Research paper
Combined inhibition of menin-MLL interaction and TGF-β signaling induces replication of human pancreatic beta cells

https://doi.org/10.1016/j.ejcb.2020.151094Get rights and content

Highlights

  • Small molecule inhibition of menin-MLL and TGF-β induced human beta cell replication.

  • Combined menin-MLL and TGF-β inhibition did not affect insulin expression.

  • menin-MLL and TGF-β inhibition associated with decreased CDK inhibitors expression.

Abstract

Both type 1 and type 2 diabetes are associated with hyperglycemia and loss of functional beta cell mass. Inducing proliferation of preexisting beta cells is an approach to increase the numbers of beta cells. In this study, we examined a panel of selected small molecules for their proliferation-inducing effects on human pancreatic beta cells. Our results demonstrated that a small molecule inhibitor of the menin-MLL interaction (MI-2) and small molecule inhibitors of TGF-β signaling (SB431542, LY2157299, or LY364947) synergistically increased ex vivo replication of human beta cells. We showed that this increased proliferation did not affect insulin production, as a pivotal indication of beta cell function. We further provided evidence which suggested that menin-MLL and TGF-β inhibition cooperated through downregulation of cell cycle inhibitors CDKN1A, CDKN1B, and CDKN2C. Our findings might provide a new option for extending the pharmacological repertoire for induction of beta cell proliferation as a potential therapeutic approach for diabetes.

Introduction

In both types 1 and 2 diabetes, there is a decrease in the functional beta cell mass that leads to insufficient insulin production and hyperglycemia (Butler et al., 2003; Ritzel et al., 2006; Stumvoll et al., 2008). Approaches to reverse diabetes progression by increasing the functional beta cell mass is, therefore, of critical importance. Pancreatic beta cell expansion in rodent models has been explored for a number of years, suggesting molecular pathways that control beta cell replication (Cozar-Castellano et al., 2008; Fiaschi-Taesch et al., 2013, 2010; Kulkarni et al., 2012; Wang et al., 2015b). Notably, previous studies, as well as our unpublished data, suggest that inhibition of transforming growth factor β (TGF-β) signaling (Dhawan et al., 2016), activation of protein kinase C (PKC) (Velazquez-Garcia et al., 2011), and inhibition of the interaction between menin protein (coded by multiple endocrine neoplasia type 1 or MEN1 gene) and mixed-lineage leukemia (MLL) family proteins (Muhammad et al., 2017) can induce beta cell replication. However, little is known about the effect of combined targeting of these pathways on adult human beta cell proliferation.

Here, we explore potential mitogenic effects of different combinations of small molecules targeting TGF-β pathway, PKC activity, and menin-MLL interaction. To provide a reference for comparing our results with previous reports, we also included control groups with a dual-specificity tyrosine-regulated kinase-1a (DYRK1A) inhibitor alone or in combination with TGF-β inhibitor, which are known to stimulate human beta cell proliferation (Wang et al., 2015a, 2019). We show that the combined inhibition of TGF-β and menin-MLL interaction results in increased ex vivo replication of adult human beta cells, suggestively through downregulation of CDK inhibitors. Taken together, we propose a novel combination of small molecules that could have therapeutic potential to expand beta cell mass for the treatment of diabetes.

Section snippets

Human islet cell preparation

The Ethics Committees of the University of Geneva (Switzerland) and Royan Institute (Iran) approved all research that pertained to the human islet samples. The islets were obtained either from the NIDDK-funded Integrated Islets Distribution Program (IIDP) at City of Hope, CA, USA or Diabetes Research Institute in Milan, Italy or Alberta Diabetes Institute (ADI) IsletCore at the University of Alberta (Canada). All human samples were checked for mycoplasma contamination.

Human islet culture

For the human intact islet

Combinations of menin-MLL inhibitor and TGF-β inhibitors induce human beta cell replication

To investigate potential effects of candidate small molecules on human beta cell proliferation, we treated human islets with small molecule inhibitor of the menin-MLL interaction: MI, PKC activator: IN, and different inhibitors of TGF-β signaling: SB431542 (SB), LY2157299 (L2), and LY364947 (L3). Previously reported small molecule inducers of human beta cell replication (Wang et al., 2015a, 2019), HA (Harmin, a DYRK1A inhibitor) and a combination of HA and L3 were also used as the positive

Discussion

In this study, we provide experimental evidence for induction of adult human beta cell replication with combined inhibition of menin-MLL and TGF-β signaling. Our results further suggested a mechanism by which menin-MLL and TGF-β inhibitors could synergize to increase the proliferation of human beta cells.

We also showed that menin-MLL and TGF-β inhibition was not associated with decreased INS expression. Since we measured normalized INS mRNA and protein expression it cannot reflect the total

Conclusion

To our knowledge, this is the first report of synergistic effect of menin-MLL and TGF-β inhibition small molecules on human beta cell replication. Importantly, we achieved this by using small molecules, which could be of potential benefit as pharmacological agents. This finding has the potential to expand the repertoire of pharmacological options for human beta cell proliferative therapies for diabetes.

Funding

Funding for the present work was provided by grants from Royan Institute, the Iran National Science Foundation (INSF), and the Department of Research Affairs of Tarbiat Modares University, to HB and M Behmanesh, and from the National Institutes of Health (NIH/NIDDK) and the Swiss National Science Foundation, to PLH.

Declaration of Competing Interest

The authors declare no competing interest.

Acknowledgment

The authors would like to thank all members of the Beta Cell and Diabetes Program at Royan Institute for their valuable comments.

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