Selective Inhibition of HDAC1 and HDAC2 As a Potential Therapeutic Option for B-ALL Matthew C

Published OnlineFirst February 16, 2015; DOI: 10.1158/1078-0432.CCR-14-1290

Cancer Therapy: Preclinical Clinical Cancer Research Selective Inhibition of HDAC1 and HDAC2 as a Potential Therapeutic Option for B-ALL Matthew C. Stubbs1, Wonil Kim1, Megan Bariteau1, Tina Davis1, Sridhar Vempati1, Janna Minehart2, Matthew Witkin2, Jun Qi3, Andrei V. Krivtsov1,2, James E. Bradner3, Andrew L. Kung1,4, and Scott A. Armstrong1,2,4

Abstract

Purpose: Histone deacetylase inhibitors (HDACi) have recent- leukemia patient samples. Assessment of isoform-specific ly emerged as efficacious therapies that target epigenetic mechan- HDACi indicated that targeting HDAC1-3 with class I isms in hematologic malignancies. One such hematologic malig- HDAC-specific inhibitors was sufficient to inhibit growth of nancy, B-cell acute lymphoblastic leukemia (B-ALL), may be B-ALL cell lines. Furthermore, shRNA-mediated knockdown of highly dependent on epigenetic regulation for leukemia devel- HDAC1 or HDAC2 resulted in growth inhibition in these cells. opment and maintenance, and thus sensitive to small-molecule We then assessed a compound that specifically inhibits only inhibitors that target epigenetic mechanisms. HDAC1 and HDAC2. This compound suppressed growth and Experimental Design: A panel of B-ALL cell lines was tested for induced apoptosis in B-ALL cell lines in vitro and in vivo, sensitivity to HDACi with varying isoform sensitivity. Isoform- whereas it was far less effective against other B-cell–derived specific shRNAs were used as further validation of HDACs as malignancies. relevant therapeutic targets in B-ALL. Mouse xenografts of B-cell Conclusions: Here, we show that HDAC inhibitors are a malignancy–derived cell lines and a pediatric B-ALL were used to potential therapeutic option for B-ALL, and that a more demonstrate pharmacologic efficacy. specific inhibitor of HDAC1 and HDAC2 could be therapeu- Results: Nonselective HDAC inhibitors were cytotoxic to a tically useful for patients with B-ALL. Clin Cancer Res; 1–11. panel of B-ALL cell lines as well as to xenografted human 2015 AACR.

Introduction Histone deacetylases (HDAC) are one such family of chromatin- modifying enzymes whose aberrant activity has been linked to There is growing evidence that epigenetics, or heritable non- hematologic malignancy (4). HDACs regulate gene expression by DNA sequence-based gene-expression alterations, and the chro- removing acetyl groups from lysine residues of numerous pro- matin modification proteins involved, are crucial players in cancer teins, including histones. In humans, there are 11 classical HDAC formation and survival (1). These chromatin-modifying enzymes isoforms, grouped into four classes. The classical HDACs (exclud- are of particular interest in leukemias, in which they have been ing Sirtuins) are in class I (HDACs 1–3, 8), II (IIa—HDACs 4, 5, 7, linked to gene-expression alterations leading to leukemogenesis 9; IIb—6, 10), and IV (HDAC11). HDACs 1–3 are enzymatically (2). As many leukemias are dependent on oncogenic fusion active members of transcriptional corepressor complexes, respon- proteins that consist of transcriptional regulators (3, 4), epigenetic sible for chromosomal compaction and gene repression through therapies could prove useful as treatment options. Therefore, the removing acetyl groups from lysine residues on histones. Inter- idea of targeting these chromatin-modifying enzymes with small- estingly, HDAC6 is mainly a cytoplasmic protein, with functions molecule inhibitors as a putative antileukemia option is growing. independent of histone deacetylation (5). Histone deacetylase inhibitors (HDACi) define a promising class of cancer drugs whose mechanism of action is not complete- 1 Division of Hematology/Oncology, Department of Pediatric Oncolo- ly understood, though they are widely touted as an epigenetic gy, Boston Children's Hospital, Dana-Farber-Cancer Institute, and fl Harvard Medical School, Boston, Massachusetts. 2Human Oncology therapy (6). Of the many possible ways HDACi in uence cell and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, survival, there are data amassing that HDACs regulate genome 3 New York, New York. Department of Medical Oncology, Dana-Farber- stability and repair (7–9). HDACi may induce apoptosis by Cancer Institute and Harvard Medical School, Boston, Massachusetts. 4Harvard Stem Cell Institute, Boston, Massachusetts. preventing chromatin compaction, facilitating an accumulation of DNA breaks that would be irreparable. Although several other Note: Supplementary data for this article are available at Clinical Cancer fi Research Online (http://clincancerres.aacrjournals.org/). mechanisms have been studied, a de nitive route to apoptosis induction is still lacking. Current address for M.C. Stubbs: Incyte Corporation, Wilmington, DE. There are more than a dozen HDACi presently being studied as Corresponding Author: Matthew C. Stubbs, Incyte Corporation, 1801 Augustine chemical probes and therapeutic agents, which may be subdi- Cut-off, Wilmington, DE 19803. Phone: 302-498-5716; Fax: 302-425-2759; vided into families based on chemical structure and biochemical E-mail: [email protected] spectrum of activity (10). The hydroxamic acid family is the most doi: 10.1158/1078-0432.CCR-14-1290 prevalent, with SAHA (Vorinostat, Zolinza; Merck) being the most 2015 American Association for Cancer Research. clinically successful as of yet. SAHA is known to inhibit the class I

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in vitro Translational Relevance necessary to prevent proliferation are in line with all other hematopoietic malignancies tested. HDAC6 inhibitors were effec- Currently, two nonspecific HDAC inhibitors are approved tive against B-ALL cells, but only at high doses exceeding the dose for use in cutaneous T-cell lymphoma, with trials underway in range in which selective inhibition of HDAC6 is observed. The several hematopoietic malignancies. However, the mecha- antiproliferative activity of class I–specific benzamide inhibitors nism of action for these histone deacetylase inhibitors prompted the study of a chemical probe that only targets HDAC1 (HDACi) is not completely understood, and may not have and HDAC2. Interestingly, selective HDAC1 and HDAC2 inhibi- a basis in epigenetics. We have found that BALLs are very tion demonstrated potent inhibition of B-ALL cell growth in vitro sensitive to an HDACi that only targets the HDAC1 and and in vivo, whereas other hematopoietic malignancies were much HDAC2 isoforms, and that other B-cell–derived malignancies less sensitive. are far less responsive to this compound. This is important for several reasons. First, new HDACi that are more isotype Materials and Methods specific should limit side effects seen with the current nonspecific HDACi. Second, inhibition of both HDACs 1 and 2 Cell lines and vectors provides more insight into the mechanisms of leukemia B-ALL, B-lymphoma, and multiple myeloma cell lines were maintenance in B-cell acute lymphoblastic leukemia (B-ALL). maintained in RPMI (Invitrogen) with 10% FCS (Invitrogen) and Third, an HDAC1 and 2 inhibitor could be a new therapeutic supplemented with 100 U/mL penicillin–streptomycin (Invitro- possibility for B-ALL that would likely act through epigenetic gen) and 2 mmol/L L-glutamine (Invitrogen). Cells (293) were regulation. maintained in DMEM (Invitrogen) supplemented with 10% FCS (Invitrogen) and 100 U/mL penicillin–streptomycin (Invitrogen). RS4;11, REH, RPMI-8226 cells were from the ATCC. The 697 and OPM-2 cells were from DSMZ. SEMK2 cells were a generous gift from the laboratory of Dr. Stanley Korsmeyer (DFCI, Boston, HDACs as well as HDAC6 at low nmol/L concentrations (11) and MA). MM1.S cells were a generous gift from Dr. Constantine is clinically approved for use in treating cutaneous T cell lym- Mitsiades (Harvard Medical School, Boston, MA). B-lymphoma phomas (CTCL). The cyclic peptide family is most well known for lines were generously given by Dr. Guo Wei (Broad Institute, the depsipeptide HDACi romidepsin (FK228, Istodax; Celgene), Cambridge, MA). Each cell line has been tested and authenticated which is also clinically approved for CTCL. Romidepsin is a by short tandem repeat at the Molecular Diagnostics Laboratories potent, class I–selective HDACi that exhibits on modest activity at Dana Farber Cancer Institute (Boston, MA). All lentiviral vectors against HDAC6 at high concentrations may have a greater spec- contained the pLKO.1 backbone and were obtained from the ificity for the class I enzymes, but also seems effective against RNAi Consortium (Broad Institute, MIT). RNAi Consortium HDAC6 (12, 13). The benzamide family of HDACi also exhibits clones were HDAC1: TRCN0000004817, TRCN0000004818; class I selectivity, with inhibition of HDAC1, 2, and 3 apparent at HDAC2: TRCN0000004819, TRCN0000004821; HDAC3: pharmacologically achievable doses. Several benzamides are pres- TRCN0000004824, TRCN0000004828. Lentiviruses were pro- ently progressing through clinical trials (14). Only recently have duced as described previously (27). The viral titer was determined selective inhibitors of HDAC6 been developed, such as tubacin, by infection of BAF3 cells. All viruses used had a viral titer of which demonstrate low potency for nuclear, class I deacetlyases approximately 5 105 infectious units per mL. and exhibit toxicity when combined with proteasome inhibitors in preclinical models of multiple myeloma (15, 16). In addition, Cell proliferation, survival, and annexin V analyses ongoing research is being performed to determine which tran- To assay for sensitivity to the HDAC inhibitors LAQ824, MS- scriptional repressor complexes associate with various inhibitors 275, WT-161, and Merck60, the colorimetric Cell Proliferation to help establish a mechanistic understanding of biologic effects Kit I—3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- observed broadly in cancer, inflammatory, and neurodegenera- mide (MTT; Roche) was used. Cells were plated at a density of 105 tive models (17). cells per 100 mL media (RPMI, Invitrogen; supplemented with We are interested in extending HDACi epigenetic therapy to B- 10% FCS and 100 U/mL penicillin–streptomycin) into 96-well acute lymphoblastic leukemia (B-ALL). Although there is evi- microtiter plates (Corning) The MTT assay was then performed as dence that nonselective HDACi may be effective against B-ALL per the manufacturer's protocol. For high-throughput HDACi (18–23), we also feel that a more isotype-specific HDACi may be screening, 1,000 cells were plated in 50 mL RPMI/10% FCS in useful against B-ALL for several reasons. First, we have shown that 384-well plates (Nunc). All drugs in the high-throughput screen fusion oncoproteins such as MLL-AF4 alter the epigenetic land- were made in house and added to cell plates using a Janus liquid scape of pro- or pre-B cells to bring about transformation (24), handler (PerkinElmer). To analyze cell fitness, CellTiter Glo was and therefore inhibiting epigenetic regulators should be useful used (Promega). EC50 values were determined using Prism therapeutically. Second, as nonselective HDACi are thought to GraphPad software. ZVAD-fmk was purchased from InvivoGen. facilitate genomic damage (25, 26), a more specific inhibitor may For cell viability assays after infection and puromycin selection function in a manner more consistent with epigenetic therapy. As (48 hours. at 2 mg/mL), cells were counted with a hemacytometer the current clinically approved cancer targets of HDACi are T cell (VWR) using light microscopy and Trypan Blue (Invitrogen) in origin, moving these drugs toward another lymphoid malig- exclusion. For apoptosis analyses, cells were harvested, washed nancy is logical. with PBS (Invitrogen), and resuspended in annexin V buffer [140 Here, we have found that B-ALLs are uniformly sensitive to class mmol/L NaCl (Fisher), 10 mmol/L HEPES (Invitrogen), 2.5 in vitro in vivo I HDAC inhibitors both and . Treated cells rapidly mmol/L CaCl2 (Fisher)] with annexin V-Cy3 reagent (Biovision) accumulate DNA damage and undergo apoptosis. The doses and analyzed on a FACSCalibur (Becton Dickenson).

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B-ALLs Are Sensitive to an HDAC1/HDAC2 Inhibitor

Western blots plementary Fig. S1B). Therefore, apoptosis is not detected until Cells were harvested 72 hours after infection and equivalent cell after a build-up of DNA damage, and until p21 reaches maximal numbers were lysed in RIPA buffer [150 mmol/L NaCl, 1% levels after roughly 18 hours of nonselective HDACi treatment. IPEGAL (Sigma), 0.5% deoxycholate (Sigma), 0.1% SDS (Invi- We next became interested in how the HDAC inhibitor sensi- trogen), 50 mmol/L Tris pH 8.0 (Sigma)] with Complete protease tivity of B-ALL cells compares with other B-cell–derived malig- inhibitor cocktail (Roche). Histones were extracted by lysing cells nancies. We treated a panel of B-cell lymphoma and multiple with Triton X-100 in PBS (Invitrogen). Nuclei were spun at 6,500 myeloma cell lines, along with other malignant cell lines with rpm for 5 minutes, and pellets containing histones were incubated LAQ824 as in Fig. 1A. We found that all lines tested were sensitive with 0.2N HCl overnight. Extracts were denatured and run on a to the drug in the same range as the B-ALL lines (Fig. 1C), with 10% Acrylamide Bis-Tris gel (Invitrogen) and transferred to nitro- EC50 values in the low nmol/L range (Supplementary Table S1). cellulose (Whatman) for Western analyses. To establish whether a nonselective HDAC inhibitor could Western blots were performed using mouse a–pan-actin (Che- decrease the leukemic burden in primary human B-ALL xeno- micon), p21 (Santa Cruz Biotechnology) gH2A.X and Histone H3 grafts, we established cohorts of mice bearing patient-derived (Abcam), HDAC1, HDAC2, HDAC3 (Cell Signaling Technology), B-ALL cells and compared treatment with 10 mg/kg LBH589 AcH3K9 and AcH3K56 (Upstate Millipore), Ac-tubulin (Sigma), (Panobinostat, Novartis), another nonselective HDACi chemical- HRP conjugated anti-mouse and anti-rabbit antibodies (Amer- ly similar to LAQ824 that is used clinically, to vehicle control. sham), and developed with the Western Lightning Enhanced Male SCID Beige mice were injected with cells from a pediatric Chemiluminescence Kit (PerkinElmer). patient with an MLL-rearranged leukemia. Engraftment was monitored by human CD45 levels in the peripheral blood. When levels In vivo drug assays reached 1%, mice were treated either with LBH589 or vehicle Bioluminescence assays were carried out as described previ- control once daily for 2 weeks. Figure 1D shows that there is a ously (28). For testing LBH589, SCID Beige mice were injected statistically significant (P < 0.0001) reduction in human leukemia with 2 105 MLL-AF4–bearing patient sample cells (B-ALL) and cells in the bone marrow of mice treated with LBH589. were treated at 10 mg/kg i.p. daily or vehicle control for 14 days. For testing Merck60, the first cohort of NOG mice was injected B-ALL cells are sensitive to class I HDAC inhibition, but not to with 7,000 MLL-AF4–bearing patient sample cells (B-ALL), and HDAC6 inhibition treated with 45 mg/kg i.p. or vehicle control for 14 days over an 18 To further characterize the response of the B-ALL cell lines to day span. Subsequent cohorts of mice were injected with 2 to 3.5 HDAC inhibition, we performed a screen to determine whether or 105 cells from B-ALL patients without MLL rearrangements and not an HDAC inhibitor that possesses class specificity would random cytogenetics. These mice were treated with 30 mg/kg i.p. effectively inhibit growth in our B-ALL lines. We assessed the Merck60 or vehicle control for 5 days, followed by 2 days off drug, antiproliferative activity of a panel of HDAC inhibitors that followed by 5 more days on drug. For both LBH589 and Merck60, possess varying degrees of isoform specificity (Supplementary þ treatment began when peripheral blood hCD45 levels exceeded Table S2) against B-ALL cell lines. We found that all pan-HDAC 1%. Peripheral blood was treated with red blood cell lysis buffer inhibitors were very effective against the B-ALL lines. Interestingly, (BD Biosciences), and white blood cells were incubated with anti- benzamide derivatives, including MS275, MGCD0103, and CI- human CD45-FITC antibody (BioLegend), and then analyzed on 994 possessed activity, indicating that inhibiting the class I iso- a FACSCalibur (BD Biosciences). forms is sufficient for growth repression in B-ALL cells. The HDAC6 inhibitor tubacin, however, lacked antiproliferative activ- Results ity within its selective dose range, only inhibiting cell growth at relatively high concentrations, where evidence of class I deacety- B-ALL cells are sensitive to pan-HDAC inhibition lase inhibition was evident by immunoblot analysis for acetylated In an effort to determine the utility of HDAC inhibitors as a histones. therapeutic option for B-ALL, we incubated the RS4;11, REH, To further verify the data that specifically targeting HDAC6 may SEMK2, and 697 cell lines in serial dilutions of the potent, not be sufficient to prevent B-ALL cell growth, we also treated our nonselective HDAC inhibitor LAQ824 (Novartis). After 48 hours, cell lines with WT161 (30), a structurally distinct HDAC6 inhib- we observed growth suppression by MTT assay in each cell line in itor. Using serial dilutions, we found that WT161 is only effective the low nmol/L range (Fig. 1A). After 18 hours, we were able to against our cell lines at doses above the range that would specif- detect increases in acetylation of histone H3 lysine 9 (H3K9) and ically target HDAC6 (Fig. 2A). Immunoblot analyses show that, acetylation of a-tubulin lysine 40, a marker of HDAC6 inhibition although tubulin is acetylated at low WT161 concentrations, (Fig. 1B). We could also detect a dose-dependent increase in p21 Histone H3 acetylation is markedly increased at 1 mmol/L, a and gH2A.X (a marker of dsDNA damage) levels, both of which concentration still below the amount necessary to inhibit growth are known to be increased in HDACi-treated cells (25, 29). at 48 hours (Fig. 2B). These data indicate that HDAC6 inhibition Induction of apoptosis was also measured by annexin V staining. alone is likely not sufficient for growth inhibition in B-ALL cell An increase in apoptosis was seen within 12 (SEMK2 cells) to 18 lines. hours of treatment with LAQ824 (Supplementary Fig. S1A). To determine the cellular effects of inhibiting class I HDACs Acetylated tubulin was detectable within 15 minutes of LAQ824 (here only referring to HDAC1, 2, and 3) on B-ALL cells, we treatment whereas Histone H3 acetylation was not detectable performed MTT assays and immunoblot analyses after treatment until 2 to 4 hours after treatment. There was also an increase in p21 with MS275 (Fig. 2C and Supplementary Fig. S2). As with in this 2 to 4 hours timeframe, but it did not reach maximal levels LAQ824, the cells treated with MS275 showed increased levels until 18 to 24 hours. Accumulation of dsDNA breaks, as marked of acetylated H3K9, p21, and gH2A.X. However, levels of acety- by gH2A.X, did not appear until 18 hours after treatment (Sup- lated tubulin did not change, even with doses of drug 10-fold over

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Figure 1. B-ALL cells are sensitive to a pan- HDAC inhibitor. A, MTT assays were carried out after treating cell lines for 48 hours with the pan-HDAC inhibitor, LAQ824 (Novartis). B, Western blots show increased levels of acetylated tubulin, acetylated H3K9, p21, and gH2A.X (DNA damage) in response to 24 hours of pan-HDAC inhibition in RS4;11 cells. C, MTT assays were carried out as in A with multiple myeloma and B lymphoma cells. The heatmap shows proliferative ability from high (red) to low (purple) with EC50 values marked by yellow boxes and given numerically on the right. D, mice treated for 14 days with LBH589 or vehicle control were sacriﬁced and bone marrow analyzed with an anti-human CD45-FITC antibody. Shown are the percentages þ of hCD45 white blood cells in the bone marrow.

the EC50 for these cell lines, indicating that HDAC6 is not lines with lentiviruses encoding shRNAs specificforHDAC1, nonspecifically targeted and does not factor into the sensitivity HDAC2, or HDAC3. Immunoblot analyses indicated that sup- of these cells to MS275 consistent with its reported biochemical pression of HDAC1 or HDAC2 levels result in increased p21, specificity (10). We extended these studies more broadly to other but little or no increase in either DNA damage (as assessed by B-lineage malignancy cell lines (Fig. 2D). Although the EC50 gH2A.X) or acetyl H3K9 accumulation, indicating that these values are not greatly disparate between leukemia, lymphoma, two isoforms may have some functional redundancy or com- and myeloma (Supplementary Table S1), it is evident that the B- pensatory abilities (Fig. 3A). This is similar to what has previ- ALL cells are more sensitive at higher dosages than the other cell ously been shown in other cell lines (31–33), where upregula- types. Possibly, the lack of HDAC6 inhibition decreases the tion of HDAC1 occurs when HDAC2 levels drop, and vice versa. sensitivities of the lymphoma and myeloma cell lines to MS- Upon reduction of HDAC3 protein levels, p21 was induced, 275. These data demonstrate that selective inhibition of class I– similar to what we saw with suppression of either HDAC1 or selective HDACi effectively inhibits proliferation in B-ALL cells. HDAC2 suppression. Acetylated-H3K9 and gH2A.X were also observed with HDAC3 suppression (Fig. 3A) with HDAC1 or 2 Suppression of specific class I HDACs induce apoptosis in B- suppression yielding a far weaker possible induction of gH2A.X ALL cells in SEMK2 cells. Interestingly, knockdown of HDAC3 does not To better understand the importance of individual class I lead to higher levels of HDAC1 or HDAC2 (Supplementary HDAC isoforms to B-ALL survival, we transduced the B-ALL cell Fig. S3A). We next monitored cell growth and apoptosis in

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Figure 2. B-ALL cell lines respond to drugs targeting class I HDACs. A, MTT assays were carried out after treating cell lines for 48 hours with the HDAC6-speciﬁc inhibitor WT161. B, Western blots show acetylated tubulin as a marker of HDAC6 inhibition and acetyl-H3K9 at higher WT161 concentrations. C, MTT assays were carried out after treating cell lines for 48 hours with a class I HDAC inhibitor MS-275 (Syndax). D, MTT assays with MS-275 were performed and are displayed as a heatmap as in Fig. 1C.

transduced B-ALL cell lines. Figure 3B shows the change in B-ALL lines are sensitive to a selective HDAC1/HDAC2 inhibitor proliferation for RS4;11 cells transduced with shRNAs targeting Having demonstrated that nonselective HDAC inhibition HDAC1, 2, or 3 as compared with control (GFP shRNA). might be a useful therapeutic option for B-ALL, we next sought Supplementary Fig. S3B shows similar data for the REH, to determine whether more specific inhibitors might demonstrate SEMK2, and 697 cells, with statistics shown in Supplementary comparable antileukemic activity. The robust antiproliferative Table S3. Suppression of HDAC1 or HDAC2 leads to a decrease activity of HDAC1 or HDAC2 suppression establishes the plau- in growth of B-ALL cells, whereas HDAC3 suppression resulted sibility that agents selective for these two isoforms might retain in complete proliferative arrest. Each of the B-ALL cell lines antileukemic activity while obviating toxicity associated with began to undergo increased apoptosis at some point later than HDAC3 inhibition. Currently, there are no available HDAC1- 2 days of HDAC1 or HDAC2 suppression, and the amount of specific or HDAC2-specific inhibitors, so we treated our cell lines apoptosis increased further after 6 days (Fig. 3C). HDAC3 with a compound capable of inhibiting both HDAC1 and HDAC2 suppression resulted in rapid induction of apoptosis, with with more than 200-fold more specificity than HDAC3 (HDAC1 essentially no viable cells remaining after 6 days. These data IC50 7 nmol/L; HDAC2 IC50 49 nmol/L; HDAC3 IC50 10 mmol/L). indicate that HDAC1 or HDAC2 suppression leads to an anti- We synthesized and characterized a chemical probe first reported proliferative phenotype, with HDAC1 knockdown likely caus- in a medicinal chemistry study by Merck Research Laboratories as ing a G1 arrest and HDAC2 knockdown yielding multiple Compound 60, which for these studies we will describe as possible growth delays (Supplementary Fig. S3C), as well as Merck60 (Fig. 4A; ref. 34), also reported as "Compound 60" an increase in apoptosis. HDAC3 suppression mimics treat- (35). We performed an MTT assay on our B-ALL cells and found ment with a nonselective HDAC inhibitor, causing nearly that treatment for only 48 hours had a marginal effect on our B- complete loss of viable cells within a few days. ALL line panel. However, consistent with our knockdown data

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Figure 3. Knockdown of individual class I HDAC isoforms results in varying cell growth inhibition and apoptosis. A, Western blots indicate that HDAC1 and HDAC2 knockdown lead to increased p21 levels. HDAC3 knockdown (below) results in increased p21 levels along with increased DNA damage as marked by gH2AX levels and increased global histone acetylation as shown by acetyl H3K9. B, growth curves of RS4;11 cells receiving GFPsh control or HDAC 1-3–specific shRNA. C, annexin V staining of B-ALL cells receiving GFPsh control or HDAC 1-3–specific shRNA at day 2 (black) or day 6 (white). Brackets indicate statistically significant (P < 0.05) differences.

where growth impairment and apoptosis were more prevalent HDACi, we sought to verify whether the gH2A.X mark was due with HDAC1 or HDAC2 knockdown after 4 to 6 days, treatment to Merck60-driven DNA damage or due to apoptosis-related DNA with Merck60 for 96 hours led to growth inhibition that was degradation. RS4;11 cells were grown in Merck60 with or without evident at doses in the low to mid nmol/L range (Fig. 4B). the caspase/apoptosis inhibitor ZVAD-fmk. Cells in ZVAD were Immunoblot analysis showed that acetylated H3K56 (AcH3K56), more viable and showed very little induction of gH2A.X (Sup- a mark known to be regulated by HDACs 1 and 2 (8), was plementary Fig. S5A). In contrast, cells treated with LAQ824 accumulating within the cells (Fig. 4C), and p21 was also induced showed an increase in gH2A.X with or without ZVAD, even when as seen with nonselective HDACi. AcH3K56 occurred within 18 cells are nearly completely viable (Supplementary Fig. S5B). hours. Interestingly, gH2A.X was also induced by Merck60. How- Therefore, Merck60 appears to be inducing apoptosis in a differ- ever, this mark did not appear until after 48 hours (Supplemen- ent manner than the nonspeciﬁc HDACi. tary Fig. S4A), corresponding with when growth suppression and To determine whether HDAC1/HDAC2 inhibition would also apoptosis began (Supplementary Fig. S4B and S4C). Although it is inhibit growth of other B-cell–derived malignancies, we per- of note that we can detect the AcH3K56 mark with 100 nmol/L formed MTT assays on our B-lymphoma and multiple myeloma Merck60 at 18 hours, and yet apoptosis is not apparent until after cell line panel as in Figs. 1C and 2E. We found that, although our 48 hours with 500 nmol/L Merck60, this disconnection is con- B-ALL lines are sensitive to Merck60 at well under 1 mmol/L sistent with what has been seen with inhibition of other epigenetic (Supplementary Table S1), our lymphoma and myeloma lines regulators as well (e.g., DOT1L inhibition; ref. 36). Because of the are far less sensitive to this drug (Fig. 5A). This is in sharp contrast later induction of gH2A.X when compared with nonspeciﬁc with our nonselective HDACi and class I HDACi experiments

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Figure 4. B-ALL cells are sensitive to an inhibitor speciﬁc for HDAC1 and HDAC2. A, structure of Merck60. B, MTT assays were carried out after treating cell lines for 48 (left) and 96 hours (right) with the HDAC1/HDAC2–selective HDAC inhibitor, Merck60 (Merck). C, Western blots show increased levels of acetyl-H3K56 (a mark speciﬁcally controlled by HDAC1/HDAC2), gH2AX (DNA damage), and p21 in response to 72 hours of Merck60 treatment.

where all lines tested were similarly sensitive. Western analyses An HDAC1/HDAC2 inhibitor reduces leukemic burden in indicate that lines that are not sensitive to Merck60 (MM1S-LN, mouse models of human primary B-ALL OPM2) still induce AcH3K56 and p21 when treated with the drug We also pursued using HDAC1/HDAC2–specific inhibition to (Fig. 5B). However, gH2A.X levels do not increase upon Merck60 inhibit growth of B-ALL patient samples. NOG mice were injected treatment, indicating a differential response between sensitive with cells from a pediatric patient with an MLL-rearranged leu- and insensitive cells (Fig. 5B). When the insensitive lines were kemia. Engraftment was monitored by human CD45 levels in the treated with the DNA-damaging agent doxorubicin or LAQ824, peripheral blood as in Fig. 1. When levels reached 1%, mice were they are able accumulate gH2A.X (Supplementary Fig. S6). These treated either with Merck60 or vehicle control. After 14 days of lines are able to show elevated gH2A.X levels, but not when treatment, there was a significant drop in the percentage of and treated with Merck60. number of human leukemia cells found in the bone marrow (Fig. Next, we wanted to investigate whether this sensitivity bias 6A), indicating that HDAC1/HDAC2–specific inhibition can occurs in vivo. Immunodeficient (NOG) mice were injected with inhibit growth of patient-derived primary leukemia cells. Marrow either SEMK2 or MM1S-LN cells that stably express firefly lucif- was harvested 2 hours after the final dose of Merck60 was erase (28). Mice with established cancer burden were treated with administered for analyses. Immunoblots revealed that marrow Merck60 (45 mg/kg daily) or vehicle control. As shown in Fig. 5C, from mice treated with Merck60 exhibited a pharmacodynamic a decrease in bioluminescence was observed in SEMK2-bearing elevation of AcH3K56 (Fig. 6B), when compared with marrow mice injected with Merck60. Mice bearing MM1S-LN tumors from mice receiving vehicle control. Merck60 was, therefore, demonstrated no response to daily therapy with Merck60 com- inhibiting HDAC1 and HDAC2 in vivo, leading to a diminished pared with vehicle controls. Thus, Merck60 was not effective leukemic burden within those mice. To verify further the ability of against the multiple myeloma cells, whereas the B-ALL cells did Merck60 to inhibit growth of primary B-ALL cells without MLL respond to HDAC1/HDAC2 inhibition in vivo. rearrangements in vivo, NSG mice were injected with cells from

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Figure 5. Other B-cell–derived malignancies are much less sensitive to an HDAC1/HDAC2–speciﬁc inhibitor than B-ALL. A, MTT assays were carried out after treating cell lines for 96 hours with the HDAC1/HDAC2–speciﬁc inhibitor, Merck60. Cell lines are B-ALL, multiple myeloma, or B lymphoma. B, Western blots indicate that the Merck60-insensitive multiple myeloma cells have increased levels of p21 and AcH3K56 in response to Merck60; however, gH2AX (DNA damage) levels remain unchanged with increasing concentrations of Merck60. C, NOG mice were injected either with SEMK2 or MM1S cells, each stably expressing the luciferase gene. After luminescence exceeded background levels, mice were treated with Merck60 or control. Mice with SEMK2-Luc cells showed a decrease in bioluminescence after Merck60 treatment, whereas mice with MM1S-Luc cells had high luminescent levels in the presence or absence of Merck60.

three separate adult B-ALL patient samples, and treated with Here, we show that B-ALL cells are sensitive to class I HDAC Merck60 or vehicle control. Figure 6C shows spleen weights as inhibition, including inhibition of only ofHDACs 1 and 2.Thisis of surrogates for leukemic response to Merck60. In two of the three particular interest in light of the fact that genetic inactivation of samples, mice treated with Merck60 showed a statistically signif- HDAC1, 2, or 3 does not hamper growth of H-Ras/T antigen– icant (P < 0.05) decrease in spleen weight when compared with transformed fibroblasts (37), yet we see growth inhibition and vehicle controls, indicating that Merck60 is also able to inhibit apoptosis in B-ALL cells upon suppression of HDAC1 or 2, and the growth of primary B-ALL cells without MLL rearrangements large-scale apoptosis from HDAC3 knockdown. This shows that in vivo. HDACs may play a much more critical role in B-ALL cells survival than in other cell types. Our experiments provide further support Discussion for this argument, as B-ALL cells are sensitive to HDAC1 and HDAC2 inhibition whereas other cell types are either far less Our goal has been to assess the potential efficacy of thera- sensitive or insensitive. Also, as compared with other B-lineage peutic approaches that target epigenetic mechanisms against B- malignancies, B-ALL cells are more sensitive to class 1–specific ALL cells. We chose to assess HDACi for several reasons. First, B- inhibitors. It is important to note that HDAC inhibitors that avoid ALL is frequently driven by transcriptionally active fusion inhibition of HDAC3 could be clinically beneficial. And as dem- oncoproteins, thus altering the transcriptional landscape might onstrated in our in vivo studies, a lack of HDAC3 inhibition does not be a useful approach in this disease. Second, knowing that appear to cause a significant loss of efficacy in B-ALL patient sample nonspecific HDACi leads to accumulation of dsDNA lesions, xenograft models (Fig. 1D vs. Fig. 6A and C).Outside of the ability and that B-ALL cells are extremely sensitive to DNA damage, we of HDAC3 to regulate genomic stability (7), mouse knockout felt this might also increase the potential efficacy of these studies have also shown that HDAC3 is necessary for maintenance inhibitors against B-ALL cells. Finally, Vorniostat (Merck) and of several metabolic functions in both cardiac and hepatic tissues Istodax (Romidepsin—Celgene) are already approved for a (38, 39). One particular problem with current clinically available lymphoid malignancy, CTCL. HDAC inhibitors is that they also bind and inhibit the hERG ion

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B-ALLs Are Sensitive to an HDAC1/HDAC2 Inhibitor

Figure 6. HDAC1 and HDAC2 inhibition reduces leukemic burden in a B-ALL xenograft model. A, NOG mice injected with an MLL rearranged pediatric B-ALL sample and receiving Merck60 or vehicle control were sacriﬁced and bone marrow analyzed with an anti- human CD45-FITC antibody. Shown are the percentages of hCD45þ white blood cells in the bone marrow (left), and the total numbers of hCD45þ cells in the bone marrow (right). B, Histones were extracted from bone marrow and immunoblot analysis showed increased acetyl-H3K56 in Merck60- treated samples. C, NSG mice injected with adult B-ALL samples that did not harbor MLL rearrangements, and were treated with Merck60 or vehicle control were sacriﬁced, and spleens were weighed as an assessment of leukemia growth.

channel, which has been linked with QT prolongation (40). This that are hyperproliferative and antiapoptotic, and are therefore off-target binding can lead to cardiac arrhythmia and is potentially less sensitive to the inhibitor. fatal. Newer inhibitors are now being screened not only for their Our data indicate that inhibition of HDACs would potentially abilities to block HDAC function, but also for their abilities to block be a useful therapeutic addition to therapies for patients with B- hERG activity as well (40, 41). An HDAC inhibitor that structurally ALL, and that an inhibitor that specifically targets HDAC1 and cannot inhibit HDAC3 and cannot bind hERG, then, would be of HDAC2 might be particularly beneficial. As such, potent and considerable therapeutic interest. selective inhibitors of HDAC1 and HDAC2 are presently being The mechanism behind HDAC1/HDAC2 inhibition and the developed in our laboratories for therapeutic application. It reasons why B-ALLs are more susceptible to HDAC1 and 2 remains to be determined whether HDAC inhibitors, broad inhibitors than other malignancies are as of yet unknown. We spectrum or specific, will synergize well with existing chemother- have seen that the kinetics of Merck60 action are much slower apeutic drugs for B-ALL. On the basis of the accumulation of DNA than that of the nonspecific HDACi, possibly due to the differing damage, as well as much prior work showing synergy between mechanisms of action between the two drugs. Here, the cell of HDACi and DNA damage–inducing agents (11), it seems highly origin of these cancer types might play an important role. HDAC1 likely that HDAC1 and 2 inhibition would fit well into existing and HDAC2 have been shown to control early B-cell development regimens. HDAC1 and 2 inhibition leads to accumulation of (33). A conditional double knockout in B-lineage cells yields a AcH3K56, a mark shown to be specifically regulated by HDAC1 differentiation block in the pre-B stage with G1 arrest. B-ALLs are and 2 (8). The AcH3K56 mark has been shown to have multiple derived from pre-B cells so there may be a necessity for HDAC1 different roles in response to DNA double-stranded breaks where and 2 for cell proliferation. The same double knockout in more the mark is either increased around sites of DNA damage (42), or mature B cells that were not proliferative had no effect until the decreased in response to DNA damage (43). HDAC inhibitors can cells were stimulated to proliferate, after which apoptosis was also cause a decrease in levels of the repair proteins RAD50 and induced. It is possible, therefore, that more mature B-cell–derived MRE11 (26) as well as homologous recombination genes (44). malignancies such as B-lymphoma and multiple myeloma have HDAC inhibitors have recently been linked to the DNA damage less requirement for functional HDAC1 and 2, possess mutations response as well as autophagy in budding yeast (9), and in-house

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Stubbs et al.

preliminary studies suggest that a functional DNA damage Acquisition of data (provided animals, acquired and managed patients, response pathway may at least partially be necessary for the provided facilities, etc.): M.C. Stubbs, W. Kim, M. Bariteau, T. Davis, growth inhibition and apoptosis caused by HDAC1- and 2- J. Minehart, M. Witkin, J.E. Bradner fi Analysis and interpretation of data (e.g., statistical analysis, biostatistics, speci c inhibition. In addition, HDAC1 and 2 have been shown computational analysis): M.C. Stubbs, W. Kim, M. Witkin, J.E. Bradner, to be necessary for dsDNA break repair by regulating the timing A.L. Kung, S.A. Armstrong and placement of nonhomologous end joining proteins (8). Writing, review, and/or revision of the manuscript: M.C. Stubbs, M. Bariteau, Therefore, under the influence of an HDAC inhibitor, this mark A.L. Kung, S.A. Armstrong will improperly accumulate throughout the genome, making it Administrative, technical, or material support (i.e., reporting or organizing plausible that in B-ALL, an HDAC inhibitor may both help bring data, constructing databases): M. Bariteau, J. Minehart, J. Qi, A.V. Krivtsov Study supervision: M.C. Stubbs, M. Bariteau, S.A. Armstrong about DNA damage, and then somehow impair the cell's ability to Other (contributed to the animal studies; daily monitoring of animal health repair it. The possibility also exists for a more specific epigenetic status, injections, harvesting of tissues/collecting relevant endpoint data, role for HDAC1/HDAC2 inhibitors giving rise to an as of yet etc.): M. Bariteau undiscovered mechanism for leading to cell death. The use of more specific HDAC inhibitors will hopefully lead to new strat- Grant Support egies for patients with B-ALL. This work was supported by NCI grant CA068484 (to S.A. Armstrong), NIH grant CA128972 (to J. Qi and J.E. Bradner), the Leukemia and Lymphoma Disclosure of Potential Conflicts of Interest Society, and the MMRF (to J.E. Bradner). M.C. Stubbs is an employee of Incyte Corporation. S.A. Armstrong is a The costs of publication of this article were defrayed in part by the consultant/advisory board member for Epizyme Inc. No potential conflicts of payment of page charges. This article must therefore be hereby marked interest were disclosed by the other authors. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors' Contributions Conception and design: M.C. Stubbs, W. Kim, A.L. Kung, S.A. Armstrong Development of methodology: M.C. Stubbs, W. Kim, S. Vempati, A.V. Krivtsov, Received May 20, 2014; revised January 9, 2015; accepted January 25, 2015; J.E. Bradner published OnlineFirst February 16, 2015.

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Selective Inhibition of HDAC1 and HDAC2 as a Potential Therapeutic Option for B-ALL

Matthew C. Stubbs, Wonil Kim, Megan Bariteau, et al.

Clin Cancer Res Published OnlineFirst February 16, 2015.

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