Selective inhibition of HDAC class IIA as therapeutic intervention for KMT2A-rearranged acute lymphoblastic leukemia

Abstract

KMT2A-rearranged acute lymphoblastic leukemia (ALL) is characterized by deregulation of the epigenome and shows susceptibility towards histone deacetylase (HDAC) inhibition. Most broad-spectrum HDAC inhibitors simultaneously target multiple human HDAC isoforms. Consequently, they often induce toxicity and especially in combination with other therapeutic agents. Therefore, more specifically targeting HDAC isoforms may represent a safer therapeutic strategy. Here we show that shRNA-mediated knock-down of the class IIA HDAC isoforms HDAC4, HDAC5, and HDAC7 results in apoptosis induction and cell cycle arrest in KMT2A-rearranged ALL cells. In concordance, the HDAC4/5 selective small molecule inhibitor LMK-235 effectively eradicates KMT2A-rearranged ALL cell lines as well as primary patient samples in vitro. However, using a xenograft mouse model of KMT2A-rearranged ALL we found that the maximum achievable dose of LMK-235 was insufficient to induce anti-leukemic effects in vivo. Similar results were obtained for the specific class IIA HDAC inhibitors MC1568 and TMP195. Finally, LMK-235 appeared to exert minimal anti-leukemic effects in vivo in combination with the BCL-2 inhibitor venetoclax, but not enough to prolong survival in treated mice. In conclusion, class IIA HDAC isoforms represent attractive therapeutic target in KMT2A-rearranged ALL, although clinical applications require the development of more stable and efficient specific HDAC inhibitors.

Fig. 1. HDAC4, HDAC5 and HDAC7 compromises cell viability upon RNAi knock-down in vitro.

a Relative mRNA expression 72 hours post puromycin selection in two KMT2A-rearranged ALL cell line models SEM and ALL-PO (n = 3). Two shRNA constructs used per class IIA HDAC, except for HDAC7. b HDAC isoform protein expression 72 hours post puromycin selection. Representative western blots shown. c Fold change viable cells (n = 3) for both cell line models assessed using daily 7-AAD viability assay starting 24 hours till 96 hours post puromycin selection. d Apoptosis analysis for SEM and ALL-PO (n = 3) 96 hours post puromycin selection upon HDAC isoform depletion using Annexin-V/7-AAD assay. e Cell cycle distribution analysis based on cell DNA content flowcytometry 96 hours post puromycin selection (n = 2). Statistics were determined using Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001 and ns for no significant p. Error bars represent S.E.M.

Fig. 2. Several class IIA HDAC inhibitors tested in KMT2A-rearranged ALL cell line models.

a Dose response curves for the class IIA HDAC inhibitors LMK-235, BRD4354, MC1568, ACY1215, TMP269 TMP195 and CAY10603 in several KMT2A-rearranged ALL cell line models (n = 5) using 4-day MTT assay. b IC₅₀ values for each class II HDAC inhibitor based on the dose response curves displayed under A). Error bars represent S.E.M.

Fig. 3. HDAC4/5-selective inhibitor LMK-235 recapitulates the knock-down phenotype.

a mRNA expression levels of HDAC4 and HDAC5 upon 100 and 200 nM LMK-235 treatment for 96 hours in three KMT2A-rearranged ALL cell line models. b HDAC isoform protein expression upon 100 and 200 nM LMK-235 treatment for 96 hours in three KMT2A-rearranged ALL cell line models. GAPDH was used as loading control. c Fold change viable cells upon 200 nM LMK-235 treatment assessed using daily 7-AAD viability assay starting 24 hours till 96 hours. d Apoptosis analysis upon LMK-235 treatment (200 nM) for 96 hours using Annexin-V/7-AAD flowcytometry analysis. e Cell cycle distribution analysis upon 96 hours LMK-235 treatment (200 nM) in three KMT2A-rearranged ALL cell line models based on cell DNA content flowcytometry. f Dose response curves for LMK-235 in KMT2A-rearranged primary patient samples from infants (n = 3) and non-leukemic whole bone marrow samples (n = 2) using 4-day MTT assay. g Cell viability upon 10, 100 and 1000 nM LMK-235 treatment using 4-day MTT assay in KMT2A-rearranged primary patient samples from infants (n = 3) and non-leukemic whole bone marrow samples (n = 2). Error bars represent S.E.M.

Fig. 4. LMK-235 monotherapy treatment is unable to reduce overall disease burden in vivo.

a Schematic of the experimental design. SEM-GFP-luc cells were injected into NRG-S mice; 7 days post-transplantation treatment was initiated after engraftment was confirmed by in vivo imaging. Mice were treated by i.p. injection with either LMK-235 (20 mg kg^-1 day^-1) or vehicle for total of 3 weeks with a 5-days on 2-days off schedule. Experimental end-point was reached directly after the last treatment cycle. b Graphical depiction of overall disease burden determined by the average radiance (photons/s/cm²/sr) emitted per mouse monitored every week until experimental end-point. Representative graphical depiction of bioluminescence imaging (n = 3, ordered vertically) for all experimental animals. Rows define experimental timepoints. c Quantification of overall disease burden determined by the average radiance (photons/s/cm²/sr) emitted per mouse monitored every week until experimental end-point. d Spleen weight (grams) upon experimental end-point. e Disease development was monitored by quantifying the fraction of human CD19-positive cells in the bone marrow f spleen and g peripheral blood, analysed after experimental animals were euthanised at the experimental end-point using flowcytometry. Statistical difference was determined by Student’s t-testing. Error bars represent S.E.M.

Fig. 5. In vivo efficacy of LMK-235/venetoclax combination treatment.

a Schematic of the experimental design. SEM-GFP-luc cells were injected into NRG-S mice and 4 days post-transplantation treatment was initiated. Mice were treated with either vehicle, LMK-235 (20 mg kg^-1 day^-1), Venetoclax (100 mg kg^-1 day^-1) or a combination for a total of 3 weeks with a 5-days on 2-days off schedule. Experimental end-point was reached after mice succumbed to leukemia. b Disease development was monitored by quantifying the fraction of human CD19/CD45-positive cells weekly in the peripheral blood. c Overall disease burden was quantified by determining the average radiance (photons/s/cm²/sr) emitted per mouse monitored weekly until experimental end-point. d Graphical depiction of bioluminescence imaging (ordered vertically) for all experimental animals. Rows define experimental timepoints. Red crosses represent deceased mice. e Survival analysis illustrated by Kaplan-Meyer plot. Statistical significance for survival was determined using log-rank testing between venetoclax monotherapy and LMK-235/venetoclax combination therapy. Statistical significance for disease burden between venetoclax monotherapy and LMK-235/venetoclax combination therapy was determined using Mann Witney-U testing. Error bars represent S.E.M.