Lack of New Therapies For AML Remains a Challenge For Oncologists

Acute myeloid leukemia (AML) has one of the highest unmet needs for new therapies out of all human cancers, according to Marcin Kortylewski, PhD, a professor in the Department of Immuno-Oncology at the City of Hope Comprehensive Cancer Center.

It is difficult to go after specific genetics of leukemic cells as their genetic background is very diverse and it develops very rapidly, explained Kortylewski. Once the strategies they have to control the leukemia fail, oncologists are left with very little to offer their patients.

Though new strategies are being researched such as chimeric antigen receptor (CAR) T-cell therapies, they are being met with challenges in targeting leukemic cells. According to Kortylewski, this is because new types of immunotherapies are directed against molecules on myeloid leukemia cells which can be shared with normal myeloid cells. Elimination of dendritic cells and macrophages can be fatal.

In an interview with Targeted OncologyTM, Marcin Kortylewski explained the need for new strategies for the treatment of AML.

Targeted Oncology: What has set the stage for this research in AML?

Kortylewski: I'm faculty at the Department of Immuno oncology, with a specific focus on pathways that suppress immune responses, and our interests are in targeting myeloid cells since they seem to be the information processing happening in the immune system. We basically are unlocking their potential to process information about the tumor specific antigens, which allows the immune system to work and allows T-cells and other immune cell populations to take action against the tumor. So traditionally, my lab has focused on targeting myeloid cells, which are dendritic cells, macrophages, in the context of solid tumors. But in this project, we have gone out of our comfort zone in that this is a project which involves collaboration with 2 excellent leukemia researchers who have an interest in acute myeloid leukemia.

Since this is a type of leukemia which are myeloid cells in origin, we decided that they might be an interesting target for our strategy, which is targeting myeloid cells. I haven't mentioned it oligonucleotide-based strategies for targeting different immune checkpoint regulating molecules in myeloid cells specifically. In this study, we were able to basically move and test these molecules in the context of acute myeloid leukemia and see whether we can achieve any of the immunostimulatory effects we have observed before in the context of solid tumors when targeting different types of myeloid cells and normal myeloid cells.

In terms of what you've seen in the lab with strategies for AML, why is there a need for new strategies in this disease?

AML probably has 1 of the highest unmet needs for new therapies in the context of all human cancers. It is a rapidly progressing disease, and it is very heterogeneous. Going after specific genetics of leukemic cells is extremely difficult since their genetic background is very diverse in this case, and there is this acuteness to this disease. At a later stage, it develops very rapidly so it's very hard to control. There are strategies to basically control leukemia at the initial occurrence, but then when these strategies fail, there is very little to offer to these patients except for bone marrow transplantation, which is also not available to everyone. So for recurrent leukemias, we don't have much to offer.

The new strategies from the range of immunotherapies, such as CAR T-cell therapies, also had an issue and met challenges in targeting leukemic cells. This is because these new types of immunotherapies usually go after very specific markers on the surface of myeloid cells, in this case leukemia cells, but these molecules are usually shared with normal myeloid cells, so eliminating leukemia also basically directs CAR T cells against normal myeloid cells. While in the case of B-cell leukemias, eliminating B-cells can be accommodated and while the patient can live with it, elimination of myeloid cells like dendritic cells, macrophages, neutrophils, can be life threatening. This is what basically prevented CAR T cells from success so far in this area. There is a need for new strategies and thinking outside of the box in case of AML.

Can you explain how your analysis was performed and what the findings of what you presented were?

We decided to target a molecule which is activated in acute myeloid leukemia cells before it was related to their proliferation survival. This is a transcription factor called STAT3. Jak-stat signaling was known to contribute to survival of leukemias, but we were more interested in another function of this molecule, which is regulating immune suppression. So STAT3 weakened immune evasion of leukemic cells and we developed a molecule that can target STAT3, specifically myeloid cells, and by gaining access to these cells through a receptor, it has allowed us to deliver this molecule specifically to myeloid leukemia cells.

What we observed and what was very encouraging in these initial results that we have already reported before are blocking STAT3 in acute myeloid leukemia cells, specifically in the model that we use, which is inversion(16) leukemia, CBFB-MYH11-positive leukemia, leads to very rapid regression of the leukemia, and this regression is caused not by direct cytotoxic effect on leukemia cells, but by immune mediated circulating tumor [ct]DNA responses. We also show some convincing data in this poster illustrating the extent of integration by key cells into leukemic cells in different organs, especially in the spleen and bone marrow. That leads to regression of completely rational leukemia in the majority of treated mice, which was very encouraging.

We wanted to know what is the mechanism of the action of this strategy, and what is actually driving immune responses. It turned out that when we looked at the leukemic cells, and we showed this data in this presentation, leukemic cells were undergoing very dramatic change. When we isolated them from mice, we could take a look at using electron microscopy, finding the subsolar structure completely changed. We have observed basically differentiation of these leukemic cells into a macrophage-like phenotype. Initially, we could speculate, and whether it's a macrophage monostatic or neutrophilic phenotype, but we basically confirmed this data later using gene expression studies. In about 10% of leukemic cells, these changes cause the differentiation and they tend to gain the ability to present the antigens. So, these cells that were originally leukemia and have markers of leukemic cells also express GFP, so we can track them, and they become GFP positive antigen presenting cells, which is interesting. This affects about 10-15% of leukemic cells in mice, but this is enough of the pool of antigen presenting cells to induce anti-tumor immune responses.

We decided to dig deeper into a gene expression and gene reprogramming of transcriptional events in those myeloid cells, using RNA sequencing analysis, looking at changes that are caused by a stopped B deletion, as well as by triggering activation of this reset to TLR9 that we use as a gateway into those cells, and by combination of these 2 effects. What was interesting was that RNA sequencing data has basically showed us that neither of these effects alone so TLR9 activation ignores STAT3 and deletion alone and had the same effect as a combination of these 2 effects. So, it seemed like a STAT3 elimination by silencing or by using a decoy molecule in our case, it was able to trigger dramatic reprogramming of the cells from leukemic cells into a macrophage-like phenotype that have been confirmed by gene expression level and by their activity and ability to phagocyte different molecules, creating a population of differentiated cells with expression of pyruvate or CvP alpha, and decreased expression of transcription factors which are normally found in leukemias such as RUNX1. These changes seem to be a driver of immune activation that we see in this model.

So, going forward, we focused on the regulation of 2 critical transcription factors for myeloid cell differentiation that turned out to be upregulated in our treated leukemic cells, the ones that differentiated. Our studies suggested that increased expression of these 2 transcription factors is actually a result of decreased methylation of their promoters. We are still confirming the data using methylome analysis, but it seems to be related to the activity of STAT3 and the interaction of STAT3, which is our target with a DNA methyltransferase such as DNMT1 or DNMT3A or DNMT3B, which under normal conditions leads to methylation of these promoters. When we eliminate STAT3, these genes can be upregulated, but they do require positive stimulus which is derived from TLR9 triggering, and these 2 functions are basically incorporated in our molecule, which is a CpG-STAT3.

We've finished our presentation by presenting some data that in fact, the transcription factor seems to be a critical driver of differentiation of those myeloid leukemia cell, so we are using usable construct inducible genetic construct in leukemic cells in mice, confirming that when I read eight and become silenced, we cannot fully induce differentiation of these cells anymore. So, it seems to be a required, necessary element of AML, and differentiation to this macrophage phenotype and driven by bio strategy.

We are close to completing this study in publication, and we want to add into it an analysis of methylation and status of different genes, including the 2 key transcription factors. We are very excited to be able to track down changes that underlie this dramatic shift in the phenotype of leukemic cells, which become an element of immune response and become able to reveal themselves and express their own antigens as a result of a single bifunctional treatment.

How do you see this research informing studies in the future? Are there any in human studies already planned for this strategy?

We do have some follow up studies as well as collaborations moving this molecule forward. City of Hope has been generous in funding this project of developing a CpG-STAT3 decoy molecule further and towards clinical studies. We have another related molecule that will be entering clinical trials in a few months at City of Hope. But in the context of B-cell lymphoma note acute myeloid leukemia in the molecule that we presented in this poster is the molecule that can be injected systemically, it can be injected intravenously, so it's relevant for treatment of acute myeloid leukemia and descending disease. We also collaborate with radiation oncologists at City of Hope, basically combining the use of CpG-STAT3 decoy molecule with potential irradiation of leukemia patients to allow for greater release of tumor new antigens and also slowing down of the disease. We plan for clinical trials in a few years, after confirming safety on these molecules, testing the context of acute myeloid leukemia patients.

What research do you think is next in the field?

There have been a lot of interest in basically 2 aspects of our molecule about immune stimulation through CpG, basically TLR9 receptor activation, as well as in blocking STAT3. There are several approaches combining CpG with the checkpoint blockade, that have been of interest in developing small molecules of STAT3 that have been relatively small. But I think from our data, what strikes me is the fact that it's really a combination of these 2 effects. STAT3 is a negative regulator of the immune system, but it's a brake. Removing STAT3 on its own, removing the brake doesn't really move us forward. TLR9, immune activation is like pressing the accelerator, but without removing the brake, it's not effective.

We do need those 2 effects to come together and that can effectively happen when a STAT3 inhibitor is incorporated into the immunostimulatory domain, like CpG and STAT3 decoy, the molecule will have this bifunctional effect of a single molecule. That actually allows us to cross the threshold of immune activation even in the context of myeloid leukemia cells. So, I think the by functionality is really the key. We need to go over the concept of single trait molecules and approaches and complex immunotherapy.