CC-401 Promotes β-Cell Replication via Pleiotropic Consequences of DYRK1A/B Inhibition

Abstract

Pharmacologic expansion of endogenous β cells is a promising therapeutic strategy for diabetes. To elucidate the molecular pathways that control β-cell growth we screened ∼2400 bioactive compounds for rat β-cell replication-modulating activity. Numerous hit compounds impaired or promoted rat β-cell replication, including CC-401, an advanced clinical candidate previously characterized as a c-Jun N-terminal kinase inhibitor. Surprisingly, CC-401 induced rodent (in vitro and in vivo) and human (in vitro) β-cell replication via dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1A and 1B inhibition. In contrast to rat β cells, which were broadly growth responsive to compound treatment, human β-cell replication was only consistently induced by DYRK1A/B inhibitors. This effect was enhanced by simultaneous glycogen synthase kinase-3β (GSK-3β) or activin A receptor type II-like kinase/transforming growth factor-β (ALK5/TGF-β) inhibition. Prior work emphasized DYRK1A/B inhibition-dependent activation of nuclear factor of activated T cells (NFAT) as the primary mechanism of human β-cell-replication induction. However, inhibition of NFAT activity had limited effect on CC-401-induced β-cell replication. Consequently, we investigated additional effects of CC-401-dependent DYRK1A/B inhibition. Indeed, CC-401 inhibited DYRK1A-dependent phosphorylation/stabilization of the β-cell-replication inhibitor p27Kip1. Additionally, CC-401 increased expression of numerous replication-promoting genes normally suppressed by the dimerization partner, RB-like, E2F and multivulval class B (DREAM) complex, which depends upon DYRK1A/B activity for integrity, including MYBL2 and FOXM1. In summary, we present a compendium of compounds as a valuable resource for manipulating the signaling pathways that control β-cell replication and leverage a DYRK1A/B inhibitor (CC-401) to expand our understanding of the molecular pathways that control β-cell growth.

Combined Inhibition of DYRK1A, SMAD, and Trithorax Pathways Synergizes to Induce Robust Replication in Adult Human Beta Cells

“…Since de-differentiation of beta cells occurs in both mice and humans with type 2 diabetes (Cinti et al, 2016; Talchai et al, The effects of harmine alone, and of various TGFbSF ligand inhibitors, some of which are specific for TGFb receptors, and others for activin, inhibin, and BMP receptors. As can be seen, and as reported previously (Aamodt et al, 2016;Abdolazimi et al, 2018;Dirice et al, 2016;Shen et al, 2015;Wang et al, 2015aWang et al, , 2016, harmine induces Ki67 labeling in approximately 2% of normal human beta cells, and TGFbSF inhibitors lead to only marginal Ki67 labeling. However, each of the TGFbSF inhibitors in combination with harmine induces striking increases in Ki67 labeling in beta cells.…”

“…While DYRK1A inhibitors such as harmine, 5-IT, INDY, and GNF4877 have been shown to induce replication in human beta cells, the ''rates'' of proliferation or labeling indices have been low, in the 1.5%-3% range in vitro (Aamodt et al, 2016;Abdolazimi et al, 2018;Dirice et al, 2016;Shen et al, 2015;Wang et al, 2015aWang et al, , 2016, and far lower in in vivo transplant models Wang et al, 2015a). Thus, while harmine analog-induced beta cell proliferation is an important advance, one might envision higher rates of proliferation as being required for therapeutic human beta cell expansion in type 1 and type 2 diabetes.…”

“…Harmine analogs derive their mitogenic effects in large part, if not exclusively, via inhibition of DYRK1A (Abdolazimi et al, 2018;Dirice et al, 2016;Shen et al, 2015;Wang et al, 2015a). To explore the presumed requirement for DYRK1A in the synergistic proliferation derived from the harmine-TGFb inhibitor combination, we employed adenoviral silencing and overexpression of DYRK1A, alone or in combination with TGFbSF inhibition .…”

“…Inhibition of the enzyme dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) in human beta cells, using drugs such as harmine, INDY, GNF4877, 5-iodotubericidin (5-IT), or CC-401, is able to induce proliferation (labeling indices) in the 1.5%-3% range, as assessed using Ki67, EdU, BrdU, and/or PCNA immunolabeling of insulin-containing cells derived from human cadaveric islets (Aamodt et al, 2016;Abdolazimi et al, 2018;Dirice et al, 2016;Shen et al, 2015;Wang et al, 2015aWang et al, , 2016. This notable accomplishment, confirmed in multi-ple laboratories, replicates the proliferation ''rate'' in human beta cells in the first year of life, the only stage of human development at which appreciable beta cell proliferation occurs (Gregg et al, 2012;Kassem et al, 2000;Meier et al, 2008;Wang et al, 2015b).…”

Structure–Activity Relationships and Biological Evaluation of 7-Substituted Harmine Analogs for Human β-Cell Proliferation

“…Recently, our group found that harmine is able to induce human β-cell proliferation and DYRK1A-NFAT pathway as being the major pathway for this cell proliferation [5]. These results have been confirmed in other labs with other DYRK1A inhibitors unrelated to the harmine scaffold, including from our own lab [6,8,[34][35][36][37]. Since, modification of harmine has not been explored previously in context of β-cell proliferation, we therefore decided to carry out structure-activity relationship studies of harmine for both DYRK1A inhibition and β-cell proliferation.…”