HDAC4 inhibition disrupts TET2 function in high-risk MDS and AML

HDACi treatment decreases total 5hmC levels and TET2 expression in a murine MDS model

We first chose to assess effects of in-vivo treatment with the pan-HDACi SAHA in NUP98-HOXD13 (NHD13) transgenic mice, which display severe symptoms resembling human MDS and die of cytopenias or transformed leukemia in 8-12 months [17]. After confirming MDS development, as evidenced by leukopenia and anemia, we treated NHD13 mice with SAHA (I.P. 50mg/kg) or vehicle control (Figure 1A) for 4 weeks. SAHA treatment did not reverse cytopenias but decreased the number of granulocytes and lymphocytes in bone marrow (BM) and peripheral blood (PB) (Figure 1B, 1C). SAHA treatment did not alter LK (Lin^-Sca1^-c-kit⁺) or LSK (Lin^-Sca1⁺c-kit⁺) frequency, but decreased frequencies of common myeloid progenitors (CMPs) and granulocyte-monocyte progenitors (GMPs) (Figure 1D). We observed a modest increase in the number of megakaryocyte erythrocyte progenitors (MEPs) and ter119⁺ erythroid cells in BM of the SAHA-treated mice, but anemia was not reversed (Figure 1C, 1D).

Figure 1. SAHA treatment reduces TET2 and 5hmC levels in HSPCs of NHD13 mice. (A) NHD13 mice were divided into two groups of matched gender and age. Peripheral blood (PB) was sampled before treatment, and mice were then treated 4 weeks with SAHA (50mg/kg/day, I.P., n = 8) or vehicle (5% DMSO in 20% cyclodextrin, I.P., n = 8). After treatment, mice were euthanized, and PB and BM were collected. (B) CBC as indicated in NHD13 mice analyzed after 4 weeks of treatment. (C) Hematopoietic lineage subset frequencies in BM of NHD13 mice after 4 weeks of indicated treatment. (D) Hematopoietic progenitor subset frequencies in BM of NHD13 mice after 4 weeks of indicated treatment. (E, F) 5hmC levels in MNCs of PB from NHD13 mice before or after treatment, as determined by ELISA (E) and dot blot (F). Methylene blue staining served as a loading control. 5hmC levels were normalized against loading of total DNA. (G) 5hmC levels in LSK cells of BM from treated NHD13 mice (n = 3), as determined by intracellular staining with a 5hmC-specific antibody. (H) Western blot showing TET2 protein levels in c-kit+ cells from BM of NHD13 mice treated as indicated. (I) RT-qPCR detection of TET2 mRNA levels in c-kit+ cells from BM of NHD13 mice treated as indicated.

We next asked whether SAHA treatment altered DNA demethylation in NHD13 mice. We found that after 4 weeks of SAHA treatment, mice showed significantly decreased 5hmC levels in PB mononuclear cells (MNCs) relative to vehicle-treated controls (Figure 1E, 1F). Moreover, intracellular staining confirmed reduced 5hmC levels in LSKs of SAHA- versus vehicle-treated NHD13 mice (Figure 1G). We further assessed TET2 protein and mRNA levels after SAHA treatment and found that both were lower in c-kit⁺ BM cells from SAHA- relative to vehicle-treated mice (Figure 1H, 1I).

HDACi treatment reduces total 5hmC and TET2 expression levels in MDS/AML lines

We next treated human MDS and AML cell lines ex-vivo with SAHA. Treatment significantly decreased total 5hmC levels in MDS-L cells (Figure 2A, 2B), a line derived from an MDS patient [18], as well as TET2 expression levels in MDS-L cells and in human AML cell lines (Figure 2C and Supplementary Figure 1A). As a positive control, SAHA treatment also increased levels of histone H3K27 acetylation (H₃K₂₇Ac) (Figure 2C, Supplementary Figure 1A). Similarly, treatment of MDS-L cells with a different pan-HDACi, Trichostatin A (TSA), reduced TET2 protein levels (Figure 2D). SAHA or TSA treatment also reduced TET2 mRNA levels (Figure 2E and Supplementary Figure 1B), indicating that HDACi treatment downregulates TET2 at the transcriptional level. Expectedly, SAHA or TSA treatment ex-vivo reduced MDS-L and Nomo-1 cell viability (Supplementary Figure 2A, 2B). Overall, these results indicate that although HDACi treatment has an anti-cancer effect, it can also significantly downregulate TET2 activity, impairing DNA demethylation capacity and potentially counteracting a therapeutic effect.

Figure 2. HDACi treatment promotes DNA hypermethylation. (A, B) MDS-L cells were treated with 2 or 4 μM SAHA for 24 hours, and then 5hmC levels were determined by ELISA (A) or dot blot (B). (C, D) MDS-L, Nomo-1 or Molm13 cells were treated with 2 or 4 μM SAHA (C) or 1 μM TSA (D) for 24 hours, and then TET2 protein levels were determined by Western blot. In (C) H3K27Ac served as a positive control indicative of HDAC inhibition. (E) TET2 mRNA levels in 2 μM SAHA- or 1 μM TSA-treated MDS-L cells, as detected by RT-qPCR.

HDAC4 activity is required to maintain TET2 levels

We next asked which HDAC is responsible for maintaining TET2 activity. Utilizing siRNAs targeting specific genes that encode HDACs, we found that only knockdown of HDAC4, a class II HDAC [13], decreased TET2 mRNA levels (Figure 3A). To validate results of the RNAi screen, we then knocked down HDAC4 in MDS-L or Nomo-1 cells by lentiviral delivery of shRNA and in both observed decreased TET2 mRNA and protein levels (Figure 3B, 3C). Next, we measured TET2 expression levels in BM of HDAC4 knockout (KO) mice and found relatively decreased TET2 protein levels (Figure 3D). To confirm that HDAC4 regulates TET2 expression, we treated MDS-L cells with the HDAC4/5 inhibitor LMK-235 and observed marked downregulation of TET2 mRNA and protein levels relative to vehicle controls. In contrast, treatment of MDS-L cells with the HDAC1/HDAC3 inhibitor MS-275 did not alter TET2 levels, although H₃K₂₇Ac levels were significantly increased relative to untreated cells (Figure 3E, 3F).

Figure 3. HDAC4 knockdown decreases TET2 expression. (A) Nomo-1 cells were transduced with siRNAs targeting indicated HDAC genes, and TET2 expression was determined by RT-qPCR. (B, C) HDAC4 knockdown using lentivirus to deliver shRNA to MDS-L and Nomo-1 cells. TET2 expression was determined by RT-qPCR (B) or Western blot (C). (D) Total BM and c-kit⁺ cells were harvested from HDAC4 WT or KO mice, and TET2 protein levels were determined by Western blot. (E) MDS-L and Nomo-1 cells were treated with 5 μM LMK-235 or 1 μM MS-275 for 24 hours, and TET2 expression was determined by RT-qPCR. (F) MDS-L cells treated with LMK-235 or MS-275 were analyzed by Western blotting for TET2 expression. H3K27Ac served as a positive control.

Notably, overexpression of HDAC4 upregulated TET2 expression levels (Figure 4A, 4B). We also observed decreased total 5hmC levels in HDAC4 knockdown MDS-L cells relative to controls (Figure 4C, 4D). MTSS1 is a reported TET2 target and tumor suppressor gene [19]. Thus, we determined MTSS1 expression levels and evaluated potential changes in 5hmC enrichment on the MTSS1 enhancer region in HDAC4 KO cells. As expected, HDAC4 KO mice showed relatively decreased 5hmC enrichment on the MTSS1 enhancer (Chr15: 58979469-58979554) (Figure 4E), a change associated with reduced MTSS1 expression (Figure 4F). Moreover, MTSS1 expression levels decreased in NHD13 mice following SAHA treatment (Figure 4G). Phenotypically, NHD13/HDAC4 KO BM cells showed significantly enhanced colony formation capacity relative to NHD13/HDAC4 WT BM cells (Figure 4H). Overall, these results indicate that HDAC4 maintains TET2 activity and that HDAC4 deficiency may promote MDS cell growth.

Figure 4. HDAC4 is required to maintain TET2 activity. (A, B) HDAC4 was overexpressed in 293T cells and TET2 expression determined by RT-qPCR (A) or Western blot (B). (C, D) After HDAC4 knockdown using lentivirus to deliver shRNA to MDS-L cells, 5hmC levels were determined by ELISA (C) or dot blot (D). (E) hMeDIP-qPCR analysis of specific 5hmC enrichment on Chr15: 58979469-58979554, enhancer region of MTSS1 in c-kit⁺ cells from BM of NHD13/HDAC4 WT or NHD13/HDAC4 KO mice. Bars represent mean enrichment over input. (F) MTSS1 mRNA levels in c-kit⁺ cells from BM of NHD13/HDAC4 WT or NHD13/HDAC4 KO mice. (G) RT-qPCR detection of MTSS1 mRNA levels in c-kit⁺ cells from BM of NHD13 mice treated with SAHA or vehicle control. (H) Serial replating of NHD13/HDAC4 WT or NHD13/HDAC4 KO cells. Total BM cells from each these mice were seeded in methylcellulose medium, and colonies were counted on day 7.

HDAC4 expression levels correlate with TET2 levels in high-risk MDS/AML

We next analyzed public accessible GEO dataset and observed a positive correlation between HDAC4 and TET2 levels within a MDS cohort (GSE13159 [20, 21]) (Figure 5A). This correlation was confirmed in another AML dataset (GSE21261) [22] (Figure 5B). To further characterize HDAC4 function in myeloid neoplasms, we analyzed HDAC4 expression levels in AML patients with normal karyotype versus healthy donors (GSE13159). Remarkably, HDAC4 expression levels in those patients were significantly lower than those in healthy individuals (Figure 5C). Importantly, lower than median levels of HDAC4 expression were associated with shorter overall patient survival (GSE12417 [23]) (Figure 5D). Taken together, these data support the idea that HDAC4 deficiency may promote disease progression in a subset of leukemia patients.

Figure 5. TET2 and HDAC4 expression is positively correlated in high-risk MDS/AML. (A, B) Microarray expression data from total BM of MDS (A, n = 206) or mononuclear AML (B, n = 96) cells. P value was calculated by Pearson r correlation. (C) HDAC4 expression data from total BM from healthy donors (n = 73) or normal karyotype AML patients (n = 351). P value was calculated by unpaired t test. (D) Overall survival in 163 normal karyotype AML patients based on clustering of HDAC4 expression. Patients were separated into 3 expression groups—low (n = 61), middle (n = 69) and high (n = 33)—based on a standard K-means clustering approach. Kaplan-Meier curves were generated and log-rank p-values calculated to assess statistical significance.

A TET2 agonist in combination with HDACi potently inhibits MDS-L cell growth in vivo

Previous studies indicate that treatment with vitamin C, a TET2 agonist, enhances TET2 activity in vivo [24, 25], promoting DNA demethylation capacity and reversing aberrant self-renewal of leukemia initiating cells [25]. Thus, to assess effects of combining HDAC inhibition with vitamin C treatment, we transplanted luciferase-expressing MDS-L cells into NSGS mice to establish a MDS line xenograft model. Once we observed basal engraftment, mice were treated with vehicle, vitamin C (Ascorbate, ASC), SAHA or a combination of each (Figure 6A). Interestingly, combined SAHA and ASC treatment promoted greater inhibition in MDS-L engraftment than did treatment with either single drug (Figure 6B, 6C). We then assessed 5hmC levels in PB of treated mice and found that although total 5hmC levels decreased upon treatment with SAHA alone, this reduction was fully rescued after SAHA/ASC combination treatment (Figure 6D).

Figure 6. TET2 agonist in combination with HDACi promotes potent anti-leukemia effects in vivo. (A) Schematic showing experimental protocol. 2.5 x 10⁶ luciferase-expressing MDS-L cells were transplanted into NSGS recipient mice intravenously. Seven days later, baseline engraftment was detected by bioluminescence imaging. Drug treatment began on D7 via I.P. injection. MDS-L engraftment was detected at D21. (B) Pseudocolored bioluminescence images of NSGS mice transplanted with MDS-L cells and subjected to indicated drug treatment. Radiance is in units of “photons/second/cm2/steradian”. One mouse in the SAHA group died during drug administration (indicated by red X). (C) Total flux (photons/sec) of NSGS mice transplanted with MDS-L cells after indicated treatments. Signal intensity was quantified from each animal shown in panel b. (D) 5hmC levels (as quantified by flow cytometry) in peripheral blood cells of mice after indicated treatments.

Mice

NUP98-HOXD13 (NHD13) mice and Vav1-Cre mice were purchased from Jackson Laboratory. HDAC4^fl/fl mice were purchased from Taconic Biosciences and bred at the City of Hope (COH) animal facility. All mice received autoclaved water and clean food, were subjected to 12-hour light/dark cycles, and kept in controlled ambient room temperature and air humidity conditions. All animal procedures were conducted in accordance with established institutional guidance and approved protocols of the Institutional Animal Care and Use Committee at COH. NHD13 mice were mated with HDAC4^fl/fl/Vav1-Cre to obtain NHD13/HDAC4^wt/fl/Vav1-Cre mice, and subsequently NHD13/HDAC4^fl/fl/Vav1-Cre (NHD13/HDAC4 KO mice).

In vivo treatment of transgenic NHD13 mice and MDS-L xenografted NSGS mice

Before drug treatment, NHD13 mice were divided into two groups with matched gender and age. Mice were then treated 4 weeks with SAHA (50mg/kg/day, I.P., n = 8) or vehicle (5% DMSO in 20% cyclodextrin, I.P., n = 8). Mice were then euthanized and PB and BM collected for further experiments. For NSGS mice, 2.5 x 10⁶ luciferase-expressing MDS-L cells were transplanted into recipients intravenously. Seven days later, basal engraftment was determined by bioluminescence imaging. Drug treatment began at day (D) 7 with vehicle (5% DMSO in 20% cyclodextrin, I.P., n = 5), ASC (4g/kg/day, I.P., n=5), SAHA (50mg/kg/day, I.P., n = 5) or SAHA + ASC (I.P., n=5). MDS-L engraftment was then evaluated at D21.

In vivo bioluminescence imaging

NSGS mice were injected intraperitoneally with 150mg/kg D-luciferin (XenoLight). Ten minutes later, mice were anesthetized and imaged with Lago X (Spectral Instruments Imaging). Bioluminescent signals were quantified using Aura imaging software (Spectral Instruments Imaging). Total flux values were determined and are presented as photons (p)/second (sec).

Flow cytometry

Mouse BM was aspirated from tibias and femurs, washed, and then filtered using 70-μm cell strainers. Cells were stained with required antibodies for 20 minutes at 4°C, washed and analyzed on a 5-laser, 15 detector LSRII. The DAPI (ThermoFisher Scientific)-negative population was selected as living cells. Mouse HSPCs were determined by staining with anti-mouse c-kit, Sca-1, CD16/32 and CD34 antibodies, biotin-linked lineage cocktails (including anti-mouse CD3, CD4, CD8, B220, IgM, CD19, CD11b, CD11c, NK1.1, Gr1, CD41 and ter119 antibodies (eBioscience)) and FITC-labeled streptavidin. The lineage^- c-kit⁺ Sca-1^- population was defined as LK, and the lineage^- c-kit⁺ Sca-1⁺ population as LSK. CMP, GMP and MEP populations were further gated from the LK population according to CD16/32 and CD34 expression. Mouse BM differentiation was evaluated using anti-mouse CD11b, Gr-1, B220, CD3 and Ter119 antibodies. Data were analyzed using BD FACS Diva or FlowJo software.

BM c-kit⁺ cell selection

BM c-kit⁺ cells were isolated using mouse CD117 MicroBeads (Miltenyi Biotec) according to the kit protocol. Briefly, total BM cells were counted and resuspended in MACS buffer. After staining with CD117 microbeads (20μL per 10⁷ total cells, 15 min at 4°C), cells were washed and applied onto MS columns, which were rinsed and placed in a magnetic field. Columns were washed three times with 500 μL buffer and then removed from the separator. Then magnetically-labeled cells were flushed out with 1mL buffer. c-kit⁺ cells were counted and used for further experiments.

Intracellular 5hmC staining

For intracellular staining, mouse BM was first stained with HSC surface markers, as described above. Peripheral blood from NSGS mice was lysed with ammonium chloride potassium (ACK) buffer, and remaining MNC cells were washed and fixed with 4% paraformaldehyde in the dark for 15 minutes and permeabilized with 0.5% Saponin permeabilization solution (BD Bioscience) for 1 hour at room temperature. After washing, cells were labeled with rabbit anti-5hmC antibody (active motif, 1/200, 30 mins at 4 °C) or isotype control and then incubated in Alexa 488-conjugated goat anti-rabbit antibody (Invitrogen, 1/1000, 30 mins at 4 °C), washed and analyzed by flow cytometry.

5hmC quantification by dot blot and ELISA

5-hmC levels in treated cells were detected by dot blots and ELISA as described [8]. For dot blots, DNA samples were isolated using a DNeasy Blood and Tissue kit (Qiagen). Denatured DNA and two-fold serial dilutions were spotted onto nitrocellulose membranes, and after UV cross-linking membranes were blocked and incubated with anti-5hmC antibody (1/1000) overnight at 4°C. Next, membranes were washed and incubated with HRP-conjugated second antibody for 1 hour at room temperature, and signal density was detected using an ECL kit (ThermoFisher Scientific). To ensure equal loading of total DNA, the same membranes were stained in 0.02% methylene blue in 0.3M sodium acetate. To quantify 5hmC, a MethylFlash Global DNA Hydroxymethylation ELISA Easy Kit (Epigentek) was used according to the suggested protocol. Briefly, equal amounts (100ng) of DNA were added, and the initial incubation time was 90 min and final developing time 10 min. Absorbance at 450 nm was determined.

Western blotting

Western blotting was performed as described [8]. Cells were lysed in CytoBuster Protein Extraction Reagent (Millipore), and boiled lysates were then resolved on 7.5% or 12% SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-Rad). The following primary antibodies were used: rabbit anti-mouse Tet2 (ab124297, Abcam), rabbit anti-human Tet2 (GTX124205, GeneTex), rabbit anti-human/mouse HDAC4 (15164, Cell Signaling Technology), rabbit anti-human/mouse H₃K₂₇ac (8173S, Cell Signaling Technology), and mouse monoclonal anti-beta-actin (sc-69879, Santa Cruz Biotechnology).

RT-qPCR analysis

Total RNA was isolated from cells using Trizol reagent (Invitrogen) according to the manufacturer's protocol. cDNA was amplified using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real time PCR was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems) with 0.2μM Taqman probe (Life Technologies). PCR amplification was carried out in a QuantStudio 7 Flex Real-Time PCR system (Life Biotechnology). Relative transcript levels were normalized to GAPDH levels.

hMeDIP-qPCR analysis

hMeDIP assays were performed based on the manufacturer’s instructions using the SimpleDIP kit, as described [8]. DNA was denatured and IP’d with anti-5hmC or IgG control antibodies and protein G magnetic beads. After three washes, 5hmC-modified DNA was eluted from beads, purified and used as the template in qPCR.

Cell culture

The human MDS cell line MDS-L was developed and provided by Dr. Kaoru Tohyama [18]. MDS-L cells were cultured in RPMI 1640 with 10% FBS and 1% penicillin-streptomycin in the presence of 30 ng/mL human recombinant human IL-3. Nomo-1 and other AML cell lines were cultured in 10% FBS/RPMI 1640 medium.

Lentivirus transduction and cell transfection

First, 293T cells were transiently transfected with pCDH plasmids and pMD2G/pSAPX2 packaging plasmids, and then 24-36 hours later, supernatants containing replication-incompetent lentiviruses were collected and concentrated using PEG-it (System Biosciences). After titration, target cells were exposed to virus (MOI=10), and 48 hours later, cells were sorted by flow cytometry based on GFP/RFP expression. For siRNA transfection, Nomo-1 cells were transfected with HDAC1-8 siRNA (Dharmacon) by nucleofection (Lonza). A FAM-conjugated siRNA control was included, and FAM positivity above 90% was considered successful transfection.

In vitro colony forming assays

NHD13/HDAC4 WT or KO BM were obtained and suspended in methylcellulose medium (M3434, StemCell Technologies) and seeded into plates (5 × 10⁴ per well) according to manufacturer’s protocol. Colonies were counted 7 days later. For replating assays, cells from each plate were harvested and replated at 5 × 10⁴ per well.

Cell viability analysis

Cell viability was determined using the Cell Titer-Glo Luminescent Cell Viability Assay Kit (G7570, Promega). Briefly, cells were cultured under different treatment conditions and transferred to 96-well plates for 30 min. Kit substrate and buffer reagents were then added to each well and mixed to allow cell lysis. Plates were read on a Microplate reader (Beckman culture DTX880). Results were expressed as a percentage of untreated cells, for three replicates.

Clinical outcome analysis

Clinical outcome analysis was performed as described [8]. Briefly, RNA sequencing data form normal karyotype AML bone marrow mononuclear cells (GSE12417, 163 patients with survival information) was obtained. Patients are separated into 3 groups based on the K-means clustering approach (Hartigan-Wong algorithm). Then, the survival curves are created using Function “survfit” (in R package “survival”) from Kaplan-Meier formula. Finally, the log-rank p-value is calculated by Function “survdiff” (in R package “survival”) to test the differences among 3 survival curves using Gp family of tests.

Statistical analysis

Data obtained from independent experiments were expressed as means ± SEM, and n represented the number of samples. Two-tailed Student’s t-tests were used to compare differences between groups. GraphPad Prism 8 software (La Jolla, CA) was used to assess statistical analyses. P < 0.05 represented a statistically significant difference.

Ethics statement

All animal procedures were conducted in accordance with established institutional guidance and approved protocols of the Institutional Animal Care and Use Committee at COH.