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Andrea Miyahira: Hi, everyone. I'm Andrea Miyahira, and I'm the Senior Director of Global Research and Scientific Communications at the Prostate Cancer Foundation.

Today I am joined by Dr. Arianna Calcinotto and Nicolo Bancaro. Dr. Calcinotto is an Assistant Professor of Biomedicine at the Università della Svizzera Italiana, and group leader of the Cancer Immunotherapy Group at the Institute of Oncology Research. She is also a 2019 PCF Young Investigator.

Nicolo Bancaro is a PhD student in the lab of Dr. Andrea Alimonti at the Institute of Oncology Research. Their group has recently published the paper, Apolipoprotein E Induces Pathogenic Senescent-like Myeloid Cells in Prostate Cancer, in Cancer Cell.
Thank you both for joining me today. I am excited to hear more about the study.

Arianna Calcinotto: Hi Andrea, and hi to all of you.

Nicolo Bancaro: Hello.

Arianna Calcinotto: I am going to show you our discoveries. Basically, we knew already that the prostate tumor microenvironment was highly infiltrated by a population of immune cells to find neutrophils. In 2018, we showed in major paper that these cells, through the production of interleukin-23, were conferring castration-resistant prostate cancer and resistance to therapy. So we aimed to block this population this pathogenic population in prostate cancer microenvironment by blocking their recruitment. Because the dogma regarding this type of cells was that they had short half-life. However, we realized and we demonstrated that these cells could persist in the tumor microenvironment longer and what we discovered here was that these cells acquire features of senescence, of aged and survive for months within the tumor microenvironment. This was completely new and undefined, unexpected from this population. But what we understood better was the mechanism by which these neutrophils, that are pathogenetic in this type of cancer, can become senescent.

Basically, we identified that prostate tumor cells produce a molecule, defined ApoE, and through these molecules, the tumor cells can directly bind TREM2 molecules, specifically expressed by these neutrophils, and through this mechanism, confer long survival, basically, to this population of immunosuppressive, pathogenic neutrophils. The next step was to dissect how we could block and maybe these pathogenic neutrophils. So to have clinical relevance, Nicolo was so smart to identify that there was histone deacetylase inhibitors, so HDAC inhibitors that through the regulation and the inhibition of the machinery of the HDAC, could inhibit the transcription of TREM2. So the inhibition of transcription of TREM2 by the neutrophils were inhibited and we helped these neutrophils to become senescent, to survive, basically.

So we then envision and tested these compounds in vitro and in vivo in a clinical trial and we basically demonstrated that the HDAC inhibitors were inhibiting the process of senescent neutrophils, but even more importantly, were inhibiting and decreasing the tumor progression and the resistance to canonical therapies, such as enzalutamide.

Andrea Miyahira: Thank you, Dr. Calcinotto, for sharing that with us. Nicolo, tell me about the normal half-life of neutrophil in a healthy individual. What mechanisms typically govern their clearance?

Nicolo Bancaro: Neutrophils, very well-known to have a short half-life, around 12 to 24 hours in blood circulation. Every day, we produce billions of these cells that at the end of the day, they undergo apoptosis or different cell death. Every day, we have this new pool of neutrophils that help us to defend from infection.

In 2013, Sebas publish a very nice Cell paper in which he show how there is this recycle of neutrophils in the bloodstream and basically when they go to die, they up-regulate some receptor that bring them to the bone marrow and get cleared by macrophages.

Andrea Miyahira: Thank you.
Dr. Calcinotto, do non-senescent neutrophils divide, and do they have any functions in normal or non-cancer tissues?

Arianna Calcinotto: Non-senescent neutrophils, so canonical neutrophils, basically do not divide. They are post-mitotic cells and in the end, they have, for this reason, also short half-lives. They are considered and they are the first line of defense in our body against natural infection. Basically, what is different in the context of cancer is that in the tumor microenvironment, they find factors in the tumor microenvironment milieu that corrupt these neutrophils to become protumoral.

Andrea Miyahira: Yeah, that kind of leads into my question.
Nicolo, do you think that the tumors are attracting senescent neutrophils or are they inducing senescence in neutrophils once they arrive?

Nicolo Bancaro: What we show is that basically the neutrophils, upon arrival in the tumor microenvironment, they are, like we said, canonical, so the normal neutrophils. Some of them, they undergo apoptosis and they die. But some other, since they can catch these ApoE produced by the tumor, they can survive longer and undergoing senescence. So at the end, it's the tumor that is producing this molecule that inside the tumor environment can induce senescence.

Andrea Miyahira: Dr. Calcinotto, did your team evaluate whether senescent neutrophil numbers or other markers correlate with clinical outcomes in patients?

Arianna Calcinotto: Yes, this is something that we are actually studying now, so it's our follow-up. What we would like to understand is if they also can predict response to therapy, the presence of such population. Because you can imagine that it's quite hard to identify the cells because we need to identify the cells through very specific markers that are expressed by the cells, for example, TREM2. So we need to have before this publication, this validation that TREM2 is a marker of senescent neutrophils. Then as a follow-up, we can use the TREM2 as a marker for senescent neutrophils and test these ideas in the patients, of course.

Andrea Miyahira: I guess, Nicolo, did you find that numbers of intratumoral, senescent neutrophils in the human prostate cancer samples that you evaluated, did they correlate with any numbers or functions of T cells, and do we know of any mechanisms that they play in T cell suppression?

Nicolo Bancaro: What we found is that there is a correlation between what we call the senescent neutrophil score and the aggressiveness of the disease. We didn't went deep into the detail of whether this is correlating with an opposite site signature of T cells, so like a T cell exhaustion or so on. In mice, what we saw is that when we were removing these cells, these senescent neutrophils, we were reactivating the normal, let's say, immune cell of the body, it was able to act through elimination of the tumor cells. But in human, we still have to go on with the studies.

Andrea Miyahira: Awesome. Yeah, I'm looking forward to learning more about the mechanisms.

Dr. Calcinotto, you did a triple combination where you looked at the combination of enzalutamide, romidepsin, and a CXCR2 inhibitor. Did you evaluate the impact of this combination on prostate cancer cells alone, and what was the effect when myeloid cells were there or not?

Arianna Calcinotto: Yes. Actually, this was one of the questions of the reviewer because they were skeptical at the beginning, and they were right, to see whether HDAC inhibitors were specific against the senescent neutrophils and did not have many other side effect on the tumors, et cetera. So Nicolo performed, actually, this experiment and we clearly showed that the HDAC inhibitors was efficacious in this system, when we had the myeloid, the senescent myeloid. So we had the specific effect on the senescent myeloid.

Andrea Miyahira: Nicolo, the next question is, how would you guys translate these findings to patients? Do you think that targeting senescent neutrophils has therapeutic potential, and how would you go about specifically targeting the neutrophils? You guys identified different signaling molecules that are active in different surface molecules.

Nicolo Bancaro: Yeah, sure. I mean, the idea at the beginning of our pre-clinical trial was to find a new therapy for prostate cancer. So actually, HDAC is really promising therapy. We should go on with the studies to understand if, actually, it's true. But what we would like to develop even further is maybe some targeting antibody against some specific marker that we found on these senescent neutrophils, and the elimination specifically of these cells in the tumor microenvironment.

Andrea Miyahira: Great. Dr. Calcinotto, your group has conducted a clinical trial testing romidepsin in metastatic CRPC patients. Are there any samples you can study to learn about the mechanism of action pertaining to the neutrophils from that study?

Arianna Calcinotto: Yeah. Actually, the clinical trial was run by Professor Johann de Bono using vorinostat, not romidepsin, that is another HDAC inhibitor.

So the idea is then to see whether there is a chance to have material from that specific trial. It's not so easy because in that trial, they were not collecting biopsies on the patients. So the idea is to enlarge this clinical trial and implement the data in the potential specimen that we can collect to validate our study, and potentially then decide and define whether romidepsin could be really a good option of combination treatment for prostate cancer patients.

Andrea Miyahira: Thank you. Nicolo, what normal roles do TREM2-expressing neutrophils play? And if we were to target them, what kind of toxicities would you expect?

Nicolo Bancaro: Thank you for the question. Actually, one of the grades, the score, in this article was actually the presence of these TREM2 neutrophils. Normally, these molecules we found on macrophages and particularly in microglia. This is a very well-known marker for Alzheimer disease, it's very well-studied in Alzheimer disease. But for prostate cancer, this is new, was a few years ago, we discover a population of macrophages to test this marker and in cancer. But about normal neutrophils, there's TREM2, we don't even know if they exist.

Andrea Miyahira: Dr. Calcinotto, what are the next steps for your group on this study?

Arianna Calcinotto: So the next step, the first idea is to organize a sort of panel meeting together with all our clinicians, collaborators, to understand whether we can really run a new clinical trial to decide whether romidepsin can be used in combination with enzalutamide. The second step will be, because there's already trials using romidepsin in other type of cancer, so we'd like to understand whether these senescent neutrophils are also present in other subtypes of cancer, breast cancer, bladder cancer, and others, so that behaves somehow similarly to prostate cancer, and to see whether these findings could be somehow used also for the potential treatment of other type of cancer. Probably so for the assessment of the new clinical trial, we will go back to Prostate Cancer Foundation, asking support for that.

Andrea Miyahira: Okay. Well, I'm really wishing your team luck with such an interesting paper on very exciting findings, so we look forward to seeing what the tread to lay still, next steps are going to be. Thank you both for joining me today.

Nicolo Bancaro: Thank you very much, Andrea.

Arianna Calcinotto: Thank you.