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. 2016 Mar 29;7(13):16650-62.
doi: 10.18632/oncotarget.7692.

The targeted histone deacetylase inhibitor tefinostat (CHR-2845) shows selective in vitro efficacy in monocytoid-lineage leukaemias

Joanna Zabkiewicz  1 , Marie Gilmour  1 , Robert Hills  1 , Pares Vyas  2 , Elizabeth Bone  3 , Alan Davidson  3 , Alan Burnett  1 , Steven Knapper  1
Affiliations

The targeted histone deacetylase inhibitor tefinostat (CHR-2845) shows selective in vitro efficacy in monocytoid-lineage leukaemias

Joanna Zabkiewicz et al. Oncotarget. .

Abstract

Tefinostat (CHR-2845) is a novel monocyte/macrophage-targeted histone deacetylase (HDAC) inhibitor which is cleaved into its active acid by the intracellular esterase human carboxylesterase-1 (hCE-1). The in vitro efficacy of tefinostat was characterised in cell lines and in a cohort of 73 primary AML and CMML samples. Dose-dependent induction of apoptosis and significant growth inhibitory effects were seen in myelomonocytic (M4), monocytic/monoblastic (M5) and CMML samples in comparison to non-monocytoid AML sub-types (p = 0.007). Importantly, no growth inhibitory effects were seen in normal bone marrow CD34+ cells exposed to AML-toxic doses of tefinostat in clonogenic assays. Expression of hCE-1 was measured by intracellular flow cytometry and immunoblotting across the cohort, with highest levels seen in M5 AML patients. hCE-1 levels correlated with significantly increased tefinostat sensitivity (low EC50) as measured by growth inhibition assays (p = 0.001) and concomitant elevation of the mature monocytoid marker CD14+. Strong induction of intracellular histone protein acetylation was observed in tefinostat-responsive samples, as were high levels of the DNA damage sensor γ-H2A.X, highlighting potential biomarkers of patient responsiveness. Synergistic interaction between tefinostat and the current standard treatment cytarabine was demonstrated in dose response and clonogenic assays using simultaneous drug addition in primary samples (median Combination Index value = 0.51). These data provide a strong rationale for the further clinical evaluation of tefinostat in monocytoid-lineage haematological neoplasms including CMML and monocyte-lineage AMLs.

Keywords: AML; CMML; HDACi; hCE-1; tefinostat.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that there are no competing financial interests in relation to the work described.

Figures

Figure 1
Figure 1. Monocytoid leukaemias show selective high sensitivity to Tefinostat
(A) Dose response plot for AML cell lines HL60 (M2), MV411 (M4), OCIAML3 (M4) and THP-1 (M5) treated with serial dilutions of Tefinostat. Tefinostat-induced Annexin V/PI incorporation measured by flow cytometry in THP1, MV411, OCIAML3 and HL60 at (B) 24 Hrs and (C) 48 Hrs post treatment. (D) Box and whiskers plots showing range of FAB type-specific MTS-derived EC50s in response to Tefinostat and the comparative non hCE-1-dependent t-butyl analogue CHR8185 in a cohort of 66 primary AML and 7 primary CMML samples. (Tefinostat EC50 M4/M5/CMML vs. M0/M1 *p = 0.009 spearman's correlation. Tefinostat vs. t-butyl analogue CHR8185, only significant in M4/M5 groups †p < 0.007).
Figure 2
Figure 2. Monocytic targeting of HDACi therapy spares normal bone marrow progenitor cells
(A) Flow cytometric analysis of viability of cell subpopulations in primary AML samples in response to Tefinostat dosing measured by 7AAD exclusion (n = 8). (B) Dose response effects of Tefinostat and CHR8185 in NBM cells (n = 4) measured by 7AAD exclusion. (C) Tefinostat effects on Colony forming units (CFUs) in NBM CD34+ cells (no significant difference, n = 4) and primary AML blasts (*p < 0.02 MWU, n = 6) following 14 days culture on methocult.
Figure 3
Figure 3. hCE-1 expression levels dictate efficacy of Tefinostat in monocytic leukaemias
(A) Correlation plot of CD14+ expression vs. hCE-1 mean fluorescence measured by intracellular flow in primary AML samples (n = 10) (B) Representative western blot of hCE-1 expression in NBM, AML and CMML patient samples. (C) Comparison of hCE-1 protein levels across FAB types (NBM vs. M5 *p = 0.01 Wilcoxon rank sum). (D) Correlation plot of hCE-1 protein levels measured by western blot (fold change relative to NBM levels, n = 3) compared to tefinostat sensitivity (EC50) in a cohort of 40 primary AML and 7 CMML samples, p = 0.001 Spearman's correlation per 10 fold increase in EC50). Dotted box represents low EC50 samples. (E) Monocytoid intracellular hCE-1 levels in tefinostat-sensitive and tefinostat-resistant primary samples gated by CD14+/CD64+ (EC50 high (n = 6) and low patients (n = 11), **p < 0.001 MWU).
Figure 4
Figure 4. Increases in intracellular acetylation and DNA damage induction are biomarkers of Tefinostat efficacy
(A) Tefinostat dose-dependent intracellular acetylation staining in a representative primary AML sample using acetylated lysine monoclonal antibody and sub-population analysis by flow cytometry. (B) Acetylation induction in Tefinostat sensitive (low EC50/CD14+ black bars, n = 8) compared to insensitive (high EC50, white bars, n = 5) primary AML samples, *p < 0.05, **p < 0.005. (C) Tefinostat dose-dependent intracellular acetylation induction in primary CMML samples (n = 3). All samples exhibit > 80% CD14+ and > 70% hCE-1 expression. (D) Representative western blot of phospho-H2A.X induction by tefinostat at 1 and 24 hours post-treatment. (E) Western blot quantification of dose-dependent tefinostat-induced γ-H2A.X induction at 24 hrs compared to vehicle treated control (n = 9 AML samples, *p < 0.01 Kruskal Wallis).
Figure 5
Figure 5. Tefinostat is synergistic with Cytarabine
(A) Dose response curves for MV411 cells treated both singly and in combination for Tefinostat and AraC (dose ratio 1:10, Tefinostat:AraC). (B) Synergy analysis of tefinostat and AraC displayed as combination index (CI) values at EC50 dose affected at an optimal ratio of 1:10 (Tefinostat:AraC) in primary patient samples set up in triplicate (AML Mean combination index (CI) = 0.51, n = 31, CMML mean CI = 0.49, n = 5). (C) Representative CI response plot from primary AML showing CI values across a range of dose effects (fraction affected). Dose response curves for simultaneous and sequential dosing of drugs in (D) OCIAML3 and (E) MV411 cell lines. (F) Clonogenic assessment of synergy interaction in primary AML blasts (n = 4) following single agent and 1:10 ratio combination pulse treatment (*p < 0.04 MWU, between both single agents and combination colony growth).
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