Efficacy of Cyclin-Dependent-Kinase 9 Inhibitors in a Murine Model Of

ORIGINAL ARTICLE Efﬁcacy of cyclin-dependent-kinase 9 inhibitors in a murine model of mixed-lineage leukemia

M-P Garcia-Cuellar1,EFu¨ ller1,EMa¨thner1, C Breitinger1, K Hetzner1, L Zeitlmann2, A Borkhardt3 and RK Slany1

Mixed-lineage leukemia fusion proteins activate their target genes predominantly by stimulating transcriptional elongation. A core component necessary for this activity is cyclin-dependent kinase 9. Here we explored the effectiveness of small molecules targeting this enzyme as potential therapeutics. A screen of seven compounds with anti-CDK9 activity applied to a panel of leukemia cell lines identified flavopiridol and the experimental inhibitor PC585 as superior in efficacy with inhibitory concentrations in the submicromolar range. Both substances induced rapid dephosphorylation of the RNA polymerase II C-terminal domain, accompanied by downregulation of CDK9-dependent transcripts for MYC and HOXA9. Global gene expression analysis indicated the induction of a general stress response program, culminating in widespread apoptosis. Importantly, colony-forming activity in leukemia lines and primary patient samples could be completely inhibited under conditions that did not affect native precursors from bone marrow. In vivo application in a mouse transplant model significantly delayed disease with PC585 showing also oral activity. These results suggest CDK9 inhibition as novel treatment option for mixed-lineage leukemia.

Leukemia (2014) 28, 1427–1435; doi:10.1038/leu.2014.40 Keywords: mixed-lineage leukemia; CDK9; inhibitor; preclinical study

INTRODUCTION complex) including P-TEFb is responsible for elongation control,5 Despite considerable progress in leukemia treatment, mixed- DotCom (DOT1L complex) methylates histone H3 at lysine 79 lineage leukemia (MLL) remains a disease with a very dismal through the catalytic activity of the histone methyltransferase 12 prognosis. Five-year survival rates for affected patients hover well DOT1L, and the MLL fusion partner ENL forms a scaffold that below 50%.1,2 MLL occurs mostly either in infants or in adults as integrates these subcomplexes and in addition it neutralizes the 13 late sequelae after previous treatment for an unrelated neoplastic repressive activity of polycomb proteins. Importantly, all these condition. The hallmark of MLL is an aberration of chromosome functions are essential for the transforming activity of MLL fusion eleven, affecting the gene coding for the histone proteins. methyltransferase MLL. As a consequence, transforming fusion In an effort to develop new therapeutics with activity against proteins are created that join part of MLL with a variety of MLL fusions, small-molecule inhibitors of the DOT1L methyltrans- different fusion partners.3,4 We and others have shown that the ferase have been designed and tested in early preclinical trials 14,15 most frequent MLL partners are members of a complex that where they showed efficacy in mouse models of MLL. The specifically stimulates transcriptional elongation.5–8 By this strategies for pharmaceutical targeting of transcriptional mechanism, MLL fusions activate an oncogenic network that elongation are represented by recent publications that use the 16–19 includes the clustered HOX-homeobox genes as important experimental inhibitors JQ1 and/or iBET. These substances determinators of hematopoietic development.9 block the activity of the BET (bromodomain and extra Control of transcriptional elongation is the predominant bromodomain) protein family (BRD2/3/4 and BRDT) by regulatory checkpoint for many genes involved in proliferation, selectively disrupting their binding to chromatin. BET proteins differentiation, and for immediate response transcripts (for are normally instrumental for the proper recruitment of P-TEFb to reviews of this topic see Adelman and Lis10 and Peterlin and acetylated histones. This mark is particularly prevalent in the area Price11). On a molecular level, the rate of elongation is determined surrounding actively initiated transcription units. Because many by the phosphorylation status of the C-terminal repeat domain of growth-related transcripts, with MYC as a prime example, depend RNA polymerase II (CTD). During initiation, TFIIH-associated on P-TEFb, the effect of BET inhibitors is pleiotropic. Consequently, kinases CDK7 and CDK8 phosphorylate Ser5 of the CTD- these molecules have been variably proposed as general heptapeptide. Subsequently, Ser2 needs to be converted to anti-cancer agents targeting mainly MYC17,18 or as a tool for phosphoserine for efficient elongation. This reaction is catalyzed intervention in MLL-fusion leukemia because of the particular by P-TEFb (positive transcription elongation factor b) a dimer of importance of P-TEFb in this disease.16 Despite their promise, CDK9 and a cyclin T. P-TEFb purifies with MLL fusion partners in a these compounds are new, with partially difficult pharmacological large macromolecular complex called EAP (ENL-interacting properties, and ‘first in man’ studies have yet to be reported. proteins or elongation-assisting proteins).6 EAP contains at least In contrast, CDK9 inhibitors have been developed for many years. three separable functional modules: SEC (super elongation Although results are mostly outstanding, flavopiridol (alvocidib),

1Department of Genetics, University Erlangen, Erlangen, Germany; 2Ingenium Pharmaceuticals AG, Munich, Germany and 3Clinic of Paediatric Oncology, Haematology and Clinical Immunology, Centre for Child and Adolescent Health, Medical Faculty, Heinrich-Heine-University Du¨sseldorf, Du¨sseldorf, Germany. Correspondence: Professor RK Slany, Deptartment of Genetics, University Erlangen, Erwin Rommel Str. 3, Erlangen 91058, Germany. E-mail: [email protected] Received 11 November 2013; revised 10 January 2014; accepted 15 January 2014; accepted article preview online 21 January 2014; advance online publication, 11 February 2014 CDK9 inhibitors in mixed-lineage leukemia M-P Garcia-Cuellar et al 1428 the best investigated CDK9 inhibitor, was already included in a enriched with 10 ng/ml IL7 (Stemcell Technologies, Grenoble, France). number of mostly phase I and phase II studies testing safety and Primary precursor cells from mouse bone marrow were isolated by magnetic response in a wide variety of solid and hematological tumors selection of CD117 (c-kit)-positive cells according to the instructions (ClinicalTrials.gov). of the manufacturer (Miltenyi Biotech, BergischGladbach, Germany). Here we examined the potential of CDK9 inhibition as potential Transplantation studies were done in a syngeneic model. Three days before transplantation Balb/C mice were prophylactically treated with therapeutic intervention for mixed-lineage leukemia. We show 6 1.1 g/l neomycin and 1 Â 10 units/l polymixinB as addition to drinking efficacy of this approach in vitro and in vivo suggesting that MLL water. Twenty-four hours after sublethal total body irradiation (6 Gy), should be included as further indication for future CDK9 inhibitor 1 Â 106 MLL-ENL-transformed cells were transplanted by retroorbital trials. injection. Treatment commenced either 2 days or 14 days after transplant by subcutaneous injection or by oral gavage. Substances for injection were dissolved in 25% DMSO/75% PBS. Suspensions for oral gavage were MATERIALS AND METHODS prepared in 0.8% methocel in H2O with 300 ml administered per individual Cell lines dose. Animals with clear signs of disease (hunched posture, ruffled fur, labored breathing) were euthanized. Spleen weight was recorded as an Human leukemia lines with MLL rearrangement (HB11;19, SEM, KOPN-8, objective surrogate marker for leukemia burden. Cohorts were followed for MV4;11, RS4,11, THP1 and Karpas45) and cells without involvement of 11q23 (HL60, K562, U937, REH and Jurkat) were obtained from the German a maximum of 120 days and surviving animals without signs of collection of microorganisms and cell cultures (DSMZ, Braunschweig, leukemia (normal physiology, spleen weighto100 mg, no infiltration Germany) or they were laboratory stocks. All human lines were kept in of leukemia cells in lymph nodes or inner organs) were censored from RPMI1640 supplemented with 10% fetal calf serum (FCS), except Karpas45 the results. Engraftment rates were generally between 80% and 100%. Animal procedures have been approved by local and institutional animal that was grown in 20% FCS. Mouse MLL-ENL-transformed lines Me2 and welfare review boards under license numbers 54–2532.1–41/11 and Me3, as well as cells used for transplantation assays were obtained by 20 54–2532.2–5/12. retroviral transduction as described. MeerIV was derived from a mouse with a germ-line integration of an inducible MLL-ENL-ER construct.21 Murine cells were cultivated in RPMI, 10% FCS and recombinant murine cytokines: 50 ng/ml stem cell factor, 5 ng/ml each of IL3, IL6 and RESULTS GM-CSF. For MeerIV cells, 100 nM 4-hydroxytamoxifen was added to Leukemia cell lines are sensitive to CDK9 inhibition in vitro activate MLL-ENL. To select for the most efficient CDK9 inhibitors, we employed seven human leukemia cell lines with MLL rearrangement (Karpas, CDK9 inhibitors, proliferation assays, antibodies THP1, RS4;11, MV4;11, SEM, KOPN-8, HB11;19) and five human cell Alsterpaullone, CDK9 inhibitorII and CDC7_CDK9 inhibitor were bought lines derived from hematological malignancies without 11q23 from Merck/Calbiochem (Darmstadt, Germany). Flavopiridol was supplied involvement (HL60, K562, U937, REH, Jurkat). In addition, we by Sigma (Taufkirchen, Germany). PC142, PC579 and PC585 are proprietary CDK9 inhibitors supplied by Ingenium Pharmaceuticals GmbH (Martinsried, created three different murine MLL lines with MLL-ENL as the sole Germany). All stock solutions were prepared in dimethyl sufoxide (DMSO). genetic abnormality either by retroviral transduction of primary To determine the inhibitor concentrations necessary for half-maximal BM precursors (Me2, Me3) or by induction of the genomic copy of inhibition of proliferation (ID50), standard MTT tests were applied an inducible Mll-ENL-ER knocked-in into the germ line of the Meer (Promega, Mannheim, Germany). In short, each cell line was incubated in mouse model21 (MeerIV). Four commercially available substances triplicate with increasing concentrations of inhibitor in the respective with a reported activity against CDK9 (alsterpaullone, CDK9 growth medium. After 48–96 h, depending on the proliferation rate, cell inhibitorII, CDC7_CDK9 inhibitor, flavopiridol) and three novel proliferation was estimated by MTT addition, solubilization and photo- experimental compounds (PC142, PC579, PC585) were tested in metric readout at 550 nm wavelength. To compare different lines, standard MTT assays (Figure 1). When plotted as normalized normalized absorptions were calculated setting the maximum measured absorption value to one unit. concentration–response curves, PC585 and flavopiridol were most Antibodies specific for the Ser2-phosphorylated version of RNA potent with IC50 values ranging around 100 nM and 50 nM, Polymerase II and for H3K27me2/3 were from ActiveMotif (LaHulpe, respectively. All other inhibitors had to be administered at Belgium). 41 mM to elicit a similar effect. Similar to the situation with BET inhibitors that impede growth of leukemia lines with various 18 Gene expression analysis etiologies, there was no significant separation of cells according Primers used for RT-qPCR were: mouse Hoxa9: fw; 50-CGGCCTTATGGCATTA to MLL status. The cohort of patient cell lines was generally more AACCTG-30; rev; 50-GAGCGAGCATGTAGCCAGTTG-30; human Hoxa9: fw; resistant, probably reflecting the fact that these cells contain an 50-CTGTCCCACGCTTGACACTCAC-30; rev; 50-CTCCGCCGCTCTCATTCTCAG-30; unknown number of additional mutations. Yet, the defined murine mouse Myc: fw; 50-CCTAGTGCTGCATGAGGAGACAC-30; rev; 50-GCACCAGAG lines, that harbor only a single oncogenic lesion clearly clustered TTTCGAAGCTGTTC-30;humanMYC:fw;50-GTCAAGAGGCGAACACACAACG-30; as most sensitive under PC585 and flavopiridol treatment. Because rev; 50-TTGGACGGACAGGATGTATGCTG-30. of their favorable concentration–response profile, we selected RNA for global gene expression analysis was isolated from two flavopiridol and PC585 for all further experiments. independent control/treated pairs of Me2 cells cultured for 6 h in 250 nM flavopiridol, PC585 or vehicle (DMSO). Hybridization to Agilent 8 Â 60 K mouse expression arrays was done commercially by OakLabs (Hennigsdorf, Rapid dephosphorylation of RNA polymerase II after blockade of Germany) according to the instructions of the manufacturer. Gene-set CDK9 activity 22 enrichment analysis was performed as described. Raw data have been Two mouse and two human cell lines with MLL rearrangement deposited to the EBI repository under accession number MEXP-3978. were chosen to further explore the biochemical consequences of flavopiridol and PC585 treatment. MV4;11 and HB11;19 represent Apoptosis, colony-forming cell (CFC) and transplantation assays myeloid and lymphoid examples of 11q23 leukemia. Transforma- Apoptosis was recorded by annexinV and propidiumiodide staining tion of primary mouse cells with MLL-ENL yields predominantly according to standard protocols. myeloid lines and two independently derived lines (Me2 and M3) For CFC assays cells were incubated in their respective growth medium were included in further experiments. Ser2-phosphorylated RNA with 100 nM of CDK9 inhibitors for the indicated time. Subsequently, an aliquot was withdrawn, washed and 5000 cells were plated in methylcellu- PolII was determined by western blotting with a specific antibody lose (R&D Systems, Wiesbaden, Germany) supplemented appropriately as in to detect the effect of CDK9 inhibition (Figure 2). After 6 h of normal growth medium. For patient material, 100 ng/ml stem cell factor, incubation with 100 nM inhibitor, the levels of this biochemical 10 ng/ml Flt3 ligand and 10 ng/ml IL7 were added to the incubation marker dropped significantly in all cells supporting the excellent medium and 1 Â 105 cells were plated in MethocultH4035 without EPO efficacy of the inhibitors at these low concentrations.

Leukemia (2014) 1427 – 1435 & 2014 Macmillan Publishers Limited CDK9 inhibitors in mixed-lineage leukemia M-P Garcia-Cuellar et al 1429

Figure 1. Dose-dependent proliferation of leukemia cell lines in the presence of CDK9 inhibitors. Human and mouse leukemia cell lines were incubated in triplicate experiments with increasing concentrations of various substances with reported activity against CDK9. Proliferation was recorded with a standard MTT test. Plotted are the average and s.d. of normalized OD550 values, where the maximal absorption was set to one unit. Cell lines are color coded with human lines without MLL involvement, blue; human MLL-rearranged lines, green; primary mouse cells transformed by MLL-ENL, red.

CDK9 inhibition efficiently blocks MYC transcription and patient cell lines and flavopiridol was slightly more potent than profoundly alters gene expression programs PC585. Whereas MYC transcription practically ceased with 100 nM Next, the consequences of an abrogation of CDK9 activity for gene inhibitor in Me2 and Me3, patient lines needed up to 500 nM to expression in MLL-rearranged cells were recorded. After treatment reach the same effect. Likewise, 250–500 nM of inhibitor were of four sample cell lines (Me2, Me3, MV4;11, HB11;19) for 6 h with necessary to record an effect on Hoxa9 steady-state RNA levels in variable concentrations of flavopiridol, PC585 or vehicle (DMSO) the murine samples, whereas this concentration was mostly RNA was isolated. Because it is well known that transcription of insufficient to cause a discernible drop of the corresponding MYC and HOXA9 is regulated to a large extent by elongation, the human transcript within the observation period. Suboptimal relative amounts of these RNAs were determined by RT-qPCR in concentrations of the compounds reversed the inhibitory effect treated and control cells (Figure 3). In the absence of active CDK9, and increased MYC as well as HOXA9 transcription. This MYC transcription generally was more severely affected than phenomenon has been observed also with BET inhibitors and is production of HOXA9 RNA. Mirroring the results of the prolifera- likely due to a transient release of CDK9 from an inactivating tion assays, murine MLL-ENL cells proved more sensitive than riboprotein complex.23

& 2014 Macmillan Publishers Limited Leukemia (2014) 1427 – 1435 CDK9 inhibitors in mixed-lineage leukemia M-P Garcia-Cuellar et al 1430 To get a more comprehensive insight into the consequences of flavopiridol, PC585 or with DMSO. RNA was isolated and CDK9 inhibition global gene expression studies were performed hybridized to expression arrays. Corresponding to the predomi- (Figure 4a). Mouse Me2 cells were treated for 6 h with 250 nM nant role of CDK9 in transcriptional control, pervasive alterations in the RNA inventory could be detected with about twice as many transcripts repressed versus increased. In agreement with its lower potency to inhibit MYC and HOXA9 expression, PC585 was somewhat less efficient with 4796 transcripts significantly down (42-fold, Po0.05) compared with 7203 for flavopiridol. Similar results were obtained for genes induced in response to treatment, which totaled 2042 transcripts for PC585 and 3809 for flavopiridol, respectively (Supplementary Table 1). Generally, there was a good qualitative concordance between PC585 and flavopiridol-trig- gered alterations. Approximately 75% of all genes downregulated by PC585 and 61% of induced genes were also contained in the flavopiridol signatures. Sentinel genes that are known to be activated in response to CDK9 inhibition are HEXIM1 or HEXIM2 (hexamethylenbis-acetamide inducible; hexamethylenbisacetamid ¼ CDK9 inhibitor) with HEXIM2 induced 69-fold under flavopiridol and 30-fold under PC585 treatment (Supplementary Table 1). Genes repressed by flavopiridol or PC585 encompassed a substantial number of direct MLL-AF9 and MLL-AF4 targets as reported by Bernt et al.24 and Gu¨nther et al.25 (Figure 4a, right panel). Also, 221 out of 491 genes that have been suggested by Wilkinson et al.26 to be under control of MLL-AF4 were repressed by flavopiridol and 156 of those were affected by PC585. Yet, CDK9 inhibition affected many genes beyond MLL fusion targets. This is concomitant with the known importance of CDK9 for transcription of genes that are not activated by MLL fusions. A quantitative comparison was performed by plotting all commonly affected genes according to their amplitude of Figure 2. Rapid dephosphorylation of RNA polymerase II after CDK9 regulation achieved by flavopiridol and PC585 (Figure 4b). This inhibition. MLL-rearranged cells as indicated were incubated for 6 h analysis revealed differences for both substances resulting in a low in the presence of 100 nM flavopiridol, PC585 or DMSO as control. correlation coefficient (Pearson r ¼ 0.17). Expectedly, most genes The amount of Ser2-phosphorylated RNA polymerase II, a major substrate of CDK9, was determined by immunoblotting with a were more severely blocked by flavopiridol than by PC585, yet the specific antibody. Actin was used as loading control. In mouse, cells rank order of transcripts was changed. This suggested that, the canonical ‘o’ (upper band) and ‘a’ (lower band) subforms of RNA although both compounds target CDK9 and affect a common polymerase II could be detected. This figure displays one of two gene repertoire, the actual biochemical mechanism must be experiments with essentially identical outcome. different. Notably, PC585 shows substantially higher selectivity for

Figure 3. Transcription of MYC and HOXA9 is dependent on CDK9. RNA was isolated from cell lines treated with various concentrations of DDCt ﬂavopiridol, PC585 or DMSO (value 0 nM) for 6 h. MYC and HOXA9 RNAs were quantiﬁed by RT-qPCR relative to actin by the 2 method. Plotted are averages and s.d. of technical triplicates as relative quantities. This experiment was repeated once with comparable results.

Leukemia (2014) 1427 – 1435 & 2014 Macmillan Publishers Limited CDK9 inhibitors in mixed-lineage leukemia M-P Garcia-Cuellar et al 1431 CDK9 compared with flavopiridol (results to be published (splenomegaly, pale organs and infiltrations of leukemic cells in separately). In contrast, the gene expression pattern induced by liver, enlarged lymph nodes). As a surrogate for leukemia burden, treatment was highly correlated (r ¼ 0.92) indicating a similar spleen weight was recorded. cellular response elicited by both substances albeit with An initial dose of 2 mg/kg flavopiridol in 25% DMSO/75% PBS or flavopiridol causing stronger effects. vehicle as control was administered by five subcutaneous Gene-set enrichment analysis yielded a strong similarity of injections every other day (Figure 6b, left panel). Under this flavopiridol inhibited genes with transcripts that get switched off treatment, a significant survival advantage was observed for the after UV irradiation (false discovery rate ¼ 9.2 Â 10 À 4) (Figure 4c). treated versus control cohort. This result was confirmed with an Owing to the weaker response amplitude, gene-set enrichment independently derived graft and a modified application schedule analysis did not find a significant overlap of transcripts blocked (Figure 6b, right panel). Five daily injections led to an improved under PC585 treatment with the UV-gene set. Still, a manual response compared to bi-daily dosage. An attempt to administer comparison showed that many hits of PC585 are also shared with 10 consecutive injections of flavopiridol was not successful, as the the UV-response pattern. For the group of genes induced by the animals showed signs of acute toxicity and the experiment had to inhibitors, a highly suggestive overlap was detected with genes be terminated (not shown). There was a trend toward lower that are marked by the repressive histone methylation H3K27me3 spleen weights under flavopiridol treatment; however, the values (false discovery rate ¼ 0). These genes are normally targets of did not reach statistical significance. polycomb complexes that silence many tumor suppressor genes. To offset the lower efficacy and the limited solubility of PC585 in Western blots of nuclear extracts showed that global H3K27me2/3 DMSO/PBS subcutaneous injections of 10 mg/kg were applied. modification was unaffected either by flavopiridol or PC585 This treatment was not efficacious when started 14 days after (Figure 4d), suggesting that this response is gene specific. transplant at a time point when leukemic cells have already fully engrafted and started to multiply. Yet, under less stringent CDK9 is necessary for survival and leukemic CFC activity conditions with treatment commencing already at day 2 after transplant (treatment ‘early’), a clear effect could be recorded To investigate the long-term outcome of CDK9 inhibition on (Figure 6c, left panel). Subcutaneous injection of PC585 caused cellular physiology, we incubated the four test lines for 18 h with localized transient ulceration indicating a suboptimal systemic varying concentrations of inhibitors and recorded cell viability and distribution of this compound. Therefore, another independent apoptosis by annexinV/propidiumiodide staining (Figure 5a). experiment was set up with oral administration of 30 mg/kg PC585 Differences in sensitivity between mouse and human cell lines suspended in methylcellulose given twice weekly with a three and became smaller after prolonged incubation. After 18 h, half- a half day interval between doses. Three animals of the cohort had maximal apoptosis was observed between 100 nM and 150 nM for to be killed after two administrations due to unexpected bleeding both inhibitors. As an exception, MV4;11 showed an even higher events (multiple petechiae and skin hematomas). Hemorrhage sensitivity toward flavopiridol (60 nM dose for 50% apoptosis). Only was most likely associated with the preceding irradiation and HB11;19 was largely resistant to PC585 at the concentrations reconstitution procedure since much higher doses of PC585 are tested. well tolerated by non-irradiated mice (not shown). The remaining Detection of apoptosis in total cell populations may be not the PC585-treated animals survived significantly longer than the optimal parameter to judge a potential therapeutic response vehicle-only group and spleen weights were also clearly reduced because it is known that a small treatment-resistant population of compared to controls. The effectiveness of oral treatment with cells (leukemia-repopulating cells) may actually reconstitute the PC585 correlated with high oral availability of the compound as disease. The remaining cells then can cause relapses even after determined in independent PK studies (results to be published successful eradication of the leukemic bulk. Therefore, we separately). performed CFC assays to detect the specific effects of CDK9 on Finally, we recorded the response of primary patient material cells with repopulating capacity (Figure 5b). In addition to toward CDK9 inhibitors. CFC assays were set up with live leukemic leukemic cell lines, native precursors isolated from mouse bone blast cells obtained from peripheral blood (Figure 6d). Primary marrow were included (c-kit-positive cells). The cells were patient material contained fewer CFCs compared with established incubated in solutions containing 100 nM flavopiridol, PC585 or cell lines. Nevertheless, similar to the previous results, CFC activity DMSO and samples were drawn at preset intervals. The cells were was largely abrogated after contact with 100 nM of CDK9 inhibitors washed and plated in methylcellulose to detect self-renewal for 16–24 h. activity. MLL-ENL-transformed populations displayed an exquisite sensitivity toward CDK9 inhibition and after 20 h of incubation with inhibitors, CFC activity was completely abolished. In stark DISCUSSION contrast, normal precursor cells could withstand 45 h in the Targeting disease specific pathways is an appealing strategy to presence of these compounds without any significant effect on treat malignant states without causing extensive collateral CFC numbers. Human patient lines proved slightly more resistant damage to normal cells. The application of ABL kinase inhibitors but most of CFC activity was lost after 30 h. This suggested an for chronic myeloid leukemia has set the precedence for this exploitable therapeutic window for CDK9 inhibitors. approach and it is still the benchmark against which all other focused therapies will be measured. The elucidation of the Blocking CDK9 activity prolongs survival in a mouse molecular underpinnings that drive mixed-lineage leukemia made transplantation model and inhibits CFC activity in primary it possible to explore new avenues for a more specific treatment. patient cells Besides inhibition of DOT1L that deposits the high levels of H3K79 To assess the feasibility of CDK9 inhibition as therapeutic option in methylation that are necessary for MLL mediated transforma- a more clinically relevant setting, a series of transplantation tion,15,24 P-TEFb and elongation stimulation have received experiments were performed (Figure 6a). Sublethally (6 Gy) heightened attention under a therapeutical aspect. Originally, irradiated BALB/C mice were transplanted with syngeneic MLL- small-molecule BET inhibitors (JQ1 and iBETs) that displace BRD4 ENL-transformed cells (1 Â 106 cells per animal). In total, three from chromatin have been developed as an anti-MYC strategy.17 different, independently generated MLL-ENL lines were used as Transcription of MYC is particularly dependent on P-TEFb function graft. Treatment was started after engraftment of leukemic cells 14 and BRD4 is essential for proper localization of this activity on the days after transplant (treatment schedule ‘late’). Moribund mice MYC transcriptional unit. With respect to mixed-lineage leukemia, were euthanized and examined for signs of advanced leukemia the seminal discovery was made in an unbiased shRNA screen.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1427 – 1435 CDK9 inhibitors in mixed-lineage leukemia M-P Garcia-Cuellar et al 1432 This experiment identiﬁed the BET protein BRD4 as essential for meantime, BRD4 was recognized to be a member of the ‘super survival of MLL-AF9-transformed cells19 paving the way for elongation complex’ that associates with MLL fusion proteins application of BET inhibitors in this leukemia subtype. In the giving a rationale for the activity of BET inhibitors in this disease

Leukemia (2014) 1427 – 1435 & 2014 Macmillan Publishers Limited CDK9 inhibitors in mixed-lineage leukemia M-P Garcia-Cuellar et al 1433 setting.16 MYC is an essential oncogenic hub in many malignancies beyond MLL, suggesting a wider applicability of BET inhibitors. Indeed, JQ1 blocked growth of many different leukemia lines of various etiology and this compound has been successfully administered in a xenograft model of Burkitt’s lymphoma.18 Similarily, flavopiridol and PC585 showed good efficacy in proliferation assays also against ‘non-MLL’ cell lines, indicating a possible wider potential for therapy. Blocking the key enzymatic activity catalyzed by CDK9 is a more direct approach to target P-TEFb. This has the advantage that CDK9 inhibitors such as flavopiridol are well studied and these substances have been validated already in clinical trials. The biochemical pathways targeted by BET inhibitors are also sensitive to CDK9 inhibition. Transcription of MYC is efficiently abrogated in the absence of active CDK9 and also MLL-transformed cells need CDK9 for self-renewal and efficient leukemogenesis. In addition, most rapidly dividing cancer cells are likely dependent on MYC and other key oncogenic drivers that are controlled by a special class of ‘super enhancers’.27 BRD4 and P-TEFb are involved in the regulation of these elements nurturing the hope that P-TEFb inhibition may specifically target cancer cells. Interestingly about half of the cellular P-TEFb is rendered inactive by storing it in a riboprotein complex containing 7SK RNA, HEXIM1/2 and LARP7.28 To become active, it has to be released and to bind either to BRD4, transcription factors or other SEC members. Paradoxically, the application of BRD4 inhibitors initially leads to a release of P-TEFb from the inactive complex, thus swamping the cell with active P-TEFb causing a transient transcriptional burst of P-TEFb-dependent RNAs.23 The equilibrium is reinstated by increased production of HEXIM till eventually all CDK9 is sequestered and repression prevails. Here we see a comparable effect with suboptimal concentrations of CDK9 inhibitors actually inducing transcription of MYC and HOXA9. In addition, HEXIM2 is strongly upregulated after flavopiridol and PC585 treatment. Because P-TEFb will be engaged by various effector molecules to drive transcription, CDK9 inhibition should have a more pleiotropic effect than disabling a single ‘recruiter’ like BRD4. This is probably the reason why no clear MYC-related signature showed up in the gene set after disabling CDK9. On the other hand, the similarity to an UV-repressed gene expression profile is

Figure 5. CDK9 inhibition causes apoptosis and loss of CFC activity in leukemia lines. (a) Result of annexinV/propidiumiodide staining of MLL leukemia cell lines after incubation with increasing concentrations of CDK9 inhibitors for 18 h. AnnexinV/propidiumiodide- negative cells were deﬁned as live (blue lines) and cells positive for either or both of these two markers are labeled apoptotic (red lines). Cells staining only for propidiumiodide have been excluded as post-apoptotic breakdown fragments. (b) CFC assays of leukemia lines. Five thousand cells were plated per well of a 24-well plate after incubation for the indicated time in DMSO or 100 nM of CDK9 inhibitors. Colonies were stained and photographed after development of macroscopically visible clusters. Normal bone marrow precursor cells (BMC) isolated by magnetic selection of CD117 (c-kit)- positive cells were used as control. This ﬁgure displays a typical example of two independent experiments.

Figure 4. Alterations of global gene expression patterns after CDK9 inhibition. RNA was isolated from murine Me2 cells after treatment with 250 nM flavopiridol, PC585 or DMSO for 6 h. RNA from duplicate samples for each control/treatment pair was hybridized to 8 Â 60 K gene expression arrays. (a) Venn diagram depicting significant gene expression changes. Left panel: effect of flavopiridol and PC585 treatment. Right panel: The flavopiridol and PC585 repressed gene signature overlaps with direct targets of MLL-AF9 according to Bernt et al.24 and MLL- AF4 as suggested by Gu¨ nther et al.25 (b) Dot plot arraying common genes affected by flavopiridol and PC585 according to their fold-change (log2) values. Pearson ‘r’ coefficients are indicated. (c) Gene-set enrichment analysis for genes repressed/activated in response to flavopiridol and PC585. (d) Western blot of nuclear extracts isolated from Me2 cells treated with 250 nM flavopiridol, PC585 or DMSO for 6 h. Global H3K27 di- and trimethylation was detected by immunostaining with total histone H3 as loading control.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1427 – 1435 CDK9 inhibitors in mixed-lineage leukemia M-P Garcia-Cuellar et al 1434

Figure 6. CDK9 inhibition is efficacious in transplantation experiments and eradicates CFC activity in primary patient material. (a) Schematic depiction of experimental protocol. Transplantation was done after sublethal irradiation (6 Gy total body irradiation). Treatment commenced either ‘early’ at day 2 after transplant or after full engraftment of the leukemic cells at day 14 after transplant. (b) Kaplan–Meier-type survival curve of flavopiridol treated and control cohorts. The inset dot-plot depicts spleen weights (in milligram) of the respective animals. P-values were calculated by T-test. n.s., not significant. Left panel: experiment done with Me2 cells. Right panel: an independently derived MLL-ENL- transformed cell line was used as graft. (c) Survival of PC585-treated cohorts as above. Left panel: Me2 cells served as transplant. Right panel: transplantation done with newly derived MLL-ENL-transformed cells. An asterisk denotes animals found dead whose spleen weight could not be determined with certainty. (d) CFC assays with primary patient material. Blast cells from peripheral blood were incubated with 100 nM of inhibitors or DMSO as control for the indicated time. A sample of 1 Â 105 cells was plated in methocel and grown till colonies were macroscopically visible. Depicted are stained colonies plated either in duplicates (left panel, patient sample A242) or, because of limited starting material, as single wells (right panel, patient sample A193).

Leukemia (2014) 1427 – 1435 & 2014 Macmillan Publishers Limited CDK9 inhibitors in mixed-lineage leukemia M-P Garcia-Cuellar et al 1435 highly suggestive, as UV treatment can release CDK9 from the 6 Mueller D, Bach C, Zeisig D, Garcia-Cuellar MP, Monroe S, Sreekumar A et al. Arole 29 inactive 7SK complex. As a consequence, HEXIM proteins are for the MLL fusion partner ENL in transcriptional elongation and chromatin produced and eventually CDK9-dependent gene expression is modification. Blood 2007; 110: 4445–4454. shut down. The simultaneous induction of genes normally marked 7 Mueller D, Garcia-Cuellar MP, Bach C, Buhl S, Maethner E, Slany RK. Misguided by H3K27 methylation hints at an hitherto unknown connection transcriptional elongation causes mixed lineage leukemia. PLoS Biol 2009; 7: e1000249. between CDK9 and the chromatin-based repression machinery. 8 Yokoyama A, Lin M, Naresh A, Kitabayashi I, Cleary ML. 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