Good morning, everyone. I'm Alexandra Byrne. I'm a reporter at GenomeWeb, and I will be your moderator for today's webinar. The title of today's webinar is "Beyond the Genome: Measuring Epigenetic Modifications Across Matrices for Biomarker and Drug Discovery," and it is sponsored by Volition. Our speakers today are Eric Ariazi, Chief Development Officer of HelioGenomics, and Mariel Herzog, Research and Development Director at Volition. You may type in a question at any time during the webinar. You can do this through the Q&A panel, which appears on the right side of the webinar presentation. If you look to the bottom tray of your window, there is a series of widgets to enhance your webinar experience. With that, I'll turn it over to Mariel.
Thank you very much. Hello, everyone. Good morning, good afternoon, good evening, wherever you might be. I'm delighted to be here with you today for this webinar focusing on Nu.Q Discover and how Nu.Q technology could help to accelerate epidrug development. About Volition, Volition is a multinational company. We have colleagues all around the world with two labs, one R&D lab and service in Belgium, and one laboratory in the U.S. in San Diego. Our mission is really to save lives and improve the outcome of millions of people worldwide. Before diving into the core of this presentation, let's take a moment to have a look at the background behind the Nu.Q technology. Nucleosomes are the fundamental structural unit of chromatin. They play a crucial role in compacting DNA into chromosomes and allowing the genome to fit into the cell nucleus.
Those nucleosomes, in case of cell death or high cell turnover, chromatin is fragmented, and the nucleosome can be released into the environment or the blood, like the blood flow. Nucleosomes are composed of approximately 140 base pairs of DNA wrapped around a histone called proteins. Histones, and especially the histone cell, are subject to histone post-translational modifications, also referred to as epigenetic modifications, such as methylation, acetylation, phosphorylation, oxygenation, and many others. Those modifications can be added by enzymes, also known as writers, could be removed by erasers, or could serve as a docking site for other proteins. Writers, erasers, and readers could be targets for epidrugs. Those modifications are key regulators of cellular processes, like regulation of gene expression, DNA damage, or cell differentiation. Alterations of nucleosomes or histone PTM levels can lead to disease, like cancer, but also inflammatory disease, autoimmune, or neurodegenerative disease.
Nu.Q assay consists of a sandwich immunoassay with a capture antibody targeting specific epigenetic features present on the nucleosome and with an antinucleosome antibody in detection. Those assays are quantitative. Results are expressed in nanograms per mL. They allow us to determine the level of circulating nucleosomes, but also to profile the nucleosomes. The assays are analytically validated in K2- EDTA plasma, but we will see in a couple of slides that they can be used in other types of matrices. Only a few microliters are required to perform the assay. Today, the Nu.Q Discover portfolio consists of 14 different assays, each assay targeting a specific epigenetic modification, like methylation, acetylation, or auto PTM. We have also a test targeting a mutation.
Those tests are highly specific, with no or very limited cross-reactivity, are highly reproducible, and can detect up to a few nanograms per mL of nucleosomes in the blood. This portfolio can be and will be expanded, and our experts are available to support you for any of your histone PTM needs. All those assays are available on an automated, 10 mm immunoassay platform using magnetic beads. Two of them are also available as a manual ELISA assay, and a point-of-care test is currently in our development pipeline. As I mentioned previously, all those Nu.Q assays can be used with multiple different types of matrices, from cell culture using supernatant or cell chromatin extract to tissue and blood, with not only plasma but also some blood cells in animal models or in humans, allowing to cover all the different steps of the drug development from discovery, preclinical to clinical.
Just two examples. The first one on a cell line, where cell lines were treated either with an HDAC inhibitor here on the left or with an EZH2 inhibitor. Cells treated appear in the red and untreated in gray. Results are expressed as a ratio of the PTM over the nucleus 2.1. As you can see, for the HDAC inhibitor treatment, we observe a clear decrease of the level of acetylated marks in the treated cells without affecting the methylation mark, K9 or K27 methylation. Whereas for EZH2 inhibitor, which targets H3K27 methylated marks, we observe a decrease of this mark in the treated cells, but specifically of the H3K27Me 3, but no effect on H3K9Me 3 or any other acetylated mark.
A second example, after chromatin extraction, Nu.Q assays were able to profile the epigenetic modifications present on different types of tissue, for example, here, lung, pancreas, prostate, and also to compare normal tissue versus a tumor. Results are represented here as a heat map, where the red shows the highest level of histone PTM, of specific histone PTM, represented as a ratio, and the blue as the lowest level. A third example, where a Nu.Q assay can track epigenetic modifications on blood cells, for example, PBMC or monocytes, and you can see that you can really have the different profiles according to the different cells. I've given you a global overview of this technology.
Now I will hand over to Eric, who will show you some studies and results illustrating how the Nu.Q Discover technology can be used, especially in plasma, to really investigate key parameters like tumor burden, dose optimization for drug discovery, pharmacodynamic toxicity, but also to evaluate predictive biomarkers or discover new biomarkers. Eric?
Thanks, Mariel. That was a nice introduction of the Volition technology, and now I can take it from here. I'm a translational cancer biologist and biotechnology executive with over 25 years of experience spanning academia and industry, advancing precision oncology and epigenetic assays from discovery through clinical development. Currently, I am the Chief Development Officer at Helio Genomics. I lead cross-functional teams to develop blood-based multi-omic cancer diagnostic assays. Some quick disclosures. I'm a former employee of ORIC Pharmaceuticals, and I have stock in ORIC Pharmaceuticals. I will be showing some data about ORIC's EED inhibitor, ORIC-944, and the case study data were originally presented from posters presented at prior AACR meetings. I'm also a former independent consultant for Volition. First, I'd like to talk to you about an overview of histone modification. As Mariel mentioned, histones can be modified by writers and erasers. Writers deposit either acetylation or methylation marks.
Acetylation marks open up the chromatin so it's more accessible to transcription factors and are associated with increased transcription. Methylation marks can either increase chromatin accessibility or decrease it to regulate transcription. For example, while H3K4 bi- and trimethylation marks usually occur at active genes, H3K9Me3 and H3K27Me3 marks are typically associated with gene repression. The Nu.Q assays for monitoring a variety of epigenetic marks are shown on the sides of the slide. At the bottom of the slide is a schematic showing the main methylation sites on histones H3 and H4. The methyl transferases are shown on top of the histone map and the demethylases at the bottom. As a case study, we will focus on the PRC2 complex with its EZH2 catalytic subunit that trimethylates H3K27. Let's start by talking about detecting cancer using these Nu.Q assays for circulating cell-free nucleosomes.
Volition collaborators took a panel of Nu.Q cell-free nucleosome assays for trimethylation of H3K27, H3K36, H3K9, and dimethylation of H3K4. They looked at the levels of these marks in non-small cell lung cancer samples versus healthy controls. They found that trimethylated H3K27 showed the highest increase of the markers in the panels, with increases of approximately 3.7 or 2.9-fold in training and validation data sets when comparing the medians between the non-small cell lung cancer group and the healthy group. Further, H3K27Me3 levels showed the best area under the receiver-operator characteristic curve, or ROC curve, with values of approximately 0.9 in the data sets and with a sensitivity of about 70% at 90% specificity. Based on these results with H3K27 trimethylation showing diagnostic potential, the Volition collaborators performed an IHC staining study in non-small cell lung cancer tissues compared to healthy tissues.
What they saw was variable H3K27Me3 staining, with increased staining associated with grade. Specifically, there was a lack of strong H3K27Me3 staining at grade 1, but increased staining, strong staining at Grade 2 and at Grade 3. Further, Grade 3 cancers no longer showed weak staining compared to Grade 1 and 2 cancers. Thus, there was a significant association between H3K27Me3 staining and increase in grade. To investigate whether circulating H3K27Me3 nucleosomes could provide additional information for monitoring the presence of non-small cell lung cancer during treatment in combination with circulating tumor DNA, that is, cell-free DNA fragments with cancer gene somatic alterations, H3K27Me3 nucleosome levels were measured in healthy subjects and then compared to that in the non-small cell lung cancer patients along with ctDNA status.
In a healthy cohort, the upper limit of H3K27Me3 nucleosomes was established at 22.5 ng/mL , based on one standard deviation above the 95% percentile. Using this cutoff, 38.8% of non-small cell lung cancer patients were categorized as H3K27Me3 positive. A next-generation sequencing analysis using a panel containing 78 cancer genes covering the majority of drivers of non-small cell lung cancer was used to detect ctDNA. The presence of at least one somatic alteration or copy number variation was considered a ctDNA positive sample. In this non-small cell lung cancer cohort, 43.1% of patients were categorized as ctDNA positive. When considering the additive value of combining H3K27Me3 status to ctDNA status, an extra 15.1% of patients could be identified as having circulating tumor material detected. This is significant because now the number of patients can be increased that can be tracked with a blood-based assay.
Moreover, as demonstrated in a ROC curve analysis, using H3K27Me3 positive status alone achieved an AUC of 0.79. Adding ctDNA positive status improved the AUC to 0.87, demonstrating that combining the two analytes further improves tracking tumor burden as defined by liquid biopsy assays. Why would we find H3K27Me3 levels to be diagnostic in non-small cell lung cancer? EZH2 is the histone methyl transferase that catalyzes trimethylation of H3K27, and its high expression is prognostic in non-small cell lung cancer and various other cancer types. Here is a meta-analysis of non-small cell lung cancer, including 10 different studies with almost 1,700 patients. What we found was a combined hazard ratio of about 1.7, indicating that EZH2 is indeed prognostic for overall survival. Likewise, in myeloid neoplasms, we have another meta-analysis in eight different studies. Again, a very significant overall hazard ratio. This time, it was 2.4.
Furthermore, EZH2 is a prognostic indicator in multiple other cancers as shown here, including breast, glioblastoma, gastric, and gynecologic cancers. Next, how can these Nu.Q assays be used in epigenetic drug development? This is where my case study with ORIC-944 comes into play. For this case study, I would like to tell you a little bit about PRC2. PRC2 is a multi-subunit complex known to play a role in regulating stem cell identity and cell differentiation through transcriptional silencing of target genes. EZH2 resides in the PRC2 complex. The PRC2 complex also contains SUZ12 and EED subunits. SUZ12 is a scaffolding subunit, while EED is the reader subunit that allosterically regulates the EZH2 subunit. While EZH2 writes or catalyzes trimethylation of H3K27, it's the EED subunit that regulates whether EZH2 is active or not.
Elevated PRC2 activity and trimethylation of H3K27 is associated with poor prognosis in patients with metastatic lesions. First-generation EZH2 inhibitors, such as tazemetostat, have been approved in epithelioid sarcoma and follicular lymphoma. Since EED regulates EZH2, EED inhibitors offer another chance to target PRC2. At ORIC , we were interested in developing the EED inhibitor ORIC-944. This EED inhibitor is orally available with picomolar activity in biochemical assays and nanomolar activity in cellular assays. At first, it was verified using the diffuse large B-cell lymphoma model, KARPAS-422, growing xenografts in immunocompromised mice. This model has been widely used to demonstrate the efficacy of tazemetostat. In this model, we looked at tazemetostat and compared it with ORIC-944. We observed that ORIC-944 at 100 mg/kg and 200 mg/kg dosed once daily was more efficacious than 200 mg/kg tazemetostat that was dosed twice daily.
Next, we were interested in metastatic castration-resistant prostate cancer. Since EZH2 has been implicated in this advanced form of prostate cancer, we tested ORIC-944 in enzalutamide-resistant 22RV1 prostate cancers. Here, we saw a strong single-agent activity ranging from 25 mg/kg- 200 mg/kg of ORIC-944 dosed once daily. For comparison, 22RV1 tumors were resistant to the androgen receptor antagonist enzalutamide, consistent with prior reports. Now, if we look at H3K27 trimethylation levels by IHC staining in the tumors itself, we can see that at baseline, there were very high levels of H3K27Me3. With increasing doses of ORIC-944, there were increasing reductions in trimethylated H3K27 levels, demonstrating target engagement. Based on these results, ORIC-944 has gone into a Phase I clinical trial sponsored by ORIC . To implement this clinical trial, we needed to have a biomarker plan. We developed three different biomarker assays in preclinical models.
The first one is a skin punch biopsy. Mouse skin was stained for H3K27Me3 by IHC. The H3K27Me3 staining was evident in the epidermal layer of the skin and in the sebaceous gland. In mice treated with ORIC-944 for seven days, the staining disappeared. The staining was quantitative, and indeed, we saw a dose-dependent reduction in H3K27Me3 labeled cells. The advantages of this assay are there was no tumor biopsy, and we did see a dose-dependent response. The assay is only semi-quantitative, and the dynamic ranges are limiting as the range of the values is large. Additionally, scoring all these positive cells takes a long time. Yet the skin punch biopsy assay has been employed in the clinic, as demonstrated by this Phase I dose escalation study with tazemetostat. Skin punch biopsies were collected from 32 patients at baseline and on treatment.
H3K27Me3 IHC staining was performed, and it was observed in the stratum spinosum layer of skin. Significant reductions were observed, and taking this data and putting it into a maximum inhibitory effect model showed that target inhibition of the H3K27 trimethylation mark was near maximum or near about 80% in the 800 mg twice daily cohort. What this model says is if you were to double the dosage to 1,600 mg twice daily, you wouldn't have much more increase in the reduction of H3K27Me3. Therefore, the skin punch biopsy assay is a viable assay to assess EZH2 inhibitor target engagement in the clinic and can be used to inform the dosage required for maximum target reductions. The skin punch biopsy assay can be challenging if you want multiple time points, and this is not comfortable for the patient.
Next, we went on to an alpha-lyasa assay to measure H3K27 in mouse monocytes. The cell input for this assay was optimized to avoid a hook effect, where excess analytes overwhelm the finite amounts of antibodies and beads in the assay. With this assay, we saw a very large reduction, almost down to baseline. Likewise, when the EZH2/EZH1 dual inhibitor CPI-0209, now known as tulmimetostat, was being evaluated in a first-in-human trial, a similar monocyte assay was employed. Again, a dramatic reduction in H3K27Me3 levels was observed in human monocytes. However, if you look at the dosages, you can see that even the lowest dose achieved a near maximal reduction, and not much more reduction was seen with increasing dose. In summary, with this monocyte assay, the advantages are, again, there's no tumor biopsy, and you can perform longitudinal sampling, but it requires monocyte isolation from whole blood.
Hence, you first need to collect whole blood from the patient and then send it to a central laboratory for monocyte isolation. Monocyte preps, after shipping whole blood overnight, even on ice, are poor because those monocytes start dying during shipping. You need to optimize the cell input into the assay to avoid a hook effect. This assay requires quite a bit of technical optimization, and then there's this lack of the dose response. After working on the monocyte assay, we wanted to look at circulating H3K27 trimethylation at the cell-free nucleosome level in plasma. For this, we used the Volition Nu.Q assay . We used immunocompromised mice implanted with 22RV1 prostate cancer cells and then treated them over the course of a week, sacrificed the animals, collected the blood, processed it to plasma, and measured H3K27 trimethylation.
After treating the mice for seven days with 10 mg/kg- 100 mg/kg ORIC-944 once daily, we saw a dose-dependent reduction in circulating levels of normalized H3K27 trimethylation. We also looked at the frequency of dosing. Over a seven-day period, we dosed for three days, five days, or seven days, and we saw a frequency-dependent decrease in normalized H3K27 trimethylation levels. We wanted to check, is H3K27Me3 coming from the tumor, or is it coming from the host cells? In essence, is it tumor selective? For this experiment, we had both tumor-bearing and non-tumor-bearing mice. They were treated again for one week. If we look at the non-tumor-bearing mice on the right side of the plot, the H3K27 trimethylation levels are already fairly low in the vehicle-treated group, and we did not see a further significant reduction in normalized H3K27 trimethylation.
When it comes to the tumor-bearing mice, the baseline levels of H3K27Me3 were elevated, and now we could see that reduction with ORIC-944. Therefore, because H3K27 levels are low in non-tumor-bearing mice but high in mice with tumors, we concluded that H3K27Me3 is indeed selectively coming from the tumor. Furthermore, we concluded this blood-based assay is capable of reading out histone post-translational modification target engagement in the tumor itself. The same exact circulating H3K27 trimethylation assay normalized to total H3.1 was used by another group of Volition collaborators that were working on the CARE clinical trial. The CARE trial is a Phase II study testing tazemetostat in combination with an anti-PD-L1 antibody. As shown in the left panel, an evaluation of 191 patients' samples across multiple time points and aggregate shows that at cycle one, day one, the H3K27Me3 over H3.1 ratio was high at 0.56.
At day one of cycle two and cycle three, the H3K27Me3 over H3.1 ratio was significantly reduced to 0.31 and was still reduced at the end of treatment. As shown on the right panel, if we take a subset of patients where all time points were valuable, we can examine the data longitudinally. This longitudinal analysis confirmed the decrease in normalized H3K27 trimethylation levels was already occurring at cycle two and was maintained throughout the end of treatment. Therefore, this assay can be used in the clinic to follow pharmacodynamic activity of EZH2 inhibitors. In conclusion, for this section, I'd like to restate that H3K27 trimethylation as a pharmacodynamic biomarker can be measured using Nu.Q assays that are both quantitative and robust. We've shown dose dependency, frequency-dependent responses, and tumor selectivity. It's non-invasive, it's already optimized, and the turnaround time is rapid, often within a week.
I'd like to point out that in the CARE trial, the trial's aim is to evaluate a combination of tazemetostat with an anti-PD-L1 antibody. Combining EZH2 inhibitors with immune checkpoint blockade or ICB therapy is a growing area of interest in the clinic because there are multiple mechanisms by which EZH2 inhibitors can augment or potentiate ICB therapy. This slide illustrates how EZH2 inhibitors can enhance cancer immunotherapy, particularly when combined with immune checkpoint blockade. Starting on the left, we see the cancer immunity cycle, the multi-step process by which the immune system detects and eliminates tumor cells. This cycle involves the expression of tumor antigens, activation of T cells, and infiltration of effector immune cells into the tumor microenvironment. EZH2 normally suppresses several steps in this cycle, such as antigen presentation, cytokine signaling, and T cell infiltration, helping tumors evade immune detection.
The right panel addresses how inhibiting EZH2 lifts this suppression. When EZH2 is inhibited, there is an increased expression of endogenous retroviruses, activation of interferon pathways, and restoration of tumor antigen visibility. This boosts the recruitment and function of CD8-positive T cells and NK cells and invariant natural killer T cells. Thus, these effects of EZH2 inhibition help convert cold tumors, those lacking immune infiltration, into hot ones that are more likely to respond to immunotherapy. By targeting EZH2, we can also disrupt immune evasion tactics by blocking regulatory T cell-mediated suppression and blocking infiltration of myeloid-derived suppressor cells. Thus, EZH2 inhibition represents a compelling approach to overcome ICB resistance and extend the reach of immunotherapy to more patients.
Here, I'd like to dive in a bit more into how EZH2 inhibition can increase the expression of endogenous retroviruses or transposable elements to trigger a viral mimicry response that promotes anti-tumor immunity. In Panel A, we see the general mechanism. Transposable elements, normally silenced by DNA methylation, become reactivated when methylation is reduced during cancer progression or treatment. This DNA hypomethylation leads to reactivation of transposable elements, which produce double-stranded RNA. This mimics a viral infection, activating sensors like IFIH1, RIG-I, and toll-like receptor 3, which in turn induce interferon beta and T cell activation. Panel B provides an example in triple-negative breast cancer, where treatment with paclitaxel alters acetylcysteine methionine metabolism and causes DNA hypomethylation. Tumors can still escape immune detection when EZH2 targets the transposable element promoters by trimethylating H3K27 to maintain their silencing despite DNA hypomethylation.
However, if we inhibit EZH2, this histone-mediated repression is lifted, allowing for transposable element re-expression and double-stranded RNA accumulation, which in turn activates the viral mimicry pathway. This enhances innate immune signaling and can make tumors more visible to the immune system, especially when combining with immunotherapy. Based on the aforementioned effects of EZH2 inhibitors helping to overcome resistance to ICB therapy, there's a growing body of clinical trial activity designed to test this in patients. The CARE trial, as shown previously and on the third trial shown here, is one such example. As the slide shows, at least six clinical trials as of 2024 are currently investigating combining an EZH2 inhibitor with an anti-PD-1, PD-L1, or CTLA4 antibody in solid tumors or in refractory diffuse large B-cell lymphoma. These trials span Phase I and II, indicating early but expanding interest in this therapeutic avenue.
This represents a strategic shift, moving from monotherapy to rational immunoepigenetic combinations to overcome resistance and broaden the reach of immunotherapy. Now I'd like to switch gears and talk about how the patient population can be expanded to offer PRC2 inhibitors to the most individuals possible. In lymphomas, the H3K27 trimethylation is elevated because there's frequently a mutation in EZH2, or there's co-expression of both EZH1 and EZH2 at very high levels that can lead to accumulation of H3K27 trimethylation. There are also other epigenetic factors that oppose PRC2, such as the SWI/SNF complex, also known as BAF and PBAF complexes in mammals, which remodel nucleosomes in an ATP-dependent fashion to regulate chromatin accessibility and allow histone acetyltransferases, or HATs, to acetylate histones.
If there are inactivating mutations in SWI/SNF subunits, such as ARID1 or SMARCA4, or in other factors like BAP1 and MLL2, then they no longer oppose PRC2, and H3K27 trimethylation accumulates. BAF and PBAF subunit members are mutated frequently in human cancers and in total are mutated in approximately 20% of all cancers. Specifically, SMARCB1 is mutated in 99% of rhabdoid tumors, a deadly pediatric cancer of the CNS, as well as frequently mutated in kidney and soft tissues, while ARID1A is also mutated in about 40% of endometrial cancers and 26% of bladder cancers. With this epigenetic antagonism between SWI/SNF and PRC2 in mind, there was a Phase II clinical trial testing the EZH1/EZH2 dual inhibitor tumametastat, where patients were recruited into six tumor cohorts. Of these, four solid tumor cohorts were based on ARID1A mutation or BAP1 loss.
Two other cohorts were lymphoma and prostate cancer because there has been activity in those indications before. Among the solid tumors, the ovarian, endometrial, and mesothelioma cohorts with ARID1A and BAP1 alterations showed good efficacy, as seen here in the waterfall plot. These cohorts achieved eligibility for Stage 2 expansion. Complete and partial responses were also observed in the lymphoma cohort. These findings support including patients with tumors containing ARID1A alterations or BAP1 loss and continuing investigations of EZH inhibitors. These clinical results bring up the question, can H3K27 trimethylation be used as a biomarker in SWI/SNF family drug development? This figure shows the BAF complex, which, as previously mentioned, is the mammalian version of the SWI/SNF complex discovered in yeast.
Considering the BAF complex, there exists a synthetic lethal relationship being exploited by pharma companies between the catalytic ATPase paralogs, BRM or SMARCA2, and BRG1 or SMARCA4, and also between the DNA targeting paralogs ARID1A and ARID1B. In the case of synthetic lethality between BRG1 and BRM, BRG1 is mutated in 10% of non-small cell lung cancer. If there is a non-small cell lung cancer with an inactivating mutation in BRG1, you can treat with an inhibitor against BRM and completely block BAF complex activity in the tumor, but not affect normal cells in the body which contain wild-type BRG1. Similarly, ARID1A is frequently mutated in up to 40% of uterine cancers. If you treat ARID1A-mutated uterine cancer with an ARID1B degrader or inhibitor, then you should again block the activity of the BAF complex in the tumor, but not in normal cells.
Now, this is important relative to H3K27 trimethylation because, as mentioned, if you completely inactivate the BAF complex in tumors, this should lead to accumulation of H3K27 trimethylation, which can be measured in circulation with a Nu.Q assay. Additionally, since SWI/SNF-mediated nucleosome remodeling also allows for recruitment of HATs to genomic regions, blocking SWI/SNF activity may also be monitored by measuring reductions in circulating acetylated marked H3K27 nucleosomes. In summary, we discussed a wide range of topics. We discussed how Nu.Q circulating nucleosomes can be used to detect non-small cell lung cancer. We examined how H3K27Me3 normalized to H3.1 can be used for pharmacodynamic monitoring of PRC2 inhibitors. We reviewed how EZH2 inhibitors could overcome ICB resistance, and we looked at how patient selection and pharma activity is expanding to SWI/SNF family member mutations, and this is another opportunity for H3K27Me3 levels to be used as a biomarker.
We've taken a lot of your time, and with that, I'd like to switch over to questions.
All right. Thank you, Mariel and Eric. As a reminder to webinar participants, if you have a question, please type it into the Q&A box in the control panel. We'd like to ask attendees to take a moment after the webinar has ended to take our exit survey to give us your feedback. We will now start the Q&A portion of the webinar. The first question will be for Mariel, which is just, where and how do I access the assays?
Thank you. You can contact directly Volition, but we are also happy to share that you can find our assay through HOLOGIC Diagenode, and you can scan this QR code to learn more how to get access through them.
All right, great. Onto the next question, which will be for Eric. You showed some data demonstrating that post-translationally modified cell-free nucleosomes could aid in the detection and monitoring of non-small cell lung cancer. Do they also serve as prognostic markers?
Thank you. I have a prepared answer for this. In the webinar, I presented several meta-analyses demonstrating that EZH2 is prognostic in non-small cell lung cancer, myeloid neoplasms, and other malignancies. Given this, it is biologically plausible that elevated levels of H3K27Me3, as a downstream product of EZH2 activity and measured on circulating nucleosomes, could also serve as a prognostic biomarker. This hypothesis is supported by a study presented at the 2025 European Lung Cancer Conference as a follow-up to the 2023 non-small cell lung cancer study shown in this webinar. An interim analysis of the follow-up study examined over 600 non-small cell lung cancer patients and demonstrated higher baseline levels of H3K27 CF nucleosomes were indeed associated with significantly worse outcomes.
Specifically, patients with high H3K27Me3 nucleosome levels had a mean survival of 13.4 months, compared to more than double the survival time of 27.4 months in patients with lower levels of this marker. The hazard ratio was 3.56, indicating a strong and statistically significant prognostic impact. It's worth noting that this analysis used a higher cutoff of 53.7 ng/mL of H3K27Me3 CF nucleosomes, as opposed to the 22.5 ng/mL threshold reported in the earlier 2023 study. This suggests that the threshold optimization may be important when applying this biomarker for prognostic purposes. Overall, these findings support the potential of H3K27Me3 cell-free nucleosomes as a non-invasive, epigenetically informed prognostic biomarker in non-small cell lung cancer. Thank you.
Great. Yeah, another question. Can you just talk about the ways in which the Nu.Q assays facilitate early toxicity detection during drug development?
Sure. The H3.1 assay as a readout of total cell-free nucleosomes in circulation can serve as an indicator of toxicity, as it provides a sensitive method for detecting systemic cell death in a patient. For instance, Volition has developed the H3.1 Nu.Q assay to detect sepsis, a condition characterized by acute cell death. The same principle applies to detecting lower levels of cell death induced by therapeutic agents. Importantly, the detection of H3.1 CF nucleosomes as a readout of cell death does not require the compound to be epigenetically active. Therefore, if a therapeutic agent induces toxicity in any tissue, cancerous or non-cancerous, it should result in increased H3.1 levels in circulation. In preclinical oncology studies, animal weight is commonly used as a surrogate indicator for toxicity. However, weight loss can also reflect other nonspecific effects, such as reduced food intake, altered metabolism, or CNS effects.
Incorporating the H3.1 Nu.Q assay alongside weight measurements can help discern true cytotoxic effects from nonspecific effects. Moreover, formal preclinical toxicity assessments can leverage this assay by measuring H3.1 CF nucleosome levels in non-tumor-bearing animals treated with an investigational compound and comparing them to vehicle-treated controls. Tissue-specific toxicity, such as hepatotoxicity, could also be evaluated using liver explant models, with H3.1 levels measured in conditioned media as a proxy for cell death. Overall, the H3.1 Nu.Q assay offers a robust and highly sensitive tool for early toxicity detection across multiple contexts in drug development. Thank you.
Yes, and then one more for you, Eric. How do the Nu.Q assays support the discovery and validation of predictive biomarkers that can identify responders and non-responders in clinical trials?
For therapeutics that target the epigenome, that is, epidrugs, it is reasonable to hypothesize that clinical responders may be enriched in the subset of patients with elevated levels of specific histone post-translational modifications targeted by the epidrug. Nu.Q assays detect specific histone PTMs, making them well-suited for patient pre-selection and predictive biomarker development. For instance, in the non-small cell lung cancer study presented in this webinar, multiple histone PTMs, including H3K27 trimethylation, were elevated in subsets of patients. In this context, Nu.Q assays could serve as minimally invasive liquid biopsy tools to pre-select those patients who are more likely to benefit from specific epidrugs.
Great, we have some questions for Mariel. I wanted to first ask, is this an immunoassay or a sequencing-based assay?
It's an immunoassay. It's a sandwich immunoassay using a capture antibody that is specific to some epigenetic feature and an anti-nucleosome antibody in detection. It's a sandwich immunoassay.
Another one for you. Someone asked, how specific is the antibody to detect H3K27Me3 versus mono and dimethylation?
Thank you. It's a very good question. In fact, you should know that one of the first experiments we did when we developed an assay is to check the specificity, and we rejected a lot of antibody there. For the H3K27Me3 Nu.Q immunoassay, we have checked the cross-reactivity with non-methylated, mono, or di, and there is no cross-reactivity with those histone modifications. H3K27 Me3 is really specific to this trimethylated mark.
One more for you, Mariel. What are the logistical considerations for sample collection, shipping, and processing, especially when using the Nu.Q platform in global clinical trials?
Yes. So indeed, there are some pre-analytical conditions or sample processing. Obviously, it will also depend on the matrices. As we mentioned at the beginning, we can work on Nu.Q assays that can be used in different types of matrices, tissue, blood, or some. Just as a first, our experts who are available to discuss with you and to guide you, but as an example for K2-EDTA plasma samples, we recommend to centrifuge your blood within the first four hours and then to store at 4°C for immediate testing. If you want to ship us, you have to be stored and shipped on ice because they have to be stored at -80°C . The other recommendation is also to avoid multiple freeze-store cycles. It's much better to have one or maximum two freeze-store cycles before analyzing your blood samples, for example.
Another for you, Mariel. Somebody asked, is the Diagenode test FDA approved?
Not yet. This test is a research-use-only test for now.
Great. We'll pop back to Eric. A question for you. How do Nu.Q assays integrate with complementary biomarker modalities such as ctDNA and circulating tumor proteins to track responses to therapeutics?
Sure. Nu.Q assays can be integrated with other biomarker platforms that use blood, such as genomic and proteomic liquid biopsy assays, and this provides a more comprehensive view of tumor biology and treatment response. A compelling example comes from the non-small cell lung cancer story presented here, where ctDNA sequencing identified 43.1% of patients undergoing treatment. In parallel, Nu.Q assays targeting histone PTMs such as H3K27Me3 detected additional patients, capturing 15.1% of cases not identified by ctDNA alone. This demonstrates Nu.Q assays can serve as complementary tools to ctDNA sequencing, improving sensitivity in detecting circulating tumor material and enhancing the overall ability to monitor tumor responses or recurrence of minimal residual disease. In addition, Nu.Q assays can complement existing circulating protein tumor markers.
For example, tumor protein markers like CEA in colorectal, breast, lung, and GI cancers, or CA-125 in ovarian cancer, or PSA in prostate cancer are commonly used to monitor treatment response. Given that EZH2 is a prognostic factor across these tumor types, the Nu.Q H3K27Me3 assay could potentially identify patients not captured by these traditional tumor markers. This combination can broaden the range of patients effectively monitored by liquid biopsy, particularly in cancers with heterogeneous expression profiles.
OK, great. I have another question for Mariel. Someone asks, is there any contamination from this free histone methylation protein other than from nucleosome? Do you need an enrichment step to enrich nucleosomes from plasma samples?
Thank you for this question. Our sandwich immunoassays are designed to detect intact circulating nucleosomes. We are not detecting any free histone. We are really detecting circulating nucleosome with specific histone PTMs. That is for the first part of the question. For the second part, no, we do not enrich our sample. You can use directly 50 microliters of plasma sample in the immunoassays. There is no enrichment. You use directly plasma.
Mariel, while I have you, can you talk a little bit about how Volition supports pharma partners in customizing these assays or developing new biomarkers that are tailored to specific drug programs?
Yeah, for sure. Another very good question. We can support and customize assays for, for example, different matrices that we have not explored yet. We are there to help to maybe optimize assays or to design protocols that would work for these new matrices, for example. We are also really open to work for and to develop new assays for new histone PTM that we have not targeted yet in our portfolio. For this, we have really a team that is dedicated to assay development. They have already developed 14 assays present in our current portfolio. They have a huge expertise to the assay development. We are really there and open to discuss with you to support your histone PTM research and really to customize any assays.
A question for Eric. How do EZH inhibitors improve the immunogenicity of tumor cells for immunotherapy?
Thank you for that question. For this, I did prepare a slide. EZH inhibitors reverse epigenetic-mediated gene repression. This leads to increased tumor visibility to the immune system and supports T cell infiltration and activation. Exciting emerging data now show that EZH inhibition also significantly enhances the efficacy of adaptive cell therapies, including CAR-T and TCR-engineered T cells across both hematologic malignancies and solid tumors. EZH inhibition does this by increasing tumor immunogenicity, which in turn supports T cell activation, persistence, and function. As shown in two recent 2025 preclinical studies published in Cancer Cell and summarized here schematically, EZH inhibition reprograms tumor cells through reduction of H3K27Me3 marks, resulting in upregulation of MHC proteins, post-stimulatory ligands, and adhesion molecules. EZH inhibition also increases inflammatory cytokine production.
These reprogramming changes make tumor cells more visible, leading to improved recruitment and engagement of CAR-T and TCR-engineered T cells, which makes the tumor cells more susceptible to T cell-mediated killing. In lymphoma models, EZH inhibition not only enhances infiltration of CAR-T cells but also prolongs interaction between T cells and tumor cells, improving cytotoxicity. Additionally, pretreating CAR-T cells with EZH2 inhibitors improves their stemness, expansion, and resistance to exhaustion, enabling better in vivo persistence and sustained anti-tumor activity. Importantly, dual EZH1/EZH2 inhibition, for instance, with tumametastat, showed even greater enhancement of adaptive cell therapy across a range of tumor antigens and cancer types, including solid tumors. Overall, EZH2/EZH inhibition acts on both tumor cells and T cells, reprogramming the tumor microenvironment to be more permissive to immune attack and supporting durable responses to adaptive cell therapies.
Ongoing clinical trials are now evaluating these combinations in relapsed refractory lymphomas and other malignancies. Thank you.
Thanks, Eric. I'm going to hop back to Mariel. We have someone in the audience who's wondering, do we need to do the centrifugation twice for plasma separation, or is one time enough?
No. For the plasma preparation, we do not need you to do twice centrifugation. You can do one or two centrifugations as you are used to. Anyway, the Nu.Q assay step includes another centrifugation step before running the assay. Basically, you can do one or two centrifugations depending on your current protocols for plasma.
Great. Thank you both. That's all the time we have for today. I'd like to thank Eric and Mariel and our sponsor, Volition. If we didn't get to your question, we'll try to follow up with our experts, and they'll be able to get back to you. Thank you, everyone, for your questions. As a reminder, please look out for the survey after you log out to provide your feedback. If you missed any part of this webinar or would like to listen to it again, an archived version will be emailed to all attendees. Thank you for joining us for this GenomeWeb webinar.