Epigenetic Drug Combination Alters Methylation Patterns in Patient-Derived Xenograft Models of KMT2A-Rearranged Pediatric AML Treated In Vivo

Pediatric acute myeloid leukemia (AML) patients possessing rearrangement of the KMT2A (previously known as MLL) gene on 11q23 constitute a subclass with a particularly poor prognosis. The five-year survival rate for these patients is only about 44% due to poor response to conventional chemotherapy and frequent early relapse. Aberrant epigenetic modifications play an important role in leukemogenesis in KMT2A-rearranged leukemia. Accordingly, several epigenome modifying drugs have been tested in preclinical studies of KMT2A-rearranged leukemia. Acknowledging the co-regulatory effects of DNA methylation and histone modifications in determining chromatin structure and governing gene expression, we combined DNA hypomethylating agent azacitidine with histone deacetylase inhibitor panobinostat in the hopes of achieving greater efficacy. We showed that this epigenetic drug combination was more efficacious than single agents using cell line derived xenograft models of pediatric AML (Gopalakrishnapillai et al., Leuk Res, 2017). We evaluated the efficacy of this epigenetic drug combination in patient-derived xenograft models of KMT2A rearranged pediatric AML and observed that similar to MV4;11 model, this combination induced complete remission in NTPL-146 model with KMT2A-MLLT1 fusion (Fig. 1A, P<0.001). We analyzed the methylome of AML cells harvested from xenografted mice treated with control, azacitidine, panobinostat, or a combination of the two. Methylation sensitive restriction endonucleases were utilized to fragment genomic DNA prior to library construction for next generation sequencing. GenPro software platform designed for highly quantitative, sensitive, and low error-rate detection of methylation at individual CpG sites was used. Methylation patterns between treatment groups were discriminated using an ordinate analysis technique of non-metric multidimensional scaling (NMDS) (Fig. 1B). CpG methylation profiles were compared among the four groups analyzed to isolate patterns conserved within groups while also differing between groups. The first two component axes were plotted to locate the individual sample points in a 2D plane. Samples from distinct PDX models undergoing similar treatment clustered together. The panobinostat-treated samples showed minimal differences compared to the control, while the azacitidine-treated samples clustered away. Interestingly, the samples treated with the combination, did not overlap with either treatment, indicating that although panobinostat alone showed minimal impact on methylation patterns, panobinostat together with azacitidine produced a distinct methylation pattern. Venn intersection sets of statistically significant differentially methylated CpG sites in the 3-way analyses derived from the control group comparisons showed 2086 CpG sites exclusively altered in the combination treatment (Fig. 1C). In order to determine the effect of the combination treatment on global methylation, the differences in methylation load (dML) per each CpG site between control and the combination treatment were summed across 1MB genome intervals and the distribution of these dML was plotted (Fig. 1D). There was a strong shift in methylation signal, with the majority of the intervals being hypomethylated in the treatment group compared to the control. Although global hypomethylation was observed in combination treatment, the most statistically significant CpG sites were hypermethylated in the combination treatment compared to the control as seen in the volcano plot in which log fold-change was plotted against the p-value (Fig. 1E). Circular ideogram presented with a mean subtraction of CpG methylation scores to calculate a summation methylation load score across chromosomal domains (Fig. 1F). The correlative association between top CpG sites is shown as arcs tracking the highest correlation for each CpG site. Gene labels indicate the positions of the top 60 CpG sites, with green and red indicating higher methylation in control and in combination treatment respectively. In conclusion, we have identified differential methylation patterns following in vivo treatment of KMT2A rearranged pediatric AML xenograft models with azacitidine and panobinostat combination compared to azacitidine alone. These methylation changes are likely to influence the increased survival seen in mice receiving combination treatment. Figure 1 Figure 1. Disclosures Gopalakrishnapillai: Geron: Research Funding. Marsh: Genome Profiling LLC: Current Employment. Barwe: Prelude Therapeutics: Research Funding.

Tracking chromatin state changes using µMap photo-proximity labeling

Interactions between biomolecules, particularly proteins, underlie all cellular processes, and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels, or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects. Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health. However, in the complex environment of the nucleus it is challenging to determine protein-protein interactions due to low abundance, transient or multi-valent binding, and a lack of technologies that are able to interrogate these interactions without disrupting the protein binding surface under study. Chromatin remodelers, modifying enzymes, interactors, and transcription factors can all be redirected by subtle changes to the microenvironment, causing global changes in protein expression levels and subsequent physiology. Here, we describe the Chroma-µMap method for the traceless incorporation of Ir-photosensitizers into the nuclear microenvironment using engineered split inteins. These Ir-catalysts can activate diazirine warheads to form reactive carbenes within a ~10 nm radius, cross-linking with proteins within the immediate microenvironment for analysis via quantitative chemoproteomics. We demonstrate this concept on nine different nuclear proteins with varied function and in each case, elucidating their microenvironments. Additionally, we show that this short-range proximity labeling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. Chroma-µMap improves our fundamental understand-ing of nuclear protein-protein interactions, as well as the effects that small molecule therapeutics have on the local chromatin environment, and in doing so is expected to have a significant impact on the field of epigenetic drug discovery in both academia and industry.

Tracking chromatin state changes using μMap photo-proximity labeling

Interactions between biomolecules, particularly proteins, underlie all cellular processes, and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels, or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects. Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health. However, in the complex environment of the nucleus it is challenging to determine protein-protein interactions due to low abundance, transient or multi-valent binding, and a lack of technologies that are able to interrogate these interactions without disrupting the protein binding surface under study. Chromatin remodelers, modifying enzymes, interactors, and transcription factors can all be redirected by subtle changes to the microenvironment, causing global changes in protein expression levels and subsequent physiology. Here, we describe the Chroma-μMap method for the traceless incorporation of Ir-photosensitizers into the nuclear microenvironment using engineered split inteins. These Ir-catalysts can activate diazirine warheads to form reactive carbenes within a ~10 nm radius, cross-linking with proteins within the immediate microenvironment for analysis via quantitative chemoproteomics. We demonstrate this concept on nine different nuclear proteins with varied function and in each case, elucidating their microenvironments. Additionally, we show that this short-range proximity labelling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. Chroma-μMap improves our fundamental understanding of nuclear protein-protein interactions, as well as the effects that small molecule therapeutics have on the local chromatin environment, and in doing so is expected to have a significant impact on the field of epigenetic drug discovery in both academia and industry.

Genetic and Epigenetic Therapies for β-Thalassaemia by Altering the Expression of α-Globin Gene

β-Thalassaemia is caused by over 300 mutations in and around the β-globin gene that lead to impaired synthesis of β-globin. The expression of α-globin continues normally, resulting in an excess of α-globin chains within red blood cells and their precursors. These unpaired α-globin chains form unstable α-hemichromes that trigger cascades of events to generate reactive oxygen species, leading to ineffective erythropoiesis and haemolysis in patients with β-thalassaemia. The clinical genetic data reported over several decades have demonstrated how the coinheritance of α-thalassaemia ameliorates the disease phenotype of β-thalassaemia. Thus, it is evident that down-regulation of the α-globin gene expression in patients with β-thalassaemia could ameliorate or even cure β-thalassaemia. Over the last few years, significant progress has been made in utilising this pathway to devise a cure for β-thalassaemia. Most research has been done to alter the epigenetic landscape of the α-globin locus or the well-characterised distant enhancers of α-globin. In vitro, pre-clinical studies on primary human erythroid cells have unveiled inhibition of histone lysine demethylation and histone deacetylation as potential targets to achieve selective downregulation of α-globin through epigenetic drug targeting. CRISPR based genome editing has been successfully used in vitro to mutate α-globin genes or enhancers of α-goblin to achieve clinically significant knockdowns of α-globin to the levels beneficial for patients with β-thalassaemia. This review summarises the current knowledge on the regulation of human α-globin genes and the clinical genetic data supporting the pathway of targeting α-globin as a treatment for β-thalassaemia. It also presents the progress of epigenetic drug and genome editing approaches currently in development to treat β-thalassaemia.

Chemoinformatic Characterization of Synthetic Screening Libraries Focused on Epigenetic Targets

The importance of epigenetic drug and probe discovery is on the rise. This is not only paramount to identify and develop therapeutic treatments associated with epigenetic processes but also to understand the underlying epigenetic mechanisms involved in biological processes. To this end, chemical vendors have been developing synthetic compound libraries focused on epigenetic targets to increase the probabilities of identifying promising starting points for drug or probe candidates. However, the chemical contents of these data sets, the distribution of their physicochemical properties, and diversity remain unknown. To fill this gap and make this information available to the scientific community, we report a comprehensive analysis of eleven libraries focused on epigenetic targets containing more than 50,000 compounds. We used well-validated chemoinformatics approaches to characterize these sets, including novel methods such as automated detection of analog series and visual representations of the chemical space based on Constellation Plots and Extended Chemical Space Networks. This work will guide the efforts of experimental groups working on high-throughput and medium-throughput screening of epigenetic-focused libraries. The outcome of this work can also be used as a reference to design and describe novel focused epigenetic libraries.

Co-Treatment with the Epigenetic Drug, 3-Deazaneplanocin A (DZNep) and Cisplatin after DZNep Priming Enhances the Response to Platinum-Based Therapy in Chondrosarcomas

Background: We have previously shown that 3-Deazaneplanocin A (DZNep) induces apoptosis in chondrosarcomas. Herein, we tested whether the combination of this epigenetic drug to a standard anticancer therapy may enhance the response to each drug in these bone tumors. Methods: Two chondrosarcoma cell lines (SW1353 and JJ012) were cultured in the presence of DZNep and/or cisplatin. Cell growth was evaluated by counting viable cells, and apoptosis was determined by Apo2.7 expression by flow cytometry. In vivo, the antitumoral effect of the DZNep/cisplatin combination was assessed through measurements of tumor volume of JJ012 xenografts in nude mice. Results: In vitro, the DZNep/cisplatin combination reduced cell survival and increased apoptosis compared to each drug alone in chondrosarcomas, but not in normal cells (chondrocytes). This enhancement of the antitumoral effect of the DZNep/cisplatin combination required a priming incubation with DZNep before the co-treatment with DZNep/cisplatin. Furthermore, in the chondrosarcoma xenograft mice model, the combination of both drugs more strongly reduced tumor growth and induced more apoptosis in tumoral cells than each of the drugs alone. Conclusion: Our results show that DZNep exposure can presensitize chondrosarcoma cells to a standard anticancer drug, emphasizing the promising clinical utilities of epigenetic-chemotherapeutic drug combinations in the future treatment of chondrosarcomas.

A novel epigenetic drug conjugating flavonoid and HDAC inhibitor confers to suppression of acute myeloid leukemogenesis

Epigenetic dysregulation has long been identified as a key driver of leukemogenesis in acute myeloid leukemia (AML). However, epigenetic drugs such as histone deacetylase inhibitors (HDACi) targeting epigenetic alterations in AML have obtained only limited clinical efficiency without clear mechanism. Fortunately, we screened out a novel epigenetic agent named Apigenin-Vorinostat-Conjugate (AVC), which provides us a possibility to handle the heterogenous malignancy. Its inhibition on HDACs was presented by HDACs expression, enzyme activity, and histone acetylation level. Its efficacy against AML was detected by cell viability assay and tumor progression of AML mouse model. Apoptosis is the major way causing cell death. We found AVC efficiently suppresses leukemogenesis whereas sparing the normal human cells. Kasumi-1 cells are at least twenty-fold higher sensitive to AVC (IC50=0.024μM) than vorinostat (IC50=0.513μM) and Ara-C (IC50=0.4366μM). Furthermore, it can efficiently regress the tumorigenesis in AML mouse model while keeping the pivotal organs safe, demonstrating a feasibility and favorable safety profile in treatment of AML. Collectively, these pre-clinical data suggest a promising potential utilizing flavonoid-HDACi-conjugate as a next-generation epigenetic drug for clinical therapy against AML.

Morphological screening of mesenchymal mammary tumor organoids to identify drugs that reverse epithelial-mesenchymal transition

The epithelial-mesenchymal transition (EMT) has been implicated in conferring stem cell properties and therapeutic resistance to cancer cells. Therefore, identification of drugs that can reprogram EMT may provide new therapeutic strategies. Here, we report that cells derived from claudin-low mammary tumors, a mesenchymal subtype of triple-negative breast cancer, exhibit a distinctive organoid structure with extended “spikes” in 3D matrices. Upon a miR-200 induced mesenchymal-epithelial transition (MET), the organoids switch to a smoother round morphology. Based on these observations, we developed a morphological screening method with accompanying analytical pipelines that leverage deep neural networks and nearest neighborhood classification to screen for EMT-reversing drugs. Through screening of a targeted epigenetic drug library, we identified multiple class I HDAC inhibitors and Bromodomain inhibitors that reverse EMT. These data support the use of morphological screening of mesenchymal mammary tumor organoids as a platform to identify drugs that reverse EMT.