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.

Figure 1.

Identification of small-molecule inducers of rat β-cell replication and their combinatorial use. (a) Summary of rat β-cell replication screening results. Compounds are clustered according to their annotated bioactivity. Supplemental Tables 1 and 2 list primary screening and confirmatory testing β-cell–replication results. (b) The β-cell replication–promoting profile of prioritized compounds. Data are presented as mean ± standard deviation (SD; n = 3 to 5 wells per treatment condition; results confirmed in n ≥ 3 independent experiments). (c) Representative images of rat islet cultures treated with vehicle (DMSO) or CC-401. (d) Representative images of compound-treated rat islet cultures. The percentage of replicating PDX1⁺ cells and compound treatments are indicated. (e) Rat islet β-cell replication indices Ki67 (blue) and PCNA (red). Compound concentrations were as indicated in (b) and was 1 μM for CHIR99021. Individual data points represent 2000 to 3000 β cells (n = 4 to 8 replicates per condition; mean ± SD shown). All compounds increased β-cell replication above DMSO (P < 0.01). All compound combinations increased β-cell replication above the relevant individual compound-treatment conditions (P < 0.01). (f) Rat islet β-cell replication index after compound treatment as indicated. Single compound treatment, CNS-1102 combinations, and BML-266 combinations are shown. All individual compound treatments increased replication (vs DMSO; P < 0.01); select combinations increased β-cell replication vs relevant individual compound-treated conditions. *P < 0.01. Individual data points represent 2000 to 3000 β cells (n = 4 to 8 replicates per condition; mean ± SD shown). Error bars represent the standard deviation of an experimental condition (n ≥ 3). cAMP, cyclic adenosine monophosphate; CAS#, Chemical Abstracts Service number; PDE, phosphodiesterase.

Figure 2.

CC-401 stimulates human β-cell replication in vitro and mouse β-cell replication in vivo. (a) Representative immunofluorescence images of vehicle- (DMSO) and CC-401–treated human islet cultures [DAPI (blue); insulin (green)]. (b) The β-cell–replication index of dispersed human islet cultures after compound treatment (48 hours). Independent treatments along with mean ± standard deviation (SD) are shown. *P < 0.05. Similar data were obtained from at least five independent islet procurements. (c) Rat β-cell–replication index of compound-treated (CC-401, 10 µM; D4476, 5 µM; and ALK5 inhibitor II, 2 µM) primary islet cultures. Independent replicates (n = 4) are shown with mean ± SD. *P < 0.01; >1000 β cells counted per data point. (d) Human β-cell–replication index of compound-treated islet cultures. Independent replicates (n = 5) are shown with mean ± SD. *P < 0.05; >1000 β cells counted per data point. (e) Representative images of pancreatic sections from 8-week female vehicle- and CC-401–treated mice stained for insulin (red), BrdU (green), and nuclei (blue). See Supplemental Fig. 2 for determination of CC-401’s in vivo half-life and in vitro potency. (f) The BrdU incorporation index (percentage of replication) of β cells (insulin⁺) and non–β cells (insulin⁻) after treatment with vehicle or CC-401 (25 mg/kg) for 1 week. Data from individual mice (n = 5) and mean ± SD shown. *P < 0.05. Error bars represent the standard deviation of an experimental condition (n ≥ 3). Two independent experiments were performed with similar results. See Supplemental Fig. 2 for in vitro replication effects on α cells, δ cells, and dermal fibroblasts. ALKV Inh. II, activin A receptor type II–like kinase inhibitor II.

Figure 3.

CC-401 promotes β-cell replication via DYRK1A/B inhibition. (a) Replication index of vehicle- and CC-401–treated fluorescence-activated cell sorting–purified rat β cells (see also Supplemental Fig. 3). Individual data points and mean ± standard deviation (SD) are shown (n = 4 wells per treatment condition with >1000 β cells per well). *P < 0.05. (b) Rat β-cell–proliferation index of JNK inhibitor–treated islet cultures. Individual wells and mean ± SD shown (n = 6 wells per treatment condition). *P < 0.05 vs DMSO treatment. (c) Percent residual kinase activity after incubation with the specified compound (10 µM). See Supplemental Table 4 for complete kinome datasets and Supplemental Fig. 4(a)–4(c) for the replication-promoting activity of additional DYRK1A/B inhibitors. (d) In vitro rat β-cell–replication response to the indicated compound treatment(s) (48 hours). Data from individual wells with mean ± SD are shown (n = 4 wells per treatment condition; >1000 β cells counted per well). *P < 0.05. (e) Fold-induction of NFAT-firefly luciferase reporter activity from cells cotransfected with Renilla (used for normalization across transfections), NFATc1, and DYRK1A expression plasmids, and treated with DMSO, CC-401, or harmine. Data from individual wells (n = 8) are presented with mean ± SD. *P < 0.05. Error bars represent the standard deviation of an experimental condition (n ≥ 3).

Figure 4.

Rationally designed CC-401 derivatives confirm DYRK1A/B as the relevant β-cell–replication target. (a) Space-filling (left) and stick (right) models of CC-401 binding to DYRK1A [4MQ2; Protein Data Bank (28)] generated with Schrodinger Glide software. Key hydrogen bond interactions are highlighted (green dashed line). (b) Chemical structures of CC-401 and synthesized derivatives. The piperidine group (red) and the triazole group (blue) are highlighted on CC-401. (c) Rat β-cell–proliferation response to STF-201161 and STF-201162. Data are presented as mean ± standard deviation (SD; n = 4 wells per treatment condition). *P < 0.05. (d) Fold-induction of NFAT-luciferase reporter activity in cells cotransfected with NFATc1, DYRK1A, and Renilla luciferase. Cells were compound treated as indicated. Data are presented as mean ± SD (n = 4 wells per experimental point). *P < 0.01 shown for STF-785 vs CC-401. A significant (P < 0.05) luciferase induction above DMSO occurred in response to CC-401 at a concentration >0.1 µM, STF-200785 (>0.05 µM), and STF-200894 (>2.0 µM). No luciferase induction occurred in response to STF-200866. (e) Recombinant DYRK1B activity measured in the presence of CC-401 or STF-200785 (n = 2 wells per experimental point). *P < 0.01. Half maximal inhibitory concentrations were not statistically different. (f) Fold increase in rat β-cell replication in response to the indicated compound treatment (n = 4 wells per treatment condition presented as mean ± SD). *P < 0.05 shown for STF-785 vs CC-401. No replication response occurred in response to STF-200866. Error bars represent the standard deviation of an experimental condition (n ≥ 3).

Figure 5.

Inhibition of DYRK1A/B promotes β-cell replication via NFAT activation, p27^kip1 destabilization, and DREAM-complex target gene derepression. (a) In vitro rat β-cell–replication indices after compound treatment as indicated. The results from individual wells (n = 4 to 7) are shown with mean ± standard deviation (SD). *P < 0.05 vs the DMSO-treated condition or as indicated. (b) Western blot analysis of phospho-S10p27, p27, and DYRK1A after in vitro incubation of recombinant p27 and DYRK1A with and without adenosine triphosphate, CC-401, or DYRK1A. Quantitation of S10 phosphorylated p27 intensity normalized to total p27 are shown in the right-side panel. (c) Representative western blot of R7T1 β-cell lysates probed for β-actin, p27, and phosphoS10-p27 after treatment of cultures with DMSO or CC-401 for the indicated time (hours). Quantitation of the relative levels of (d) phosphoS10-p27 and (e) total p27 obtained from western blots (n = 3) performed as in (c). Protein levels were normalized to β-actin levels. *P < 0.05 vs DMSO condition at the relevant time point. (f) The average fluorescence intensity of p27 and phospho-S10p27 measured in β cells (PDX1⁺) from human islet cultures treated with DMSO or CC-401. Individual wells (n = 4; >1500 β cells per well) are shown with mean ± SD. *P < 0.01. (g) Enriched gene ontology categories (left) and transcription factor networks (right) identified by MetaCore analysis of differentially expressed genes from RNA sequencing performed on fluorescence-activated cell–sorted β cells after vehicle- and CC-401–treatment (48 hours); see Supplemental Table 4 for complete results. (h) Relative gene expression levels determined by qPCR (normalized to Gapdh) after treatment of growth-arrested R7T1 β-cell cultures with DMSO or CC-401 for 48 hours. (i) Relative gene expression levels determined by qPCR (normalized to GAPDH) after treatment of human islet cultures with DMSO or CC-401 for 48 hours. Error bars represent the standard deviation of an experimental condition (n ≥ 3). ATP, adenosine triphosphate.