The background is that venetoclax is a drug that is an oral agent, is approved for treatment of newly diagnosed patients with acute myeloid leukaemia. As a single agent it has a very limited activity in this patient population so it’s approved in combination with other agents, namely azacitidine, decitabine or low dose cytarabine. In the patients with the relapsed/refractory setting it essentially has, again, very limited activity, underscoring the need for some novel therapy.

In our previous study we showed that in a drug, the name is Erwinia crisantaspase, or actually the short acting form of that, the asparaginase Erwinia chrysanthemi, in patients with relapsed/refractory AML depleted plasma glutamine levels. So my research is on the glutamine addiction, which is an amino acid, the building block of protein, of acute myeloid leukaemia cells. So the hypothesis was that if we combine the specific asparaginase that can deplete the plasma glutamine level can we see if there’s some synergy between the venetoclax and glutamine.

What we did is that we had the first in vitro study in the tissue culture become minus and we saw that the very low dose of the PegC or Peg Erwinia, pegcrisantaspase, that has been tested in children with ALL, acute lymphoblastic leukaemia, can specifically enhance the cell kill activity of venetoclax. So then we moved on and we did some very robust mechanistic study. We looked at the genes that are overexpressed or underexpressed after each of the treatment groups. So we had four treatment groups, one was control essentially, DMSO or the [?? 1:54], one was pegcrisantaspase, one was venetoclax and one was combination. We saw that in the combination group the number of genes that they are upregulated or downregulated was significantly more than any other group.

Then we looked at what genes are upregulated or downregulated. One of the genes that was up was the gene that is important for the protein translation. What is protein translation? In the dogma of the genetic we have a DNA, that the DNA gets transcribed to mRNA and that mRNA gets translated to protein and protein makes us us, essentially does the functioning. From the transcription to translation is a very complex machinery, how our gene codes get converted to the amino acid which is the important thing. So we did some very robust mechanisms, again in the transcriptome and also the translatome, and we found that this combination of the pegcrisantaspase and venetoclax significantly interfered with what we’re calling a cap complex formation. Cap is when the ribosome, which is the machinery you’re making protein is bound to the mRNA which is coming from the DNA and starts the protein. So the initiation of protein translation essentially was shut down in these cells that were treated with venetoclax and pegcrisantaspase. That was a very, very robust finding and we proved it in multiple different ways.

One important thing is one of the proteins that is downstream of this protein translation, the name is MCL1 which is a protein that if it’s overexpressed prevents the cells to die. That’s one of the mechanisms of resistance to venetoclax which is an approved drug. So we saw that this combination significantly inhibited the MCL1 as is controlled by the protein translation we’re calling EIF4E, which is one of the proteins that starts the initiation.

So this is all very good, so we’ve had an in vitro study, we had a very robust mechanism of action study, we said, ‘Can this work in humans or, sorry, actually in an animal?’ So we combined this combination in animal, we saw that no animal had lost weight. Losing weight more than 20% of the weight is one of the major toxicities in the in vivo or animal model. Also we did not see a very significant change in the serum chemistry and white blood cell was decreased which is kind of the mechanism of the venetoclax but it wasn’t that much compared to many other combinations.

Then we moved on to the efficacy data. We had a cell line that is a very, very complex karyotype. Having a complex karyotype or a complex cytogenetic is, by far, the worst prognosis in patients for acute myeloid leukaemia. So we had this cell from a patient that had a relapsed AML after some myelodysplastic syndrome and the patient had a very complex karyotype. We turned those cells into a cell that we can inject into mice and then we added some chemical, the name is luciferase. Essentially the leukaemia cells, when they grow, you can monitor them by imaging the animal. So we carried this study for almost six months in the animal model which is very interesting, a lot of the time you don’t go for six months, and we saw that the Ven-PegC, for example by 36[?], as in the abstract, we did not find any detectable disease in the combined group compared to each of the other groups as well as the control group. Then we saw that other animals in other groups started dying versus no animal in the Ven-PegC group reached the level that they died by their leukaemia. We showed this in the photon intensity, all of them were highly statistically significant, showing essentially this combination, which I coined as a new regimen that’s never been tested before, before this EHA. So this is essentially proposed to the world this combination that I coined the name Ven-PegC, venetoclax and pegcrisantaspase, we saw that this is a far superior regimen compared to any other control in a very, very highly resistant model of leukaemia.

Importantly, we said that this by chance or not so we repeated the study and we saw again the same things. In the second cohort, again in the Ven-PegC group, essentially there was negligible leukaemia. In the second cohort what we did after, so this time we didn’t go that long, after about a month we sacrificed all of the animals and then we isolated their bone marrow cells and we took those and did the same mechanistic study. The mechanistic study is showing that the mechanism that we saw in vivo was exactly similar to the mechanism that we saw in vitro in tissue culture. So that validated our mechanism of action. The second part was the pharmacodynamics. I told you that I’m a glutamine person, so we measured amino acid levels in the plasma of these animals and we saw that by day 33 essentially there was zero glutamine and zero asparagine, which is completely going by the mechanism of action of the pegcrisantaspase. So essentially we depleted the plasma glutamine and asparagine. As the glutamine gets converted to glutamate and ammonia we see that glutamine goes zero, the level of glutamate goes up.

So, in conclusion, essentially this Ven-PegC, it’s a completely new regimen that in vivo is extremely promising. Essentially in the preclinical model the story cannot get better than this. It’s everything makes sense, from the mechanism of action – in this combination it’s essentially targeting the ribosomal protein synthesis which is the major drug resistance mechanism. We showed that it’s tolerable, we don’t kill animals, and we are hoping that very soon we are planning to have a phase I study in patients with relapsed refractory AML. Particularly I’m very interested to see this result in those patients with the complex karyotype, which unfortunately we see a lot of those patients, and those patients, again, after the transplant still the mortality rate is very, very high.

So this is a very novel regimen, in summary, that I’m hopeful that we can apply that to a very resistant type of AML.