therapy can potentially reverse this resistance and break that tolerance.

Finally, a synthesis of all the above events with epigenetic therapies influence how immune cells physically interact with tumor cells. Prior to seeing immune checkpoint therapy, cancers such as NSCLC have different states relative to these interactions which determine whether the tumor is classified as having a “cold” versus a “hot” immune state. In the former, the cancer contains very few key immune cells in the region, or, when these cells are present, they surround the beds of tumor cells rather than infiltrating them. In a hot state, immune cells are embedded in and around the tumor cells creating a state favorable for potential response to the immune therapy.

In the laboratory, our group found in mouse studies that epigenetic therapy could convert a cold to a hot tumor and increase the number of immune cells that bring them into the bed of tumor cells. Now in ongoing clinical trials for NSCLC we have mounting evidence that this can occur in patients, giving us hope that this reflects true potential for epigenetic therapy to enhance immune checkpoint therapy and even reverse prior resistance to the latter in patients.

PerkinElmer: Can you explain how epigenetic processes sensitize tumors to immune checkpoint therapy?

Dr. Baylin: The above series of interactions between tumor and immune cells is the predicted dynamic through which it is hoped that epigenetic therapy may sensitize tumors to immune checkpoint therapy. Much remains to uncover all facets of this possibility and realize through clinical trials, bringing such combinational therapy to patient management. From a laboratory standpoint, specific molecular aspects are being uncovered.

One key feature is that when epigenetic therapy upregulates the tumor cell inflammatory signals, this stimulates the cancer cells to secrete cytokines that signal immune cells and draws them to the tumor. For example, CCL-5 is one cytokine that can directly signal immune cells and there are others as well.

But, with Dr. Feyruz Rassool’s group at the University of Maryland, we found that the immune signaling from tumors treated with epigenetic therapy is a broader activity than we had expected. The events occur within a process termed the “inflammasome” and when this response is generated, the end result is not just strictly immune. The upregulation also causes a defect in cancer cells for their ability to repair DNA damage. One such result is creation of what is termed a homologous recombination defective state, or HRD. An important class of cancer drugs, PARP inhibitors, “feed off of” HRD to inhibit the survival of cancer cells. A classic example of this occurs in women with breast cancers that harbor a BRCA mutation which genetically creates HRD, so PARP inhibitors are often an effective treatment for such tumors.

A clinical trial is ongoing to test whether epigenetic therapy can create HRD in non-BRCA mutant breast cancers to sensitize the tumors to a PARP inhibitor. Such efficacy may also provide a therapy to further enhance immune checkpoint therapy in this setting.

PerkinElmer: How do DNMTi upregulate immune signaling in cancer? And a follow-up question: what effect do HDACi have on DNMTi function?

Dr. Baylin: HDACi’s open up chromatin, and this is synergistic with reducing DNA methylation at gene start sites to enhance gene expression. We first found this combinatorial effect in 1999 for DNA hypermethylated genes. This is what led us to use that combination to try to enhance immune checkpoint efficacy. We have explored the potential combination of drugs in more depth over the last several years in clinical trials and in the lab. Others have recently found HDACi plus DNA demethylating drugs given alone invoke a very robust response in patients with peripheral T cell cutaneous lymphomas. This approach has not yet gained FDA approval for human cancers, but continued results such as those mentioned above should increase this possibility.

PerkinElmer: Do any examples come to mind of promising clinical data on how different therapeutic combinations benefit the patient?

Dr. Baylin: There are many, many trials going on in the area of epigenetic therapy and newer drugs are coming into play. One example involves the targeting of the polycomb-group of proteins which can silence many tumor suppressor genes. Normally, such as in development, these proteins can repress such genes in the absence of start site DNA methylation but allow them to be activated when needed. A key protein for polycomb repression of gene expression is the EZH2 enzyme and EZH2 inhibitors are in clinical trials with the hope of lifting the polycomb-related gene suppression. An EZH2 inhibitor has been FDA approved for a rare form of pediatric muscle sarcoma and this is the first epigenetic therapy approved for a solid, non hematologic tumor.

Yet another example is the LSD1 molecule. There have been studies that show that LSD1 will upregulate the viral defense response, and investigators are using LSD1 inhibitors that could be combined with DNMTi to improve immune checkpoint therapy. There is a growing series of molecules in the chromatin space that could produce similar results. The trials are ongoing and over the next few years it could be that other drugs will be doing the same thing we're aiming at. The entire epigenetic therapy research field needs to use clinical trial findings to direct our next steps with all of the new drugs and combination possibilities.

PerkinElmer: What has been your most interesting or surprising finding?

Dr. Baylin: Over the years, there has been a continued series of surprises. Most recently, the cancer cell inflammasome activation mentioned earlier and how it encompasses our viral defense response was a big surprise to us. How far we can go with it, I don't know, but it's been used to create the PARP inhibitor approach that we never would have thought about.

PerkinElmer: Finally, what are your next steps and future plans?

Dr. Baylin: Our quest right now is for the use of epigenetic therapy to enhance immune checkpoint inhibitor therapies. In the lung cancer therapy trials that we're doing, we’re banging hard on the genomics that may hopefully identify clues to markers which correlate with the best responses and the longest disease free durations that we're seeing after combining epigenetic with immune checkpoint therapy. A lot of where we're going in the immediate future relies on the next few months and how all these data sort out. We’ll be pounding hard on genome-expression data to see if we get the DNA methylation changes we’re targeting, synthesizing everything and asking every question we can at the epigenome and pathways level.

If the data convinces us that the therapy is working, then we want to focus on personalizing the therapy. That is the next step in our future plans because we may be able to provide the combination therapy to those patients most likely to benefit and this will be a big step if we can achieve it. We also have hopes for combining epigenetic therapy with PARP inhibitors as was discussed earlier.

PerkinElmer: Thank you for your time, Dr. Baylin.

For Further Reading:

The emerging role of epigenetic therapeutics in immuno-oncology. https://doi.org/10.1038/s41571-019-0266-5

Inhibiting DNA methylation causes an interferon response in cancer via dsRNA including endogenous retroviruses. http://dx.doi.org/10.1016/j.cell.2015.07.011

Epigenetic therapy ties MYC depletion to reversing immune evasion and treating lung cancer. https://doi.org/10.1016/j.cell.2017.10.022

Pharmacologic induction of innate immune signaling directly drives homologous recombination deficiency. http://www.pnas.org/cgi/doi/10.1073/pnas.2003499117