Inhibition of c-Kit Is Not Required for Reversal of Hyperglycemia by Imatinib in NOD Mice

<div><p>(1) Aim/Hypothesis</p><p>Recent studies indicate that tyrosine kinase inhibitors, including imatinib, can reverse hyperglycemia in non-obese diabetic (NOD) mice, a model of type 1 diabetes (T1D). Imatinib inhibits c-Abl, c-Kit, and PDGFRs. Next-generation tyrosine kinase inhibitors for T1D treatment should maintain activities required for efficacy while sparing inhibition of targets that might otherwise lead to adverse events. In this study, we investigated the contribution of c-Kit inhibition by imatinib in reversal of hyperglycemia in NOD mice.</p><p>(2) Methods</p><p>The T670I mutation in c-Kit, which confers imatinib resistance, was engineered into the mouse genome and bred onto the NOD background. Hematopoietic stem cells (HSCs) from NOD.c-Kit^T670I mice and NOD.c-Kit^wt littermates were expanded in the presence or absence of imatinib to verify imatinib resistance of the c-Kit^T670I allele. Diabetic mice were treated with imatinib at the onset of hyperglycemia for three weeks, and blood glucose was monitored.</p><p>(3 )Results</p><p><i>In vitro</i> expansion of HSCs from NOD.c-Kit^wt mice was sensitive to imatinib, while expansion of HSCs from NOD.c-Kit^T670I mice was insensitive to imatinib. However, <i>in vivo</i> treatment with imatinib lowered blood glucose levels in both strains of mice.</p><p>(4) Conclusions/Interpretation</p><p>The HSC experiment confirmed that, in NOD.c-Kit^T670I mice, c-Kit is resistant to imatinib. As both NOD.c-Kit^T670I and NOD.c-Kit^wt mice responded comparably to imatinib, c-Kit inhibition does not substantially contribute to the efficacy of imatinib in T1D. Thus, we conclude that inhibition of c-Kit is not required in next-generation tyrosine kinase inhibitors for T1D treatment, and may be selected against to improve the safety profile.</p></div

Diabetic NOD.c-Kit^T670I mice are sensitive to imatinib treatment.

<p>Blood glucose values for individual diabetic NOD.c-Kit^wt mice treated with either PBS (<b>A</b>) or imatinib (<b>B</b>), in comparison to diabetic NOD.c-Kit^T670I mice treated with either PBS (<b>C</b>) or imatinib (<b>D</b>) for 21 consecutive days. <b>E.</b> Average blood glucose values in diabetic NOD.c-Kit^wt or NOD.c-Kit^T670I mice treated with either PBS ((thick, solid line) NOD.c-Kit^wt; (thin, solid line) NOD.c-Kit^T670I) or imatinib ((thick, dashed line) NOD.c-Kit^wt; (thin, dashed line) NOD.c-Kit^T670I) for 21 consecutive days (n = 5–10 mice/group; *** p<0.0001 between PBS vs. imatinib within each mouse group for entire data set, as determined by one-way ANOVA). Note there was no statistically significant difference between NOD.c-Kit^wt and NOD.c-Kit^T670I within imatinib treatment groups. <b>F.</b> Area under curve for diabetes progression over 21 days comparing PBS ((open, white square) NOD.c-Kitwt; (closed, black square) NOD.c-Kit^T670I) or imatinib ((grey, slashed square) NOD.c-Kit^wt; (grey, closed square) NOD.c-Kit^T670I) treated mice. (* p≤0.01 between PBS vs. imatinib within each mouse group, as determined by one-way ANOVA).</p

NOD.c-Kit^T670I mice are imatinib resistant and develop diabetes.

<p>A. Targeting strategy for generation of NOD.c-Kit^T670I mice. PCR genotyping (<b>B</b>) and sequence traces (<b>C</b>) of wild-type (wt) and mutant (mut) mice using primers F and R to generate fragments of 795 bp and 967 bp for wt and mut alleles, respectively. The T670I codon change and accompanying introduction of <i>Bgl-II</i> restriction site are highlighted. <b>D.</b> Expansion of c-Kit⁺/Sca-1⁺ murine HSCs from either NOD.c-Kit^T670I (black bar) or NOD.c-Kit^wt (white bar) littermates in the presence or absence of 5 µM imatinib. (*** p = 0.0002, as determined by two-way ANOVA). <b>E.</b> NOD.c-Kit^T670I (black square) mice develop diabetes comparably to NOD.c-Kit^wt (white circle) littermates (n = 41–45 mice/group; average age of diabetes onset = 14.5 weeks in NOD.c-Kit^wt mice and 15.5 weeks in NOD.c-Kit^T670I mice).</p

Lack of negative masking in mRGC ablated mice.

<p>Representative wheel running activity profile of DT-treated (A) <i>Opn4^Cre/+;R26^iDTR/+</i> and (B) WT mice held under ultradian cycle of 4 h light and 4 h darkness are shown. The sessions of darkness are shown by shaded area. Activity profile is plotted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002451#pone-0002451-g005" target="_blank">Figure 5A</a>. (C) Average percentage (±SEM, n = 3 to 6 mice) of daily activity during the bouts of light sessions (on left) or during darkness (on right) for five different groups of mice are shown. Groups with percent activity during light phase significantly different (Student's <i>t</i> test, <i>p</i><0.05) from that of the DT-treated WT mice are shown with asterisk.</p

Strategy for fluorescent labeling or inducible ablation of mRGC lineage in the mouse retina.

<p>(A) The cellular circuitry underlying the rod/cone and mRGC contribution to visual responses. Thickness of the filled arrows roughly highlights the relative strength of information flow. (B) Strategy to fluorescently label mRGCs by breeding a mouse carrying a <i>Cre</i> recombinase “knocked-in” to the <i>melanopsin</i> promoter to <i>Z/EG</i> mouse that allows Cre-dependent expression of GFP from chicken beta-actin promoter. (C) Strategy to achieve inducible and specific ablation of mRGCs. <i>Opn4^Cre/+</i> mouse was bred to a mouse expressing <i>Cre</i>-dependent expression of simian diphtheria toxin receptor. The resulting progeny develop normal mRGCs expressing DTR, which allows specific ablation of these cells by DT administration. Schematic of two targeting vectors used to achieve inducible mRGC ablation are shown in (D). The <i>Cre</i> knock-in cassette for targeted insertion to <i>melanopsin</i> locus also carried coding sequences for CRE dependent expression of βTau-eYFP. However, fluorescence from βTau:eYFP was undetectable in retina from <i>Opn4^Cre/+</i> mice (data not shown). The targeting vector and generation of <i>R26^iDTR/+</i> mice are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002451#pone.0002451-Buch1" target="_blank">[20]</a>. A schematic of the targeting vector is shown here.</p

mRGC ablation does not alter the normal retina architecture and image-forming responses.

<p>(A) Hematoxylin and Eosin staining of 5 μm thick paraffin embedded sections of retina from <i>Opn4^Cre/+;R26^iDTR/+</i> mice without and with DT injection. DT application had no detectable adverse effect on the normal stratification of the retina (outer segment (OS), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL)). (B) Representative full-field ERG of WT and DT-treated <i>Opn4^Cre/+;R26^iDTR/+</i> mice showing rod, cone and maximal combined responses. Responses from both eyes were simultaneously measured and plotted. Quantitative analysis of magnitude and timing of a-wave, b-wave and oscillatory potentials of these two genotype groups (3 mice each) showed no significant difference (data not shown). (C) Image forming visual function as assessed by the visual cliff test was unaffected by mRGC ablation. Average percentage (+SEM, n = 5 to 13 mice) of positive choice in 10 trials for each mouse are shown. Mice with outer retina degeneration (<i>rd/rd</i>) made random choices while stepping down from the platform and were significantly different (Student's <i>t</i> test, <i>p</i><0.05; red asterisk) from the other four groups. No significant difference in test performance was found among native or DT-treated WT or <i>Opn4^Cre/+;R26^iDTR/+</i> mice.</p

Cre-dependent GFP labeling and inducible ablation of mRGCs.

<p>Retina of adult <i>Opn4^Cre/+;Z/EG</i> mouse probed with (A) anti-OPN4 or (B) anti-GFP antibodies show staining of a small subset of cells. (C) Only a subset of GFP positive cells also stained with anti-OPN4 antibody. A section of the flat mount retina containing a cell (marked with an arrow) that stained with melanopsin antibody, but did not express detectable level of GFP is shown. (D) Retina of <i>Opn4^Cre/+;R26^iDTR/+</i> showed normal melanopsin immunostaining in a small fraction of RGCs. (E) Two weeks after DT administration, the number of melanopsin-immunoreactive cells were significantly reduced. (F) Average melanopsin immunoreactive cell density in WT and <i>Opn4^Cre/+;R26^iDTR/+</i> retina was comparable. Two weeks after DT administration, the number of cells in WT retina remained unchanged, while that in <i>Opn4^Cre/+;R26^iDTR/+</i> retina reduced from 62.3 mm⁻² to 2.8 mm⁻². No melanopsin immunostaining was observed in <i>Opn4^−/−</i> mice. It is important to note that retina from all DT-treated mice tested by immunostaining still retained a few melanopsin staining cells (arrows), implying incomplete expression of Cre in all mRGCs and/or insufficient level of bioavailable DT. Average cell counts (+SEM, n = 3 to 5 retinas) from each genotype/treatment group are shown. Significant difference in cell numbers (Student's <i>t</i> test, <i>p</i><0.05) between mRGCs without and with DT was highlighted with an asterisk.</p

Necessity of mRGCs for PLR.

<p>DT injection severely attenuates pupil constriction in response to 20 μW of monochromatic blue light (470 nm) in <i>Opn4^Cre/+;R26^iDTR/+</i> mice (A), but does not affect in such PLR response in WT mice (B). Normalized pupil constriction (-SD; n = 3 mice) measured one day prior to or every day following DT injection for up to 8 days are shown. There was variability in the rate of loss in PLR response among <i>Opn4^Cre/+;R26^iDTR/+</i> as reflected in the larger error bars. (C) Pupil constriction in response to varying irradiance levels over 5 log units shows the necessity of mRGC for PLR. Average (+SEM, n = 5 to 6 mice) and fitted sigmoid curves for WT, <i>Opn4^Cre/+;R26^iDTR/+</i>, <i>Opn4^−/−;rd/rd</i> and DT-treated <i>Opn4^Cre/+;R26^iDTR/+</i> mice are shown. (D) Representative frozen video images showing dark adapted pupil and pupil under low (10¹¹ photons.cm⁻².s⁻¹) or high intensity light (10¹⁵ photons.cm⁻².s⁻¹) of 470 nm are shown. Notice the complete lack of pupil constriction in <i>Opn4^−/−;rd/rd</i> and in DT-treated <i>Opn4^Cre/+;R26^iDTR/+</i> mice. For each genotype representative images of the same eye under three different conditions are shown.</p

mRGCs are necessary for light adaptation of circadian wheel running activity rhythm.

<p>Representative daily wheel running activity profile of an (A) <i>Opn4^Cre/+;R26^iDTR/+</i> and (B) WT mouse under 12 h light∶12 h dark (LD), constant dark (DD) or constant light (LL) are shown. A week after DT injection (red arrows) the wheel running activity of <i>Opn4^Cre/+;R26^iDTR/+</i> began to “free run” with a periodicity similar to that under no light. Constant light had no effect on the daily drift in activity onset in this mouse. The wheel running activity of the WT mouse remained entrained to the LD cycle even after DT injection and was lengthened under constant light. Daily wheel running activity profile of mice were binned in 6 min and double plotted such that the activity from consecutive days are plotted to the right and beneath the data from previous day. Periods of darkness are shown in shaded area. (C) Genotypes of mice and their respective average (±SEM, n = 3 to 6 mice) period length of wheel running activity rhythm under conditions of LD, DD and LL as determined by periodogram analysis in Clocklab software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002451#pone.0002451-Siepka1" target="_blank">[41]</a> are shown. Within each lighting or treatment group (separated by solid box) significant difference (Student's <i>t</i> test, <i>p</i><0.05) from WT group is shown by red asterisk. (D) Anterograde CTB-Alexa Fluor 488 tracing in the optic tract (OT) is intact, but is completely abolished in the SCN of DT treated <i>Opn4^Cre/+;R26^iDTR/+</i> mice. (E) Staining in both regions are left intact in WT mice treated with DT.</p

Itpkb inhibitors block rat antigen-induced arthritis.

<p>(A) Lewis rats were immunized intra-dermally on Day -21 and -14 with methylated BSA (mBSA), followed by daily oral dosing of GNF362, or dexamethasone (Dex) as a positive control. On Day 0, the rats received an intra-articular injection of mBSA into the right knee joint. (B) Serum was sampled on Day +7, and IgG antibody titers to mBSA were determined by ELISA. The antibody titers were calculated by determining the average dilution at which half-maximal absorbance is detected after subtraction of background. Fold reduction in antibody titer over vehicle is shown in the table. Data shown is one representative experiment. (C) The diameters of the right and left knees were measured on Days 0, 2, 4, and 7, and the ratio of right over left knee diameters (R/L) is shown. (D) Histological analysis of the knee joint was performed blindly and scored on a 5-point scale at the termination of the study. Data shown is one representative experiment. *, P < 0.05; **, P < 0.01.</p