The role of CLL cells, T-cells and macrophages and their effects on the tumour microenvironment

Prof Arnon Kater - Academic Medical Center, Amsterdam, The Netherlands

Talking about your abstract that you’re going to be presenting later on today, can you tell me what led you to research this topic and a bit about the background behind it?

Yes, the background is what we are studying actually in this research that we’re going to discuss today is that this microenvironment is very important in chemoresistance. We knew that already for quite some years that if you activate cells, so CD40 activated cells which are present in this microenvironment, that those cells become anti-apopototic and protected from chemotherapy. Interestingly we very recently published a paper in Haematologica where we showed that also on these newer drugs like ABT-199, which is a specific BCL2 inhibitor, also there, at least in vitro, you can see that if you put cells or CD40 ligands, so actually activated T-cells, you also make the cells resistant. So I think it’s still valid and very important as of today also with these new drugs to look further into this mechanism of resistance in this microenvironment and how to resolve that.

So previously we have mostly focused on T-cells but, you see this at the conference, a lot of groups actually now focus also on the monocyte macrophage. So one of the key questions we had is…  so actually we had three questions that we discussed today. The first one is how do monocytes actually get attracted to this tumour microenvironment? Is it a passive activation or do CLL cells really are able to recruit those monocytes from the peripheral blood into this tumour microenvironment? So what we did is we grew culture cells with T-cells or CD40 activated cells, so to activate them, and then what we saw is that those cells are going to produce chemo-attractants, so chemokines that actually induce the migration of monocytes from the peripheral blood to the lymph node environment. To test that we did migration assays, so we did chemotaxis. So we put monocytes in a top well which is like a transfer system; in the bottom we put CLL cells either or not activated by CD40 ligands, so by activated T-cells. And we indeed saw an increased migration from the monocytes towards the CLL compartments. Interestingly, we could also inhibit that by blocking the receptors that are actually key molecules for these chemokines. So we first found that they produced more chemokines by CD40 activation and secondly we found that indeed this production of chemokines results in increased chemotaxis.

So then the next question was if those monocytes are attracted to these activated CLL cells, what’s the phenotype? Is that an anti-tumour or more a pro-tumour, tumour supportive, phenotype? So we looked at the phenotype and, I don’t know if you’re aware of that, but you have two types of macrophages, an M1 macrophage, actually there are more but these are the most predominant ones, and an M2 macrophage. An M1 macrophage has more an anti-tumour, a more Th1 type macrophage, and a M2 macrophage is a more pro-tumour phenotype. Both by flow cytometry but also on immunohistochemistry of lymph nodes derived from CLL patients we found indeed that those monocytes that were attracted to this tumour microenvironment, to the lymph node, get a more M2 type phenotype.

So the third and last question that we then addressed is, OK, so these monocytes are actively attracted to the CLL microenvironment so does this draw any survival advantage of the CLL cells to have these macrophages in proximity. What we did is we co-cultured the cells with activated monocytes and macrophages and we saw indeed that they give a significant induced survival advantage. Then we looked to cellular components, molecular components of the apoptotic machinery to see why are those cells better at survival. What we found is that of all the anti-apoptotic molecules that you have, BCL2, BCL-xL, BCL-W, and only MCL1 was induced to express by macrophages. Then the question was why does it happen? What is the reason for this increased expression of MCL1, of this anti-apoptotic molecule in CLL cells post macrophage stimulation? So we have three ways, a cell has three ways, to increase expression of a protein: it can either be increased transcription so you have more RNA transcribed, or you have decreased. So you can have either increased translation or you have a decreased turnover. MCL1 is a labile protein so it turns over pretty rapidly. One of the mechanisms could be that the turnover could be inhibited by the macrophage. Both were not seen, so both translation and turnover was completely equal in stimulated and unstimulated CLL cells. So then the third one that was left over was increased translation. The translation means that there is more RNA actually is used for a protein synthesis. Indeed, what we found is that there was more RNA bound, polysomal RNA, meaning RNA that has ribosomes on top of it so actually it’s actively transcribed into protein, that was found and we confirmed that it could be inhibited by some key molecules that are actually activated by macrophages, including phospho-Akt which could be inhibited by PI3K inhibitors like we have in the clinic now, like idelalisib, but also by inhibitors of mTOR.

So, to round it up, what we found is that macrophages are actively attracted to the CLL microenvironment, that it can be inhibited by inhibiting chemokines that are produced actually by the CLL cells. And, third, that the increased survival is supported by increased expression of MCL1 which is on the translational regulation level and that this can be inhibited by either Akt inhibitors, PI3K inhibitors or mTOR inhibitors.

What do you think the next steps in your research will be? Where will you take this next?

Yes, I think it would be of interest indeed to… What we don’t know yet, more from a biology perspective, is we know that this macrophage is doing all those things, it’s increasing MCL1 by activating the phosphor-Akt levels so activating mTOR but we don’t know which receptor is responsible for that. So what we do know now is that it’s context bound so if you just take the supernatant of macrophages and you put them to CLL cells much less of an effect is seen when you put them into direct contact. So there is a contact due factor and a receptor ligand interaction but still we don’t know exactly that is. So we will try to find that now. I think that’s a way to go and the other interesting thing is, indeed, if those drugs that we have already in the clinic, idelalisib or maybe mTOR inhibitors, are indeed effective by inhibiting this MCL1 which induces the macrophages that’s still the proof that we don’t have so far.

For CLL patients, what will this mean down the track if things go to plan?

I think that all those new drugs, like the kinase inhibitors but also BCL2 inhibitors, are extremely potent but don’t provide a cure. For BCL2 inhibitors I think that the research that we did points that you always might keep residual cells in the tumour microenvironment to overexpress MCL1 or BCL-xL or other molecules that are not targeted by the BCL2 molecule. I think if we could better understand how we could inhibit those signals, for instance in the case of macrophages by maybe inhibiting translation, by inhibiting kinases etc., instrumental for translation that those drugs may even become more effective. For a clinical goal I think it’s much better if we do get combination therapies that we are able to stop treatment and don’t continue treatment until we have a relapse or such refractory disease that we can’t do anything anymore.

Is there a take home message you’d like to give?

Yes, I think the take home message is that we start to unravel much much more about the tumour microenvironment and that’s actually the key target of therapies. By understanding more of those microenvironments we will be better in really inhibiting the different pathways that are involved so we can indeed come up with smart rational combination therapies which we will test in the clinic.