Promising preclinical data on selective HDAC11 inhibition and MPN improvement

Histone deacetylases (HDACs) are enzymes that remove acetyl groups from histones and non-histone proteins and are divided in four classes (Class I, IIa, IIb, IV).¹ Preclinical observations have indicated that HDACs are overexpressed in myeloproliferative neoplasms (MPNs), with many being involved in the regulation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling.² Multiple pan-HDAC inhibitors have been examined in clinical trials demonstrating limited efficacy combined with systemic toxicities.^2,3 Nevertheless, the therapeutic potential of specifically targeting distinct HDACs has not yet been investigated.

During the 61st American Society of Hematology (ASH) Annual Meeting & Exposition (2019), Vasundhara Sharma presented results from preclinical studies inhibiting distinct HDAC proteins in various models of MPN. The article below is summarizing the data presented at the ASH meeting and may supersede those in the published abstract.

Study design

HDAC inhibitor high-throughput screen

• More than 200 inhibitors were screened for full-length human HDAC protein selectivity, with electrophoretic mobility shift assays
• Human HDAC protein was expressed in baculoviruses

MPN cell lines & primary cells

• Two JAK2 V617F harbouring MPN cell lines were used: SET2 and HEL
• Human MPN primary cells were used to assess ex vivo erythropoietin-independent colony formation

Murine MPN models

• The authors created their own HDAC11 deficient MPN mouse model by first transducing HDAC11 knockout hematopoietic stem cells (HSCs) with a virus expressing the MPL^W515L oncogene, and then engrafting these cells in irradiated wildtype (wt) mice. Equivalent control animals were also generated for the transduction stage with HDAC11 wt HSCs.

Results

In vitro results

• When investigating the roles of individual HDACs in MPN, using an HDAC inhibitor compound screen, only HDAC11, the sole member of Class IV HDACs, was identified leading to therapeutic vulnerability in MPN cell lines
• Using the HDAC11 and HDAC8-selective inhibitor, FT895, the in vitro MPN cell viability was reduced, while HDAC6-specific inhibition did not affect the MPN cell viability
• This was further confirmed using an inducible shRNA HDAC11 knockdown in MPN cell lines; HDAC11 shRNA silencing significantly decreased SET2 cell viability
• In primary human MPN cells, HDAC11 inhibition with selective HDAC inhibitors, FT234 and FT895, led to significant reduction in erythropoietin-independent colony formation, which was statistically more pronounced than the reduction in colony formation obtained with the ruxolitinib treatment

In vivo results

• HDAC11 deficient mice transplanted with MPL^W515L transduced stem cells, were compared to transplanted wt mice showing:
  • Significantly reduced spleen size, white blood cell (WBC) counts, platelet (PLT) counts and red blood cell counts
  • Improved histological bone marrow architecture (reduced bone marrow fibrosis)
  • Prolonged mice survival
• In a serial transplantation assay, where equal numbers of wt and HDAC11 knockout MPL^W515L transduced HSCs were isolated from the primary transplantation recipient, purified, and re-transplanted to a second recipient mouse for survival data:
  • Second transplant recipient mice receiving HDAC11 deficient cells showed 100% survival rates, while mice receiving wt cells died within 10–20 days after transplantation
• Interestingly, under steady state conditions, the HDAC11 deficient mice did not display any disease symptoms (normal WBC, PLT levels, normal HSCs). Only when their stem cells had a MPN-like pathology, the cell survival was reduced, and apoptosis was increased

Mechanistic HDAC11 model

• To further investigate the molecular targets affected by HDAC inhibition, the authors performed a global acetylomic analysis on HEL cells. This showed that:
  • Glycolytic enzymes, like alpha enolase (ENO1), were hyperacetylated (suppressed) in cells treated with an HDAC11 inhibitor
  • HDAC11 is a regulator of metabolism in MPN cells and that HDAC inhibition leads to suppression of glycolysis
• Mutations of the various lysine residues on ENO1 in vitro, showed that the main lysine substrate of HDAC11 is lysine 343 (K343)
  • Deacetylation-mimicking mutations of K343 (similar to an active HDAC) led to increased glycolysis in vitro; while
  • Acetylation-mimicking mutations of K343 (similar to HDAC inhibition) led to suppressed glycolysis in vitro

Conclusions

The above preclinical data show that HDAC11 inhibition supresses the viability and metabolic activity of MPN cells in vitro, by inhibiting glycolysis. In vivo, HDAC11 inhibition improved MPN-related symptoms and survival in both primary and secondary MPN HSC transplantation recipients. Overall, the results indicate that HDAC11 might be a novel therapeutic target for MPN and warrant the further investigation of selective HDAC11 inhibitors in a clinical trial setting.