HDAC6 inhibition effectively reverses chemotherapy-induced peripheral neuropathy

Abstract

Chemotherapy-induced peripheral neuropathy is one of the most common dose-limiting side effects of cancer treatment. Currently, there is no Food and Drug Administration-approved treatment available. Histone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase whose function includes regulation of α-tubulin-dependent intracellular mitochondrial transport. Here, we examined the effect of HDAC6 inhibition on established cisplatin-induced peripheral neuropathy. We used a novel HDAC6 inhibitor ACY-1083, which shows 260-fold selectivity towards HDAC6 vs other HDACs. Our results show that HDAC6 inhibition prevented cisplatin-induced mechanical allodynia, and also completely reversed already existing cisplatin-induced mechanical allodynia, spontaneous pain, and numbness. These findings were confirmed using the established HDAC6 inhibitor ACY-1215 (Ricolinostat), which is currently in clinical trials for cancer treatment. Mechanistically, treatment with the HDAC6 inhibitor increased α-tubulin acetylation in the peripheral nerve. In addition, HDAC6 inhibition restored the cisplatin-induced reduction in mitochondrial bioenergetics and mitochondrial content in the tibial nerve, indicating increased mitochondrial transport. At a later time point, dorsal root ganglion mitochondrial bioenergetics also improved. HDAC6 inhibition restored the loss of intraepidermal nerve fiber density in cisplatin-treated mice. Our results demonstrate that pharmacological inhibition of HDAC6 completely reverses all the hallmarks of established cisplatin-induced peripheral neuropathy by normalization of mitochondrial function in dorsal root ganglia and nerve, and restoration of intraepidermal innervation. These results are especially promising because one of the HDAC6 inhibitors tested here is currently in clinical trials as an add-on cancer therapy, highlighting the potential for a fast clinical translation of our findings.

Figure 1. Structure, selectivity and pharmacokinetics of ACY-1083

(A) The inhibitory effects of ACY-1083 on the enzymatic activities of HDAC1-HDAC9. The Y-axis represents the percentage of basal enzymatic activity corresponding to various concentrations of ACY-1083. The curve fit was generated by a 4-parameter, nonlinear regression in Graph Pad Prism. (B) The effects of various concentrations of ACY-1083 on the acetylation level of the HDAC6 substrate α-tubulin and on histone, which is not an HDAC6 substrate in SK-N-BE2 cells. (C) Structure and pharmacokinetics of ACY-1083. ACY-1083 is present at biologically active concentrations 8 hours after dosing in the plasma of mice received ACY-1083 i.p. (n=3/group).

Figure 2. HDAC6 inhibition prevents and reverses cisplatin-induced mechanical allodynia

Mechanical allodynia was measured using von Frey hairs, and the 50% paw withdrawal threshold was calculated by the up–down method. (A) Mice were administered with 2 rounds of cisplatin treatment; 3 days after the last cisplatin dose, mice received ACY-1083 (10 mg/kg or 3 mg/kg) for 7 days. Two-way repeated-measure ANOVA revealed a main effect for time (P<0.01), group (P<0.01), and a group-by-time interaction (P<0.01). Tukey post-hoc analysis was used to determine differences between groups at specified time points: ^***P<0.001 between cisplatin + vehicle vs. saline + vehicle; ^^^P<0.001 between cisplatin + ACY-1083 (10mg/kg) vs. cisplatin + vehicle. n=10–12/group. (B) Two-way repeated-measure ANOVA showed a main effect for time (P<0.05) and a group-by-time interaction (P<0.05). Tukey post-hoc analysis for cisplatin + ACY-1083 (10 mg/kg) vs. cisplatin + vehicle: ^***P<0.001. n= 6–8/group. (C) Mice received cisplatin daily for 5 days. ACY-1083 (10 mg/kg or 3 mg/kg) was administered i.p. 1 hour prior to each cisplatin injection and for 2 days after the last cisplatin injection. Two-way repeated-measure ANOVA showed a main effect for time (P<0.0001), group (P<0.03), and a group-by-time interaction (P<0.03). Tukey post-hoc analysis for ACY-1083 (10 mg/kg) + cisplatin vs. vehicle + cisplatin: ^***P<0.001. n= 6–8/group. (D) Rats were treated with paclitaxel for 13 days. Two daily doses of ACY-1083 (3 mg/kg) were given orally for 7 days. Mechanical allodynia were measured at baseline, post-paclitaxel treatment (day 14), after completion of ACY-1083 treatment (day 20) and 3 days after completion of ACY-1083 treatment (day 23). Two-way ANOVA with Tukey’s post-hoc analysis showed a significant treatment effects at day 20 and day 23: ^***P<0.001. n= 11–12/group.

Figure 3. Effect of repeated or prolonged ACY-1083 treatment on symptoms of cisplatin-induced peripheral neuropathy

(A) Mice were administered with 2 rounds of cisplatin treatment; 3 days after the last cisplatin dose, mice received ACY-1083 (10 mg/kg) for 7 days followed by 7 days rest and then another 7 days of cisplatin treatment. Alternatively, mice received ACY-1083 (10 mg/kg) for 14 consecutive days. Mechanical allodynia was measured using von Frey hairs, and the 50% paw withdrawal threshold was calculated by the up–down method. Two-way repeated-measure ANOVA revealed a main effect for time (P<0.01), a group effect (P<0.01), and a group-by-time interaction (P<0.01). Tukey post-hoc analysis was used to determine differences between groups at specified time points. ^***P<0.001 between cisplatin + vehicle vs. saline + vehicle; ^^P<0.01 between cisplatin + ACY-1083 vs. cisplatin + vehicle. n=6–14/group. (B) Mice were administered with 2 rounds of cisplatin treatment; 3 days after the last cisplatin dose, mice received ACY-1215 (30 mg/kg) orally for 14 days. Mechanical allodynia was measured using von Frey hairs, and the 50% paw withdrawal threshold was calculated by the up–down method. Two-way repeated-measure ANOVA revealed a main effect for time (P<0.01), a group effect (P<0.01), and a group-by-time interaction (P<0.01). Tukey post-hoc analysis was used to determine differences between groups at specified time points. ***P<0.001 between cisplatin + vehicle vs. saline + vehicle; ˆˆˆP<0.01 between cisplatin + ACY-1215 vs. cisplatin + vehicle. n=6–8/group. (C) Spontaneous pain was measured by the conditioned place preference test after 2 weeks of ACY-1083 treatment. The Y-axis indicates the change in time spent in light chamber. Two-way ANOVA revealed a signification interaction (P<0.05). Tukey post-hoc analysis revealed significant differences between groups: ^*P<0.05. n=6–8 mice/group. (D) Cisplatin-induced numbness was measured by the adhesive removal test. Mice were tested in week 5 for cisplatin-induced numbness. Statistical analysis using Two-way ANOVA revealed a significant interaction (P<0.05). Tukey post-hoc analysis revealed significant differences between groups: ^*P<0.05. n=4–10/group.

Figure 4. Effect of ACY-1083 on α-tubulin acetylation

Acetylated α-tubulin levels in tibial nerves from mice that had received 2 rounds of cisplatin and 11 doses of ACY-1083 were assessed by Western blot analysis. ACY-1083 treatment induced α-tubulin acetylation. Two-way ANOVA revealed a significant treatment (ACY-1083) effect (P<0.05). Tukey post-hoc analysis revealed significant differences between groups: ^*P<0.05. n=4 mice/group.

Figure 5. ACY-1083 enhances mitochondrial bioenergetics and contents in the tibial nerve of cisplatin-treated mice

(A) Mitochondrial bioenergetics in tibial nerves from mice that had received 2 rounds of cisplatin treatment and 11 injections of ACY-1083. Two-way ANOVA revealed a signification interaction (P<0.05) for baseline respiration, ATP-coupled respiration, proton leak, and maximal respiratory capacity (MRC). Tukey post-hoc analysis revealed significant differences between groups: ^*P<0.05. n=6–8 mice/group. (B) Tibial nerves retrieved from the XF-analysis were used for Western blot analysis of mitochondrial marker protein Cox IV. Two-way ANOVA revealed a signification interaction (P<0.05). Tukey post-hoc analysis revealed significant differences between groups: *P<0.05. n=6–8 mice/group. (C) Tibial nerves from mice received 2 rounds of cisplatin treatment and 11 injections of ACY-1083 were used for Western blot analysis of additional mitochondrial marker protein SDHA and VDAC. (D) Mitochondrial transport was determined with rat DRG neuron cultures in vitro. The percent of moving mitochondria was determined and shown in the Y-axis. Two-way ANOVA revealed a signification interaction (P<0.05). Tukey post-hoc analysis revealed significant differences between groups: *P<0.05. n=3–4/group.

Figure 6. ACY-1083 enhances mitochondrial bioenergetics in DRG neurons of cisplatin-treated mice

(A) Mitochondrial bioenergetics in DRG neurons from mice that had received 2 rounds of cisplatin treatment and 11 injections of ACY-1083. Two-way ANOVA revealed a signification difference for cisplatin + vehicle and cisplatin + ACY-1083 versus saline + vehicle and saline + ACY-1083 for maximal respiratory capacity: *P<0.05. (B) Mitochondrial bioenergetics in DRG neurons from mice received 2 rounds of cisplatin and two weeks of ACY-1083, tissues were taken two weeks after the last ACY-1083 treatment. Two-way ANOVA revealed a signification difference for cisplatin + vehicle versus all three other groups for maximal respiratory capacity: *P<0.05.

Figure 7. ACY-1083 reverses the loss of intra-epidermal nerve fibers in cisplatin-treated mice

Paw biopsies were obtained from the hind paws of mice that received 2 cycles of cisplatin and 11 doses of ACY-1083. Tissues were stained with antibodies for IENFs (PGP9.5; red) and collagen (green). Representative images from saline + vehicle-treated mice: (A) saline + vehicle; (B) cisplatin + vehicle; (C) saline + ACY-1083; and (D) cisplatin + ACY-1083. The basement membrane is indicated by the dashed lines, the nerve fibers crossing the basement membrane are indicated by arrows. (E) Quantification of IENF density expressed as the number of nerve fibers crossing the basement membrane/length of the basement membrane (mm), scale bar = 20 μm, magnification 40×. Two-way ANOVA revealed a signification interaction (P<0.05). Tukey post-hoc analysis revealed significant differences between groups: *P<0.05. n=4 mice/group.