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. 2015 Nov 5;17(5):597-610.
doi: 10.1016/j.stem.2015.08.004. Epub 2015 Sep 18.

HDAC8 Inhibition Specifically Targets Inv(16) Acute Myeloid Leukemic Stem Cells by Restoring p53 Acetylation

Jing Qi  1 , Sandeep Singh  1 , Wei-Kai Hua  1 , Qi Cai  1 , Shi-Wei Chao  2 , Ling Li  1 , Hongjun Liu  1 , Yinwei Ho  1 , Tinisha McDonald  1 , Allen Lin  1 , Guido Marcucci  1 , Ravi Bhatia  1 , Wei-Jan Huang  2 , Chung-I Chang  3 , Ya-Huei Kuo  4
Affiliations

HDAC8 Inhibition Specifically Targets Inv(16) Acute Myeloid Leukemic Stem Cells by Restoring p53 Acetylation

Jing Qi et al. Cell Stem Cell. .

Abstract

Acute myeloid leukemia (AML) is driven and sustained by leukemia stem cells (LSCs) with unlimited self-renewal capacity and resistance to chemotherapy. Mutation in the TP53 tumor suppressor is relatively rare in de novo AML; however, p53 can be regulated through post-translational mechanisms. Here, we show that p53 activity is inhibited in inv(16)(+) AML LSCs via interactions with the CBFβ-SMMHC (CM) fusion protein and histone deacetylase 8 (HDAC8). HDAC8 aberrantly deacetylates p53 and promotes LSC transformation and maintenance. HDAC8 deficiency or inhibition using HDAC8-selective inhibitors (HDAC8i) effectively restores p53 acetylation and activity. Importantly, HDAC8 inhibition induces apoptosis in inv(16)(+) AML CD34(+) cells, while sparing the normal hematopoietic stem cells. Furthermore, in vivo HDAC8i administration profoundly diminishes AML propagation and abrogates leukemia-initiating capacity of both murine and patient-derived LSCs. This study elucidates an HDAC8-mediated p53-inactivating mechanism promoting LSC activity and highlights HDAC8 inhibition as a promising approach to selectively target inv(16)(+) LSCs.

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Conflict of interest statement

The authors declared that no conflicts of interest exist.

Figures

Figure 1
Figure 1. CM fusion protein binds to p53 and impairs p53 acetylation
A. Western blot time course analysis of Ac-p53, p53, CM, CBFβ, β-actin after IR (3 Gy) in CM-expressing or control BM cells. B. Western blot time course analysis of Ac-p53, p53, CM, CBFβ, HDAC8 and β-actin after IR (3Gy) in 32D-CM or 32D-CBFβ cells. C. Western blot of Ac-p53, p53, CM, CBFβ, β-actin in Cbfb56M/+ BM progenitor cells transduced with MIG-Cre and stimulated with IR (3Gy, 6 h). D. Western blot of Ac-p53, p53, CM, CBFβ, β-actin in 32D-CM cells expressing control (Ctrl)-shRNA or CM-shRNA (A3, D4) 6 h after IR. E. Western blot of CM and β-actin (top) and relative expression of p53 target genes in sorted AML cells transduced with ctrl-shRNA or CM-shRNA (A3, D4) and induced with IR (bottom). Shown are fold change (mean ± SD) relative to ctrl-shRNA-expressing cells, performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001. F. Co-IP and immunoblot (IB) analysis in 32D-CM (top) or 32D-CBFβ (bottom) cells using anti-p53 or anti-mouse IgG for IP, and anti-p53 (left) or anti-CBFβ (right) for IB. G. Representative images of Duolink in situ PLA using mouse anti-CBFβ, rabbit anti-p53 antibodies and PLA probes. Red foci indicate CM-p53 interactions (left), DAPI-stained nuclei are in blue (center) and GFP+ indicates transduced cells (right); scale bar, 10 μm. H. Co-IP (anti-CBFβ) and IB (anti-p53 or anti-CBFβ) analysis in control BM or CM BM cells with or without IR (3Gy). I. Co-IP (anti-p53 or IgG) and IB (anti- CBFβ or anti-p53) in inv(16)+ AML (163, 987) or non-inv(16) AML (467, 865) CD34+ cells 3h after IR. See also Figure S1.
Figure 2
Figure 2. CM fusion protein recruits HDAC8 and p53 in a protein complex and promotes the deacetylation of p53 by HDAC8
A. Sequential co-IP (1° IP anti-HDAC8 or anti-p53, 2° IP anti-CBFβ) and IB (anti-p53 or anti-HDAC8) analysis in 32D-CBFβ or CM cells that were not IR (left) or IR (3 Gy)-treated (right). B. Illustration of CM deletion variants (left) used in the co-IP (anti-p53) and IB (anti-CBFβ or anti-p53) analysis (right). Red arrows indicate the expected size of CM variants. C. Representative images of in situ PLA in 32D FL-CM, d134, d179, ΔC95, Ctrl- or Hdac8-shRNA (sh1 or sh2) expressing cells using mouse anti-CBFβ, rabbit anti-p53 and PLA probes. CM-p53 interactions are shown in red (top), DAPI staining is in blue (center) and the GFP reporter indicates transduced cells (bottom); scale bar, 10 μm. D. Co-IP (IgG or anti-p53) and IB (anti-CBFβ or anti-p53) in 32D-CM cells expressing Ctrl- or Hdac8-shRNA (sh1 or sh2). E. Western blotting of Hdac8, Ac-p53 (K379), p53 and β-actin in 32D-CM cells expressing Ctrl- or Hdac8-shRNA after IR (3Gy, 6h). F. Western blotting of Ac-p53, p53 in CM-, ΔC95-, CBFβ- or FLAG-expressing 32D cells before or after IR (3Gy). G. Fold induction of p53 target genes in CBFβ, CM or CM-ΔC95 expressing cells, 24h after 3Gy IR. Bars represent mean ± SD duplicated assays and two experiments. *P < 0.05; **P < 0.01; ***P < 0.001. See also Figure S2.
Figure 3
Figure 3. Hdac8 deletion diminishes CM-induced LSC transformation and promotes p53 activation
A. Western blot analysis of Hdac8, CM, CBFβ in BM cells from Cbfb56M/+/Mx1-Cre (CM) or CM/Hdac8KO mice 2 weeks after 7 doses of pIpC treatment. B. Western blotting of Ac-p53, p53 before or after (2h) IR (3Gy) in pre-leukemic CM or CM/Hdac8KO BM cells. C. Kaplan-Meier survival curve of induced CM (n=23; median survival 122 days) or CM/Hdac8KO mice (n=56) mice monitored up to 1 year. D. Western blotting of Ac-p53, p53, Hdac8 and β-actin in 32D-CM cells treated with indicated dose of HDAC8i PCI-34051 (μM), 22d (μM) or Nutlin-3 (2.5 μM) for 6 h. E. Western blotting of Ac-p53, p53, Ac-Smc3, Smc3, and β-actin in 32D-CM cells treated with 22d (5 μM), MS-275 (2.5 μM), TV6 (2.5 μM), PCI-24781 (200 nM) or Nutlin-3 (2.5 μM). F. Fold activation of p53 targets in 32D-CM cells treated with HDAC8i PCI-34051 (10 μM) or 22d (10 μM) for 16 h, relative to levels in vehicle-treated cells (dashed line). Shown are mean ± SD of triplicated qRT-PCR assays and two experiments. G. Fold activation of p53 targets in primary CM (n=4) or CM/Hdac8KO (n=5) progenitors cells treated with the HDAC8i 22d (10 μM) for 16 h, relative to levels in vehicle treated cells (dashed line). Mean ± SD of triplicated qRT-PCR assays are shown. H. Survival inhibition dose-response curve and IC50 of HDAC8i 22d for 32D-CBFβ, 32D-CM or 32D-CM cells overexpressing HDAC8. I. Western blot analysis of Ac-p53, p53, Ac-SMC3, SMC3, HDAC8 and β-actin in 32D-CM cells with or without HDAC8 overexpression (O/E) treated with 22d (0, 2.5, 5, 10 μM). J. Fold induction of luciferase reporter under the control of p53 responsive elements in K562 cells co-transfected with CM and p53-WT or p53-8KR mutant, and treated with 22d (10 μM). Dashed line indicates levels in vehicle treated cells. Mean ± SD of four assays in two experiments are shown. *P < 0.05; **P < 0.01; ***P < 0.001; see also Figure S3.
Figure 4
Figure 4. Inhibition of HDAC8 selectively induces p53-dependent apoptosis in inv(16)+ AML stem/progenitor cells
A. Relative expression of HDAC8 in normal (NL) PBSC (n=7; mean indicated by dashed line), inv(16)+ AML (n=7) or non-inv(16)+ AML (n=19) CD34+ cells. Each dot represents the average of triplicate qRT-PCR assays from an individual patient and line indicates the mean ± SEM of all samples. B. Percent survival of NL (n=7), inv(16)+ AML (n=12), t(8;21) AML (n=3) or other non-inv(16) AML (n=4) CD34+ cells treated with HDAC8i 22d for 48 h, measured by Annexin V labeling. Each dot represents an individual subject and lines indicate the mean ± SEM. C. Survival inhibition dose-response curve and IC50 of HDAC8i 22d for NL, inv(16)+ AML, t(8;21) AML or other non-inv(16) AML CD34+ cells as described in B. D. Western blots of Ac-p53 (K382), p53 and β-actin in inv(16)+ AML CD34+ cells treated with 22d (10 μM) or Nutlin-3 (2.5 μM) for 6 h. Shown are representative results from four patients. E. Fold induction of p53 target genes in inv(16)+ AML CD34+ (n=9–13) or NL CD34+ (n=7) cells treated with 22d (10 μM) for 16 h, relative to the levels in vehicle-treated cells (dashed line). Each dot represents the mean of triplicated qRT-PCR assay for an individual subject and the lines indicate the mean ± SEM. F. Representative FACS plot of Annexin V/DAPI labeling in inv(16)+ AML CD34+ cells expressing control (ctrl) or p53 shRNA (GFP+) and treated with 22d for 48 h. G. Relative survival of sorted GFP+ inv(16)+ AML CD34+ cells expressing ctrl- or p53-shRNA treated with 22d for 48 h (top). Each dot represents an individual patient and the lines indicate mean ± SEM. Dose-response curve and IC50 of shctrl- (solid line) or shp53-cells (dashed line) treated with 22d (bottom). H. Percent survival of inv(16)+ AML (n=3–7) CD34+ cells treated with 22d (5 or 10 μM) or combined with DNR (50 or 100 nM) or Ara-C (0.2, 1, 5μM). *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant. See also Figure S4 and Table S1.
Figure 5
Figure 5. Ex vivo treatment of HDAC8i 22d diminishes AML engraftment and leukemia initiation
A. Schematic illustration of the experimental design. AML cells were isolated from pIpC-induced Cbfb+/56MMx1Cre/tdTomato+ moribund mice, treated with 22d or vehicle for 48 or 72 h and transplanted into congenic mice. Engraftment of AML in PB was monitored over time (4, 8 weeks). Mice were analyzed for BM and spleen engraftment at 8 weeks or monitored for leukemia onset and survival up to 8 months. B. Engraftment of AML cells in the PB at 4 (n=7; P=0.0006) or 8 weeks (vehicle, n=5; 22d, n=7; P=0.0025). Shown are mean ± SEM. C. Representative images of spleens from recipients of vehicle-treated (top) or 22d-treated cells (bottom) at 8 weeks. D. Representative FACS plots and frequencies of engrafted dTomato+/cKit+ AML cells in the BM at 8 weeks. E. Frequency of AML cells in the BM of recipients of vehicle treated (n=5) or 22d treated cells (n=7). Each dot represent results from an individual mouse and lines indicate mean ± SEM. **P=0.0025 F. Frequency of AML cells in the spleen of recipients of vehicle-treated (n=5) or 22d-treated cells (n=7). **P=0.0025 G. Survival curve of mice transplanted with AML cells treated with 22d (n=8) or vehicle (n=11; ***P=0.0007). H. The frequency of dTomato+ cells in the PB 8 weeks after transplantation of 2×106 live AML cells treated with 22d or vehicle ex vivo for 48 h (n=4). Each dot represents results from individual mice and line indicate mean ± SEM. *P=0.0357 I. The frequency of dTomato+/ckit+ cells in the BM 28 weeks after transplantation of 2×106 live cells treated with 22d (n=4) or vehicle (n=2; another 2 had died of AML prior to analysis) ex vivo for 48 h (n=4). Each dot represents results from individual mice and line indicate mean ± SEM. *P=0.0206 J. Survival curve of mice transplanted with 2×106 live AML cells treated with 22d or vehicle (n=4; **P=0.0091). See also Figure S5.
Figure 6
Figure 6. Administration of HDAC8i 22d in vivo effectively reduces AML burden and abrogates LSC activity
A. Schematic illustration of the experimental design. CM/tdTomato+ AML cells (2×106) were transplanted into cohorts of congenic mice. After 5–6 weeks, mice were treated with vehicle or 22d (50mg/kg/dose) twice a day for 2 weeks. AML engraftment was analyzed at the end of treatment and BM cells were transplanted into second recipients, which were analyzed for engraftment at 8 weeks or monitored for leukemia onset and survival monitored up to 1 year. B. Western blot analysis of Ac-p53, Ac-SMC3 levels in BM cells from mice treated with HDAC8i 22d or vehicle. C. Representative FACS plots showing gating and frequency of dTomato+/cKit+ AML cells in the BM of vehicle- (top) or 22d (bottom)-treated mice. D. Frequency of AML cells in the BM of mice treated with vehicle (n=13) or 22d (n=13) for 2 weeks. Each dot represents results from an individual mouse and lines indicate mean ± SEM. **P=0.0097 E. Total number of AML cells in the BM of mice treated with vehicle (n=13) or 22d (n=13) for 2 weeks. *P=0.01 F. Frequency of AML cells in the BM of secondary transplant recipients of BM from vehicle- (n=5) or 22d-treated (n=4) mice. ***P<0.0001 G. Total number of AML cells in the BM of second transplant recipients of vehicle (n=5) or 22d treated (n=4) BM. ***P=0.0006 H. Spleen weight of second transplant recipients of vehicle (n=5) or 22d treated (n=4) BM. *P=0.0159 I. Survival curve of second transplant recipients of vehicle- (n=5) or 22d-treated (n=4) BM. **P=0.0049
Figure 7
Figure 7. Propagation of primary inv(16)+ AML is diminished by in vivo HDAC8i 22d treatment
A. Schematic illustration of the experimental design. Primary inv(16)+ AML cells from patient AML1070 (see Table S1) were depleted of T cells and directly injected intrafemorally into irradiated (300cGy) NSGS mice (1×106 cells/mouse). Human AML hCD45+ cells were selected from the leukemic BM for transplantation (2×106 hCD45+ cells/mouse) into larger cohorts of NSGS mice. When AML progression is evident, we began treatment with 22d (50mg/kg/dose) or vehicle twice daily for 2 weeks. Treated mice were then analyzed for AML burden and BM cells were transplanted into secondary recipients. B. Representative FACS plots showing gating and frequency of hCD45+ (left) and hCD45+/CD34+ (right) cells in mice received vehicle (top) or 22d (bottom) treatment for 2 weeks. C. Frequency of hCD45+ AML cells in the PB of vehicle or 22d treated mice (n=5; P=0.0159). D. Frequency of hCD45+ AML cells in the BM of vehicle or 22d treated mice (n=5; P=0.0079). E. Frequency of hCD45+/CD34+ AML cells in the BM of vehicle or 22d treated mice (n=5; P=0.0079). F. The spleen weight of mice treated with vehicle (n=5) or 22d (n=5). *P=0.0159 G. Representative FACS plots of hCD45+/CD34+ AML cell frequency in BM of vehicle- or 22d-treated secondary transplants 12 weeks after transplantation. H. Frequency of hCD45+ AML cells in BM of vehicle- or 22d-treated secondary transplants (n=4; P=0.0007). I. Frequency of hCD45+/CD34+ AML cells in BM of vehicle- or 22d-treated secondary transplants (n=4; P=0.0006). J. Frequency of immunophenotypic populations within hCD45+ cells in BM of vehicle- or 22d-treated secondary transplants (n=4; *P<0.05; ***P<0.001). K. Survival curve of 2nd transplant recipients of vehicle- (n=4) or 22d-treated (n=3) BM up to 5 months after transplantation. *P=0.0285 See also Figure S6.
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