With a projected increase in the incidence of diabetes, finding a cure for insulin-dependency is imperative to improving the quality of life for millions worldwide. Currently, the only restorative treatment remains the transplantation of whole-pancreases or more commonly islet transplants. However, these are limited by the number of donors available. Therefore, finding therapeutics that can replace the endocrine β-cell mass is crucial for the treatment of insulin-dependent diabetics (1, 2).

 Although the presence of pancreatic progenitors within the ductal niche of the adult is still a matter of debate, previous studies have shown the ability of the pancreas to regenerate following extensive injury either through pancreatomies or chemical ligation. This was an interesting avenue to explore given the potential of endogenously stimulating the restoration of insulin-producing cells.

Previous work from members in our group demonstrated that not only were α-cells capable of giving rise to β-cells, but that these cells arose from the ductal niche, hinting at their progenitor status.  This work was conducted in mice that were reprogrammed to overexpress the β-cell gene Pax4 or downregulate the α-cell gene Arx. Importantly, we were able to show that a key gene involved in initiating endocrine development, NGN3, was differentially methylated, hinting at an epigenetic barrier to the process of regeneration (3). However, the epigenetic mechanisms that govern endocrine progenitor regeneration in humans are poorly understood.

The EZH2 protein is responsible for trimethylation of lysine 27 on the histone 3 (H3K27me3) of chromatin, a mark that is associated with silencing of genes associated. While the protein plays an important role in the development of the pancreas, it has also been shown to maintain stemness of progenitors in other systems. The rare opportunity to examine fresh tissue resected from a donor allowed us to show that it is now possible to partly restore insulin gene expression from pancreatic ductal cells by converting the refractory nature of chromatin using GSK126, an FDA-approved EZH2 inhibitor. However, given the n=1 nature of the study, questions into the generalisability of the study, as well as the extent of regeneration remained. Moreover, questions persisted on the significance of default suppression and whether reducing H3K27me3 to restore gene expression are sufficient to influence protein expression in situ.

Our current work was prompted by the death of a juvenile (7 yrs of age and 1 month diabetes duration) with newly diagnosed T1D along with a long-term adult T1D (61 yrs of age and 33 yrs diabetes duration) and a healthy non-diabetic, which allowed us to gain further insight into the regenerative process, as well as conduct functional studies to ascertain the maturity of the newly formed insulin-producing cells. Furthermore, to characterise regenerative outcomes, we subsequently evaluated Tazemetostat, a selective-competitive inhibitor of EZH2. Building upon recent and previous studies, we expand age-independent endocrine reprogramming of exocrine tissue isolated from type 1 diabetics.

In this study, gene expression analysis of GSK126 or Taz stimulated cells from a juvenile T1D donor demonstrated elevated expression of the master regulator of pancreatic endocrine cells, NGN3. Indeed, the transcriptomic analyses of our study identify genes critical for pancreatic function, β-cell development and insulin regulation in the juvenile T1D. RNA sequencing of cells stimulated with GSK126 and Taz in juvenile T1D donor showed elevated expression of genes that is indicative of coordinated β-cell neogenesis, as well as genes involved in the preservation of progenitor cells, and the differentiation and maturation of β-cells. Strikingly, this was correlated with not only the synthesis of insulin, as determined by immunofluorescence, but the secretion of insulin in response to high glucose conditions. Indeed, these studies suggest for the first time the regenerated β-cell-like cells ability to respond dynamic changes to glucose levels.

The ability to reactivate transcriptional activity of key regenerative genes by EZH2 inhibition in human pancreatic ductal epithelial cells is in accordance with chromatin modification and reduced H3K27me3 (4). Whereas bivalency protects reversibly repressed genes from default silencing, EZH2 inhibition effectively elevated H3K4me3 thereby influencing regenerative competence. Collectively, our findings from human ductal cells not only shine a light on the regulatory mechanisms governing pancreatic function but also underscore the plasticity of ductal cells derived from the pancreas. Although other regulatory factors will need to be considered, including more efficient methods of regeneration, our data reveal details of transcriptional control by targeting EZH2 to adopt a β-cell-like cell phenotype and contribute to insulin secretion.

Despite drug free conditions at 96 h, robust transcriptional output remains and closely corresponds with reduced H3K27me3 gene content. The therapeutic implications of these findings for β-cell regeneration are clearly complicated. First, the human data supports targeting EZH2 to influence regenerative indices associated with β-cell development. However, the reversibility of the effects upon drug removal highlights the importance of sustained modulation of the epigenetic landscape to achieve long-lasting therapeutic outcomes. Moreover, it underscores the need for further studies to understand the temporal dynamics of EZH2 inhibition and its potential impact on β-cell differentiation.

While chromatin coupled regulation of differentiation was confirmed, we cannot rule out sub-optimal in-situ conditions used did not parallel the exact nature of the pancreatic exocrine milieu. An undefined fraction of responsive ductal progenitors remains possible (5). Nevertheless, it is likely that this second case report of T1D including non-diabetic donors and the detailed studies outlined will open a window to examine the refractory nature of chromatin mediated H3K27me3 silencing which can be reversibly targeted to restore transcriptionally permissive H3K4me3 gene content and β-cell differentiation.

References

1.          Gruessner RWG. The current state of clinical islet transplantation. Lancet Diabetes Endocrinol. 2022 Jul;10(7):476-478. doi: 10.1016/S2213-8587(22)00138-3.

2.          Shapiro AM, Pokrywczynska M, Ricordi C. Clinical pancreatic islet transplantation. Nat Rev Endocrinol. 2017 May;13(5):268-277. doi: 10.1038/nrendo.2016.178.

3.          Al-Hasani K, Khurana I, Mariana L, Loudovaris T, Maxwell S, Harikrishnan KN, Okabe J, Cooper ME, El-Osta A. Inhibition of pancreatic EZH2 restores progenitor insulin in T1D donor. Signal Transduct Target Ther. 2022 Jul 22;7(1):248. doi: 10.1038/s41392-022-01034-7.

4.          Naina Marikar S, Al-Hasani K, Khurana I, Kaipananickal H, Okabe J, Maxwell S, El-Osta A. Pharmacological inhibition of human EZH2 can influence a regenerative β-like cell capacity with in vitro insulin release in pancreatic ductal cells. Clin Epigenetics. 2023 Jun 12;15(1):101. doi: 10.1186/s13148-023-01491-z.

5.          Bonner-Weir S, Toschi E, Inada A, Reitz P, Fonseca SY, Aye T, Sharma A. The pancreatic ductal epithelium serves as a potential pool of progenitor cells. Pediatr Diabetes. 2004;5 Suppl 2:16-22. doi: 10.1111/j.1399-543X.2004.00075.x.