Targeting DNA Damage Repair Functions of Two Histone Deacetylases, HDAC8 and SIRT6, Sensitizes Acute Myeloid Leukemia to NAMPT Inhibition

Acute myeloid leukemia (AML) is the most commonly diagnosed leukemia in adults (1, 2). The overall prognosis of the disease remains poor, with a 5-year survival rate of less than 10% for patients more than 60 years old. AML cells are addicted to nicotinamide phosphoribosyltransferase (NAMPT)-mediated salvage pathway for the biosynthesis of NAD⁺ (3, 4), which serves as a coenzyme for redox reactions and substrate for PARP, regulating DNA damage repair (DDR) gene expression and stress responses. Thus, targeting the NAMPT-dependent NAD⁺ generation has gained attention as a potential therapeutic strategy in AML and NAMPT inhibitors (NAMPTi) and have moved to phase I trials. Our previous studies demonstrated preclinical efficacy of targeting NAMPT on eliminating AML in vitro and in vivo by employing a potent NAMPTi, KPT-9274 (5). Although our preclinical data are compelling, we recognize that NAMPTis will likely be ineffective as monotherapies based upon AML metabolic plasticity ultimately permitting resistance (6). In addition, dose-limiting toxicities pose another barrier for clinical success of the inhibitor (7, 8). In clinical trials with the first-generation NAMPTis, dose-limiting toxicities were observed, such as thrombocytopenia and gastrointestinal, retinal, and cardiac toxicities (9). Unlike first-generation NAMPTis, KPT-9274 was better tolerated in phase I trials on solid tumor and non-Hodgkin lymphoma, but drug-related adverse events, including anemia and fatigue, were reported. Therefore, strengthening the clinical efficacy of NAMPTis through rational combination therapies represents an unmet need in AML.

To address this, we utilized an unbiased genome-wide CRISPR screen to identify genes that upon depletion confer sensitivity to KPT-9274 in AML cells. Among the top ranked hits, we identified two genes encoding NAD⁺-dependent histone deacetylases (HDAC), namely HDAC8 and SIRT6, both with known roles in compensatory DDR pathways. We next determined the efficacy of combined NAMPTi with genetic or pharmacologic inhibition of HDAC8 or SIRT6 in AML cell lines and primary patient samples, and investigated the mechanisms that may contribute to the effect. We hypothesize that HDAC8 inhibitor (HDAC8i)- or SIRT6 inhibitor (SIRT6i)-mediated DDR deficiencies sensitize AML cells to synthetic lethality orchestrated by NAMPTi. Therefore, concurrent inhibition of NAMPT and factors involved in compensatory DDR pathways may achieve our goals of improving the therapeutic index and effectiveness of NAMPTis in AML.

The Human GeCKOv2 CRISPR Knockout Library was obtained from Addgene. The library was amplified in bacteria and packaged into viral particles in HEK293FT cells. MOLM13 cells were transduced with lentiviral particles at a predetermined ratio in the presence of polybrene and spinoculated at 450 × g for 90 minutes. Puromycin selection was initiated after 48 hours and continued for 7 days to eliminate cells with essential gene-targeting single-guide RNA (sgRNA) and nontransduced cells. The transduced cells were cultured for 3 days in the presence of 50 nmol/L KPT-9274 or DMSO to negatively select sgRNAs that sensitize resistant cells to KPT-9274. Cells were collected on days 0 and 3 and subject to P5/P7 barcoding and deep sequencing using Illumina HiSeq4000 sequencer to detect the abundance of each sgRNA. Sequencing data were analyzed using MAGeCK VISPR algorithm to discover sgRNAs that were negatively or positively selected with KPT-9274.

KPT-9274, AR-42, AG221, AG120, and PCI-34051 were purchased from Selleckchem. For in vivo study, AR-42 was formulated in 0.5% methylcellulose (w/v) and 0.1% Tween‐80 (volume/volume) in sterile water.

MOLM13 (catalog no. ACC-554, RRID:CVCL_2119), MV4-11 (catalog no. ACC-102 RRID:CVCL_0064), and OCI-AML3 (catalog no. ACC-582, RRID:CVCL_1844) were purchased from DSMZ and cultured in RPMI1640 (Gibco) supplemented with 10% FBS. Kasumi-1 cells (catalog no. CRL-2724, RRID:CVCL_6911) were purchased from the ATCC and cultured in RPMI1640 (Gibco) supplemented with 20% FBS. Isocitrate dehydrogenase2 (IDH2)^R140Q (catalog no. CRL-2003IG, RRID:CVCL_UE10) and wild-type (WT) TF-1 cells (catalog no. CRL-2003, RRID:CVCL_0559) were obtained from the ATCC and cultured in RPMI1640 (Gibco) supplemented with 10% FBS and 2 ng/mL recombinant human GM-CSF. MOLM13-luciferase cells were a kind gift from Dr. Ramiro Garzon [Ohio State University (OSU), Columbus, OH]. HEK293FT cells were obtained from Life Technologies and cultured in DMEM (Gibco) with 10% FBS. All cell lines were used between passages three and 20 and routinely tested negative for Mycoplasma contamination with universal Mycoplasma detection kit (ATCC 30-1012K). Cells were authenticated with microsatellite genotyping (short tandem repeat analysis by the OSU Genomic Services Core, Columbus, OH). For shRNA transfection, shRNA oligos for knockdown of SIRT6, HDAC8, DCPS, PTGS1, HEXA, and IMPA2 in lentiviral vector pLKO.1 were purchased from Sigma-Aldrich. shRNA sequences are listed in Supplementary Materials and Methods. All viruses were produced using the HEK293FT cells with packaging and envelope plasmids, psPax2 and VSVG. Cells were transduced by spinofection at 1,500 rpm for 90 minutes in the presence of polybrene. Seventy-two hours after transduction, puromycin was added to the culture to select shRNA stable clones.

AML patient and normal donor samples were obtained from OSU (Columbus, OH) Leukemia Tissue Bank. Informed written consent was obtained from each subject or each subject's guardian under an institutional review board–approved protocol according to the Declaration of Helsinki.

AML cell lines or patient-derived cells were treated in a 96-well plate at 0.2–6 × 10⁵ cells per well for 24–72 hours. Patient-derived CD34⁺ primary cells were cultured in 96-well plates coated with collagen in the presence of 10 ng/mL IL3, 10 ng/mL IL6, 10 ng/mL SCF, and 10 ng/mL GM-CSF. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Promega] was incubated with drug-treated cells according to the manufacturer's instructions. Plates were read by using a BioTek Synergy H4 Hybrid Multimode Microplate Reader (Thermo Fisher Scientific) at 490 nm. Combenefit software was used to calculate synergy scores for pairwise combinations of drug doses.

For colony-forming unit (CFU) assays, viable cells were plated at optimal densities in MethoCult H0435 (StemCell Technologies) in the presence of DMSO, 0.1 μmol/L KPT-9274, 0.8 μmol/L AR-42, or combination of inhibitors. Formed colonies were counted blindly after 7–14 days and replated at 10,000 cells/well. The images of colonies were captured with Echo revolving microscope. The images of Giemsa staining of cytospin preparations were acquired by CX43 Olympus inverted microscope.

EJDR reporter cells (10) were plated at 5 × 10⁵ cells per well in 10% FBS and RPMI1640. After drug treatments, cells were grown in 10% Tet-free FBS (100–800; Gemini) and RPMI1640. Incorporated I-Sce1 was induced with Shield1 (632189, Clontech) and triamcinolone (T6510, Sigma-Aldrich) ligands for 24 hours. Nonhomologous end joining (NHEJ) and homologous recombination (HR) repair activities were assessed 48 hours after induction by quantification of DsRed- and GFP-positive cells on Cytomics FC 500 Flow Cytometer (Beckman Coulter).

Limiting cell–RNA sequencing (LC-RNA-seq) was performed as described previously (11). Briefly, patient cells were cultured in the presence of cytokines and treated with vehicle, single agents, or drug combination. A total of 300 viable leukemia-initiating cells (LIC) from treated patient samples were sorted directly into SMART-seq lysis buffer with FACSAria Fusion (BD) based on putative stem cell markers, CD34⁺CD38⁻ (12). Initiating/stem cells were enriched in sorted CD34⁺CD38⁻ cells, as CD34⁺CD38⁻ subpopulations grew significantly more colonies than CD34⁺CD38⁺ subpopulations in CFU assays (unpublished observation). The Clontech SMARTer v4 Kit (Takara Bio USA, Inc.) was used for preamplifying samples prior to library construction with the Nextera XT DNA Library Prep Kit (Illumina, Inc.). Samples were sequenced to a depth of 15–20 million 2 × 150 bp clusters with the Illumina HiSeq 4000 platform. After quality control with CLEAR selection process and size normalization, differentially expressed genes (DEG) were called with DESeqs with (FDR) P_adj < 0.05. Principle component analysis (PCA) plots were created from count tables, which were normalized by size, and transformed. For pathway analysis, the list of DEGs, containing gene IDs and corresponding expression values, was uploaded into the ingenuity pathway analysis Software (IPA, Qiagen, RRID:SCR_008653). The “core analysis” function in the software was employed to interpret the differentially expressed data and identify top enriched pathways.

All experiments were approved by the OSU (Columbus, OH) Institutional Animal Care and Use Committee. Male NOD-Prkdc^em26Cd52Il2rg^em26Cd22/NjuCrl (NCG) mice (RRID:IMSR_CRL: 572; ages 6–8 weeks) were obtained from Charles River Laboratories. Mice were group-housed under conditions of constant photoperiod (12-hour light/12-hour dark), temperature, and humidity with ad libitum access to water and irradiated standard pelleted chow. A total of 1 × 10⁴ luciferase-tagged MOLM13 cells were injected via tail vein into NCG mice. On day 5 post-engraftment, mice were randomized to receive vehicle once daily, 100 mg/kg of KPT-9274 once daily, 20 mg/kg AR-42 every other day, or the combination of KPT-9274 and AR-42 via oral gavage. Overall survival was the primary endpoint for the majority of the mice. A separate cohort of mice per group was used to monitor leukemic progression using IVIS imaging. Mice were monitored by animal technicians who were blinded to the treatment groups and determined when mice met early removal criteria (20% weight loss, lethargy, hunching, and poor body condition). Mice were euthanized by CO₂ inhalation.

Data are presented as the mean ± SEM of independent experiments, unless otherwise specified. Statistical analyses were performed with GraphPad Prism 7.0 (GraphPad Prism, RRID:SCR_002798) or SAS/STAT software (version 9.0) with ANOVA with Tukey post-test correction for multigroup comparisons or a two-tailed Student t test for two-group comparisons, unless otherwise specified in the figure legends. A P value of less than 0.01 or 0.05 was considered significant. With 8 mice per group, there was 80% power to detect a difference between treated versus control group at a 5% significance level. For survival analyses, a Cox proportional hazard model was used to determine statistical significance. For CFU measurements, a negative binomial model was used to fit data.

CRISPR screening and RNA sequencing data are available in Gene Expression Omnibus (accession nos., GSE162473 and GSE161397).

To perform the CRISPR-Cas9 screen, we used the lentivirus-based GeCKOv2 library, which contains approximately 130,000 sgRNAs targeting approximately 20,000 protein-coding genes and miRNAs(∼6 sgRNAs/gene; ref. 13). The library was packaged and transduced into Cas9-expressing MOLM13 cells (5). Then, transduced cells were selected in puromycin and received sublethal dose of 50 nmol/L KPT-9274 or DMSO control for 3 days. After mapping sgRNA sequence reads, changes in abundance of each sgRNA were assessed using the MAGeCK program (Fig. 1A; ref. 14). Coverage (400×) of the library was achieved with 95% of the sgRNA sequences being retained in all samples, ensuring the sufficient sgRNA representation. By analyzing the sgRNAs that were negatively selected in the presence of KPT-9274, several genes were identified as top hits with small P values (Fig. 1B; Table 1). We prioritized top hits with available preclinical or clinical inhibitors (i.e., HDAC8, SIRT6, HEXA, IMPA2, PTGS1, and DCPS) for validation. For these hits, high proportion of sgRNAs targeting the same gene exhibited large fold depletion following KPT-9274 treatment (Fig. 1C). In this unbiased manner, gene candidates whose depletions allowed the broader therapeutic index and overcome NAMPTi insensitivity were nominated.

To validate the results of the CRISPR screen, we first demonstrated genetically the drug coessential nature of these dropout genes on KPT-9274–sensitive cell line, MOLM13, and KPT-9274–insensitive cell line, Kasumi-1. Four CDS-targeting shRNAs and one 3′ untranslated region (UTR)-targeting shRNA were designed per gene. CDS-targeting oligos were pooled and packaged into lentivirus. After puromycin selection, HDAC8-CDS–targeting shRNAs effectively reduced protein expression in Kasumi-1 and MOLM13 cells, while 3′ UTR–targeting oligo only marginally decreased HDAC8 abundance (Fig. 2A). SIRT6 3′ UTR–targeting shRNA decreased the SIRT6 level by 80%.

shHDAC8-CDS or shSIRT6–3′ UTR transfection reduced IC₅₀ doses of KPT-9274 for MOLM13 and Kasumi-1, resulting in a significant reduction of cell survival (Fig. 2B). The vulnerabilities of shHDAC8–3′ UTR–transfected cells to KPT-9274 treatment were comparable with those of scramble-transfected cells, mirroring the low knockdown efficiency of this shRNA construct. These results imply that genetic depletion of HDAC8 decreased tumor survival in the presence of KPT-9274 proportional to the reduction in HDAC8 abundance. Knockdown of IMPA2, HEXA, PTGS1, and DCPS did not demonstrate strong synergies with KPT-9274 treatment, suggesting that they might be false-positive hits from the screen (Supplementary Fig. S1A). In the presence of KPT-9274 at sublethal IC₂₀ doses, HDAC8 depletion resulted in a significant increase in the percentage of apoptotic cells in MOLM13 and Kasumi-1 cell lines (Fig. 2C). Likewise, SIRT6 depletion and KPT-9274 treatment cooperated to enhance apoptosis of AML cells. These results provided additional evidence about HDAC dependency of KPT-9274–treated AML cells for survival.

Next, we examined the serial replating potentials of shRNA stable cell lines following NAMPT inhibition. In the scramble groups, the colony numbers formed by cells treated with sublethal doses of KPT-9274 and vehicle control were comparable (Fig. 2D). On the contrary, in HDAC8- or SIRT6-depleted MOLM13 and Kasumi-1 cells, KPT-9274 treatment suppressed colony formation and decreased long-term self-renewal cell populations (Fig. 2D). Knockdown of HDAC8 or SIRT6 reduced the size of colonies and rendered them more compact with KPT-9274 (Fig. 2E). To evaluate the effect of treatments on cell differentiation, we stained cytospin preparations of cells derived from CFU assays. KPT-9274 treatment and depletion of HDAC8 or SIRT6 cooperatively differentiated AML cells (Fig. 2E). The blasts morphologically became more mature with condensed chromatin and fragmented nuclei.

To find translational relevance, we pharmacologically inhibited HDAC8 with HDAC8-specific inhibitor, PCI-34051, and assessed potential synergistic effects of this inhibitor with KPT-9274 on AML cells (15). PCI-34051 and KPT-9274 synergistically reduced cell survival, as assessed by the HSA independence model (Supplementary Fig. S2A–S2C). Drug synergistic effect was most pronounced for 50 μmol/L PCI-34051 in combination with 0.01 μmol/L KPT-9274 in MV4-11 cells and 0.1 μmol/L KPT-9274 in Kasumi-1 cells. MOLM13 cells exhibited synergy over a broader range, as the combination of PCI-34051 at 50 μmol/L and KPT-9274 at doses from 0.01 to 0.1 μmol/L displayed the strongest synthetic lethality. It was reported that unlike the pan-HDAC inhibitor, PCI-24781, PCI-34051 at a concentration as high as 50 μmol/L did not induce significant tubulin acetylation, implying that it did not exert its cytotoxicity through inhibition of other HDAC isoforms (15).These data provide evidence that HDAC8 inhibition functions synergistically with NAMPT inhibition to reduce AML viability. Nevertheless, the human equivalent dose of 50 μmol/L of PCI-34051 is not considered to be clinically tolerable. It was reported that PCI-34051 failed to induce AML death at low micromolar doses, although it caused apoptosis on T-cell leukemia in a caspase-dependent manner (15). It is possible that PCI-34051 was not metabolized effectively in AML to hit HDAC8 targets.

AR-42 is a potent inhibitor for class I/II HDACs, including HDAC8 (16). AR-42 entered phase I clinical trials in combination with decitabine in AML and was shown to eradicate leukemia stem cells (17–19). Depletion of HDAC1, HDAC3, or HDAC6 with CRISPR did not have significant impact on KPT-9274 sensitivity (Supplementary Fig. S1B). This excluded the possibility of involvement of other HDAC isoforms in AR-42 efficacy. Therefore, we employed AR-42 as a tool compound to delineate the effect of HDAC8 inhibition on the sensitivity of AML to KPT-9274 treatment. The highest synergistic scores were observed at combination doses of 0.1 μmol/L KPT-9274 and 0.8 μmol/L AR-42 for MOLM13 and of 0.1 μmol/L KPT-9274 and 0.4 μmol/L AR-42 for Kasumi-1 (Fig. 3A). Cotreatment of KPT-9274 and AR-42 also exhibited synergy on other cell lines, although to a lesser extent (Supplementary Fig. S2D and S2E). To determine the effect of combined treatment on mitochondria respiration, we measured mitochondria membrane potential of treated cells with TMRM staining. In comparison with single agents, combined treatment attenuated TMRM fluorescence intensity and concomitantly elevated Annexin V staining in MOLM13, Kasumi-1, and MV4-11 (Supplementary Fig. S3A–S3C). This suggests that disruption of mitochondria function may account for the observed synergism between KPT-9274 and AR-42 on AML. AR-42 and KPT-9274 cotreatment also significantly increased the percentage of apoptotic cells in a dose-dependent manner (Supplementary Fig. S3D).

It was reported that IDH1 and IDH2 mutations inhibit the expressions of BRCA1/2 and ATM proteins and induce HR repair defects by producing 2-hydroxyglutarate(2-HG), leading to synthetic lethality triggered by PARP inhibitors (20). Here, we evaluated the effect of IDH1 or IDH2 mutation on cellular sensitivity to combined treatment of KPT-9274 and AR-42 by employing IDH2-mutant (IDH2^mut) and -WT (IDH2^WT) TF-1 cell lines as testing system. The most synergistic area was achieved when 0.1–1 μmol/L KPT-9274 was combined with 0.8 μmol/L of AR-42 in IDH2^mut cells (Fig. 3C). IDH2^mut cells were more sensitive to the combination treatment than IDH2^WT cells, suggesting that cotreatment with AR-42 and KPT-9274 resulted in a synergistic cell death in an IDH2-dependent manner (Fig. 3B). We then tested the ability of the drug combination to inhibit the growth of primary AML patient cells. In accordance with findings in cell lines, exposure to AR-42 sensitized primary AML cells to KPT-9274 treatment (Fig. 3C; Table 2). Combination treatment resulted in higher maximum synergy scores in IDH1^mut patient cells than in IDH1^WT patient cells (max synergy score, 48 vs. 26). Mutant IDH1/2 inhibitors protected IDH1/2^mut AML cells against combined treatment, suggestive of a causal relationship existing between IDH1/2^mut and this sensitization to AR-42/KPT-9274 combination (Supplementary Fig. S4A–S4D). The effect of mutant IDH1/2 on drug sensitivity was 2-HG dependent, as 2-HG treatment induced stronger synergism of AR-42/KPT-9274 combination on IDH^WT cells (Supplementary Fig. S4B).

To further characterize the synergistic effect of combined treatment on the functional subsets of AML cells, we performed CFU assays on primary cells. Compared with single agents, the drug combination significantly diminished the colony-forming and long-term replating potentials of AML blasts from patients (Fig. 3D; Table 3A and B). In contrast, the colony-forming capacities of CD34⁺ hematopoietic stem cells from the bone marrow of age-matched healthy donors were not substantially affected by combined treatment. Therefore, AR-42 and KPT-9274 synergistically reduced self-renewal capacities of AML cells while sparing normal human bone marrow cells.

To determine the in vivo relevance of the drug combination, we tested the efficacy of cotreatment of AR-42 and KPT-9274 in the MOLM13 xenograft model. We transplanted 1 × 10⁴ luciferase-transfected MOLM13 cells by tail vein injection into NCG mice (Fig. 4A). Five days after engraftment, mice were randomized to receive vehicle, KPT-9274 alone (100 mg/kg daily), AR-42 alone (20 mg/kg every other day), or combination regimen. The 20 mg/kg dose of AR-42 was tolerated according to an earlier pharmacokinetics study (21). On the basis of IVIS bioluminescence imaging, the disease burden was lower in the drug combination group than in either single agent or vehicle group over the course of the experiment (Fig. 4B). Survival of vehicle-treated mice began to decline by week 3 (Fig. 4C). KPT-9274 or AR-42 as a monotherapy added little to this effect, whereas mice in drug combination arm had a significantly longer survival. Specifically, mice in the KPT-9274 and AR-42 monotherapy groups had median survival times of 27 and 29 days, respectively (Supplementary Fig. S5A). Combined therapy prolonged the lifespan, conferring survival benefits on these MOLM13-engrafted mice (median survival time, 41 days). Histopathology of a variety of organs, including bone marrow, liver, lymph node, and spleen, revealed that animals in combination groups had the least infiltrating neoplastic blasts and most differentiated hematopoietic cells (myeloid, erythroid, and megakaryocytic lineages; Fig. 4D). Mice in the combination group had vacuolation of the testes with atrophy of the seminiferous tubules and a lack of spermatogenesis (Supplementary Fig. S5B). No overlapping toxicities between AR-42 and KPT-9274, like cytopenia, kidney injury, and liver damage, were evident (Fig. 4D). In addition, we did not observe any noticeable weight loss in the AR-42 and drug combination groups over the course of the study (Supplementary Fig. S5C).

Because combined treatment of AR-42 and KPT-9274 abolishes the self-renewal potentials of patient LICs (Fig. 3D), the transcriptomes of LICs could be modulated by the drug treatments. To gain insights into the mechanism by which inhibition of HDAC8 overcomes KPT-9274 insensitivity, we performed RNA sequencing on patient LICs. Bone marrow cells from 5 patients were treated with vehicle, single agents, or drug combination ex vivo for a short time period (12 hours). Treated LICs were analyzed for transcriptional differences, with DNA repair mechanisms being highlighted. Although the transcriptomes from 5 patients were quite heterogeneous, combination therapy–treated LICs were clustered separately from vehicle-treated populations in PCA (Fig. 5A). Consistent with PCA, the divergent responses of patient LICs to drug combination treatments were observed for transcriptome patterns and DEGs. As displayed by volcano plot, genes promoting DNA repairs, like XRCC5, XRCC6, NBN, and RBBP8, were differentially downregulated, while genes involved in transferase activity (MAST3) and serine/threonine phosphatase (PPM1J) were differentially upregulated (Fig. 5B). Hierarchical clustering and heatmap analysis revealed that combined treatment induced a transcriptional response profile of HR and NHEJ pathways closely related to that of AR-42 or KPT-9274 treatment, but distinct from that of vehicle treatment in LIC compartments (Fig. 5C).

AR-42 alone or in combination with KPT-9274 downregulated the mRNA levels of HR genes, like NBN, RBBP8, RAD51, and BRCA1; D-NHEJ genes, like XRCC4 and XRCC5; and genes in ATM pathway (CHEK1 and CHEK2; Fig. 5D; Supplementary Fig. S6A). Interestingly, BRCA1 and XRCC4 were upregulated by KPT-9274 alone in some patients. KPT-9274 treatment decreased the yields of D-NHEJ transcripts, like XRCC6 and PRKDC, and B-NHEJ gene, PARP1, in all patients. On the basis of these observations, we performed gene set enrichment analysis on drug combination–treated samples relative to control. Among the significantly enriched pathways were “the roles of BRCA1 in DNA damage response” and “ATM signaling,” suggestive of a potential impact of the combination therapy on DDR (Fig. 5E). Taken together, AR-42 and KPT-9274 synergistically impair HR, NHEJ, and ATM pathways by abolishing gene transcription. This suggests that synthetic lethality may be due to ineffective DNA damage response.

To further explore the mechanistic basis of synthetic lethality of AR-42 and KPT-9274, we detected treatment-induced changes of intracellular phospho-H2A.X levels. The drug combination created significantly more unrepaired double-strand breaks (DSB) as indicated by an increase in phospho-H2A.X staining than either AR-42 or KPT-9274 alone in MOLM13 cells (Fig. 6A). Although AR-42 or KPT-9274 slightly increased phospho-H2A.X staining in IDH2^WT TF-1 cells, the drug combination failed to further upregulate phospho-H2A.X levels. In contrast, the drug combination enhanced phospho-H2A.X staining on top of the effects of single agents in IDH2^mut TF-1 cells, suggesting that IDH2 mutation synergizes with HDAC8/NAMPT inhibition to impair DDR system. This effect might be attributed to HR deficiency caused by IDH2 mutation, which may render cells more vulnerable to combined treatment.

We next examined HR and NHEJ activities in AML cells exposed to these treatments using the EJDR reporter stable U2OS cells and established OCI-AML3-DR and OCI-AML3-EJ cell lines (Fig. 6B; Supplementary Fig. S6B and S6C). KPT-9274 treatment did not impact HR activity, whereas AR-42 exposure significantly reduced I-SceI–induced HR. AR-42 treatment was also associated with NHEJ deficiency, as measured by the reduction of the percentage of DsRed-positive cells, but residual NHEJ activity was consistently detectable in AR-42–treated cells. KPT-9274 treatment caused a more robust reduction of NHEJ activity when applied alone or in combination with AR-42. Consistent with these findings, immunoblotting further revealed that 0.4 and 0.8 μmol/L AR-42 treatment markedly reduced the levels of HR components, CtIP, Rad51, and BRCA1, as well as phosphorylated ATM, as a monotherapy or in combination with KPT-9274 in MOLM13 cells (Fig. 6C). The expression of D-NHEJ pathway factors, Ku70 and DNA-PKcs, and ATM pathway mediators, CHEK1 and CHEK2, was abolished by AR-42/KPT-9274 cotreatment. This effect was accompanied by induction of phospho-H2A.X. Knockdown of HDAC8 phenocopied the magnitude of reduction in the levels of HR mediators seen in AR-42–treated cells, while shHDAC8 and KPT-9274 synergistically downregulated the abundance of D-NHEJ and ATM pathway mediators (Fig. 6D). Therefore, suppression of HDAC8 can attenuate HR and D-NHEJ gene expressions, resulting in NAMPTi sensitivity. To elucidate the mechanism of synergism between shSIRT6 and KPT-9274, we measured the accumulation of phospho-H2A.X. In the presence of KPT-9274, SIRT6-depleted cells exhibited more severe DNA damage as evidenced by increased phospho-H2A.X staining compared with controls (Fig. 6E). KPT-9274 enhanced mono-ADP-ribosylation of PARP1, which was eradicated by knockdown of SIRT6, suggesting that SIRT6 was responsible for PARP1 mono-ADP-ribosylation (Fig. 6F). This observation provided justification for exploring the DDR-targeting mechanism of synthetic lethal effect exerted by simultaneous inhibition of NAMPT and HDAC8 or SIRT6 in AML.

In this study, we employed a CRISPR screen to identify genes that upon depletion, increase KPT-9274 sensitivity with the ultimate goal of developing synthetic lethal therapies that allow for lowering dosage of NAMPTis. We found that depletion of the screen's top hits, HDAC8 and SIRT6, conferred sensitivity to KPT-9274 treatment. Cotreatment with AR-42 (HDACi) and KPT-9274 resulted in a dramatic reduction of AML viability. In addition, combination therapy showed superior efficacy in a MOLM13 xenograft. Strikingly, AR-42/KPT-9274 combination abrogated the self-renewal of patient LICs by shutting down HR and NHEJ pathways. HDAC8i, shSIRT6, and NAMPTi simultaneously suppressed compensatory DSB repair processes through the regulation of transcription and posttranslational modifications (Fig. 6G). As inhibition of HDACs and associated DNA repair machinery as a strategy to enhance NAMPTi efficacy has not been reported previously, our combination strategy represents a novel regimen to improve efficacy and tolerability of KPT-9274.

Because of the lack of potent HDAC8-specific inhibitors, AR-42 was employed in this study for in vivo and in vitro target validation. Although AR-42 targets multiple HDAC isoforms, the observed synergistic effect with KPT-9274 was most likely attributed to its activity toward HDAC8, given that HDAC8 was the highest ranked hit among all HDACs (the only one with P < 0.01 in CRISPR screen). In addition, knockout of other major AR-42 targets, HDAC1, HDAC3, or HDAC6, did not display any synergy with KPT-9274. Simultaneous AR-42 and KPT-9274 treatment resulted in the accumulation of phospho-H2A.X and lethal DSBs. HDAC inhibitors disable functional HR by controlling the activities and expression of HR-related genes (22, 23). The roles of HDAC8 in HR were reported, as HDAC8 is associated with Rad51 and MRE11a and HDAC8 depletion leads to a decrease in Rad51 levels in multiple myeloma (24, 25). Consistent with these findings, we detected substantial changes in the abundance of transcripts in HR pathway, like NBN and RBBP8(CtIP), in patient LICs upon AR-42 treatment (Fig. 5C and D). We also observed a remarkable reduction of transcripts in D-NHEJ and ATM pathways with cooperative actions of AR-42 and KPT-9274. Immunoblotting results on cell lines recapitulate our patient transcriptome data by showing that AR-42 or shHDAC8 primarily abrogated the expression of HR genes, while KPT-9274 and HDAC8 inhibition synergistically downregulated the components involved in D-NHEJ and ATM pathways at protein levels, sustaining DSBs. Our findings provide evidence that HDAC8i treatment suppresses HR and D-NHEJ (“BRCAness/DNA-PKness” phenotype), which, in combination with NAMPTi, causes synthetic lethality in AML cells due to accumulation of lethal DSBs beyond the reparable threshold.

IDH1/2 mutations exacerbate the HR deficiency by upregulating 2-HG production. This “BRCAness” phenotype of IDH^mut cells renders tumors exquisitely sensitive to PARP1 inhibitors (20). In this study, we found that treatment of IDH1/2-deficient AML cell lines or patient cells with the drug combination resulted in elevated accumulation of γH2A.X and greater inhibition of cell growth compared with IDH^WT cells. It was not our focus to delineate the mechanism of increased sensitivity of IDH1/2^mut cells to AR-42/KPT-9274 combination. But it is likely that IDH1/2 mutation may induce deficiency in some compensatory repair pathways, which cooperates with AR-42–induced HR and KPT-9274–induced NHEJ deficiencies to induce synergistic cell apoptosis. It was reported that 2-HG accumulation induced by IDH1(R132) mutation in AML inhibits the function of histone demethylases (KDM4A and KDM4B) that are critical for HR function and consequently TIP60 and ATM activities are also decreased (26, 27). This effect is likely independent of HDAC inhibitor–mediated suppression of CtIP and BRCA1 functions. This reasoning provides a plausible explanation for why IDH1/2^mut cells are more sensitive to NHEJ and HR inhibition than IDH1/2^WT cells. It is conceivable that inhibition of mutant IDH may attenuate 2-HG–mediated DNA repair defects and antagonize the vulnerability of AML to drug combination treatment. Therefore, cotreatment of AR-42 and KPT-9274 may benefit patients with mutant IDH1/2 in a 2-HG–dependent manner in clinical settings and our combination strategy should not be applied concomitantly with IDH inhibitors. Therefore, IDH2 could be used as a precision medicine marker for identifying patients with AML that may benefit from a therapeutic regimen combining NAMPTi and HDAC8i.

LICs are associated with relapse and drug resistance, due to their tolerance to DNA damage (28–32). Here, we report that AR-42 and KPT-9274 cooperated to shut down multiple DDR pathways and decreased self-renewal of LICs, but not normal hematopoietic stem cells. AR-42 was reported to induce death of LICs by triggering caspase-dependent apoptosis (18). However, it is the first time to show that AR-42 can eradicate LICs in a combination therapy at sublethal doses. By probing LIC transcriptomes, we also found that a strong DDR suppressive effect was achieved through combined treatments, while LICs treated with KPT-9274 alone augmented the levels of HR genes in some patients. This raises the possibility that KPT-9274–treated LIC populations could be particularly vulnerable to this anti-HR strategy. This is supported by the observation that inhibition of NAMPT downstream molecule, PARP1, induces accumulation of Rad51 and preserves HR responses (33).

Studies showed that there exists a poor prognostic subset of patients with AML relying on SIRT6 and NHEJ to compensate for DNA replication stress (1, 34). SIRT6 inhibition was shown to compromise the ability of leukemia cells to repair DSBs that, in turn, increases their sensitivity to daunorubicin and Ara-C (34). SIRT6 is known to stimulate NHEJ in the absence of DNA-PKcs, the D-NHEJ enzyme. Independent of its deacetylase function, SIRT6 mono-ADP ribosylates lysine 521 of PARP1, thereby stimulating its poly-ADP ribosylation activity (35, 36). This modification is required for SIRT6-mediated stimulation of DSB repair, cooperating with NAMPT to fully unlock PARP1-mediated B-NHEJ. Our discovery provides a rationale of developing therapies targeting mono-ADP-ribosylation activity of SIRT6 while sparing deacetylase function to minimize toxicity for healthy cells.

The limitations of our study are that we only tested a limited number of AML cell lines and patient samples. On the basis of our previous and current studies, while KPT-9274 as a monotherapy is more efficacious toward patients with NPM1, DNMT3A, and NRAS mutations, patients with NPM1, DNMT3A, and IDH1 comutation manifest high sensitivity to combined treatment. These results advocate in favor of clinical trials of HDAC8i/NAMPTi in these patients. It was reported that NMRK1 plays a critical role in bypassing NAMPT dependence (6). AML cells vary by the degrees of NMRK1 expression. It would be of interest to study the synergistic effect of NMRK1 and NAMPT coinhibition on the growth of NMRK1^high cells in vitro and in vivo. Of note, we conducted our CRISPR screen on MOLM13, which carries MLL-AF9 translocation with truncated MLL1 (KMT2A). Future screen/validation study design may take into account KMT2A dependency in NAMPTi sensitivity with non-MLL-AF9 cells. Other factors influencing cell sensitivity to combined treatments may include different expressions and mutations in genes encoding drug transporters and metabolic enzymes. But it is expected that our combined therapy can be extended to a broad spectrum of AML subtypes. In addition, future studies may be needed to nominate resistance dependency by using an essential screen in a resistant cell line in combination with a drug screen in a sensitive cell line. Given that HDAC8, SIRT6, and NAMPT govern different DDR pathways, it would be of great translational interest to develop a triple-combination therapy of HDAC8i, SIRT6i, and NAMPTi in the future. Testing drug efficacy on patient-derived xenograft or other spontaneous leukemia models warrants further investigations.

In summary, we validated selected HDACs as attractive targets for inhibition along with NAMPTi. On the basis of these findings, we speculate that AML cells are particularly sensitive to concurrent silencing of multiple DDR pathways, which causes cell death and lowers the risk of overdose toxicity of KPT-9274. Combined AR-42/KPT-9274 therapy may be most beneficial for patients carrying IDH mutations. Our study sheds light into the mechanistic basis of synthetic lethality and the potential of development of HDAC8i/NAMPTi or SIRT6i/NAMPTi as a novel therapy for a broad spectrum of AML subtypes, especially those manifesting resistance to traditional therapies.

P. Zhang reports a patent for T2021-096 pending. L.T. Brinton reports grants from NIH during the conduct of the study and personal fees from ZielBio outside the submitted work. S. Orwick reports grants from NIH during the conduct of the study. C.C. Coss reports a patent for method of use for AR-42 pending, licensed, and with royalties paid from Recursion Pharmaceuticals. S.K. Kulp reports being a creator of intellectual property related to AR-42 that was recently licensed by The Ohio State University to Recursion Pharmaceuticals. S. Mitchell reports grants from NIH outside the submitted work. D. Sampath reports grants from NCI during the conduct of the study. J.S. Blachly reports personal fees from AbbVie, AstraZeneca, INNATE Pharma, and KITE Pharma and other from Mingsight Pharmaceuticals outside the submitted work, as well as a patent for leukemia diagnostic device pending to none. R. Lapalombella reports grants from NIH during the conduct of the study, a patent T2021-096 pending, and U.S. provisional patent application no. 63/113,821 filed November 13, 2020 entitled “Methods and compositions for cancer therapy.” No disclosures were reported by the other authors.

P. Zhang: Conceptualization, software, formal analysis, investigation, methodology, writing–original draft, writing–review and editing. L.T. Brinton: Software, formal analysis, investigation. K. Williams: Resources, methodology. S. Sher: Resources, methodology. S. Orwick: Resources, methodology. L. Tzung-Huei: Resources, methodology. A.S. Mims: Resources, methodology. C.C. Coss: Resources. S.K. Kulp: Resources, methodology. Y. Youssef: Resources, methodology. W.K. Chan: Resources, methodology. S. Mitchell: Resources, methodology. A. Mustonen: Resources, methodology. M. Cannon: Resources, methodology, writing–review and editing. H. Phillips: Resources, methodology. A.M. Lehman: Resources, methodology. T. Kauffman: Resources, methodology. L. Beaver: Resources, methodology. D. Canfield: Resources, methodology. N.R. Grieselhuber: Conceptualization, resources, data curation, supervision, funding acquisition, validation, investigation, methodology, project administration, writing–review and editing. L. Alinari: Conceptualization, resources, data curation, supervision, funding acquisition, validation, investigation, methodology, project administration, writing–review and editing. D. Sampath: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, project administration, writing–review and editing. P. Yan: Resources, methodology. J.C. Byrd: Conceptualization, supervision, funding acquisition, investigation, methodology, writing–review and editing. J.S. Blachly: Conceptualization, software, investigation, methodology, writing–review and editing. R. Lapalombella: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, project administration, writing–review and editing.

The authors are grateful for the patients and healthy volunteers who provided blood and tissue samples for these studies and to the OSU Comprehensive Cancer Center Leukemia Tissue Bank (supported by the NIH, NCI P30 CA016058) for sample procurement. We also thank Drs. David Lucas and Chis Manning for their help in identification and management of primary AML samples at The Ohio State University Leukemia Tissue Bank. We thank the OSUMC genomics core facility for its help with LC-RNA-seq studies. We are grateful for Dr. Feng Zhang's laboratory for CRISPR GeCKOv2 library. This work was supported by the NIH, NCI (R35 CA197734 and R01 CA223165), the OSU Comprehensive Cancer Center using the Pelotonia Foundation funds, and further research support to the Byrd laboratory from the Harry Mangurian Foundation and the D. Warren Brown Foundation.

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